Neurotrophin-3 produced by motor neurons non-cell autonomously regulate the development of pre-motor interneurons in the developing spinal cord

  1. Andrea Angla-Navarro
  2. Ana Dominguez Bajo
  3. Mathilde Toch
  4. Cédric Francius
  5. Maria Hidalgo-Figueroa
  6. Jingwen Zhang
  7. Olivier Schakman
  8. Manon Martin
  9. Xiuqian Mu
  10. René Rezsohazy
  11. Françoise Gofflot
  12. Frédéric Clotman

  • Curated by eLife

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    eLife Assessment

    This is a useful study that seeks to elucidate the molecular mechanisms underlying spinal motor circuit assembly. The authors demonstrate that loss of Onecut transcription factors in spinal motor neurons affects the size and spatial distribution of pre-motor interneurons. However, the study in its current form is incomplete: the data and analyses do not fully support the main conclusion that Onecut acts through Neurotrophin-3 to regulate interneuron development in a non-cell autonomous manner. The work will be of broad interest to cell and developmental biologists.

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Abstract

The development of multicellular organisms requires proper interplays between cell-autonomous genetic programs controlled by combinations of transcription factors that regulate the differentiation of distinct cell populations and non-cell autonomous processes that coordinate the proliferation, the fate, the survival, the respective location, and the proper interactions of these populations. During the development of the nervous system, non-cell autonomous mechanisms determine neuronal fate, survival, distribution, axon guidance, and connectivity. Although similar processes are suggested to be at work in the formation of spinal motor circuits, the molecular mechanisms involved remain mostly elusive. Here, we provide evidence that the Onecut transcription factors regulate a non-cell autonomous mechanism that modulate pre-motor interneuron development. We show that conditional inactivation of the Onecut factors in spinal motor neurons affects the differentiation and the positioning of pre-motor interneuron populations. We identify that Neurotrophin-3 produced by motor neurons under the control of the Onecut factors non-cell autonomously regulate the production and the distribution of pre-motor interneuron populations. Thus, we elucidated one of the non-cell autonomous mechanisms that coordinate the formation of the spinal motor circuits.

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  1. Version published to 10.7554/elife.110788.1 on eLife
  2. Version published to 10.7554/elife.110788 on eLife
  3. eLife

    eLife Assessment

    This is a useful study that seeks to elucidate the molecular mechanisms underlying spinal motor circuit assembly. The authors demonstrate that loss of Onecut transcription factors in spinal motor neurons affects the size and spatial distribution of pre-motor interneurons. However, the study in its current form is incomplete: the data and analyses do not fully support the main conclusion that Onecut acts through Neurotrophin-3 to regulate interneuron development in a non-cell autonomous manner. The work will be of broad interest to cell and developmental biologists.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    In this study, Angla et al investigate the basis of observations made from previous studies where loss of Onecut (OC) transcription factors leads to changes in spinal interneuron populations that do not themselves normally express OC. The authors hypothesize that OC expression in spinal motor neurons has non-cell-autonomous effects on pre-motor interneuron (V1, V2a/b/c) population size and distribution. By knocking out OC in the motor neuron lineage (i.e., downstream of Olig2, a motor neuron progenitor marker gene), they indeed show that motor neuron-specific loss of OC expression decreases V2c interneuron number and alters the spatial distribution of V1, V2a, V2b, and V2c populations. Using bulk RNA-sequencing of WT and OC conditional knockout (cKO) motor neurons, the authors identify that the neurotrophic factor Ntf3 is downregulated by OC expression. They subsequently hypothesize that the non-cell-autonomous effects observed by loss of OC expression in motor neurons can be explained by de-repression of Ntf3. To test this, the authors conditionally knock out Ntf3 downstream of Olig2 and show that this leads to increased interneuron numbers and alters their spatial distribution, ultimately leading to dysregulation of spinal motor circuits and motor activity.

    Strengths:

    The authors use sophisticated genetic tools to precisely remove OC and Ntf3 expression in a lineage-specific manner and comprehensively assess the downstream effects across brachial, thoracic, lumbar levels of the spinal cord, as well as at two developmental timepoints, E12.5 and E14.5.

    Weaknesses:

    There are two main concerns that are not fully addressed:

    (1) Based on the effects observed with OC vs. Ntf3 cKO, it is unclear whether OC is indeed exerting its non-cell-autonomous effects via Ntf3. Knocking out both Ntf3 and OC and comparing the effects to those seen with just OC cKO alone could provide more insight on this point. Also, a quantitative summary of the effects of Ntf3 overexpression in motor neurons in the chick is lacking.

    (2) How the authors assess changes in the spatial distribution of interneurons is unclear. In Figures 2 and 4, the control distributions (despite reporting the same populations in the same regions) look different, suggesting large sample-to-sample variance in distribution. Although the authors report that several sections in each level were taken from at least three animals for each condition, it's unclear how variance within WT or cKO sections was accounted for in the final statistical evaluation. It seems at a glance that a comparison between control samples in Figure 2 and Figure 4 could report statistically significant differences, which would be problematic. A more rigorous report of sample-to-sample variance and a more in-depth explanation of the statistical methods are needed.

  5. eLife

    Reviewer #2 (Public review):

    The study by Angla et al. proposes a model in which NT-3 produced by motor neurons regulates interneuron numbers and distribution in a non-cell autonomous manner. The authors demonstrate that ablation of motor neurons (MNs) and global and conditional deletion of OC transcription factors lead to changes in interneuron distribution. They identify that NT3 is upregulated after MN-specific OC deletion in RNA-seq experiments and show that olig2-cre mediated NT3 deletion leads to increased ventral interneuron numbers, altered distribution, and defects in locomotor behavior. The authors conclude that MN-derived NT3, under OC control, regulates interneuron development. While this is an intriguing hypothesis, additional experiments are needed to support it and strengthen the link between the different experiments described here.

    (1) The study primarily quantifies interneuron numbers and distribution at different levels of the spinal cord and under different genetic manipulations. Experimental details are lacking, defining how many sections were analyzed (several are noted in the methods) and how the rostrocaudal levels of the spinal cord were precisely aligned. In different figures, the values and distributions shown for controls vary quite a lot. For example, in Figure 2B vs Figure 4B, the number of FoxP2+ V1 neurons at brachial levels is ~350 vs 125. Similarly, the control distributions in 2I and 4I are quite different. This makes it challenging to determine whether the conclusions regarding the impact of each genetic manipulation on interneuron numbers and distribution are valid.

    (2) The relationship between OC and NT3 deletion data is not entirely clear. Both deletions presumably lead to changes in interneuron distribution, but is there any reverse relationship between the two that relates to relative changes in NT3 levels? The authors do not directly compare NT3 and OC KO IN distributions. Similarly, one might expect a decrease in interneuron numbers in OC mutants, which is only reported for V2c neurons. However, the image presented in Figure 2G shows an equal number of V2c INs in control and mutant.

    (3) It is not clear that the behavioral phenotypes seen in the olig2-cre mediated deletion of NT3 can be attributed to changes in interneuron development. How about a role of NT3 in oligodendrocytes? There is a big gap between the embryonic changes shown here and behavior, with no in-between circuit-level changes in locomotor circuits shown. A more restricted manipulation would be deleting TrkC from specific interneuron populations. Related to this, although TrkC is shown to be broadly expressed in ventral interneurons, it is not shown specifically to colocalize with any of the interneuron markers. The authors should validate that the receptor is expressed in the subsets that they are investigating.

    (4) The rationale for following up on NT3 seems to be the chick electroporation experiments; however, no changes in distribution are shown in those experiments, and only a very minor decrease in Chx10 interneurons. Shouldn't NT3 overexpression lead to substantial decreases in IN numbers according to the authors' model? The "data not shown", which presumably refers to distribution, would be important to show here, to further support this rationale.

    (5) The idea that NT3 downregulation causes an increase in IN numbers is not intuitive. Also, considering the DTA experiments in Figure 1, showing that MN ablation leads to a decrease in several IN subtypes and no changes in V2a neurons. It would be helpful for the reader if the authors could synthesize their results in the discussion and reconcile their experimental findings.

  6. eLife

    Reviewer #3 (Public review):

    This manuscript aims to investigate cell extrinsic mechanisms that regulate the differentiation and distribution of interneuron types in the spinal cord. The authors demonstrate that the loss of motor neurons leads to changes in the number and distribution of different interneuron types, specifically V0v, V1, and V2b (but not V2a). The authors then hypothesize that this phenotype may be controlled by the action of Onecut (OC) transcription factors in motor neurons. Conditional knockout of OC1 + OC2 in motor neurons using Olig2-Cre, however, does not lead to significant changes in the numbers of V1, V2a, and V2b interneurons, although there is a change in their spatial distribution. While the authors do not check V0v neurons in OC mutants, they do check V2c, which show a reduction in number and change in distribution. Why the same neurons are not checked across experiments is unclear. The authors then analyze existing RNA-seq data to identify factors that could be mediating the effects of the OC factors in motor neurons. They identify Ntf3 as a candidate and confirm that it is upregulated in OC mutants. Conditional loss of function of Ntf3 (Olig2-Cre) leads to increases in V1, V2a, and V2b (but not V2c) interneurons and changes in the distribution of all four interneuron types. Finally, the authors demonstrate that these Ntf3 conditional mutants have major defects in motor function.

    The conclusions of this manuscript are not well supported by the data for the reasons listed below, making it difficult to assess the impact of this work on the field.

    (1) The manuscript relies heavily on quantifying numbers and the spatial distribution of interneuron populations. However, these do not seem to be consistent in control animals across experiments, making it difficult to interpret any changes observed in genetic manipulations. Specifically, in Figures 2 and 4, the same markers are being used to quantify V1, V2a, V2b, and V2c interneurons in controls vs. OC (Figure 2) or Ntf3 (Figure 4) conditional knockouts, but the numbers of neurons and their distribution in control animals are variable between these two figures. For example, there seems to be a mean of >300 V1 neurons in E12.5 brachial sections of Fig. 2 controls, but this number is <150 in Fig. 4 controls. The cell distribution scoring is similarly variable between these controls without any explanation. The same is true for E14.5 controls used in Figure S1 vs. Figure S3.

    (2) Neurotrophic factors generally promote neuronal survival. However, in this study, the loss of Ntf3 leads to increased numbers of interneurons. This finding is in disagreement with previous observations in slice cultures of spinal cords, as stated in the discussion. This discrepancy makes it even more important that the cell counts reported in the figures discussed above are robust.

    (3) The claim that phenotypes are non-cell autonomously driven by motor neurons is not well supported. In Olig2-Cre conditional knockouts of Onecut and Ntf3, there is no confirmation that the loss of these factors is specific to motor neurons. Therefore, it cannot be ruled out that other cell populations may be mediating the phenotypes.

    (4) The claim that interneuron development is regulated by OC control of Ntf3 expression in motor neurons is not well supported. The authors show that loss of OC1/2 leads to an increase in Ntf3 expression in motor neurons. If this pathway were controlling interneurons, loss of OC function and overexpression of Ntf3 would have the same phenotype, which is not the case. Additionally, it would also be expected that loss of OC function and loss of Ntf3 function would have inverse phenotypes, which is also not the case. The phenotypes from OC loss of function and Ntf3 loss of function seem distinct from one another. The authors state that too little and too much Ntf3 are both bad for interneuron development, but there is no data to support their claim that OC1/2 mutants have altered interneuron development because of higher Ntf3 expression.

    (5) It is not clear that interneurons being studied express the Ntf3 receptor TrkC, which makes it difficult to assess whether changes in Ntf3 signaling are directly responsible for the phenotype.

    (6) While the behavioral phenotypes are consistent with Ntf3 playing a role in motor circuits, there is no evidence to suggest that Ntf3's influence on premotor interneurons being studied is driving or contributing to this phenotype, as discussed by the authors.

  7. eLife

    Author response:

    Public Reviews:

    Reviewer #1 (Public review):

    (1) Based on the effects observed with OC vs. Ntf3 cKO, it is unclear whether OC is indeed exerting its non-cell-autonomous effects via Ntf3. Knocking out both Ntf3 and OC and comparing the effects to those seen with just OC cKO alone could provide more insight on this point.

    In this study, we did not intend to demonstrate that Onecut transcription factors exert their non-cell autonomous action on spinal interneuron development by regulating Ntf3 expression, and we do not state in the manuscript that this is the case. We only show that Onecut factors and Ntf3, the expression of which they regulate, contribute to the non-cell autonomous regulation of spinal interneuron development by the motor neurons. We are convinced that Onecut factors could regulate multiple independent factors and pathways involved in extrinsic regulation of interneuron development, as supported by the regulation of multiple secreted factor or membrane protein expression in motor neurons detected in the reported RNA-sequencing experiment (this manuscript and [1]). This possibly also includes, as demonstrated in cell culture for multiple homeoproteins including human Onecut factors [2], the intercellular transfer of the Onecut homeoproteins during spinal cord development, a process that we are currently investigating. Knocking out both OC and Ntf3 in the motor neurons, beyond being technically extremely challenging (1/64 probability to obtain triple-mutant embryos), would not enable to address this question, as it will simply results in the addition of two different defects.

    Also, a quantitative summary of the effects of Ntf3 overexpression in motor neurons in the chick is lacking.

    A quantitative summary of the effects of Ntf3 overexpression in the chicken embryonic spinal cord is provided in Figure S2.

    (2) How the authors assess changes in the spatial distribution of interneurons is unclear. In Figures 2 and 4, the control distributions (despite reporting the same populations in the same regions) look different, suggesting large sample-to-sample variance in distribution. Although the authors report that several sections in each level were taken from at least three animals for each condition, it's unclear how variance within WT or cKO sections was accounted for in the final statistical evaluation. It seems at a glance that a comparison between control samples in Figure 2 and Figure 4 could report statistically significant differences, which would be problematic. A more rigorous report of sample-to-sample variance and a more in-depth explanation of the statistical methods are needed.

    The experimental procedure to analyze the spatial distribution of spinal interneurons at different stages of development is described in details in the “Statistical analyses” paragraph of the Materials and Methods section of the manuscript, and has been repeatedly used by ourselves [3,4] and by others (see for example [5-7]) to conduct similar analyses.

    We also noticed that the distribution of the different analyzed interneuron populations in the control embryos showed some differences between the cOc1Oc2-/- and the cNtf3-/- lines. Several parameters can account for this observation. First, this study has been conducted over a period of 15 years, different investigators each contributing to different steps of the analysis. Second, the genetic background of these two lines is not identical, impacting both the duration of the gestation (hence, the embryonic stage of the performed analyses, even if the embryos were collected on the same gestation day) and possibly the distribution of some interneuron populations. Third, because of evolutions in the availability of the primary antibodies used to label the interneuron populations of interest, the same antibodies were not used throughout the study, as stated in the Materials and Methods section, although the same antibody was used by the same investigator to label the same interneuron population in each mouse line at each developmental stage.

    A detailed description of the number of sections and embryos included in each analysis as well as the whole statistical workflow that was used for the distribution analyses, which takes into account variance within control or mutant samples, will be provided in the revised version of the manuscript.

    Reviewer #2 (Public review):

    (1) The study primarily quantifies interneuron numbers and distribution at different levels of the spinal cord and under different genetic manipulations. Experimental details are lacking, defining how many sections were analyzed (several are noted in the methods) and how the rostrocaudal levels of the spinal cord were precisely aligned.

    A detailed description of the number of sections and embryos included in each analysis as well as the whole statistical workflow that was used for the distribution analyses will be provided in the revised version of the manuscript. The rostrocaudal levels of the spinal cord were precisely aligned using the distribution of Foxp1 in the Lateral Motor Columns (LMCs) at brachial or lumbar levels of the spinal cord [8,9], which will also be indicated in the revised version.

    In different figures, the values and distributions shown for controls vary quite a lot. For example, in Figure 2B vs Figure 4B, the number of FoxP2+ V1 neurons at brachial levels is ~350 vs 125. Similarly, the control distributions in 2I and 4I are quite different. This makes it challenging to determine whether the conclusions regarding the impact of each genetic manipulation on interneuron numbers and distribution are valid.

    Multiple factors may explain these observations. First, this study spans a 15-year period, with different researchers contributing to various stages of the analysis. Second, the genetic backgrounds of the two mouse lines are not identical, affecting both gestation length (thus influencing the embryonic stage at which analyses were performed, even when embryos were collected on the same gestational day) and potentially the distribution of certain interneuron populations. Third, due to changes in the availability of primary antibodies used to label the targeted interneuron populations, the same antibodies were not consistently employed throughout the study as noted in the Materials and Methods section though each investigator used the same antibody for a given interneuron population and developmental stage within each mouse line.

    (2) The relationship between OC and NT3 deletion data is not entirely clear. Both deletions presumably lead to changes in interneuron distribution, but is there any reverse relationship between the two that relates to relative changes in NT3 levels? The authors do not directly compare NT3 and OC KO IN distributions. Similarly, one might expect a decrease in interneuron numbers in OC mutants, which is only reported for V2c neurons. However, the image presented in Figure 2G shows an equal number of V2c INs in control and mutant.

    This study was not designed to demonstrate that Onecut transcription factors influence spinal interneuron development in a non-cell-autonomous manner through Ntf3 regulation, nor do we claim this in the manuscript. Instead, we show that Onecut factors and Ntf3, whose expression they control contribute to the non-cell-autonomous regulation of spinal interneuron development by motor neurons. We believe Onecut factors may regulate multiple independent factors and pathways involved in the extrinsic control of interneuron development. For instance, as noted earlier [2], we observed intercellular transfer of Onecut homeoproteins during spinal cord development, suggesting alternative mechanisms for non-cell-autonomous regulation.

    The two mouse lines studied here consist, on the one side, in a combination of OC inactivation and Ntf3 increased expression, and, on the other side, in Ntf3 inactivation. Therefore, a reverse relationship between the changes in interneuron distribution is not expected. Furthermore, gain-of-function and loss-of-function experiments in mouse models frequently generate phenotypes that are not inverse to each other [10-13].

    (3) It is not clear that the behavioral phenotypes seen in the olig2-cre mediated deletion of NT3 can be attributed to changes in interneuron development. How about a role of NT3 in oligodendrocytes? There is a big gap between the embryonic changes shown here and behavior, with no in-between circuit-level changes in locomotor circuits shown.

    We agree, the motor behavior changes that we recorded in Ntf3 conditional mutant mice are, as stated, “consistent with the hypothesis that Ntf3 produced by MNs is required to generate locomotor circuits with properly coordinated activity” but do not demonstrate a direct causal relationship. However, investigating the intrinsic activity of the spinal locomotor circuits, independently from, for example, oligodendrocyte contribution may prove to be extremely challenging and was beyond the scope of this study. In addition, to our best knowledge, Ntf3 has not been shown to be expressed in healthy oligodendrocytes in vivo, and TrkC has not been reported to be displayed by these cells in the same conditions.

    A more restricted manipulation would be deleting TrkC from specific interneuron populations. Related to this, although TrkC is shown to be broadly expressed in ventral interneurons, it is not shown specifically to colocalize with any of the interneuron markers. The authors should validate that the receptor is expressed in the subsets that they are investigating.

    We agree, investigating the consequences of inactivating the TrkC receptor in specific interneuron populations would be extremely informative. However, this experiment is also very challenging to perform, as most of the driver lines available to target spinal interneuron populations additionally target multiple neuronal populations outside of the spinal cord that are also involved in the control of movements and could therefore induce confounding effects on motor behavior analyses [14-20].

    We thank the reviewer for suggesting to investigate in more details the interneuron populations that display TrkC receptors, this will be include in the revised version of the manuscript.

    (4) The rationale for following up on NT3 seems to be the chick electroporation experiments; however, no changes in distribution are shown in those experiments, and only a very minor decrease in Chx10 interneurons. Shouldn't NT3 overexpression lead to substantial decreases in IN numbers according to the authors' model? The "data not shown", which presumably refers to distribution, would be important to show here, to further support this rationale.

    Chicken spinal cord electroporation only enables to study spinal cord development in a limited time-window, given the high mortality rate observed after longer incubation. At the stage we collected the electroporated embryos for analyses, interneuron migration has barely been initiated, and distribution cannot be studied yet. Consistently, we are not aware of any report of interneuron distribution analysis in electroporated chicken embryonic spinal cord, as compared to mouse embryos [3-7].

    (5) The idea that NT3 downregulation causes an increase in IN numbers is not intuitive. Also, considering the DTA experiments in Figure 1, showing that MN ablation leads to a decrease in several IN subtypes and no changes in V2a neurons. It would be helpful for the reader if the authors could synthesize their results in the discussion and reconcile their experimental findings.

    We agree, this will be included in the revise version of the manuscript.

    Reviewer #3 (Public review):

    (1) The manuscript relies heavily on quantifying numbers and the spatial distribution of interneuron populations. However, these do not seem to be consistent in control animals across experiments, making it difficult to interpret any changes observed in genetic manipulations. Specifically, in Figures 2 and 4, the same markers are being used to quantify V1, V2a, V2b, and V2c interneurons in controls vs. OC (Figure 2) or Ntf3 (Figure 4) conditional knockouts, but the numbers of neurons and their distribution in control animals are variable between these two figures. For example, there seems to be a mean of >300 V1 neurons in E12.5 brachial sections of Fig. 2 controls, but this number is <150 in Fig. 4 controls. The cell distribution scoring is similarly variable between these controls without any explanation. The same is true for E14.5 controls used in Figure S1 vs. Figure S3.

    We indeed observed variations in the quantifications and distributions of the analyzed interneuron populations in control embryos between the cOc1/Oc2⁻/⁻ and cNtf3⁻/⁻ lines. Several factors may explain this discrepancy. First, the study was carried out over 15 years, with different investigators contributing to distinct stages of the analysis—meaning interneuron distribution was not assessed by the same researchers in both lines. Second, the genetic backgrounds of the two lines differ, affecting gestation length (and thus the embryonic stage at analysis, even when embryos were collected on the same gestational day) as well as potentially altering the distribution of certain interneuron populations. Third, changes in the availability of primary antibodies targeting the interneuron populations of interest led to inconsistencies in antibody use across the study, as detailed in the Materials and Methods section. However, each investigator consistently used the same antibody for a given interneuron population and developmental stage within each mouse line.

    (2) Neurotrophic factors generally promote neuronal survival. However, in this study, the loss of Ntf3 leads to increased numbers of interneurons. This finding is in disagreement with previous observations in slice cultures of spinal cords, as stated in the discussion. This discrepancy makes it even more important that the cell counts reported in the figures discussed above are robust.

    Considering that neurotrophic factors only support neuronal survival would strongly neglect their important function in neuronal differentiation, which has been broadly demonstrated. Severe immunotoxic ablation of motor neurons or anti-serum blockade of Ntf3 activity severely depleted inhibitory, but not excitatory, interneurons in a highly apoptotic-prone organotypic culture model of embryonic rat spinal cord slices, which was rescued by Ntf3 in the first model [21]. Opposite results were obtained in vivo by other researchers using mouse models lacking almost all MNs due to the elimination of skeletal muscles, where the number of spinal INs remained unaffected [22,23]. Combined to our results, these in vivo observations suggest that Ntf-3 is involved in interneuron differentiation rather in their survival. Consistently, Ntf3 has been shown to promote neuronal differentiation [24].

    (3) The claim that phenotypes are non-cell autonomously driven by motor neurons is not well supported. In Olig2-Cre conditional knockouts of Onecut and Ntf3, there is no confirmation that the loss of these factors is specific to motor neurons. Therefore, it cannot be ruled out that other cell populations may be mediating the phenotypes.

    Combined conditional inactivation of Oc1 and Oc2 has been reported in [1]. Conditional inactivation of Ntf3 only impacts motor neurons as it is the only cell population in the ventral spinal cord wherein this factor is produced (this study and [25-27]). Furthermore, Olig2-Cre has been shown to be active in motor neurons and in V3 interneurons (see for example [10]), which, for this reason, have not been studied in the frame of this project as stated in the manuscript.

    (4) The claim that interneuron development is regulated by OC control of Ntf3 expression in motor neurons is not well supported. The authors show that loss of OC1/2 leads to an increase in Ntf3 expression in motor neurons. If this pathway were controlling interneurons, loss of OC function and overexpression of Ntf3 would have the same phenotype, which is not the case. Additionally, it would also be expected that loss of OC function and loss of Ntf3 function would have inverse phenotypes, which is also not the case. The phenotypes from OC loss of function and Ntf3 loss of function seem distinct from one another. The authors state that too little and too much Ntf3 are both bad for interneuron development, but there is no data to support their claim that OC1/2 mutants have altered interneuron development because of higher Ntf3 expression.

    This study was not aimed at proving that Onecut transcription factors mediate their non-cell-autonomous effects on spinal interneuron development through Ntf3 regulation, nor do we make this claim in the manuscript. Rather, we demonstrate that Onecut factors and Ntf3, whose expression they control—participate in the non-cell-autonomous regulation of spinal interneuron development by motor neurons. We propose that Onecut factors likely modulate multiple independent factors and pathways involved in the extrinsic regulation of interneuron development, as evidenced by the regulation of various secreted factors and membrane proteins in motor neurons observed in our RNA-sequencing data (this study and [1]). This may also involve intercellular transfer of Onecut homeoproteins during spinal cord development—a mechanism previously shown in cell culture for several homeoproteins, including human Onecut factors [2] and which we are currently exploring.

    (5) It is not clear that interneurons being studied express the Ntf3 receptor TrkC, which makes it difficult to assess whether changes in Ntf3 signaling are directly responsible for the phenotype.

    Immunofluorescence experiment in Figure 3C shows that TrkC receptor is present in cell populations surrounding motor neurons at e12.5, a stage where only the pre-motor interneuron populations reported in the manuscript are present. However, we thank the reviewer for suggesting to investigate in more details the interneuron populations that display TrkC receptors, this will be include in the revised version of the manuscript.

    (6) While the behavioral phenotypes are consistent with Ntf3 playing a role in motor circuits, there is no evidence to suggest that Ntf3's influence on premotor interneurons being studied is driving or contributing to this phenotype, as discussed by the authors.

    We acknowledge that the motor behavior changes observed in Ntf3 conditional mutant mice—as noted—are “consistent with the hypothesis that MN-derived Ntf3 is necessary for the formation of locomotor circuits with properly coordinated activity,” but they do not establish a direct causal link. However, analyzing the intrinsic activity of spinal locomotor circuits was beyond the scope of this study.

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  8. Version published to 10.1101/2025.07.27.665825 on bioRxiv