peerj_json_files/PeerJ_Json_100.json

{
    "v1_Abstract": "While female mate preference is very well studied, male preference has only recently begun to receive significant attention. Its existence is found in numerous taxa, but empirical research has mostly been limited to a descriptive level and does not fully address the factors influencing its evolution. We attempted to address this issue using preference functions by comparing the strength of male preference for females of different sizes in nine populations of four poeciliid species. Due to environmental constraints (water toxicity and surface versus cave habitat), females from these populations vary in the degree to which their size is correlated to their fecundity. Hence, they vary in how their size signals their quality as mates. Since female size is strongly correlated with fecundity in this subfamily, males were sequentially presented with conspecific females of three different size categories and the strength of their preference for each was measured. Males preferred larger females in all populations, as predicted. However, the degree to which males preferred each size category, as measured by association time, was not correlated with its fecundity. In addition, cave males discriminated against smaller females more than surface males. Assuming that male preference is correlated with female fitness, these results suggest that factors other than fecundity have a strong influence on female fitness in these species.",
    "v2_Abstract": "While female mate preference is very well studied, male preference has only recently begun to receive significant attention. Its existence is found in numerous taxa, but empirical research has mostly been limited to a descriptive level and does not fully address the factors influencing its evolution. We attempted to address this issue using preference functions by comparing the strength of male preference for females of different sizes in nine populations of four poeciliid species. Due to environmental constraints (water toxicity and surface versus cave habitat), females from these populations vary in the degree to which their size is correlated to their fecundity. Hence, they vary in how their size signals their quality as mates. Since female size is strongly correlated with fecundity in this subfamily, males were sequentially presented with conspecific females of three different size categories and the strength of their preference for each was measured. Males preferred larger females in all populations, as predicted. However, the degree to which males preferred each size category, as measured by association time, was not correlated with its fecundity. In addition, cave males discriminated against smaller females more than surface males. These results suggest that factors other than fecundity have a stronger influence on female fitness in these species.",
    "v1_text": "materials & methods : Species and populations: Nine populations of four poeciliid species representing different habitat types were used (summarized in Table 1). Gambusia eurystoma is a surface species endemic to the sulfidic Ba\u00f1os del Azufre in Tabasco, Mexico (Tobler et al. 2008c). Limia sulphurophila is another surface fish living in a sulfidic habitat, but it is endemic to a small pool in the island of Hispaniola (Rivas 1984). The population of G. sexradiata used lives in non-sulfidic surface waters. The six populations of P. mexicana used in this study live in different habitats. The Oxolotan population is named after the non-sulfidic, surface river it originates from. The PS0 population also lives in a surface creek, but whose water is sulfidic. The water from this creek, named El Azufre, originates from Cueva del Azufre, a sulfidic cave from which three of the other populations originated. These populations, inhabiting a dark and toxic environment, are the PSV, PSX, and PSXIII populations (For a schematic map of the region and of the cave, see Plath et al. 2010, and Tobler et al. 2006). They are named after the chamber of the cave in which they live. The sixth population is the Luna population, which originates from a non-toxic cave of the same name (Tobler et al. 2008c). Fish from all of the populations are maintained in flow-through stock tanks in the Aquatic Research Facility at the University of Oklahoma, and have been in captivity for varying lengths of time (Table 1). These tanks were the immediate source of the fish used in this study. All are maintained in nonsulfidic, common garden conditions inside a greenhouse that receives natural light. Fish from the stock tanks were caught with a small seine and segregated by sex. Mature females were then selected using minimum standard length (tip of the snout to the end of the vertebral column) as the criterion for sexual maturity. Due to natural size differences between species, the exact criterion used varied (P. mexicana: 29mm for the Luna population, and 30mm for all others; G. eurystoma: 22mm; G. sexradiata: 18mm; and L. sulphurophila: 21mm). The 4 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 8 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t mature females were then sorted into roughly equally sized groups according to size (small, medium, and large), and were then placed in different 38L stock tanks. Males were randomly assigned an ID number that determined the order in which they would be tested, and they were housed in individual 5L tanks that were out of sight of the females. Experimental Setup: Preference functions are established by measuring the amount of time a focus individual spends in association with different stimulus fish. These stimulus fish are presented sequentially and differ in the variable in question. In this case, females of different sizes were sequentially presented to a male, and the time that the male spent with each female was recorded. To do this, a 76L aquarium, with gravel spread evenly to reduce potential bias from fish being distracted by uneven gravel, was divided lengthwise into three equal sections with two vertical lines drawn on the glass (Fig 2). The outer two sections were considered the \"preference zones\", while the central section was considered a \"neutral zone\". Three hollow square prisms (or \"cylinder\") made out of clear plexiglass were located in the center of each section of the tank. The cylinder in the center of the tank had solid walls, while the two outer cylinders were perforated with seven circular holes 6mm in diameter to allow for chemical and mechanosensory signals. Chemical and mechanosensory signals have been found to be important factors in poeciliid mating behavior, influencing the repeatability of individual preferences as well as the overall preference (Coleman 2011; Hoysak & Godin, 2007; Plath et al. 2006; R\u00fcschenbaum & Schlupp 2013). All three cylinders were 8.5cm long by 8.5cm wide, and tall enough to stick through the water. To reduce visual distractions, three sides of the tank were covered. The observer sat in a chair 2m from the tank and observed the fish through the front pane of the tank. A light with a 60W, \"soft white\" light bulb was placed 30cm above the center of the tank for illumination. Testing Procedure: A randomly selected female from the predetermined size category (small, medium, or large) was placed in the cylinder in one of the outer preference zones. The order of the females which each male would be presented with, as well as the side in which each female would be placed, were randomly determined. A male was then placed in the central cylinder for 5 minutes. 5 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 10 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t After the 5 minutes of acclimation, the cylinder around the male was gently removed. Using two stopwatches, the amount of time the male spent in the preference zone containing the female was measured by the observer. This was done for 5 minutes, after which the fish were removed from the tank. The water was then stirred to homogenize any lingering chemical signals from affecting the results of future trials. Another pair of fish was then placed in their corresponding cylinders to acclimate. Every three pairs, a partial water change was also made previous to the acclimation period of the next fish. Male weight and standard length were also measured and used as covariates, but were not included in the final model because neither was significant. Statistical analysis: After checking the assumptions, a mixed between-within subjects ANOVA was performed to determine the effect of two habitat variables on male preference for small, medium, and large females. The two habitat variables used were \"cave habitat\", whether the population originated from a cave or from a surface stream, and \"toxicity\", whether the population originated from a toxic or non-toxic stream. Because the raw results were not normally distributed, the male preference variables were reflected and square root transformed to meet the normality assumption. Experiments were approved by the University of Oklahoma IACUC (R09-030). Results: All statistical assumptions were met after the data transformation, with the exception of homogeneity of variances for the time males spent with medium females (p = 0.026). However, this violation was not deemed to be severe enough to invalidate the ANOVA. As expected, there was a significant main effect for time spent with larger females, regardless of the habitat of origin (Wilks' Lambda = 0.911, F(2, 123)= 5.99, p= 0.003, p 2h = 0.089). The main between-subjects effect comparing toxicity was not significant (F(1, 124)= 0.047, p= 0.829, p 2h = 0.000), nor was the main effect for cave habitat (F(1, 124)= 1.043, p= 0.309, p 2h = 0.008), or the interaction between cave habitat and toxicity (F(1, 124)= 0.011, p= 0.917, p 2h = 0.000). These results suggest that, correcting for the effect of female size, habitat type does not affect male preference values. There was also a significant interaction between cave habitat and the time males spent with females from different size categories (Wilks' Lambda= 0.934, F(2, 123)= 4.36, p= 0.015, p 2h = 6 132 134 136 138 140 142 144 146 148 150 152 154 156 158 160 12 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t 0.066; Fig 3). However, there was no significant interaction between the time males spent with females of different sizes and the toxicity of the habitat from which they originated (Wilks' Lambda = 0.996, F(2, 123)= 0.26, p= 0.77, p 2h = 0.004; Fig 4). There was also no significant interaction between time, cave habitat, and toxicity together (Wilks' Lambda= 0.956, F(2, 123)= 8.86, p= 0.06, p 2h = 0.044). These results suggest that the presence or absence of hydrogen sulfide in the population's habitat of origin does not influence males' preference for female size, but the cave habitat does. Descriptive statistics are summarized in Table 2. Discussion: As predicted, males did exhibit a general preference for larger females when all populations were considered in aggregate. This result is consistent with previous dichotomous-test studies finding preference for larger females in poeciliids (Bisazza et al. 1989; Herdman et al. 2004; Hoysak & Godin 2007; Jeswiet & Godin 2011; Plath et al. 2006) and indicates that absolute preference functions are an accurate tool to study individual preferences. Because preference functions can be used to compare preferences between individuals, they can also be used to address a more specific and broader range of questions than is possible using only dichotomous choice tests. In addition to an overall preference for larger females, the strength of male preference should reflect how tightly correlated female size is with female fecundity. The populations tested originated from habitats with different combinations of two variables- water toxicity as a result of the presence or absence of H2S (\"toxicity\"), and epigean or hypogean habitat (\"cave habitat\"). Previous studies have shown that in P. mexicana (Riesch et al. 2009b; Riesch et al. 2010c), as well as in G. sexradiata and G. eurystoma (Riesch et al. 2010b), female fecundity is strongly correlated with toxicity. Females from toxic habitats have much larger, but fewer, offspring. Because of this, there is a larger change in fecundity from small to large females in nontoxic habitats. The main hypothesis was therefore that the preference function of males from toxic habitats would be less steep than that of males from nontoxic habitats. The results did not support our hypothesis, as there was no significant interaction between time spent with a female and the toxicity of the habitat the male originated from. This result suggests that the change in female fecundity experienced from benign to toxic habitats is only weakly correlated with the change female quality. The reason for this is unclear and could be due to a combination of factors. It is possible that female size in nontoxic habitats is 7 162 164 166 168 170 172 174 176 178 180 182 184 186 188 190 192 14 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t correlated with increased female mortality and/or with a decrease in offspring quality. An alternative possibility is that female size and quality are highly correlated, but that there has been insufficient time for male preference to change as a response to female adaptation. It is currently unknown how long the populations in toxic habitats have been adapting to their environments, and how recently the changes in female fecundity have evolved. Since male preference is likely under weaker selection than other traits, it is possible that males from toxic populations have either not had a sufficient amount of time to adapt, or that the amount of gene flow from nontoxic populations has been able to counteract the effects of selection. It is currently unknown how much gene flow there is between toxic (P. mexicana: PS0, G. sexradiata: populations not used in the present study) and nontoxic (P. mexicana: Oxolotan, G. sexradiata) surface populations. While the amount of gene flow between toxic and non-toxic populations still needs to be determined, it is known that there is very little gene flow between P. mexicana populations from the Cueva del Azufre (PSV, PSX, and PSXIII) and those in the surface (Plath et al., 2010). The genetic isolation of cave fish from surface fish might be an important reason why cave habitat did have a significant effect on male preference. This difference seems to be mainly derived from cave males' relatively low preference for small females (Fig 3), as the change in preference for medium to large females is nearly identical in males from both habitats. This suggests that males are not responding to fecundity or offspring size per se, since one would expect that the change of preference from medium to large females would differ between the two environments. This same logic also suggests that the difference is not due to cave males being choosier than surface males. If males preferred larger females due to the cave habitat leading to a greater cost in male effort, males would disproportionally prefer large females over medium females as well. Instead, these results indicate that there is a relative disadvantage for cave males to mate with smaller females, which could result if small cave females have a lower fitness than small surface females. A fitness difference could occur if mortality rates for small cave female are higher than those of medium or large females, perhaps as a result of differential predation pressures. No direct evidence exists on the relative predation pressures between the two habitats, but there is reason to believe that this possibility might be true. Cave populations of P. mexicana are known to be preyed upon by predators which hunt by sensing tactile and/or chemical signals from fish in close proximity (Horstkotte et al. 2010; Tobler et al. 2008a; Tobler et al. 2009), and may prey disproportionally upon smaller females. At the same time, surface populations experience very 8 194 196 198 200 202 204 206 208 210 212 214 216 218 220 222 16 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t different predation pressures: surface fish are preyed upon by large visual predators (Riesch et al. 2009a, 2010a; Tobler et al. 2006) that target larger fish (Trexler et al. 1994). Thus, medium and large surface females are likely to experience greater predation pressure than small females. In summary, we have shown that (1) male preference for larger female size exists. This is consistent with previous research, indicating that absolute preference functions are a valid approach in this system. (2) Hydrogen sulphide does not affect the shape of male's preference function for female size in this system. Since H2S greatly affects female fecundity, this suggests that inter-population differences of fecundity are not very highly correlated with inter-population differences in fitness. Alternatively, there has not been enough time or enough selection pressure to allow male preference to evolve as a response to changes in female fecundity, or gene flow has been large enough to negate the effects of these pressures. (3) Cave habitat, independent of water toxicity, does affect male preference. Cave males had a relative lack of preference for small females. We suggest that this could be a result of differences in predation pressure which could lead to relatively increased mortality for small females in the caves, relatively increased mortality for medium and large females in the surface, or both. If true, this would highlight the role that predators play in the evolution of male mate choice. Acknowledgements: We thank Millard L. Henry for his help with the experiments, and Sam B. Rhodes and Amber M. Makowicz for their help with fish care. We are grateful to Dr. Constantino Mac\u00edas Garcia and an anonymous reviewer for comments on a previous version of the manuscript. 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Compensatory behavior in response to sulphide-induced hypoxia affects time budgets, feeding efficiency, and predation risk. 13 350 352 354 356 358 360 362 364 366 368 370 372 374 376 378 26 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Evolutionary Ecology Research. 11: 935\u2013948. Tobler M, Schlupp I, Heubel KU, Riesch R, Garc\u00eda de Le\u00f3n FJ, Giere O, Plath M. 2006. Life on the edge: hydrogen sulfide and the fish communities of a Mexican cave and surrounding waters. Extremophiles. 10: 577-585. Trexler JC, Tempe RC, Travis J. 1994. Size\u2013selective predation of sailfin mollies by two species of heron. Oikos. 69: 250\u2013258. Trivers RL. 1972. Parental investment and sexual selection. In: B. Campbell, ed. Sexual selection and the Descent of Man. Chicago: Aldine Publishing Company, 136\u2013179. Van den Berghe EP, Warner RR. 1989. The effects of mating system on male mate choice in a coral reef fish. Behavioral Ecology and Sociobiology. 24: 409-415. Verrell PA. 1985. Male mate choice for large, fecund females in the red-spotted newt, Notophthalmus viridescens: how is size assessed? Herpetologica. 41: 382-386. Wagner WE Jr. 1998. Measuring female mating preferences. Animal Behaviour. 55: 1029-1042. Wong BBM, Fisher HS, Rosenthal GG. 2005. Species recognition by male swordtails via chemical cues. Behavioral Ecology. 16: 818\u2013822. 14 380 382 384 386 388 390 392 394 396 398 400 28 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Figure 1 Effect of habitat toxicity on the size/fecundity relationship size and her fecundity in toxic and non-toxic habitats. PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Table 1(on next page) experimental setup. : Figure 2: Schematic representation of the experimental setup during acclimation period. Gravel and cylinder perforations were omitted for clarity. PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Figure 3 Male preference for female size (cave vs. surface) Figure 3: Average transformed male preference for female size in cave vs. surface habitats. There is a significant difference between the two preference functions. PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Figure 4 Male preference for female size (toxic vs. non-toxic) Figure 4: Average transformed male preference for female size in toxic vs. benign habitats. Preference functions are not significantly different from each other. PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Table 2(on next page) introduction: : The existence and evolution of female mate choice has received substancial attention in both theoretical and empirical grounds. However, male mate choice has been comparatively neglected until recently. This is because females typically have a larger a priori investment in any given mating event, and they are also inherently limited in the number of offspring they are able to produce (Trivers 1972). The selective pressures giving rise to female mate choice are therefore obvious. However, while these pressures are often stronger in females, similar pressures are also experienced by males in many species. Males are limited in the proportion and quality of females they are able to fertilize, and can therefore maximize their fitness by selectively allocating their resources towards certain females. Theory thus predicts that male mate choice can be selected for under the following circumstances: 1) There is substantial male effort in terms of searching, courtship, mating, and mate guarding (Pomiankowski 1987); 2) Females are scarce due to a biased operational sex ratio (van den Berghe & Warner 1989); 3) Female quality varies (Johnstone et al. 1996); 4) Males invest in parental care (Sargent et al. 1986); and 5) Males' mating opportunities are limited and/or insemination success varies between different females (Nakatsuru & Kramer 1982; Verrell 1985; also see reviews of Bonduriansky 2001; and Edward & Chapman 2011). There is some evidence that these factors have indeed resulted in male preference in species ranging from sexually cannibalistic arthropods, fish and birds with heavy parental investment, and polygynous species without parental care (see reviews by Amundsen 2000; Bonduriansky 2001; Edward & Chapman 2011). However, it difficult to determine the specific factors driving the evolution of male choice in these systems since multiple factors predicted to drive male mate choice evolution are present in these species. Previous empirical research has often been limited to demonstrating the existence of male mate choice and describing its manifestation in particular species. We are not aware of research examining the evolution of the strength of male preference in response to specific selective pressures. We attempted to address this by comparing poeciliid populations in which variation in female quality is likely to be the main driving force behind male choice evolution. Poeciliids are a family of internally-fertilizing, promiscuous fish that form mixed-sex shoals and give birth to live young. Previous studies have demonstrated male preference for larger females in many species (Abrahams 1993; Bisazza et al. 1989; Dosen & Montgomerie 2 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 4 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t 2004a; Gumm & Gabor 2005; Herdman et al. 2004; Hoysak & Godin 2007; Jeswiet & Godin 2011; Plath et al. 2006; Ptacek & Travis 1997b). This is likely a result of the strong correlation between female size and fecundity (Herdman et al. 2004; Hughes 1985; Marsh-Matthews et al. 2005; Milton & Arthington 1983; Reznick & Endler 1982; Riesch et al. 2009b), suggesting that size is used as a signal of female quality and has played a role in the evolution of male mate choice. While previous studies have shown that males prefer more fecund females, how the strength of this preference changes as female fecundity evolves has not been investigated. To address this, we selected a number of populations from four poeciliid species (Poecilia mexicana, Limia sulphurophila, Gambusia sexradiata, and Gambusia eurystoma) that exhibit different relationships between female size and fecundity. These different relationships evolved as a response to living in different habitats (Fig 1), and have been found to persist even in fish raised in common garden conditions (Riesch et al. 2009b, Riesch et al. submitted). Living in a toxic habitat or living in a cave independently led to larger and fewer offspring; in other words, larger and fewer offspring are found in toxic habitats (whether on the surface or in a cave), as well as cave habitats (whether toxic or nontoxic); smaller and more numerous offspring are found in nontoxic surface habitats (Riesch et al. 2009b; Riesch et al. 2010b; Riesch et al. 2010c). Because female size and fecundity decoupled from each other in this system, it is possible to comparatively determine how female fecundity affects the evolution of male preference. Mate preference is most commonly studied using dichotomous choice tests, where focal individuals are given a choice between two stimuli (Ritchie 1996). While this is a powerful approach to assess mate preferences within populations, this approach makes it difficult to compare between populations. Absolute preference functions are an alternative method that allows the preferences between populations to be compared (Wagner 1998). Absolute preference functions measure the preference of individual males for females varying in a continuous trait. This is done by sequentially presenting individual females to each male, allowing the shape of a male's preference for that trait to be quantified. The resulting correlation can thus be thought as being the probability that a given male will accept a particular female trait (Ritchie 1996). Such association preferences are commonly used to study male mating preferences in poeciliids and have been shown to correlate well with actual mating choices (Dosen & Montgomerie 2004b; Plath et al. 2006; Schlupp & Ryan 1997; Wong et al. 2005). 3 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 6 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t The present study had two goals; 1) to test whether male preferences can be detected using preference functions, and 2) to see if male preference tracks changes in female fecundity in these populations. Our prediction was that male preference for larger females would be stronger in populations from nontoxic environments, where the relative increase in female fecundity is higher as compared to populations from toxic environments. title page: : Luis R. Arriaga Department of Biology, University of Oklahoma, Norman OK, USA. Ingo Schlupp Department of Biology, University of Oklahoma, Norman OK, USA. Corresponding author: Luis Arriaga, Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, USA. E-mail: ouroboricalmus@yahoo.com 1 2 4 6 2 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t populations used : Table 1: Collection details and habitats characteristics of the populations from which the individuals used originated. All individuals were descendants of these original populations, and were raised in common garden conditions. PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Population Source location Year Toxic/non-toxic Cave/Surface P. mexicanaOxolotan 17\u00b026'55\"N 92\u00b045'55\"W 2005 non-toxic Surface P. mexicanaPS0 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Surface P. mexicanaLuna 17\u00b026'35\"N 92\u00b046'39\"W 2006 non-toxic Cave P. mexicanaPSV 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Cave P. mexicanaPSX 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Cave P. mexicanaPSXIII 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Cave G. eurystoma 17\u00b033'10\"N 92\u00b059'51\"W 2006 Toxic Surface G. sexradiata 17\u00b059'56\"N 93\u00b0 8'11\"W 2006 non-toxic Surface L. sulphurophila 18\u00b023'52''N 71\u00b034'12''W 2006 Toxic Surface PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t Figure 2 descriptive statistics : Table 2: Sample size, average time, and standard deviation that males from each of the populations spent with each female size category. PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t N Mean association time (s) Std. Deviation Small Female Cave Surface Nontoxic Toxic 56 72 43 85 191.4 205.1 199.5 198.8 74.9 59.1 75.7 61.9 Medium Female Cave Surface Nontoxic Toxic 56 72 43 85 218.1 206.9 206.8 214.3 59.4 50.9 62.4 50.8 Large Female Cave Surface Nontoxic Toxic 56 72 43 85 233.2 215.1 219.1 225.0 62.2 62.2 56.4 65.7 PeerJ reviewing PDF | (v2013:06:590:1:0:NEW 21 Jul 2013) R ev ie w in g M an us cr ip t",
    "v2_text": "materials & methods : Species and populations: Nine populations of four poeciliid species representing different habitat types were used (summarized in Table 1). Gambusia eurystoma is a surface species endemic to the sulfidic Ba\u00f1os del Azufre in Tabasco, Mexico (Tobler et al. 2008c). Limia sulphurophila is another surface fish living in a sulfidic habitat, but it is endemic to a small pool in the island of Hispaniola (Rivas 1984). The population of G. sexradiata used lives in non-sulfidic surface waters. The six populations of P. mexicana used in this study live in different habitats. The Oxolotan population is named after the non-sulfidic, surface river it originates from. The PS0 population also lives in a surface creek, but whose water is sulfidic. The water from this creek, named El Azufre, originates from Cueva del Azufre, a sulfidic cave from which three of the other populations originated. These populations, inhabiting a dark and toxic environment, are the PSV, PSX, and PSXIII populations (For a schematic map of the region and of the cave, see Plath et al. 2010, and Tobler et al. 2006). They are named after the chamber of the cave in which they live. The sixth population is the Luna population, which originates from a non-toxic cave of the same name (Tobler et al. 2008c). Fish from all of the populations are maintained in flow-through stock tanks in the Aquatic Research Facility at the University of Oklahoma, and have been in captivity for varying lengths of time (Table 1). These tanks were the immediate source of the fish used in this study. All are maintained in nonsulfidic, common garden conditions inside a greenhouse that receives natural light. Fish from the stock tanks were caught with a small seine and segregated by sex. Mature females were then selected using minimum standard length (tip of the snout to the end of the vertebral column) as the criterion for sexual maturity. Due to natural size differences between species, the exact criterion used varied (P. mexicana: 29mm for the Luna population, and 30mm for all others; G. eurystoma: 22mm; G. sexradiata: 18mm; and L. sulphurophila: 21mm). The 4 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t mature females were then sorted into roughly equally sized groups according to size (small, medium, and large), and were then placed in different 38L stock tanks. Males were randomly assigned an ID number that determined the order in which they would be tested, and they were housed in individual 5L tanks that were out of sight of the females. Experimental Setup: Preference functions are established by measuring the amount of time a focus individual spends in association with different stimulus fish. These stimulus fish are presented sequentially and differ in the variable in question. In this case, females of different sizes were sequentially presented to a male, and the time that the male spent with each female was recorded. To do this, a 76L aquarium, with gravel spread evenly to reduce potential bias from fish being distracted by uneven gravel, was divided lengthwise into three equal sections with two vertical lines drawn on the glass (Fig 2). The outer two sections were considered the \"preference zones\", while the central section was considered a \"neutral zone\". Three hollow square prisms (or \"cylinder\") made out of clear plexiglass were located in the center of each section of the tank. The cylinder in the center of the tank had solid walls, while the two outer cylinders were perforated with seven circular holes 6mm in diameter to allow for chemical and mechanosensory signals. Chemical and mechanosensory signals have been found to be important factors in poeciliid mating behavior, influencing the repeatability of individual preferences as well as the overall preference (Coleman 2011; Hoysak & Godin, 2007; Plath et al. 2006; R\u00fcschenbaum & Schlupp 2013). All three cylinders were 8.5cm long by 8.5cm wide, and tall enough to stick through the water. To reduce visual distractions, three sides of the tank were covered. The observer sat in a chair 2m from the tank and observed the fish through the front pane of the tank. A light with a 60W, \"soft white\" light bulb was placed 30cm above the center of the tank for illumination. Testing Procedure: A randomly selected female from the predetermined size category (small, medium, or large) was placed in the cylinder in one of the outer preference zones. The order of the females which each male would be presented with, as well as the side in which each female would be placed, were randomly determined. A male was then placed in the central cylinder for 5 minutes. 5 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t After the 5 minutes of acclimation, the cylinder around the male was gently removed. Using two stopwatches, the amount of time the male spent in the preference zone containing the female was measured by the observer. This was done for 5 minutes, after which the fish were removed from the tank. The water was then stirred to homogenize any lingering chemical signals from affecting the results of future trials. Another pair of fish was then placed in their corresponding cylinders to acclimate. Every three pairs, a partial water change was also made previous to the acclimation period of the next fish. Male weight and standard length were also measured and used as covariates, but were not included in the final model because neither was significant. Statistical analysis: After checking the assumptions, a mixed between-within subjects ANOVA was performed to determine the effect of two habitat variables on male preference for small, medium, and large females. The two habitat variables used were \"cave habitat\", whether the population originated from a cave or from a surface stream, and \"toxicity\", whether the population originated from a toxic or non-toxic stream. Because the raw results were not normally distributed, the male preference variables were reflected and square root transformed to meet the normality assumption. Experiments were approved by the University of Oklahoma IACUC (R09-030). Results: All statistical assumptions were met after the data transformation, with the exception of homogeneity of variances for the time males spent with medium females (p = 0.026). However, this violation was not deemed to be severe enough to invalidate the ANOVA. As expected, there was a significant main effect for time spent with larger females, regardless of the habitat of origin (Wilks' Lambda = 0.911, F(2, 123)= 5.99, p= 0.003, p 2h = 0.089). The main between-subjects effect comparing toxicity was not significant (F(1, 124)= 0.047, p= 0.829, p 2h = 0.000), nor was the main effect for cave habitat (F(1, 124)= 1.043, p= 0.309, p 2h = 0.008), or the interaction between cave habitat and toxicity (F(1, 124)= 0.011, p= 0.917, p 2h = 0.000). These results suggest that, correcting for the effect of female size, habitat type does not affect male preference values. There was also a significant interaction between cave habitat and the time males spent with females (Wilks' Lambda= 0.934, F(2, 123)= 4.36, p= 0.015, p 2h = 0.066; Fig 3). However, there 6 132 134 136 138 140 142 144 146 148 150 152 154 156 158 160 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t was no significant interaction between the time males spent with females and the toxicity of the habitat from which they originated (Wilks' Lambda = 0.996, F(2, 123)= 0.26, p= 0.77, p 2h = 0.004; Fig 4). There was also no significant interaction between time, cave habitat, and toxicity together (Wilks' Lambda= 0.956, F(2, 123)= 8.86, p= 0.06, p 2h = 0.044). These results suggest that the presence or absence of hydrogen sulfide in the population's habitat of origin does not influence males' preference for female size, but the cave habitat does. Descriptive statistics are summarized in Table 2. Discussion: As predicted, males did exhibit a general preference for larger females when all populations were considered in aggregate. This result is consistent with previous dichotomous-test studies finding preference for larger females in poeciliids (Bisazza et al. 1989; Herdman et al. 2004; Hoysak & Godin 2007; Jeswiet & Godin 2011; Plath et al. 2006) and indicates that absolute preference functions are an accurate tool to study individual preferences. Because preference functions can be used to compare preferences between individuals, they can also be used to address a more specific and broader range of questions than is possible using only dichotomous choice tests. In addition to an overall preference for larger females, the strength of male preference should reflect how tightly correlated female size is with female fecundity. The populations tested originated from habitats with different combinations of two variables- water toxicity as a result of the presence or absence of H2S (\"toxicity\"), and epigean or hypogean habitat (\"cave habitat\"). Previous studies have shown that in P. mexicana (Riesch et al. 2009b; Riesch et al. 2010c), as well as in G. sexradiata and G. eurystoma (Riesch et al. 2010b), female fecundity is strongly correlated with toxicity. Females from toxic habitats have much larger, but fewer, offspring. Because of this, there is a larger change in fecundity from small to large females in nontoxic habitats. The main hypothesis was therefore that the preference function of males from toxic habitats would be less steep than that of males from nontoxic habitats. The results did not support our hypothesis, as there was no significant interaction between time spent with a female and the toxicity of the habitat the male originated from. This result suggests that the change in female fecundity experienced from benign to toxic habitats is only weakly correlated with the change female quality. The reason for this is unclear and could be due to a combination of factors. It is possible that female size in nontoxic habitats is 7 162 164 166 168 170 172 174 176 178 180 182 184 186 188 190 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t correlated with increased female mortality and/or with a decrease in offspring quality. An alternative possibility is that female size and quality are highly correlated, but that there has been insufficient time for male preference to change as a response to female adaptation. It is currently unknown how long the populations in toxic habitats have been adapting to their environments, and how recently the changes in female fecundity have evolved. Since male preference is likely under weaker selection than other traits, it is possible that males from toxic populations have either not had a sufficient amount of time to adapt, or that the amount of gene flow from nontoxic populations has been able to counteract the effects of selection. It is currently unknown how much gene flow there is between toxic (P. mexicana: PS0, G. sexradiata: populations not used in the present study) and nontoxic (P. mexicana: Oxolotan, G. sexradiata) surface populations. While the amount of gene flow between toxic and non-toxic populations still needs to be determined, it is known that there is very little gene flow between P. mexicana populations from the Cueva del Azufre (PSV, PSX, and PSXIII) and those in the surface (Plath et al., 2010). The genetic isolation of cave fish from surface fish might be an important reason why cave habitat did have a significant effect on male preference. This difference seems to be mainly derived from cave males' relatively low preference for small females (Fig 3), as the change in preference for medium to large females is nearly identical in males from both habitats. This suggests that males are not responding to fecundity or offspring size per se, since one would expect that the change of preference from medium to large females would differ between the two environments. This same logic also suggests that the difference is not due to cave males being choosier than surface males. If males preferred larger females due to the cave habitat leading to a greater cost in male effort, males would disproportionally prefer large females over medium females as well. Instead, these results indicate that there is a relative disadvantage for cave males to mate with smaller females, which could result if small cave females have a lower fitness than small surface females. A fitness difference could occur if mortality rates for small cave female are higher than those of medium or large females, perhaps as a result of differential predation pressures. No direct evidence exists on the relative predation pressures between the two habitats, but there is reason to believe that this possibility might be true. Cave populations of P. mexicana are known to be preyed upon by predators which hunt by sensing tactile and/or chemical signals from fish in close proximity (Horstkotte et al. 2010; Tobler et al. 2008a; Tobler et al. 2009), and may prey disproportionally upon smaller females. At the same time, surface populations experience very 8 192 194 196 198 200 202 204 206 208 210 212 214 216 218 220 222 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t different predation pressures: surface fish are preyed upon by large visual predators (Riesch et al. 2009a, 2010a; Tobler et al. 2006) that target larger fish (Trexler et al. 1994). Thus, medium and large surface females are likely to experience greater predation pressure than small females. In summary, we have shown that (1) male preference for larger female size exists. This is consistent with previous research, indicating that absolute preference functions are a valid approach in this system. (2) Hydrogen sulphide does not affect the shape of male's preference function for female size in this system. Since H2S greatly affects female fecundity, this suggests that inter-population differences of fecundity are not very highly correlated with inter-population differences in fitness. Alternatively, there has not been enough time or enough selection pressure to allow male preference to evolve as a response to changes in female fecundity, or gene flow has been large enough to negate the effects of these pressures. (3) Cave habitat, independent of water toxicity, does affect male preference. Cave males had a relative lack of preference for small females. We suggest that this could be a result of differences in predation pressure which could lead to relatively increased mortality for small females in the caves, relatively increased mortality for medium and large females in the surface, or both. If true, this would highlight the role that predators play in the evolution of male mate choice. Acknowledgements: We thank Millard L. Henry for his help with the experiments, and Sam B. 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Ritchie MG. 1996. The shape of female mating preferences. Proceedings of the National Academy of Sciences of the United States of America. 9325: 14628\u201314631. Riesch R, Tobler M, Plath M, Schlupp I. 2009b. Offspring number in a livebearing fish Poecilia mexicana, poeciliidae: reduced fecundity and reduced plasticity in a population of cave mollies. Environmental Biology of Fishes. 84: 89-94. 12 318 320 322 324 326 328 330 332 334 336 338 340 342 344 346 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Rivas LR. 1980. Eight new species of poeciliid fishes of the genus Limia from Hispaniola. Northeast Gulf Science. 41: 28-38. Rivas LR. 1984. Comments on Briggs 1984: Freshwater Fishes and Biogeography of Central America and the Antilles. Systematic Zoology. 35: 633-639. Sargent RC, Gross M, van den Berghe EP. 1986: Male mate choice in fishes. Animal Behaviour. 34: 545-550. Schlupp I, Ryan MJ. 1997. 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Male mate choice for large, fecund females in the red-spotted newt, Notophthalmus viridescens: how is size assessed? Herpetologica. 41: 382-386. Wagner WE Jr. 1998. Measuring female mating preferences. Animal Behaviour. 55: 1029-1042. Wong BBM, Fisher HS, Rosenthal GG. 2005. Species recognition by male swordtails via chemical cues. Behavioral Ecology. 16: 818\u2013822. 14 380 382 384 386 388 390 392 394 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Figure 1 Effect of habitat toxicity on the size/fecundity relationship size and her fecundity in toxic and non-toxic habitats. PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Table 1(on next page) experimental setup. : Figure 2: Schematic representation of the experimental setup during acclimation period. Gravel and cylinder perforations were omitted for clarity. PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Figure 3 Male preference for female size (cave vs. surface) Figure 3: Average transformed male preference for female size in cave vs. surface habitats. There is a significant difference between the two preference functions. PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Figure 4 Male preference for female size (toxic vs. non-toxic) Figure 4: Average transformed male preference for female size in toxic vs. benign habitats. Preference functions are not significantly different from each other. PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Table 2(on next page) introduction: : The existence and evolution of female mate choice has received substancial attention in both theoretical and empirical grounds. However, male mate choice has been comparatively neglected until recently. This is because females typically have a larger a priori investment in any given mating event, and they are also inherently limited in the number of offspring they are able to produce (Trivers 1972). The selective pressures giving rise to female mate choice are therefore obvious. However, while these pressures are often stronger in females, similar pressures are also experienced by males in many species. Males are limited in the proportion and quality of females they are able to fertilize, and can therefore maximize their fitness by selectively allocating their resources towards certain females. Theory thus predicts that male mate choice can be selected for under the following circumstances: 1) There is substantial male effort in terms of searching, courtship, mating, and mate guarding (Pomiankowski 1987); 2) Females are scarce due to a biased operational sex ratio (van den Berghe & Warner 1989); 3) Female quality varies (Johnstone et al. 1996); 4) Males invest in parental care (Sargent et al. 1986); and 5) Males' mating opportunities are limited and/or insemination success varies between different females (Nakatsuru & Kramer 1982; Verrell 1985; also see reviews of Bonduriansky 2001; and Edward & Chapman 2011). There is some evidence that these factors have indeed resulted in male preference in species ranging from sexually cannibalistic arthropods, fish and birds with heavy parental investment, and polygynous species without parental care (see reviews by Amundsen 2000; Bonduriansky 2001; Edward & Chapman 2011). However, it difficult to determine the specific factors driving the evolution of male choice in these systems since multiple factors predicted to drive male mate choice evolution are present in these species. Previous empirical research has often been limited to demonstrating the existence of male mate choice and describing its manifestation in particular species. We are not aware of research examining the evolution of male preference in response to specific selective pressures. We attempted to address this by comparing poeciliid populations in which variation in female quality is likely to be the main driving force behind male choice evolution. Poeciliids are a family of internally-fertilizing, promiscuous fish that form mixed-sex shoals and give birth to live young. Previous studies have demonstrated male preference for larger females in many species (Abrahams 1993; Bisazza et al. 1989; Dosen & Montgomerie 2 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t 2004a; Gumm & Gabor 2005; Herdman et al. 2004; Hoysak & Godin 2007; Jeswiet & Godin 2011; Plath et al. 2006; Ptacek & Travis 1997b). This is likely a result of the strong correlation between female size and fecundity (Herdman et al. 2004; Hughes 1985; Marsh-Matthews et al. 2005; Milton & Arthington 1983; Reznick & Endler 1982; Riesch et al. 2009b), suggesting that size is used as a signal of female quality and has played a role in the evolution of male mate choice. While previous studies have shown that males prefer more fecund females, how the strength of this preference changes as female fecundity evolves has not been investigated. To address this, we selected a number of populations from four poeciliid species (Poecilia mexicana, Limia sulphurophila, Gambusia sexradiata, and Gambusia eurystoma) that exhibit different relationships between female size and fecundity. These different relationships evolved as a response to living in different habitats (Fig 1). Living in a toxic habitat or living in a cave independently led to larger and fewer offspring; in other words, larger and fewer offspring are found in toxic habitats (whether on the surface or in a cave), as well as cave habitats (whether toxic or nontoxic); smaller and more numerous offspring are found in nontoxic surface habitats (Riesch et al. 2009b; Riesch et al. 2010b; Riesch et al. 2010c). Because female size and fecundity decoupled from each other in this system, it is possible to comparatively determine how female fecundity affects the evolution of male preference. Mate preference is most commonly studied using dichotomous choice tests, where focal individuals are given a choice between two stimuli (Ritchie 1996). While this is a powerful approach to assess mate preferences within populations, this approach makes it difficult to compare between populations. Absolute preference functions are an alternative method that allows the preferences between populations to be compared (Wagner 1998). Absolute preference functions measure the preference of individual males for females varying in a continuous trait. This is done by sequentially presenting individual females to each male, allowing the shape of a male's preference for that trait to be quantified. The resulting correlation can thus be thought as being the probability that a given male will accept a particular female trait (Ritchie 1996). Such association preferences are commonly used to study male mating preferences in poeciliids and have been shown to correlate well with actual mating preferences (Dosen & Montgomerie 2004b; Plath et al. 2006; Schlupp & Ryan 1997; Wong et al. 2005). 3 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t The present study had two goals; 1) to test whether male preferences can be detected using preference functions, and 2) to see if male preference tracks changes in female fecundity in these populations. Our prediction was that male choice would be more acute in populations from toxic environments, where the relative increase in female fecundity is higher as compared to surface populations. title page: : Luis R. Arriaga Department of Biology, University of Oklahoma, Norman OK, USA. Ingo Schlupp Department of Biology, University of Oklahoma, Norman OK, USA. Corresponding author: Luis Arriaga, Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, USA. E-mail: ouroboricalmus@yahoo.com 1 2 4 6 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t populations used : Table 1: Collection details and habitats characteristics of the populations from which the individuals used originated. All individuals were descendants of these original populations, and were raised in common garden conditions. PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Population Source location Year Toxic/non-toxic Cave/Surface P. mexicanaOxolotan 17\u00b026'55\"N 92\u00b045'55\"W 2005 non-toxic Surface P. mexicanaPS0 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Surface P. mexicanaLuna 17\u00b026'35\"N 92\u00b046'39\"W 2006 non-toxic Cave P. mexicanaPSV 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Cave P. mexicanaPSX 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Cave P. mexicanaPSXIII 17\u00b026'30\"N 92\u00b046'30\"W 2005 Toxic Cave G. eurystoma 17\u00b033'10\"N 92\u00b059'51\"W 2006 Toxic Surface G. sexradiata 17\u00b059'56\"N 93\u00b0 8'11\"W 2006 non-toxic Surface L. sulphurophila 18\u00b023'52''N 71\u00b034'12''W 2006 Toxic Surface PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t Figure 2 descriptive statistics : Table 2: Sample size, average time, and standard deviation that males from each of the populations spent with each female size category. PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t N Mean association time (s) Std. Deviation Small Female Cave Surface Nontoxic Toxic 56 72 43 85 191.4 205.1 199.5 198.8 74.9 59.1 75.7 61.9 Medium Female Cave Surface Nontoxic Toxic 56 72 43 85 218.1 206.9 206.8 214.3 59.4 50.9 62.4 50.8 Large Female Cave Surface Nontoxic Toxic 56 72 43 85 233.2 215.1 219.1 225.0 62.2 62.2 56.4 65.7 PeerJ reviewing PDF | (v2013:06:590:0:1:NEW 16 Jun 2013) R ev ie w in g M an us cr ip t",
    "url": "https://peerj.com/articles/142/reviews/",
    "review_1": "M Fabiana Kubke \u00b7 Aug 4, 2013 \u00b7 Academic Editor\nACCEPT\nI would like to thank the authors for their cooperation through the processing of the manuscript. The article represents a valuable contribution to the field.\n\nThere are a few very small editorial changes that I will ask the authors to make prior to final publication:\n\n1. Page 3, last sentence of first paragraph.\nThe sentence: \u201cProprioception is mediated by sensory neurons that project through the dorsal spinal cord to an intermediate zone which in turn projects to the ventral spinal cord where direct connection is made to motoneurons (Brown, 1981; for review: Caspary & Anderson 2003)\u201d\nConsider changing to: \u201c [\u2026] to the ventral spinal cord where [a] direct connection is made [with or onto] motoneurons [\u2026]\u201d\nThe sentence also needs a period at the end.\n\n2. Page 6, second paragraph:\nA period appears to be missing before the sentence starting \u201dThe presence of a wild-type allele was detected using \u2026\u201d\n\n3. Page 6 last paragraph:\nThe molarity of PBS is expressed as PBS 1x \u2013 I still don\u2019t know what that is \u2013 please include the actual molarity at this stage, (eg PBS 1x (XXXX M, pH 7.5) and then continue using 1x thereafter if desired.\n\n4. Page 9 first sentence:\n\u201c(rpm) in 5 min.\u201d I am not sure but should this be \u201cover\u201d 5 min?\n\n5. The ages at which the gait was analysed is confusing \u2013 It is first said that it is analysed at 10 wk of age (p. 8), then at 4-6 wk of age (p.11) and then the onset is described at P16-17 (p.19). To avoid confusion, please specify the age used for the data in Table 1, Figure 2 and supplementary movie (eg add age in the figure legend and into the table).\n\n6. Page 8, First paragraph, second/third line\nThe statement \u201cand mouse anti-islet \u2026\u201d Suggest changing [and] and substituting with [for] \u2013 am assuming the different antibodies were done in different sections and not in the same section, which is what \u201cand\u201d seems to suggest.\n\n7. Figure 5 and Figure 5 legend:\nThe abbreviation BN is used for \u201cwhite noise\u201d (assuming you mean \u201cblanche\u201d?) \u2013 I suggest changing this to WN, since English speakers might confuse this with Brown Noise (which is what the abbreviation BN is usually used for).",
    "review_2": "M Fabiana Kubke \u00b7 Jun 5, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThank you very much for the resubmission, and also for your effort addressing the issues raised. I want to once again emphasise that I consider this to be a valuable set of experiments that deserves to be published, and I hope you will be able to address some remaining issues that prevent me from accepting the manuscript at the time.\n\nOne important issue still remains regarding the analysis of the GABA cells, which is of great importance since this seems to be at the centre of the interpretation of the behavioural phenotype.\n\n1. I was surprised that Dr Dembele suggested a paired t-test since I couldn\u2019t understand what criterion/justification was used to pair the animals from the two genotypes (and I couldn\u2019t find where one was provided). I have consulted with a colleague of mine in the statistics department (Dr Russell) who suggested that since you have 2 genotypes and 3 observations in each of 3 animals per genotype, the data would be best analysed with a 2 way mixed model anova with the first variable as treatment effect (fixed effect ie, genotype_ and a second variable as random effect of repeat observations on the same individual. He provided me these resources\nhttp://www.statisticshell.com/docs/mixed.pdf and http://www.math.montana.edu/~cherry/st412/pdf_files/CourseNotes16.pdf\nPlease note that the same criticisms apply to the data on Figure S4.\n2. I am also surprised that the authors were unable to find any references to the switch of GABA from depolarising to hyperpolarising to speculate at what age this might be happening in the dorsal horn at the levels of the spinal cord where the GABA phenotype is described. My search returned a few articles that the authors may want to consider: Stein, V., Hermans-Borgmeyer, I., Jentsch, T. J., & H\u00fcbner, C. A. (2004) J Comp Neurol 468(1), 57\u201364 sets the shift at around P7-P10 at least for hippocampal and spinal cord motoneurons in mouse; Cordero-Erausquin et al (2005) J Neurosci 25(42):9613-8623 show similar time course for the switch of GABA in neurons of LI in the dorsal horn (albeit in rats) while Sibilla, S., & Ballerini, L. (2009). Progress in Neurobiology, 89(1), 46\u201360 report these changes as happening within the first two weeks of age. This would suggest (if I have interpreted the articles correctly) that the switch may be over by the time that the phenotype becomes expressed (P16-17). The authors may also want to consider when incorporating this to their discussion that a depolarising effect of GABA does not necessarily means it is excitatory (some depolarising GABA has been shown to be inhibitory, eg. Viemari, J.-C et al (2011). Importance of chloride homeostasis in the operation of rhythmic motor networks. In Progress in Brain Research (Vol. 188, pp. 3\u201314). ).\n3. The statement : \u201cAs expected, natural cell death occurs in the developing spinal ganglia (Fig. 9A, arrow; Paschaki et al. 2012) but not in the spinal cord\u201d and what follows:\nThe reference does not support the statement, since it does not constitute a study of normal cell death in mice, and, within that same reference it appears to me that apopototic labeling in the ventral spinal cord at 12.5 can be seen (Figure 4I). There are a number of studies looking at normal cell death in mouse spinal cord, and my interpretation of some of those is that one would expect to see cell death in the spinal cord as well as the DRG at least in your E12.5 and E 14.5 material. (eg, White et al Journal of Neuroscience, February 15, 1998, 18 (4):1428); Yamamoto, Y., & Henderson, C. E. (1999). Developmental biology, 214(1), 60\u201371). It would be useful to discuss why it might be the case that no TUNEL labeling is visible in your material at those ages, which is crucial to validate the results.\n4. I will also ask that those data where the statistical analysis failed to show a statistical significance are clearly described as such. Eg the sentence : \u201cSuch abnormal sensory processing is suggested at least for thermal stimuli, as Gbx1-/- mice displayed increased latency suggesting reduced pain in the hot plate test (thermosensory functions)\u201d. Should reflect that this is only true in one sex. There are several instances of similar reporting throughout the manuscript.\n5. An issue also remains as to the need to discuss how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system which, as the authors state, projects to the ventral and intermediate spinal cord, not the dorsal horn which was examined in this study. The explanation :\u201cwe cannot exclude abnormal functions of those afferents caused by reduced GABAergic cell number in superficial dorsal horn (see below), which is the transition and/or adjacent region for proprioceptive afferents. Such proprioceptive-like deficits may be also secondary to a defect in sensory perception or expression of such sensory response, as indicated by sensory deficits in Gbx1-/- mice and abnormal development of the dorsal horn, the main target of sensory afferents.\u201d . I don\u2019t find the explanation convincing, and perhaps adding support for such mechanism from the literature might help make your case. Regardless, the statement needs to be amended for accuracy:\na. This study has provided evidence for a reduction in the proportion (not number) of GABA neurons (also please correct other references to number of GABAergic cells elsewhere in the manuscript, eg, in page 1 and 12, please check for other instances.)\nb. The data analysis failed to show significant sensory deficits in most sets of data. Other than the latency in the beam test in females (proprioceptive test) all other measures for proprioceptive defects were NS (This also applies to the sentence: \u201cImportantly, abnormal gait resemble proprioceptive-like deficits, which was further suggested by deficits in beam-walking, the test used for evaluation of proprioceptive functions\u201d) and \u201cThis abnormal gait, documented by a series of behavioral tests, is not due to deficits in muscle strength or motor coordination, but may be related to proprioceptive deficits suggested by reduced performance in beam walking, a test used in studies of proprioception.\u201d Please check for other possible similar isntances.\nc. The projection patterns of proprioceptive afferents were also described as normal, so it is unclear why a case is made for a defect in proprioceptive function due to possible measured GABA deficits in a region that in not recipient of those proprioceptive afferents. Perhaps this argument could be strengthened by providing evidence from the literature that might support this case.\nd. For the most part development of the spinal cord was normal (based on the description of Nissl staining, and several markers: Lbx1, Lmx1b, Netrin-1, Drg11, etc.) other than the GABAergic phenotype\ne. Similarly, \u201cThis abnormal gait, documented by a series of behavioral tests, is not due to deficits in muscle strength or motor coordination, but may be related to proprioceptive deficits suggested by reduced performance in beam walking, a test used in studies of proprioception. (in the conclusion)\u201d needs to accurately reflect the results from the proprioceptor tests.\n\nOther:\n\n6. Please amend the materials and methods which says that the histological analysis was done in 3 sections per animal, in 3 animals of each genotype since some of your figures the legend states that the number of animals is 2, not 3. The N for the results of Figure S3 are not provided.\n7. Panel D in Figure 8 appears to be flipped horizontally\n8. Please provide the molarity and pH of all buffers used.\n9. \u201cunmasking in citrate 0.1 M (pH 6)\u201d do you mean citrate buffer?\n10. What were the solutions used to dilute the primary antibodies?\n11. \u201cFurthermore, electromyography (EMG) measurements revealed that the sensory nerve conduction\u201d Do you mean SNCV?\n\nOther suggestions:\n1. Please amend the statement: \u201d In contrast to our observations, those mutants show disorganized peripherin expression, together with a decrease of Islet1-expressing cells in the ventral horn of the lumbar spinal cord (Buckley et al. 2013).\u201d See Figures 9 (showing change at E14.5-15.5) and Fig 5 (no change at E11.5). Please specify that the statement is correct for ages comparable to yours (not for younger embryos).\n2. The sentence \u201cAt E12.5, the expression domains of Gbx1 and Gbx2 overlap, both being expressed in the ventricular and mantle zones of the dorsal spinal cord (Rhinn et al. 2004; Waters, Wilson & Lewandoski 2003). As Gbx2 expression is downregulated fter E12.5, both genes are only transiently coexpressed in dorsal spinal cord progenitor cells, and Gbx1 is the only Gbx gene persistently expressed during later dorsal horn development (John, Wildner & Britsch 2005).\u201d Which appears under the hindbrain heading would more appropriately be placed in the next section under the spinal cord heading.\n3. I am also confused as to the statement \" This may reflect an abnormal development or survival of GABAergic neurons, which in consequence coud lead to abnormal control of neuronal network in dorsal horn, possibly affecting inhibitory circuits throughout the spinal cord.\" that is presented in the context of the GABA reduction \u2013 since the authors go on to show in the following paragraph that cell death as a factor could be ruled out and instead a change of phenotype to glutamate was proposed as a more likely explanation.\n4. The sentence \"Proprioceptive neuron afferents form two types of termination zones, one in the intermediate spinal cord and one in the ventral spinal cord where they are directly connected to motoneurons (Brown, 1981)\" which was added in page 17: I think a similar description of the targets of proprioceptive afferents should be added to the description of afferents in the first paragraph of the Introduction where the rest of the afferent projections to the SC are described.",
    "review_3": "M Fabiana Kubke \u00b7 May 1, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nPlease address the following remaining issues, as well as other issues as per our offline correspondence. Please note that the reviewer's comments are incorporated into the Editor's comments, so there is no need to respond to those separately.\n\n1. Can the authors please comment in the manuscript on the possible implications of the subtle increase of Gbx2 expression at E12.5-E14.5\n\n2. Reviewer #1: Quantifications of number of stained cells for each molecular marker displayed in fig 8 should be accompanied by an appropriate statistical analysis to assess the significance of the differences observed.\n\nIf the authors can argue why this statistical analysis cannot be performed, these limitations, and the limitations on the interpretation of the data need to be made explicit.\n\n3. To address the query about the possible fate of GABAergic neurons the authors complemented their GABAergic phenotype studies with examination of a glutamatergic marker at E18.5 and a cell death marker (at E12.5, 14.5, 16.5, 18.5). I think that it would be valuable to include these figures in the main article rather than in the supplementary material.\n\n4. Could the authors also please discuss how the ages at which the TUNEL was examined correspond to expected apoptosis through naturally occurring cell death in the mouse lumbosacral spinal cord and how this may (or may not) have an impact on your data results and interpretation.\n\n5. To address the concerns about how valid it is to compare the embryological data to the adult phenotype, the authors have added to the histological examination at E18.5 the observation that the onset of defects coincides with the switch of GABA from excitatory to inhibitory. These observations are described as being obtained by checking pups visually around weaning. Can you please provide the specific ages and frequency with which this monitoring was done, the number of animals and if possible the male to female ratio. Also, please double check reference to Ben-Ari et al. \u201cwhich coincides with the time point at which the GABA pathway switches from excitatory to inhibitory (Ben-Ari et al. 2007).\u201d I was unable (albeit through a quick superficial scan) to find in that paper mention of a switch from excitatory to inhibitory in mice spinal cord at that age.\n\n6. Reviewer #1: Moreover, the present data differ from the Buckley\u2019s data in that the expression of peripherin and Islet1 was not changed in the Gbx1 -/- model. This discrepancy was attributed to the use of different time points. Although I don\u2019t think this may be the reason, if the authors insist in this explanation they should evaluate the expression of those markers at the same time points. In addition, it would be very interesting to analyse their expression at postnatal stages, when the locomotor defect is detected, as requested on our previous revision\n6.1. Regarding the discrepancies between this and Buckley\u2019s results on the peripherin staining, it might be useful if the authors used an image of peripherin at E16.5, since they already have that material and the age is closer to that of Buckley et al. (E14.5 and E15.5) to allow for a better age-matched comparison. I think this would satisfy the reviewer\u2019s request.\n6.2. Regarding the discrepancies in Islet1+ results between the current work and that of Buckley et al., please amend the statement: \u201d In contrast to our observations, those mutants show disorganized peripherin expression, together with a decrease of Islet1-expressing cells in the ventral horn of the lumbar spinal cord (Buckley et al. 2013).\u201d See Figures 9 (showing change at E14.5-15.5) and Fig 5 (no change at E11.5). Since Buckley et al. show a decrease of ISL1 cells at E14.5 and E15.5, it might be useful to include images and the quantification for E16.5 in your material for a better age-matched comparison. If you do not have that quantification, please amend the sentence \u201cNo differences in the number of Islet1+ cells within the lumbar ventral spinal cord were found at E14.5, E16.5 or E18.5\u201d by removing the E16.5.\n\n7. For figure 7 the text states that there are \u201cno defects in patterning of sensory afferent fiber projections\u201d but this conclusion is based mainly on the staining of a subset of sensory afferents (those labeled with calbindin) and on the apparent lack of differences in expression of Drg11. I think the resolution of the images in panels A and B is too low for the reader to assess any possible differences and/or ectopic projections, and cannot differentiate between afferent inputs and neuropil stained originating from what appear as calbindin+ neurons in the SC. Also, the markers used cannot rule out other types of patterning differences from primary afferents that do not label with calbindin or possible patterning differences at ages other than the one provided. Please address these limitations.\n\n8. Reviewer #1: On page 9 the authors say \u201cAltogether the behavioural data revealed that the Gbx1 -/- animals \u2026.because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test\u201d. This paragraph should be revised correcting the hot plate test observation (underlined) and should be better explained how animals with less pain sensitivity in the hot plate could suffer from pain on movement.\n\n9. Reviewer #1: In the conclusion section, in the end of first paragraph the sentence \u201c\u2026 as mutant mice also display a significant reduced response time in the hot plate (thermosensory) test.\u201d Is in contradiction with the affirmation in the third paragraph \u201c\u2026 as Gbx1 -/- mice displayed significantly increased response time in the hot plate test.\u201d\n\n10. The sentence \u201cWe cannot exclude that the proprioceptive defects may be secondary to a defect in sensory pathways (for example, caused by pain on movement), because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test.\u201d Should be corrected to reflect that only males showed a significant difference and this was an increase (not decrease) in the response latency.\n\n11. In resonse to the request for details on the methods by which abnormal gait was assessed, please indicate whether observers were familiar with normal gait patterns in normal mice prior to the observational classification\n\n12. [R#1: In the Buckley study as in the study under revision, it is not discussed how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system. The authors should address this point in the discussion, as requested in our first revision of the paper. ]\n12.1. The authors might also want to make explicit that changes in the GABA phenotype were not analysed outside of the dorsal horn, and that contribution of inhibitory circuits elsewhere in the spinal cord cannot be excluded.\n12.2. In the statement: \u201c\u201cWe cannot exclude that the proprioceptive defects may be secondary to a defect in sensory pathways (for example, caused by pain on movement), because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test.\u201d Please amend the sentence to reflect that only males displayed a significantly reduced response time.\n12.3. In the statement: \u201cAltogether, the behavioral data revealed that the Gbx1-/- animals have apparent proprioceptive defects and/or altered sensory abilities.\u201d I am still uncertain as to how after failing to show significance on sensory features measured (other than conduction velocity in females and hotplate in males) this statement can be justified.\n\n13. The statement \u201cThis is supported by data from the beam walking test showing that at least Gbx1-/- females required longer time to cross the beam distance and tended to have higher number of slips (not statistically significant)\u201d if it is NS it is not supported by the data \u2013 the trend you see might indicate that a higher power test might uncover this, but it is speculation.\n\n14. The statement: \u201cElectrophysiological measurements showed that Gbx1-/- females had increased sensory nerve conduction velocity, measured in the caudal nerve, supporting the altered sensory functions observed in the behavioral tests.\u201d Not sure what sensory functions are being referred to here. Responses in the hot plate, startle, beam slips and sensorimotor all failed to show an effect on phenotype.\n\n15. The statement \u201cWe found that both Gbx1-/- males and females show reduced locomotor activity in different situations.\u201d, but legend to figure 4 says \u201cThe distance traveled over the 20 min period of test reflects locomotor activity\u201d and shows no difference, and on page 11 this is described as \u201c. When considering each gender separately, both Gbx1-/- males and females tended to have reduced locomotor activity over the testing period (although not statistically significant, p=0.09) (Fig. 4).\u201d\n\nGeneral corrections:\n\n16. R#1: In results section when describing Drg11 expression they should mention (Fig 7C,D and data not shown)\n16.1. I found similar instances throughout the manuscript. Please differentiate between the data that was used for the analysis but that is not shown and that which is shown: Lbx1 analysis reported at E12.5, 14.5, 16.5, 18.5 but only shown at E16.5. Lmx1b analysis reported at E12.5, 14.5, 16.5, 18.5 but shown at E16.5; etc. In the text: \u201cor at the level of fibers that enter into the spinal gray matter, at E16.5 or E18.5 (arrows in Fig. 7E,F).\u201d not clear which age is shown in the figure.\n\n17. P16: The sentence \u201cImportantly, Gad67 expression was reduced in the dorsal spinal cord of Gbx1 mutant mice (Fig. 8A-D), i.e. there was a 16% decrease in the number of Gad67-expressing cells in comparison to WT mice (Fig. 8I).\u201d should be corrected \u2013 there was a 16% decrease in the proportion of GAD67 expressing cells. The same applies to sentence \u201cThis finding was strengthened by analysis of Pax2, another gene expressed in GABAergic cells in the spinal cord (Cheng et al. 2004), with cell countings corroborating the decrease in the number of GABAergic cells\u201d Similarly in the legend to Figure 8 the statement \u201cCountings (percentages of labelled vs. total cells) revealed that the numbers of Gad67+ cells are diminished by 16% in Gbx1-/- mice\u201d should be corrected to \u201crevealed that the proportion of GAD67+ cells\u201d. The same applies to \u201cnumber of Pax2 cells\u201d and \u201cnumbers of Slc17a6+ cells\u201d\n\n18. There is a discrepancy in the reporting of the Netrin-1 material \u2013 the text says the image shown is E18.5 whereas the figure legend suggests it is E16.5\n\n19. On Figure 7, Please specify the age at which the Drg11 and Peripherin staining shown was performed.\n\n20. P11: \u201cchecked the expression of Gbx2 from E12.5 onwards\u201d specify ages\n21. P11: \u201cunnevenless\u201d check spelling\n\nReferences\n22. \u201cWaters, Wilson & Lewandoski 2004\u201d. Please double check \u2013 PubMed cites as Gene Expr Patterns. 2003 Jun;3(3):313-7.\n23. Reference to Nagy et al. 2003 does not seem to be in the reference list\n\n24. In order to adhere to PeerJ\u2019s editorial policy: \u201cMethods should be described with enough information to be reproducible by another investigator\u201d and that \u201cthe data should be robust, statistically sound, and controlled\u201d I will ask the authors to make the following amendments:\n\n1. Further details on how cell counting was performed need to be provided \u2013 eg what does using ImageJ mean? Was a threshold applied for counting, or just used to track cells? Were the sections counterstained, and if not, how were the unlabelled cells visualised. Unlike the case of the material shown in S4, it is difficult to see from the images provided how the authors were able to count labelled and unlabelled cells.\n2. Please make sure that all measurements in the tables and graph axes are accompanied by the appropriate measuring units\n3. Please try to track down which of the two forms of the DSHB Islet1 antibody was used for these studies\n4. Please go through the M+M to make sure all buffers and solutions are specified as well as possible (e.g., P6- L2: PBS what molarity?; P6- L4: PFA in what buffer?; P6- L4: 20% sucrose in what buffer?; P7- L2: dehydrated how?; 3% hydrogen peroxide in what buffer, etc)\n5. Please provide surgical, electrodes etc and other details used for the electrophysiological studies.\n6. In all figures (or figure legends), where appropriate, please report the N, and whether the data represents mean/median, SD/SEM etc.",
    "review_4": "Reviewer 1 \u00b7 Apr 23, 2013\nBasic reporting\nWe are pleased to verify that the authors have addressed several of our comments, which contributed to increase the quality of the manuscript.\nHowever, very recently another study on the characterization of a Gbx1 -/- allele was published (Buckley et al, 2013) demonstrating a a locomotive defect affecting the hindlimb gait and stating that this phenotype results from abnormal formation of the proprioceptive sensorimotor circuit. In the Burckley\u2019s study, as in the study under revision, it is not discussed how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system. The authors should address this point in the discussion, as requested in our first revision of the paper.\nMoreover, the present data differ from the Burckley\u2019s data in that the expression of peripherin and Islet1 was not changed in the Gbx1 -/- model. This discrepancy was attributed to the use of different time points. Although I don\u2019t think this may be the reason, if the authors insist in this explanation they should evaluate the expression of those markers at the same time points. In addition, it would be very interesting to analyze their expression at postnatal stages, when the locomotor defect is detected, as requested in our previous revision.\nMinor comments:\n- Quantifications of number of stained cells for each molecular marker displayed in fig 8 should be accompanied by an appropriate statistical analysis to assess the significance of the differences observed.\n- On page 9 the authors say \u201cAltogether, the behavioral data revealed that the Gbx1-/- animals \u2026 because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test.\u201d This paragraph should be revised correcting the hot plate test observation (underlined) and should be better explained how animals with less pain sensitivity in the hot plate test could suffer from pain on movement.\n- In results section when describing Drg11 expression they should mention (Fig. 7C,D and data not shown)\n- In the conclusion section, in the end of first paragraph the sentence \u201c\u2026as mutant mice also display a significant reduced response time in the hot plate (thermosensory) test.\u201d Is in contradiction with the affirmation in the third paragraph \u201c\u2026as Gbx1-/- mice displayed significantly increased response time in the hot plate test\u2026\u201d.\nExperimental design\nNothing to add\nValidity of the findings\nNothing to add\nAdditional comments\nThis is an interesting paper that would benefit with the ameliorations suggested\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord (v0.3)\". PeerJ https://doi.org/10.7287/peerj.142v0.3/reviews/1",
    "pdf_1": "https://peerj.com/articles/142v0.5/submission",
    "pdf_2": "https://peerj.com/articles/142v0.4/submission",
    "review_5": "M Fabiana Kubke \u00b7 Mar 31, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nI have now gone through the revised manuscript and my comments are listed below. My overall impression is that some of the issues that were raised during the first round of review still need to be further addressed. My comments below reflect my views on the current version as well as how well I consider the issues raised by the first round of review were addressed.\n\n\nResults reporting:\n\n1. In response to Reviewer\u2019s #1 request to address compensatory expression of GBx2, Fig S1 appears to satisfy that request. However, the data is interpreted as only showing a \u201cpossible subtle increase at E14.5\u201d, but in the images provided there also seems (to me) to be an increase in expression at E12.5, and some staining pattern differences at other ages. Were these data quantified or was this a qualitative analysis. If the latter, what were the criteria used to assess differences between and within groups? Can the authors please comment on the possible implications of these differences?\n\n2. Report on motor performance: Paragraph 3 states that in the beam walking test the latency to cross the beam was significantly increased, but the figure shows this to be true only in females. I suggest this be qualified in the text. Similarly \u201cthe number of slips was slightly increased\u201d is misleading, since it is not significantly different. \u201cMales were less affected than females\u201d suggests that males are affected, which is not what the data shows. I understand your suspicions regarding the power of some of the analysis, the analysis provided shows no significant differences, and it might be worthwhile to design a repeat of the experiment with more power to determine whether these tendencies are significant or not. Similarly in paragraph 5 of discussion: \u201ctended to have higher number of slips\u201d need to qualify this was NS.\n\n3. Report on motor performance: \u201cmales and females tended to have reduced locomotor activity [\u2026] p=0.09 . Isn\u2019t this NS?\n\n4. Report on motor performance: \u201creduced pain sensitivity\u201d Not sure the sensitivity to pain, but rather the response to pain is what is measured. If the argument is made that it is the reduction of GABA cells in the DSC that are responsible for the motor deficits, I am not sure how this relates to reduced sensitivity at the primary afferent level \u2013 I still fail to see why the conclusion is that there are proprioceptive defects and/or altered sensory abilities. Are there no alternative explanations? Though this might be simply a semantic issue. The way I read it is that the argument is made that the deficits are in the sensory periphery/primary afferents.\n\n5. Hindbrain analysis: In response to Reviewer #1 an analysis at E18.5 was performed in the hindbrain using GAD67 and reported as no difference between wild type and mutant \u2013 Figure S3 G,H appears to show a reduced expression of GAD67 in cerebellum., and perhaps some increase in the mutants in dorsal hindbrain (Panels E/F). Of course, I am not able to look at the entire section set. Was this quantified, or is this a qualitative assessment? If the latter, were any criteria used?\n\n6. Development of spinal cord dorsal horn:The text says that Nissl staining of E18.5 mice \u2026 (Fig 6) but the Figure legend says it is E 16.5.\n\n7. For figure 4 the text states that there are \u201cno defects in patterning of sensory afferent fiber projections\u201d but this conclusion is based mainly on the staining of a subset of sensory afferents (that are labeled with calbindin) and on the apparent lack of differences in expression of Drg11. I think the resolution of the images in panels A and B is too low for the reader to assess any possible differences and/or ectopic projections. Also, the markers used cannot rule out other types of patterning differences from primary afferents that do not label with calbindin or possible patterning differences at ages other than the one provided. The statement that follows regarding that males show longer withdrawal to the hotplate cannot be easily correlated with the images shown since it is not known whether the tissue presented corresponds to males or female animals, and this limitation should be made explicit. It would be useful to refer to Buckley et al.\u2019s results that indicate that indeed there are abnormal proprioceptive projections.\n\n8. In panels EF of Figure 4 the text reports no differences in the patterns of staining for peripherin. There appears to be a down regulation of peripherin at least in the most medial ventral spinal cord, a finding that is consistent with those of Buckley (2013). Were you able to find ectopic projecting axons such as those described by Buckely et al?\n\n9. The statement that \u201cno differences in the number of Islet1+ cells were found in the ventral spinal cord - was unable to find this quantification \u2013 or was this a qualitative assessment? If so, can you please describe how this was done.\n\n10. I am also surprised that the differences in ages between the Buckley study and the present study are not considered as a source of discrepancy.\n\n11. Regarding Reviewer#1 request to look at the fate of neurons that do not become GAD67 positive \u2013 Is there any reason why the apoptosis analysis is not shown or details of how this was analysed provided?\n\n12. In response to Reviewer #1\u2019s request to address post natal expression abnormalities at the spinal cord level, the argument is made that these will be part of a separate study. While I understand why the choice of separating these two studies may be appropriate, it does not address the reviewer\u2019s concerns regarding how appropriate it is to draw causal conclusions on mature motor performance based on embryonic GABAergic phenotype. The authors might want to consider discussing this limitation in more detail in their discussion.\n\n13. In response to reviewer #2\u2019s query about the onset of the motor deficits, you provide a statement that the deficit first appears at P16-17. Could you please provide details on how this was analysed.\n\n14. In response to the editor\u2019s summary you state that you have repeated analyses at several developmental stages. I am unable to find those data, other than for the additional material provided in the supplementary figures (for the additional Gbx2 expression, the analysis of hindbrain and the expression of Islet in the SC). However, the main ISH and ICChem data on which the main conclusions are based seem to only be described at a single time point.\n\nMethods reporting:\n\n15. Can you please provide a reference to the methods used for obtaining the blastocysts, etc. \u2013 I understand this might be standard, but I would imagine the source would be needed for any attempts of replication.\n\n16. In response to Reveiwer#2\u2019s request to clarify how abnormal gait was assessed, I am unable to find a description in the material and methods for other groups to be able to replicate your work. Could you please provide the details including what criteria were used for the assessment of what was \u201cnormal\u201d or not. I am assuming observers were familiar with normal gait patterns in normal mice prior to the observational classification? Did both observers always agree on the classification?\n\n17. In response to Reviewer #1 regarding details on the cell counting method: I still don\u2019t think that enough detail has been provided for an independent replication of the data. The figure legend says the areas shown in panels C and D were the ones used for counting \u2013 but there are areas where there is no tissue included in those areas \u2013 Was there a criterion as to where the counting box was placed, that is, over what anatomical area were the cells counted? Also could you please provide information as to the objective used (eg NA) to get a sense of resolution, and whether the cells counted under the microscope or from printed photographs? Like the reviewer, I still fail to see in the images the reduction that is reported in the graphs.\n\n18. It also seems to me that if one is counting cells over a defined area (which according to your figure excludes some tissue where GAD cells are present too), what is being provided is an estimate of cell density rather than actual cell number .\n\n19. I am also confused in the graph what the % of cells refers to, and hence what the reported percentage reduction refers to. Can this be clarified further please? Also, if 3 sections of 3 animals were used for counting for each, the n for each group would be 3, not 9 \u2013 I am not convinced a t test is appropriate, and wouldn\u2019t think it is appropriate to plot the data as means and standard deviations. These issues also apply to the quantification of Pax2 and VGLUT.\n\n20. The mouse anti-islet1 antibody is reported as being used at 1:100 \u2013 please indicate whether this was the supernatant or the concentrate form. Please provide the concentration used for the secondary antibodies. Can you please provide the constitution of the buffers in which antibodies were diluted and washes made. Could you please also provide (or specify) your experimental controls results.\n\n21. Rotarod test: I am not sure what the \u201clatency\u201d in the test is. Am I right to assume this is the time it takes for them to fall? Please specify.\n\n22. Electrophysiological measures: I am still not sure how the stimulating electrode was placed on the sciatic nerve, or how the distance between the stimulating electrode was measured (and with what accuracy). I am also no sure how these latencies were actually measured or how the data were analysed. I am also unsure how many recordings per animal were done, etc. Can you please also provide the dosage of anaesthesia.\n\nThere are also a few typographical/grammatical errors in the manuscript.",
    "pdf_3": "https://peerj.com/articles/142v0.3/submission",
    "pdf_4": "https://peerj.com/articles/142v0.2/submission",
    "pdf_5": "https://peerj.com/articles/142v0.1/submission",
    "review 6": "M Fabiana Kubke \u00b7 Jan 18, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nI have now received 2 reviewer\u2019s reports for your manuscript and reviewed it myself. Overall, I find the work to be valuable, but there are some issues that I think are important to be addressed before the manuscript is accepted for publication. While some comments suggest that the work would benefit from additional experiments, I understand this is not always possible. However, I think the issues raised are valid and should be at least addressed in the text of the manuscript. General Interpretation of the data: Gbx1 -/- phenotype: One of the reviewers asks whether there might be a compensatory expression of Gbx2 as a result of loss of function of Gbx1. I think that it would be useful to address this in the discussion. Anatomical/histological analysis: It seems the data for ICChem and ISH is based on a single time point which is also different from the age at which motor function was assessed. Both reviewers highlighted the limits of how these data can be extrapolated to relate it to the behavioural results. One reviewer asks what the expression patterns observed with ICChem and ISH might be in the spinal cord in more mature rats to warrant the link made between GABAergic phenotype and behavioural deficits. I would agree that showing that the differences observed in embryos persist beyond the age study would be useful. One reviewer and I also wonder about the use of ISH GAD-67 used as a proxy for GABA-ergic phenotype (suggesting the use of a second marker such as Pax2). I also wonder what the expression patterns of GAD-65 might be. Is it possible that those neurons that are not expressing GAD-67 might still express a GABAergic phenotype? Or is it possible that the differences observed might reflect a difference in the time course of expression of the GABAergic phenotype? One of the reviewers also queries whether the onset of locomotor deficits with respect to the transition of GABA between inhibitory to excitatory. Could this be added to the general discussion? One reviewer also queries whether the lack of obvious abnormalities in the hindbrain at E9.5 is sufficient evidence to rule this possibility out. Is the GABA phenotype, for example, normal in hindbrain at later stages, or are there deficits similar to those described in the SC? I also wonder whether it might be useful to show the hindbrain data on which these conclusions are based. It seems that this is important data that is substantial to the interpretation of the behavioural data (as highlighted in pp. 12-13). Similarly, on P 14 the statement is made that \u201cAltogether, these data suggest that there are not defects in patterning\u2026\u201d My impression is that what is provided is a very broad morphological analysis on a single time point. I am weary as to the degree of confidence with which that statement can be made, eg., do these data show with enough detail layer-specific projections, and is a single time point sufficient to rule out defects in patterning? Pain sensitivity: One reviewer questions the interpretation of the possible role of allodynia. I also wondered why the argument is made that the Gbx1 -/- might have reduced thermal pain sensitivity \u2013 an alternative is that they have issued processing reflexes involved in thermal pain which may not necessarily be accompanied by a reduced sensitivity per se.\nIn the results, the statement \u201cshowed a tendency for higher latencies than WT, but the difference between genotypes was not significant\u201d should be corrected. If there is no statistical difference, then I don\u2019t think the implication that the latencies may be higher is not justified. Similarly on Page 12 \u201cat least Gbx1 -/- females [..] tended to have higher number of slips\u201d these differences were not shown to be significant and \u201cGbx1-/- mutants displayed impaired rotarod behavior [\u2026]\u201d but table 1 and p10 this is described as NS. General Methodological queries and replicability. Both reviewers identify areas where further methodological information would be useful for the interpretation of data, and further descriptions are needed to ensure replicability of these studies. Please provide the N for all experiments- for example, tissue collection and sample preparation does not say how many pregnant females were used and how many embryos from each were harvested. This is important when considering the actual size of the sample analysed for histology and to infer possible litter effects in the results. It was also not clear to me in the behavioural phenotyping experiments whether the cohorts described mean that each cohort was put through a given paradigm, or whether there were several cohorts each tested through the set of behavioural paradigms. Please provide a description of how the blastocysts were obtained (Page 6, second paragraph). Please indicate whether behavioural experiments were performed during the day or night cycle of the rats. Cell counting: One of the reviewers queries about how GAD-67 + cells were counted, and whether the images shown are representative of the data. Cell counts are described as being made in 9 sections for each animal and 3 animals of each genotype. I am assuming the data in the plot in Figure 8 represents an average of the 27 sections for each genotype, but this could lead to pseudoreplication. The n for each genotype should be 3, each represented by the average of the 9 sections (which would probably preclude from using the parametric t-test). It is also not reported whether the tissue used for this study was from males and females, and given the male-female differences seen in some of the behavioural tests and the sensory conduction velocity this is an important factor to take into account. Assessment of motor deficits: One of the reviewers queries about what is meant by \u201cduck type gait\u201d and \u201clack of fluidity\u201d in movement. I wonder whether providing a movie (if available) might aid in the description. The reviewer also queries it would be useful to look at time s. pent in extension and flexion. Electromyography: The description of the electrode placed at the \u201cbase of the tail\u201d is not too clear. I am assuming since it is described as an EMG it would be a muscle, but I fail to see how recording from a muscle would provide a measure of the sensitive (do you mean sensory?) nerve conduction velocity. I am also not sure how these latencies were actually measured. How much precision was there in the placement of the stimulating electrode at 20 mm away from the recording electrode? It is also not stated how the data was analysed \u2013 how many animals were used, how many recordings per animal, etc. Would be worthwhile to show an image of these recordings, especially given the differences between males and females? General corrections: Reference to Table 1 at the bottom of Page 11 should be Table 2? There is also a discrepancy with the text saying females had increased conduction velocity, but in the table it marks the males as being significantly different. Similarly re: latency (top of p 12) the text says there is N.S. but the table suggests there is a significant difference in females. Results for rotarod need to be checked for accuracy. The statement \u201cmales and females tended to have reduced locomotor activity over the testing period (p=0.09). Is this p value correct? Figure 7 also needs correcting. General suggestions I am surprised that the sexual dimorphism in the Gbx1-/- phenotype is not addressed. Would it be useful to speculate on its significance?\nIn the introduction (first paragraph) tactile projections are described as projecting to laminae III, IV and V and temperature and pain as projecting to lamina I, II and III, whereas in page 14 the former are said to project mainly to laminae III, IV and V and the latter to lamina I/II. Perhaps it should be made clear in the introduction the degree of projections to lamina III from pain and temperature receptors. I am not sure I follow the argument on P12 \u201cGbx1-/- females had increased sensory nerve conduction velocity [..] supporting the altered sensory deficits observed in the behavioural tests\u201d nor in P14 paragraph 2, I suggest revising the wording. P14 par 1 Can you provide references for the statements on the second half of the paragraph? Please add the names of people providing supplies (for immuno and in situ) to the acknowledgements and obtain permission for that. Please declare if these supplies would be available for replication studies.\nAnimal Ethics Statement is presented under the generation of genotyping of chimeric and mutant mice \u2013 I would assume that ethical approval has been sought for the entirety of the project. If so, it might be made more explicit by placing the animal ethics statement at the beginning (or at the end) of the M+M. . Deolinda Lima \u00b7 Jan 11, 2013\nBasic reporting\n- In figure 1 it would be interesting to look for the expression of Gbx2 to see if there is a compensatory expression of Gbx2 due to the loss-of- function of Gbx1 in mutant mice, especially in developmental ages from E12.5 onwards.\n- No obvious developmental abnormalities were found at the hindbrain in E9.5 Gbx1-ko mice. The authors should look for alterations at later developmental ages, using terminal differentiation markers (as it was done at the spinal cord level) to completely exclude the involvement of hindbrain in the observed mutant phenotype.\n- The authors discuss that allodynia cannot be discarded as a cause for the altered gait phenotype. In the hot plate analysis, Gbx1 ko mice (particularly the males) displayed a reduced response in hot plate test indicating that mutant mice feel less pain. Therefore, allodynia does not seem a possible scenario to reconcile with the observed hot plate performance.\n- In the characterization of the spinal cord dorsal horn of ko mice, there are several weaknesses that compromise one of the main conclusions of the manuscript, which concerns the cell specification defect in the dorsal spinal cord (see at \"Experimental Design\").\nMinor corrections:\n- Figure 7 C,D and E,F does not correspond to text description.\n- In the text the results of increased sensory nerve conduction velocity are stated in Table2 and not in Table 1.\n- In Table 1 \u2013 rotarod, results for Gbx1-KO females does not seem correct.\n- On page 4 it is mentioned that Drg11 is expressed in late born derived cells from dI5 neurons but the reference mentioned is not fully correct. Authors should consider adding the studies by Rebelo et al (2010 Dev Dyn).\nExperimental design\n1- It is not clear how counting of Gad67-positive cells was performed (a full description is need in the Materials and Methods section) and, in figure 8, Gad67 in situ staining pictures do not reflect the cell counting shown in the graph. As the difference is not so marked, the authors should use other GABAergic markers, such as Pax2, to corroborate the result of a reduction of GABAergic neuronal population.\n2- If these Gad67-positive neurons are missing, it is important to address whether these neurons died, change to a glutamatergic phenotype or possibly there is simply a Gad67 expression down regulation. Therefore, neuronal death markers and glutamatergic markers should be looked up. Indeed, it may be relevant to double check a possible difference in the Drg11 expression (Figure 7 EF) since at sight there seems to be a difference in expression between wild type and Gbx1-knockout embryos at E16.5.\n3- It would be also important to address post natal expression abnormalities at the spinal cord level atfer the first two or three weeks after birth.\nValidity of the findings\n- The authors conclude that Gbx1 has a role in the specification of a subset of GABAergic dorsal horn interneurons involved in the control of hindlimb movement, however it\u2019s not clear how this assumption could be made. It is well established that limb movement is controlled by neuronal circuits located at ventral spinal cord and that somatosensory information is integrated and conveyed to the brain by the dorsal spinal cord. Therefore, it\u2019s no clear how authors could do such a conclusion.\nAdditional comments\nIn order to find a physiological function for Gbx1, the authors generated and characterized the phenotype of the Gbx1-knockout (ko) mice at two levels: molecularly during development and behaviorally. After a battery of behavioral tests the most striking phenotype was the altered gait during forward locomotion affecting the hindlimbs. The authors then proceeded trying to correlate this observation with a molecular defect at the neuroaxis.\nThis paper requires, however, significant revision.\nCite this review as\nLima D (2013) Peer Review #1 of \"The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord (v0.1)\". PeerJ https://doi.org/10.7287/peerj.142v0.1/reviews/1 Reviewer 2 \u00b7 Dec 26, 2012\nBasic reporting\nIt seems odd to end the introduction with a reference based on the content of the sentence?\nExperimental design\nNo comments\nValidity of the findings\npg. 10- the description of a \"duck type\" gait needs explanation. It was not immediately apparent what was meant by this. Describing the specific sequence of hyper-flexion and hyper-extension behaviors would be more useful to the reader. pg. 10- For the following statement \"However, many of the Gbx1-/- mutants showed significantly abnormal gait (\u03c72 \u22655.20, p<0.05). Indeed, 43% of Gbx1 mutant males and 63% of Gbx1 mutant females displayed lack of fluidity in movement\" clarify the test that was used to demonstrate gait was abnormal and motion was not fluid.\nAdditional comments\n1) It would be interesting to determine whether behavioral/locomotor deficits coincide with change of GABA from excitatory to inhibitory which (I think) happens around P17 in mouse- well after the onset of weight bearing locomotion. If the locomotor deficits began around this time it would really strengthen the paper by directly linking them to GABA. 2) The conclusion really needs a discussion of the experiments required to determine the potential source of the locomotor deficit (central vs proprioceptive deficit). Some discussion of the locomotor CPG (role of other genetically defined populations and how they might be connected to GABAergic neurons) as well as the role of proprioceptive input in generating locomotor outputs needs be included in this section. 3) It would be useful to quantitate the amount of time spent in flexion (paw raised) vs time spent in extension (paw on ground) and compare to wt. In \"regular\" mammalian locomotion as speed of locomotion increases the proportion of time spent in the flexor phase (swing) increases. Is this the case in the Gbx1-/-?\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord (v0.1)\". PeerJ https://doi.org/10.7287/peerj.142v0.1/reviews/2",
    "all_reviews": "Review 1: M Fabiana Kubke \u00b7 Aug 4, 2013 \u00b7 Academic Editor\nACCEPT\nI would like to thank the authors for their cooperation through the processing of the manuscript. The article represents a valuable contribution to the field.\n\nThere are a few very small editorial changes that I will ask the authors to make prior to final publication:\n\n1. Page 3, last sentence of first paragraph.\nThe sentence: \u201cProprioception is mediated by sensory neurons that project through the dorsal spinal cord to an intermediate zone which in turn projects to the ventral spinal cord where direct connection is made to motoneurons (Brown, 1981; for review: Caspary & Anderson 2003)\u201d\nConsider changing to: \u201c [\u2026] to the ventral spinal cord where [a] direct connection is made [with or onto] motoneurons [\u2026]\u201d\nThe sentence also needs a period at the end.\n\n2. Page 6, second paragraph:\nA period appears to be missing before the sentence starting \u201dThe presence of a wild-type allele was detected using \u2026\u201d\n\n3. Page 6 last paragraph:\nThe molarity of PBS is expressed as PBS 1x \u2013 I still don\u2019t know what that is \u2013 please include the actual molarity at this stage, (eg PBS 1x (XXXX M, pH 7.5) and then continue using 1x thereafter if desired.\n\n4. Page 9 first sentence:\n\u201c(rpm) in 5 min.\u201d I am not sure but should this be \u201cover\u201d 5 min?\n\n5. The ages at which the gait was analysed is confusing \u2013 It is first said that it is analysed at 10 wk of age (p. 8), then at 4-6 wk of age (p.11) and then the onset is described at P16-17 (p.19). To avoid confusion, please specify the age used for the data in Table 1, Figure 2 and supplementary movie (eg add age in the figure legend and into the table).\n\n6. Page 8, First paragraph, second/third line\nThe statement \u201cand mouse anti-islet \u2026\u201d Suggest changing [and] and substituting with [for] \u2013 am assuming the different antibodies were done in different sections and not in the same section, which is what \u201cand\u201d seems to suggest.\n\n7. Figure 5 and Figure 5 legend:\nThe abbreviation BN is used for \u201cwhite noise\u201d (assuming you mean \u201cblanche\u201d?) \u2013 I suggest changing this to WN, since English speakers might confuse this with Brown Noise (which is what the abbreviation BN is usually used for).\nReview 2: M Fabiana Kubke \u00b7 Jun 5, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThank you very much for the resubmission, and also for your effort addressing the issues raised. I want to once again emphasise that I consider this to be a valuable set of experiments that deserves to be published, and I hope you will be able to address some remaining issues that prevent me from accepting the manuscript at the time.\n\nOne important issue still remains regarding the analysis of the GABA cells, which is of great importance since this seems to be at the centre of the interpretation of the behavioural phenotype.\n\n1. I was surprised that Dr Dembele suggested a paired t-test since I couldn\u2019t understand what criterion/justification was used to pair the animals from the two genotypes (and I couldn\u2019t find where one was provided). I have consulted with a colleague of mine in the statistics department (Dr Russell) who suggested that since you have 2 genotypes and 3 observations in each of 3 animals per genotype, the data would be best analysed with a 2 way mixed model anova with the first variable as treatment effect (fixed effect ie, genotype_ and a second variable as random effect of repeat observations on the same individual. He provided me these resources\nhttp://www.statisticshell.com/docs/mixed.pdf and http://www.math.montana.edu/~cherry/st412/pdf_files/CourseNotes16.pdf\nPlease note that the same criticisms apply to the data on Figure S4.\n2. I am also surprised that the authors were unable to find any references to the switch of GABA from depolarising to hyperpolarising to speculate at what age this might be happening in the dorsal horn at the levels of the spinal cord where the GABA phenotype is described. My search returned a few articles that the authors may want to consider: Stein, V., Hermans-Borgmeyer, I., Jentsch, T. J., & H\u00fcbner, C. A. (2004) J Comp Neurol 468(1), 57\u201364 sets the shift at around P7-P10 at least for hippocampal and spinal cord motoneurons in mouse; Cordero-Erausquin et al (2005) J Neurosci 25(42):9613-8623 show similar time course for the switch of GABA in neurons of LI in the dorsal horn (albeit in rats) while Sibilla, S., & Ballerini, L. (2009). Progress in Neurobiology, 89(1), 46\u201360 report these changes as happening within the first two weeks of age. This would suggest (if I have interpreted the articles correctly) that the switch may be over by the time that the phenotype becomes expressed (P16-17). The authors may also want to consider when incorporating this to their discussion that a depolarising effect of GABA does not necessarily means it is excitatory (some depolarising GABA has been shown to be inhibitory, eg. Viemari, J.-C et al (2011). Importance of chloride homeostasis in the operation of rhythmic motor networks. In Progress in Brain Research (Vol. 188, pp. 3\u201314). ).\n3. The statement : \u201cAs expected, natural cell death occurs in the developing spinal ganglia (Fig. 9A, arrow; Paschaki et al. 2012) but not in the spinal cord\u201d and what follows:\nThe reference does not support the statement, since it does not constitute a study of normal cell death in mice, and, within that same reference it appears to me that apopototic labeling in the ventral spinal cord at 12.5 can be seen (Figure 4I). There are a number of studies looking at normal cell death in mouse spinal cord, and my interpretation of some of those is that one would expect to see cell death in the spinal cord as well as the DRG at least in your E12.5 and E 14.5 material. (eg, White et al Journal of Neuroscience, February 15, 1998, 18 (4):1428); Yamamoto, Y., & Henderson, C. E. (1999). Developmental biology, 214(1), 60\u201371). It would be useful to discuss why it might be the case that no TUNEL labeling is visible in your material at those ages, which is crucial to validate the results.\n4. I will also ask that those data where the statistical analysis failed to show a statistical significance are clearly described as such. Eg the sentence : \u201cSuch abnormal sensory processing is suggested at least for thermal stimuli, as Gbx1-/- mice displayed increased latency suggesting reduced pain in the hot plate test (thermosensory functions)\u201d. Should reflect that this is only true in one sex. There are several instances of similar reporting throughout the manuscript.\n5. An issue also remains as to the need to discuss how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system which, as the authors state, projects to the ventral and intermediate spinal cord, not the dorsal horn which was examined in this study. The explanation :\u201cwe cannot exclude abnormal functions of those afferents caused by reduced GABAergic cell number in superficial dorsal horn (see below), which is the transition and/or adjacent region for proprioceptive afferents. Such proprioceptive-like deficits may be also secondary to a defect in sensory perception or expression of such sensory response, as indicated by sensory deficits in Gbx1-/- mice and abnormal development of the dorsal horn, the main target of sensory afferents.\u201d . I don\u2019t find the explanation convincing, and perhaps adding support for such mechanism from the literature might help make your case. Regardless, the statement needs to be amended for accuracy:\na. This study has provided evidence for a reduction in the proportion (not number) of GABA neurons (also please correct other references to number of GABAergic cells elsewhere in the manuscript, eg, in page 1 and 12, please check for other instances.)\nb. The data analysis failed to show significant sensory deficits in most sets of data. Other than the latency in the beam test in females (proprioceptive test) all other measures for proprioceptive defects were NS (This also applies to the sentence: \u201cImportantly, abnormal gait resemble proprioceptive-like deficits, which was further suggested by deficits in beam-walking, the test used for evaluation of proprioceptive functions\u201d) and \u201cThis abnormal gait, documented by a series of behavioral tests, is not due to deficits in muscle strength or motor coordination, but may be related to proprioceptive deficits suggested by reduced performance in beam walking, a test used in studies of proprioception.\u201d Please check for other possible similar isntances.\nc. The projection patterns of proprioceptive afferents were also described as normal, so it is unclear why a case is made for a defect in proprioceptive function due to possible measured GABA deficits in a region that in not recipient of those proprioceptive afferents. Perhaps this argument could be strengthened by providing evidence from the literature that might support this case.\nd. For the most part development of the spinal cord was normal (based on the description of Nissl staining, and several markers: Lbx1, Lmx1b, Netrin-1, Drg11, etc.) other than the GABAergic phenotype\ne. Similarly, \u201cThis abnormal gait, documented by a series of behavioral tests, is not due to deficits in muscle strength or motor coordination, but may be related to proprioceptive deficits suggested by reduced performance in beam walking, a test used in studies of proprioception. (in the conclusion)\u201d needs to accurately reflect the results from the proprioceptor tests.\n\nOther:\n\n6. Please amend the materials and methods which says that the histological analysis was done in 3 sections per animal, in 3 animals of each genotype since some of your figures the legend states that the number of animals is 2, not 3. The N for the results of Figure S3 are not provided.\n7. Panel D in Figure 8 appears to be flipped horizontally\n8. Please provide the molarity and pH of all buffers used.\n9. \u201cunmasking in citrate 0.1 M (pH 6)\u201d do you mean citrate buffer?\n10. What were the solutions used to dilute the primary antibodies?\n11. \u201cFurthermore, electromyography (EMG) measurements revealed that the sensory nerve conduction\u201d Do you mean SNCV?\n\nOther suggestions:\n1. Please amend the statement: \u201d In contrast to our observations, those mutants show disorganized peripherin expression, together with a decrease of Islet1-expressing cells in the ventral horn of the lumbar spinal cord (Buckley et al. 2013).\u201d See Figures 9 (showing change at E14.5-15.5) and Fig 5 (no change at E11.5). Please specify that the statement is correct for ages comparable to yours (not for younger embryos).\n2. The sentence \u201cAt E12.5, the expression domains of Gbx1 and Gbx2 overlap, both being expressed in the ventricular and mantle zones of the dorsal spinal cord (Rhinn et al. 2004; Waters, Wilson & Lewandoski 2003). As Gbx2 expression is downregulated fter E12.5, both genes are only transiently coexpressed in dorsal spinal cord progenitor cells, and Gbx1 is the only Gbx gene persistently expressed during later dorsal horn development (John, Wildner & Britsch 2005).\u201d Which appears under the hindbrain heading would more appropriately be placed in the next section under the spinal cord heading.\n3. I am also confused as to the statement \" This may reflect an abnormal development or survival of GABAergic neurons, which in consequence coud lead to abnormal control of neuronal network in dorsal horn, possibly affecting inhibitory circuits throughout the spinal cord.\" that is presented in the context of the GABA reduction \u2013 since the authors go on to show in the following paragraph that cell death as a factor could be ruled out and instead a change of phenotype to glutamate was proposed as a more likely explanation.\n4. The sentence \"Proprioceptive neuron afferents form two types of termination zones, one in the intermediate spinal cord and one in the ventral spinal cord where they are directly connected to motoneurons (Brown, 1981)\" which was added in page 17: I think a similar description of the targets of proprioceptive afferents should be added to the description of afferents in the first paragraph of the Introduction where the rest of the afferent projections to the SC are described.\nReview 3: M Fabiana Kubke \u00b7 May 1, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nPlease address the following remaining issues, as well as other issues as per our offline correspondence. Please note that the reviewer's comments are incorporated into the Editor's comments, so there is no need to respond to those separately.\n\n1. Can the authors please comment in the manuscript on the possible implications of the subtle increase of Gbx2 expression at E12.5-E14.5\n\n2. Reviewer #1: Quantifications of number of stained cells for each molecular marker displayed in fig 8 should be accompanied by an appropriate statistical analysis to assess the significance of the differences observed.\n\nIf the authors can argue why this statistical analysis cannot be performed, these limitations, and the limitations on the interpretation of the data need to be made explicit.\n\n3. To address the query about the possible fate of GABAergic neurons the authors complemented their GABAergic phenotype studies with examination of a glutamatergic marker at E18.5 and a cell death marker (at E12.5, 14.5, 16.5, 18.5). I think that it would be valuable to include these figures in the main article rather than in the supplementary material.\n\n4. Could the authors also please discuss how the ages at which the TUNEL was examined correspond to expected apoptosis through naturally occurring cell death in the mouse lumbosacral spinal cord and how this may (or may not) have an impact on your data results and interpretation.\n\n5. To address the concerns about how valid it is to compare the embryological data to the adult phenotype, the authors have added to the histological examination at E18.5 the observation that the onset of defects coincides with the switch of GABA from excitatory to inhibitory. These observations are described as being obtained by checking pups visually around weaning. Can you please provide the specific ages and frequency with which this monitoring was done, the number of animals and if possible the male to female ratio. Also, please double check reference to Ben-Ari et al. \u201cwhich coincides with the time point at which the GABA pathway switches from excitatory to inhibitory (Ben-Ari et al. 2007).\u201d I was unable (albeit through a quick superficial scan) to find in that paper mention of a switch from excitatory to inhibitory in mice spinal cord at that age.\n\n6. Reviewer #1: Moreover, the present data differ from the Buckley\u2019s data in that the expression of peripherin and Islet1 was not changed in the Gbx1 -/- model. This discrepancy was attributed to the use of different time points. Although I don\u2019t think this may be the reason, if the authors insist in this explanation they should evaluate the expression of those markers at the same time points. In addition, it would be very interesting to analyse their expression at postnatal stages, when the locomotor defect is detected, as requested on our previous revision\n6.1. Regarding the discrepancies between this and Buckley\u2019s results on the peripherin staining, it might be useful if the authors used an image of peripherin at E16.5, since they already have that material and the age is closer to that of Buckley et al. (E14.5 and E15.5) to allow for a better age-matched comparison. I think this would satisfy the reviewer\u2019s request.\n6.2. Regarding the discrepancies in Islet1+ results between the current work and that of Buckley et al., please amend the statement: \u201d In contrast to our observations, those mutants show disorganized peripherin expression, together with a decrease of Islet1-expressing cells in the ventral horn of the lumbar spinal cord (Buckley et al. 2013).\u201d See Figures 9 (showing change at E14.5-15.5) and Fig 5 (no change at E11.5). Since Buckley et al. show a decrease of ISL1 cells at E14.5 and E15.5, it might be useful to include images and the quantification for E16.5 in your material for a better age-matched comparison. If you do not have that quantification, please amend the sentence \u201cNo differences in the number of Islet1+ cells within the lumbar ventral spinal cord were found at E14.5, E16.5 or E18.5\u201d by removing the E16.5.\n\n7. For figure 7 the text states that there are \u201cno defects in patterning of sensory afferent fiber projections\u201d but this conclusion is based mainly on the staining of a subset of sensory afferents (those labeled with calbindin) and on the apparent lack of differences in expression of Drg11. I think the resolution of the images in panels A and B is too low for the reader to assess any possible differences and/or ectopic projections, and cannot differentiate between afferent inputs and neuropil stained originating from what appear as calbindin+ neurons in the SC. Also, the markers used cannot rule out other types of patterning differences from primary afferents that do not label with calbindin or possible patterning differences at ages other than the one provided. Please address these limitations.\n\n8. Reviewer #1: On page 9 the authors say \u201cAltogether the behavioural data revealed that the Gbx1 -/- animals \u2026.because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test\u201d. This paragraph should be revised correcting the hot plate test observation (underlined) and should be better explained how animals with less pain sensitivity in the hot plate could suffer from pain on movement.\n\n9. Reviewer #1: In the conclusion section, in the end of first paragraph the sentence \u201c\u2026 as mutant mice also display a significant reduced response time in the hot plate (thermosensory) test.\u201d Is in contradiction with the affirmation in the third paragraph \u201c\u2026 as Gbx1 -/- mice displayed significantly increased response time in the hot plate test.\u201d\n\n10. The sentence \u201cWe cannot exclude that the proprioceptive defects may be secondary to a defect in sensory pathways (for example, caused by pain on movement), because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test.\u201d Should be corrected to reflect that only males showed a significant difference and this was an increase (not decrease) in the response latency.\n\n11. In resonse to the request for details on the methods by which abnormal gait was assessed, please indicate whether observers were familiar with normal gait patterns in normal mice prior to the observational classification\n\n12. [R#1: In the Buckley study as in the study under revision, it is not discussed how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system. The authors should address this point in the discussion, as requested in our first revision of the paper. ]\n12.1. The authors might also want to make explicit that changes in the GABA phenotype were not analysed outside of the dorsal horn, and that contribution of inhibitory circuits elsewhere in the spinal cord cannot be excluded.\n12.2. In the statement: \u201c\u201cWe cannot exclude that the proprioceptive defects may be secondary to a defect in sensory pathways (for example, caused by pain on movement), because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test.\u201d Please amend the sentence to reflect that only males displayed a significantly reduced response time.\n12.3. In the statement: \u201cAltogether, the behavioral data revealed that the Gbx1-/- animals have apparent proprioceptive defects and/or altered sensory abilities.\u201d I am still uncertain as to how after failing to show significance on sensory features measured (other than conduction velocity in females and hotplate in males) this statement can be justified.\n\n13. The statement \u201cThis is supported by data from the beam walking test showing that at least Gbx1-/- females required longer time to cross the beam distance and tended to have higher number of slips (not statistically significant)\u201d if it is NS it is not supported by the data \u2013 the trend you see might indicate that a higher power test might uncover this, but it is speculation.\n\n14. The statement: \u201cElectrophysiological measurements showed that Gbx1-/- females had increased sensory nerve conduction velocity, measured in the caudal nerve, supporting the altered sensory functions observed in the behavioral tests.\u201d Not sure what sensory functions are being referred to here. Responses in the hot plate, startle, beam slips and sensorimotor all failed to show an effect on phenotype.\n\n15. The statement \u201cWe found that both Gbx1-/- males and females show reduced locomotor activity in different situations.\u201d, but legend to figure 4 says \u201cThe distance traveled over the 20 min period of test reflects locomotor activity\u201d and shows no difference, and on page 11 this is described as \u201c. When considering each gender separately, both Gbx1-/- males and females tended to have reduced locomotor activity over the testing period (although not statistically significant, p=0.09) (Fig. 4).\u201d\n\nGeneral corrections:\n\n16. R#1: In results section when describing Drg11 expression they should mention (Fig 7C,D and data not shown)\n16.1. I found similar instances throughout the manuscript. Please differentiate between the data that was used for the analysis but that is not shown and that which is shown: Lbx1 analysis reported at E12.5, 14.5, 16.5, 18.5 but only shown at E16.5. Lmx1b analysis reported at E12.5, 14.5, 16.5, 18.5 but shown at E16.5; etc. In the text: \u201cor at the level of fibers that enter into the spinal gray matter, at E16.5 or E18.5 (arrows in Fig. 7E,F).\u201d not clear which age is shown in the figure.\n\n17. P16: The sentence \u201cImportantly, Gad67 expression was reduced in the dorsal spinal cord of Gbx1 mutant mice (Fig. 8A-D), i.e. there was a 16% decrease in the number of Gad67-expressing cells in comparison to WT mice (Fig. 8I).\u201d should be corrected \u2013 there was a 16% decrease in the proportion of GAD67 expressing cells. The same applies to sentence \u201cThis finding was strengthened by analysis of Pax2, another gene expressed in GABAergic cells in the spinal cord (Cheng et al. 2004), with cell countings corroborating the decrease in the number of GABAergic cells\u201d Similarly in the legend to Figure 8 the statement \u201cCountings (percentages of labelled vs. total cells) revealed that the numbers of Gad67+ cells are diminished by 16% in Gbx1-/- mice\u201d should be corrected to \u201crevealed that the proportion of GAD67+ cells\u201d. The same applies to \u201cnumber of Pax2 cells\u201d and \u201cnumbers of Slc17a6+ cells\u201d\n\n18. There is a discrepancy in the reporting of the Netrin-1 material \u2013 the text says the image shown is E18.5 whereas the figure legend suggests it is E16.5\n\n19. On Figure 7, Please specify the age at which the Drg11 and Peripherin staining shown was performed.\n\n20. P11: \u201cchecked the expression of Gbx2 from E12.5 onwards\u201d specify ages\n21. P11: \u201cunnevenless\u201d check spelling\n\nReferences\n22. \u201cWaters, Wilson & Lewandoski 2004\u201d. Please double check \u2013 PubMed cites as Gene Expr Patterns. 2003 Jun;3(3):313-7.\n23. Reference to Nagy et al. 2003 does not seem to be in the reference list\n\n24. In order to adhere to PeerJ\u2019s editorial policy: \u201cMethods should be described with enough information to be reproducible by another investigator\u201d and that \u201cthe data should be robust, statistically sound, and controlled\u201d I will ask the authors to make the following amendments:\n\n1. Further details on how cell counting was performed need to be provided \u2013 eg what does using ImageJ mean? Was a threshold applied for counting, or just used to track cells? Were the sections counterstained, and if not, how were the unlabelled cells visualised. Unlike the case of the material shown in S4, it is difficult to see from the images provided how the authors were able to count labelled and unlabelled cells.\n2. Please make sure that all measurements in the tables and graph axes are accompanied by the appropriate measuring units\n3. Please try to track down which of the two forms of the DSHB Islet1 antibody was used for these studies\n4. Please go through the M+M to make sure all buffers and solutions are specified as well as possible (e.g., P6- L2: PBS what molarity?; P6- L4: PFA in what buffer?; P6- L4: 20% sucrose in what buffer?; P7- L2: dehydrated how?; 3% hydrogen peroxide in what buffer, etc)\n5. Please provide surgical, electrodes etc and other details used for the electrophysiological studies.\n6. In all figures (or figure legends), where appropriate, please report the N, and whether the data represents mean/median, SD/SEM etc.\nReview 4: Reviewer 1 \u00b7 Apr 23, 2013\nBasic reporting\nWe are pleased to verify that the authors have addressed several of our comments, which contributed to increase the quality of the manuscript.\nHowever, very recently another study on the characterization of a Gbx1 -/- allele was published (Buckley et al, 2013) demonstrating a a locomotive defect affecting the hindlimb gait and stating that this phenotype results from abnormal formation of the proprioceptive sensorimotor circuit. In the Burckley\u2019s study, as in the study under revision, it is not discussed how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system. The authors should address this point in the discussion, as requested in our first revision of the paper.\nMoreover, the present data differ from the Burckley\u2019s data in that the expression of peripherin and Islet1 was not changed in the Gbx1 -/- model. This discrepancy was attributed to the use of different time points. Although I don\u2019t think this may be the reason, if the authors insist in this explanation they should evaluate the expression of those markers at the same time points. In addition, it would be very interesting to analyze their expression at postnatal stages, when the locomotor defect is detected, as requested in our previous revision.\nMinor comments:\n- Quantifications of number of stained cells for each molecular marker displayed in fig 8 should be accompanied by an appropriate statistical analysis to assess the significance of the differences observed.\n- On page 9 the authors say \u201cAltogether, the behavioral data revealed that the Gbx1-/- animals \u2026 because mutant mice also display a significantly reduced response time in the hot plate (thermosensory) test.\u201d This paragraph should be revised correcting the hot plate test observation (underlined) and should be better explained how animals with less pain sensitivity in the hot plate test could suffer from pain on movement.\n- In results section when describing Drg11 expression they should mention (Fig. 7C,D and data not shown)\n- In the conclusion section, in the end of first paragraph the sentence \u201c\u2026as mutant mice also display a significant reduced response time in the hot plate (thermosensory) test.\u201d Is in contradiction with the affirmation in the third paragraph \u201c\u2026as Gbx1-/- mice displayed significantly increased response time in the hot plate test\u2026\u201d.\nExperimental design\nNothing to add\nValidity of the findings\nNothing to add\nAdditional comments\nThis is an interesting paper that would benefit with the ameliorations suggested\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord (v0.3)\". PeerJ https://doi.org/10.7287/peerj.142v0.3/reviews/1\nReview 5: M Fabiana Kubke \u00b7 Mar 31, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nI have now gone through the revised manuscript and my comments are listed below. My overall impression is that some of the issues that were raised during the first round of review still need to be further addressed. My comments below reflect my views on the current version as well as how well I consider the issues raised by the first round of review were addressed.\n\n\nResults reporting:\n\n1. In response to Reviewer\u2019s #1 request to address compensatory expression of GBx2, Fig S1 appears to satisfy that request. However, the data is interpreted as only showing a \u201cpossible subtle increase at E14.5\u201d, but in the images provided there also seems (to me) to be an increase in expression at E12.5, and some staining pattern differences at other ages. Were these data quantified or was this a qualitative analysis. If the latter, what were the criteria used to assess differences between and within groups? Can the authors please comment on the possible implications of these differences?\n\n2. Report on motor performance: Paragraph 3 states that in the beam walking test the latency to cross the beam was significantly increased, but the figure shows this to be true only in females. I suggest this be qualified in the text. Similarly \u201cthe number of slips was slightly increased\u201d is misleading, since it is not significantly different. \u201cMales were less affected than females\u201d suggests that males are affected, which is not what the data shows. I understand your suspicions regarding the power of some of the analysis, the analysis provided shows no significant differences, and it might be worthwhile to design a repeat of the experiment with more power to determine whether these tendencies are significant or not. Similarly in paragraph 5 of discussion: \u201ctended to have higher number of slips\u201d need to qualify this was NS.\n\n3. Report on motor performance: \u201cmales and females tended to have reduced locomotor activity [\u2026] p=0.09 . Isn\u2019t this NS?\n\n4. Report on motor performance: \u201creduced pain sensitivity\u201d Not sure the sensitivity to pain, but rather the response to pain is what is measured. If the argument is made that it is the reduction of GABA cells in the DSC that are responsible for the motor deficits, I am not sure how this relates to reduced sensitivity at the primary afferent level \u2013 I still fail to see why the conclusion is that there are proprioceptive defects and/or altered sensory abilities. Are there no alternative explanations? Though this might be simply a semantic issue. The way I read it is that the argument is made that the deficits are in the sensory periphery/primary afferents.\n\n5. Hindbrain analysis: In response to Reviewer #1 an analysis at E18.5 was performed in the hindbrain using GAD67 and reported as no difference between wild type and mutant \u2013 Figure S3 G,H appears to show a reduced expression of GAD67 in cerebellum., and perhaps some increase in the mutants in dorsal hindbrain (Panels E/F). Of course, I am not able to look at the entire section set. Was this quantified, or is this a qualitative assessment? If the latter, were any criteria used?\n\n6. Development of spinal cord dorsal horn:The text says that Nissl staining of E18.5 mice \u2026 (Fig 6) but the Figure legend says it is E 16.5.\n\n7. For figure 4 the text states that there are \u201cno defects in patterning of sensory afferent fiber projections\u201d but this conclusion is based mainly on the staining of a subset of sensory afferents (that are labeled with calbindin) and on the apparent lack of differences in expression of Drg11. I think the resolution of the images in panels A and B is too low for the reader to assess any possible differences and/or ectopic projections. Also, the markers used cannot rule out other types of patterning differences from primary afferents that do not label with calbindin or possible patterning differences at ages other than the one provided. The statement that follows regarding that males show longer withdrawal to the hotplate cannot be easily correlated with the images shown since it is not known whether the tissue presented corresponds to males or female animals, and this limitation should be made explicit. It would be useful to refer to Buckley et al.\u2019s results that indicate that indeed there are abnormal proprioceptive projections.\n\n8. In panels EF of Figure 4 the text reports no differences in the patterns of staining for peripherin. There appears to be a down regulation of peripherin at least in the most medial ventral spinal cord, a finding that is consistent with those of Buckley (2013). Were you able to find ectopic projecting axons such as those described by Buckely et al?\n\n9. The statement that \u201cno differences in the number of Islet1+ cells were found in the ventral spinal cord - was unable to find this quantification \u2013 or was this a qualitative assessment? If so, can you please describe how this was done.\n\n10. I am also surprised that the differences in ages between the Buckley study and the present study are not considered as a source of discrepancy.\n\n11. Regarding Reviewer#1 request to look at the fate of neurons that do not become GAD67 positive \u2013 Is there any reason why the apoptosis analysis is not shown or details of how this was analysed provided?\n\n12. In response to Reviewer #1\u2019s request to address post natal expression abnormalities at the spinal cord level, the argument is made that these will be part of a separate study. While I understand why the choice of separating these two studies may be appropriate, it does not address the reviewer\u2019s concerns regarding how appropriate it is to draw causal conclusions on mature motor performance based on embryonic GABAergic phenotype. The authors might want to consider discussing this limitation in more detail in their discussion.\n\n13. In response to reviewer #2\u2019s query about the onset of the motor deficits, you provide a statement that the deficit first appears at P16-17. Could you please provide details on how this was analysed.\n\n14. In response to the editor\u2019s summary you state that you have repeated analyses at several developmental stages. I am unable to find those data, other than for the additional material provided in the supplementary figures (for the additional Gbx2 expression, the analysis of hindbrain and the expression of Islet in the SC). However, the main ISH and ICChem data on which the main conclusions are based seem to only be described at a single time point.\n\nMethods reporting:\n\n15. Can you please provide a reference to the methods used for obtaining the blastocysts, etc. \u2013 I understand this might be standard, but I would imagine the source would be needed for any attempts of replication.\n\n16. In response to Reveiwer#2\u2019s request to clarify how abnormal gait was assessed, I am unable to find a description in the material and methods for other groups to be able to replicate your work. Could you please provide the details including what criteria were used for the assessment of what was \u201cnormal\u201d or not. I am assuming observers were familiar with normal gait patterns in normal mice prior to the observational classification? Did both observers always agree on the classification?\n\n17. In response to Reviewer #1 regarding details on the cell counting method: I still don\u2019t think that enough detail has been provided for an independent replication of the data. The figure legend says the areas shown in panels C and D were the ones used for counting \u2013 but there are areas where there is no tissue included in those areas \u2013 Was there a criterion as to where the counting box was placed, that is, over what anatomical area were the cells counted? Also could you please provide information as to the objective used (eg NA) to get a sense of resolution, and whether the cells counted under the microscope or from printed photographs? Like the reviewer, I still fail to see in the images the reduction that is reported in the graphs.\n\n18. It also seems to me that if one is counting cells over a defined area (which according to your figure excludes some tissue where GAD cells are present too), what is being provided is an estimate of cell density rather than actual cell number .\n\n19. I am also confused in the graph what the % of cells refers to, and hence what the reported percentage reduction refers to. Can this be clarified further please? Also, if 3 sections of 3 animals were used for counting for each, the n for each group would be 3, not 9 \u2013 I am not convinced a t test is appropriate, and wouldn\u2019t think it is appropriate to plot the data as means and standard deviations. These issues also apply to the quantification of Pax2 and VGLUT.\n\n20. The mouse anti-islet1 antibody is reported as being used at 1:100 \u2013 please indicate whether this was the supernatant or the concentrate form. Please provide the concentration used for the secondary antibodies. Can you please provide the constitution of the buffers in which antibodies were diluted and washes made. Could you please also provide (or specify) your experimental controls results.\n\n21. Rotarod test: I am not sure what the \u201clatency\u201d in the test is. Am I right to assume this is the time it takes for them to fall? Please specify.\n\n22. Electrophysiological measures: I am still not sure how the stimulating electrode was placed on the sciatic nerve, or how the distance between the stimulating electrode was measured (and with what accuracy). I am also no sure how these latencies were actually measured or how the data were analysed. I am also unsure how many recordings per animal were done, etc. Can you please also provide the dosage of anaesthesia.\n\nThere are also a few typographical/grammatical errors in the manuscript.\nReview 6: M Fabiana Kubke \u00b7 Jan 18, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nI have now received 2 reviewer\u2019s reports for your manuscript and reviewed it myself. Overall, I find the work to be valuable, but there are some issues that I think are important to be addressed before the manuscript is accepted for publication. While some comments suggest that the work would benefit from additional experiments, I understand this is not always possible. However, I think the issues raised are valid and should be at least addressed in the text of the manuscript. General Interpretation of the data: Gbx1 -/- phenotype: One of the reviewers asks whether there might be a compensatory expression of Gbx2 as a result of loss of function of Gbx1. I think that it would be useful to address this in the discussion. Anatomical/histological analysis: It seems the data for ICChem and ISH is based on a single time point which is also different from the age at which motor function was assessed. Both reviewers highlighted the limits of how these data can be extrapolated to relate it to the behavioural results. One reviewer asks what the expression patterns observed with ICChem and ISH might be in the spinal cord in more mature rats to warrant the link made between GABAergic phenotype and behavioural deficits. I would agree that showing that the differences observed in embryos persist beyond the age study would be useful. One reviewer and I also wonder about the use of ISH GAD-67 used as a proxy for GABA-ergic phenotype (suggesting the use of a second marker such as Pax2). I also wonder what the expression patterns of GAD-65 might be. Is it possible that those neurons that are not expressing GAD-67 might still express a GABAergic phenotype? Or is it possible that the differences observed might reflect a difference in the time course of expression of the GABAergic phenotype? One of the reviewers also queries whether the onset of locomotor deficits with respect to the transition of GABA between inhibitory to excitatory. Could this be added to the general discussion? One reviewer also queries whether the lack of obvious abnormalities in the hindbrain at E9.5 is sufficient evidence to rule this possibility out. Is the GABA phenotype, for example, normal in hindbrain at later stages, or are there deficits similar to those described in the SC? I also wonder whether it might be useful to show the hindbrain data on which these conclusions are based. It seems that this is important data that is substantial to the interpretation of the behavioural data (as highlighted in pp. 12-13). Similarly, on P 14 the statement is made that \u201cAltogether, these data suggest that there are not defects in patterning\u2026\u201d My impression is that what is provided is a very broad morphological analysis on a single time point. I am weary as to the degree of confidence with which that statement can be made, eg., do these data show with enough detail layer-specific projections, and is a single time point sufficient to rule out defects in patterning? Pain sensitivity: One reviewer questions the interpretation of the possible role of allodynia. I also wondered why the argument is made that the Gbx1 -/- might have reduced thermal pain sensitivity \u2013 an alternative is that they have issued processing reflexes involved in thermal pain which may not necessarily be accompanied by a reduced sensitivity per se.\nIn the results, the statement \u201cshowed a tendency for higher latencies than WT, but the difference between genotypes was not significant\u201d should be corrected. If there is no statistical difference, then I don\u2019t think the implication that the latencies may be higher is not justified. Similarly on Page 12 \u201cat least Gbx1 -/- females [..] tended to have higher number of slips\u201d these differences were not shown to be significant and \u201cGbx1-/- mutants displayed impaired rotarod behavior [\u2026]\u201d but table 1 and p10 this is described as NS. General Methodological queries and replicability. Both reviewers identify areas where further methodological information would be useful for the interpretation of data, and further descriptions are needed to ensure replicability of these studies. Please provide the N for all experiments- for example, tissue collection and sample preparation does not say how many pregnant females were used and how many embryos from each were harvested. This is important when considering the actual size of the sample analysed for histology and to infer possible litter effects in the results. It was also not clear to me in the behavioural phenotyping experiments whether the cohorts described mean that each cohort was put through a given paradigm, or whether there were several cohorts each tested through the set of behavioural paradigms. Please provide a description of how the blastocysts were obtained (Page 6, second paragraph). Please indicate whether behavioural experiments were performed during the day or night cycle of the rats. Cell counting: One of the reviewers queries about how GAD-67 + cells were counted, and whether the images shown are representative of the data. Cell counts are described as being made in 9 sections for each animal and 3 animals of each genotype. I am assuming the data in the plot in Figure 8 represents an average of the 27 sections for each genotype, but this could lead to pseudoreplication. The n for each genotype should be 3, each represented by the average of the 9 sections (which would probably preclude from using the parametric t-test). It is also not reported whether the tissue used for this study was from males and females, and given the male-female differences seen in some of the behavioural tests and the sensory conduction velocity this is an important factor to take into account. Assessment of motor deficits: One of the reviewers queries about what is meant by \u201cduck type gait\u201d and \u201clack of fluidity\u201d in movement. I wonder whether providing a movie (if available) might aid in the description. The reviewer also queries it would be useful to look at time s. pent in extension and flexion. Electromyography: The description of the electrode placed at the \u201cbase of the tail\u201d is not too clear. I am assuming since it is described as an EMG it would be a muscle, but I fail to see how recording from a muscle would provide a measure of the sensitive (do you mean sensory?) nerve conduction velocity. I am also not sure how these latencies were actually measured. How much precision was there in the placement of the stimulating electrode at 20 mm away from the recording electrode? It is also not stated how the data was analysed \u2013 how many animals were used, how many recordings per animal, etc. Would be worthwhile to show an image of these recordings, especially given the differences between males and females? General corrections: Reference to Table 1 at the bottom of Page 11 should be Table 2? There is also a discrepancy with the text saying females had increased conduction velocity, but in the table it marks the males as being significantly different. Similarly re: latency (top of p 12) the text says there is N.S. but the table suggests there is a significant difference in females. Results for rotarod need to be checked for accuracy. The statement \u201cmales and females tended to have reduced locomotor activity over the testing period (p=0.09). Is this p value correct? Figure 7 also needs correcting. General suggestions I am surprised that the sexual dimorphism in the Gbx1-/- phenotype is not addressed. Would it be useful to speculate on its significance?\nIn the introduction (first paragraph) tactile projections are described as projecting to laminae III, IV and V and temperature and pain as projecting to lamina I, II and III, whereas in page 14 the former are said to project mainly to laminae III, IV and V and the latter to lamina I/II. Perhaps it should be made clear in the introduction the degree of projections to lamina III from pain and temperature receptors. I am not sure I follow the argument on P12 \u201cGbx1-/- females had increased sensory nerve conduction velocity [..] supporting the altered sensory deficits observed in the behavioural tests\u201d nor in P14 paragraph 2, I suggest revising the wording. P14 par 1 Can you provide references for the statements on the second half of the paragraph? Please add the names of people providing supplies (for immuno and in situ) to the acknowledgements and obtain permission for that. Please declare if these supplies would be available for replication studies.\nAnimal Ethics Statement is presented under the generation of genotyping of chimeric and mutant mice \u2013 I would assume that ethical approval has been sought for the entirety of the project. If so, it might be made more explicit by placing the animal ethics statement at the beginning (or at the end) of the M+M. . Deolinda Lima \u00b7 Jan 11, 2013\nBasic reporting\n- In figure 1 it would be interesting to look for the expression of Gbx2 to see if there is a compensatory expression of Gbx2 due to the loss-of- function of Gbx1 in mutant mice, especially in developmental ages from E12.5 onwards.\n- No obvious developmental abnormalities were found at the hindbrain in E9.5 Gbx1-ko mice. The authors should look for alterations at later developmental ages, using terminal differentiation markers (as it was done at the spinal cord level) to completely exclude the involvement of hindbrain in the observed mutant phenotype.\n- The authors discuss that allodynia cannot be discarded as a cause for the altered gait phenotype. In the hot plate analysis, Gbx1 ko mice (particularly the males) displayed a reduced response in hot plate test indicating that mutant mice feel less pain. Therefore, allodynia does not seem a possible scenario to reconcile with the observed hot plate performance.\n- In the characterization of the spinal cord dorsal horn of ko mice, there are several weaknesses that compromise one of the main conclusions of the manuscript, which concerns the cell specification defect in the dorsal spinal cord (see at \"Experimental Design\").\nMinor corrections:\n- Figure 7 C,D and E,F does not correspond to text description.\n- In the text the results of increased sensory nerve conduction velocity are stated in Table2 and not in Table 1.\n- In Table 1 \u2013 rotarod, results for Gbx1-KO females does not seem correct.\n- On page 4 it is mentioned that Drg11 is expressed in late born derived cells from dI5 neurons but the reference mentioned is not fully correct. Authors should consider adding the studies by Rebelo et al (2010 Dev Dyn).\nExperimental design\n1- It is not clear how counting of Gad67-positive cells was performed (a full description is need in the Materials and Methods section) and, in figure 8, Gad67 in situ staining pictures do not reflect the cell counting shown in the graph. As the difference is not so marked, the authors should use other GABAergic markers, such as Pax2, to corroborate the result of a reduction of GABAergic neuronal population.\n2- If these Gad67-positive neurons are missing, it is important to address whether these neurons died, change to a glutamatergic phenotype or possibly there is simply a Gad67 expression down regulation. Therefore, neuronal death markers and glutamatergic markers should be looked up. Indeed, it may be relevant to double check a possible difference in the Drg11 expression (Figure 7 EF) since at sight there seems to be a difference in expression between wild type and Gbx1-knockout embryos at E16.5.\n3- It would be also important to address post natal expression abnormalities at the spinal cord level atfer the first two or three weeks after birth.\nValidity of the findings\n- The authors conclude that Gbx1 has a role in the specification of a subset of GABAergic dorsal horn interneurons involved in the control of hindlimb movement, however it\u2019s not clear how this assumption could be made. It is well established that limb movement is controlled by neuronal circuits located at ventral spinal cord and that somatosensory information is integrated and conveyed to the brain by the dorsal spinal cord. Therefore, it\u2019s no clear how authors could do such a conclusion.\nAdditional comments\nIn order to find a physiological function for Gbx1, the authors generated and characterized the phenotype of the Gbx1-knockout (ko) mice at two levels: molecularly during development and behaviorally. After a battery of behavioral tests the most striking phenotype was the altered gait during forward locomotion affecting the hindlimbs. The authors then proceeded trying to correlate this observation with a molecular defect at the neuroaxis.\nThis paper requires, however, significant revision.\nCite this review as\nLima D (2013) Peer Review #1 of \"The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord (v0.1)\". PeerJ https://doi.org/10.7287/peerj.142v0.1/reviews/1 Reviewer 2 \u00b7 Dec 26, 2012\nBasic reporting\nIt seems odd to end the introduction with a reference based on the content of the sentence?\nExperimental design\nNo comments\nValidity of the findings\npg. 10- the description of a \"duck type\" gait needs explanation. It was not immediately apparent what was meant by this. Describing the specific sequence of hyper-flexion and hyper-extension behaviors would be more useful to the reader. pg. 10- For the following statement \"However, many of the Gbx1-/- mutants showed significantly abnormal gait (\u03c72 \u22655.20, p<0.05). Indeed, 43% of Gbx1 mutant males and 63% of Gbx1 mutant females displayed lack of fluidity in movement\" clarify the test that was used to demonstrate gait was abnormal and motion was not fluid.\nAdditional comments\n1) It would be interesting to determine whether behavioral/locomotor deficits coincide with change of GABA from excitatory to inhibitory which (I think) happens around P17 in mouse- well after the onset of weight bearing locomotion. If the locomotor deficits began around this time it would really strengthen the paper by directly linking them to GABA. 2) The conclusion really needs a discussion of the experiments required to determine the potential source of the locomotor deficit (central vs proprioceptive deficit). Some discussion of the locomotor CPG (role of other genetically defined populations and how they might be connected to GABAergic neurons) as well as the role of proprioceptive input in generating locomotor outputs needs be included in this section. 3) It would be useful to quantitate the amount of time spent in flexion (paw raised) vs time spent in extension (paw on ground) and compare to wt. In \"regular\" mammalian locomotion as speed of locomotion increases the proportion of time spent in the flexor phase (swing) increases. Is this the case in the Gbx1-/-?\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord (v0.1)\". PeerJ https://doi.org/10.7287/peerj.142v0.1/reviews/2\n",
    "LLM_extracted_review": "['**Review 1:**\\n1. The article represents a valuable contribution to the field.\\n2. There are a few very small editorial changes that I will ask the authors to make prior to final publication.\\n3. The ages at which the gait was analysed is confusing \u2013 please specify the age used for the data in Table 1, Figure 2 and supplementary movie.\\n4. The abbreviation BN is used for \u201cwhite noise\u201d \u2013 I suggest changing this to WN, since English speakers might confuse this with Brown Noise.\\n\\n**Review 2:**\\n1. One important issue still remains regarding the analysis of the GABA cells, which is of great importance since this seems to be at the centre of the interpretation of the behavioural phenotype.\\n2. I was surprised that Dr Dembele suggested a paired t-test since I couldn\u2019t understand what criterion/justification was used to pair the animals from the two genotypes.\\n3. The reference does not support the statement regarding natural cell death in the spinal cord, and it would be useful to discuss why it might be the case that no TUNEL labeling is visible in your material at those ages.\\n4. I will also ask that those data where the statistical analysis failed to show a statistical significance are clearly described as such.\\n5. An issue also remains as to the need to discuss how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system.\\n\\n**Review 3:**\\n1. Can the authors please comment in the manuscript on the possible implications of the subtle increase of Gbx2 expression at E12.5-E14.5?\\n2. Quantifications of number of stained cells for each molecular marker displayed in fig 8 should be accompanied by an appropriate statistical analysis to assess the significance of the differences observed.\\n3. It would be valuable to include figures examining a glutamatergic marker and a cell death marker in the main article rather than in the supplementary material.\\n4. Could the authors also please discuss how the ages at which the TUNEL was examined correspond to expected apoptosis through naturally occurring cell death in the mouse lumbosacral spinal cord?\\n5. The authors should evaluate the expression of those markers at the same time points to address discrepancies with previous studies.\\n\\n**Review 4:**\\n1. The authors should address how a defect in dorsal spinal neurons could interfere with the proprioceptive afferent system.\\n2. Quantifications of number of stained cells for each molecular marker displayed in fig 8 should be accompanied by an appropriate statistical analysis.\\n3. The paragraph regarding behavioral data should be revised correcting the hot plate test observation.\\n4. The authors should clarify the discrepancies in the expression of peripherin and Islet1 between studies.\\n\\n**Review 5:**\\n1. The data is interpreted as only showing a \u201cpossible subtle increase at E14.5,\u201d but there also seems to be an increase in expression at E12.5.\\n2. The statement regarding the latency to cross the beam should be qualified in the text.\\n3. The authors should clarify the interpretation of reduced pain sensitivity in relation to the Gbx1 -/- phenotype.\\n4. The statement regarding no defects in patterning of sensory afferent fiber projections should be supported by more detailed analysis.\\n\\n**Review 6:**\\n1. The authors should address the compensatory expression of Gbx2 due to the loss of function of Gbx1.\\n2. The authors should look for alterations at later developmental ages in the hindbrain.\\n3. The argument regarding allodynia as a cause for the altered gait phenotype should be reconsidered.\\n4. The authors should provide a full description of how counting of Gad67-positive cells was performed.']"
}