Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling
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Evaluation Summary:
This manuscript examines how retinoid acid signaling controls the timing of neural crest production in avian embryos. The authors propose that local production of retinoid acid signaling activates the expression of BMP inhibitors in the dorsal neural tube. Disruption of BMP signaling results in the termination of neural crest migration and the establishment of the definite neural plate.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Production and emigration of neural crest cells is a transient process followed by the emergence of the definitive roof plate. The mechanisms regulating the end of neural crest ontogeny are poorly understood. Whereas early crest development is stimulated by mesoderm-derived retinoic acid, we report that the end of the neural crest period is regulated by retinoic acid synthesized in the dorsal neural tube. Inhibition of retinoic acid signaling in the neural tube prevents the normal upregulation of BMP inhibitors in the nascent roof plate and prolongs the period of BMP responsiveness which otherwise ceases close to roof plate establishment. Consequently, neural crest production and emigration are extended well into the roof plate stage. In turn, extending the activity of neural crest-specific genes inhibits the onset of retinoic acid synthesis in roof plate suggesting a mutual repressive interaction between neural crest and roof plate traits. Although several roof plate-specific genes are normally expressed in the absence of retinoic acid signaling, roof plate and crest markers are co-expressed in single cells and this domain also contains dorsal interneurons. Hence, the cellular and molecular architecture of the roof plate is compromised. Collectively, our results demonstrate that neural tube-derived retinoic acid, via inhibition of BMP signaling, is an essential factor responsible for the end of neural crest generation and the proper segregation of dorsal neural lineages.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Rekler and Kalcheim examines the role of neural tube-derived retinoic acid (RA) in neural crest development. They observe that the onset of expression of the RA-synthesizing enzyme RALDH2 in the dorsal neural tube coincides with the end of neural crest production. The authors propose that this local source of RA is essential to activate the transcription of Bambi other BMP inhibitors, leading to the disruption of BMP signaling. Loss of BMP activity at the dorsal neural tube would halt neural crest production, leading to the establishment of the definite roof plate. Thus, precise temporal regulation of RALDH2 in the dorsal neural tube would dictate the timing of neural crest production and the segregation of PNS and CNS progenitors.
Previous studies have already …
Author Response
Reviewer #1 (Public Review):
The manuscript by Rekler and Kalcheim examines the role of neural tube-derived retinoic acid (RA) in neural crest development. They observe that the onset of expression of the RA-synthesizing enzyme RALDH2 in the dorsal neural tube coincides with the end of neural crest production. The authors propose that this local source of RA is essential to activate the transcription of Bambi other BMP inhibitors, leading to the disruption of BMP signaling. Loss of BMP activity at the dorsal neural tube would halt neural crest production, leading to the establishment of the definite roof plate. Thus, precise temporal regulation of RALDH2 in the dorsal neural tube would dictate the timing of neural crest production and the segregation of PNS and CNS progenitors.
Previous studies have already identified a role for RA in the control of the timing of neural crest production. MartinezMorales et al (JCB 2011) have shown that during early trunk development, mesoderm-derived RA works with FGF signaling to jumpstart the BMP/Wnt cascade that drives neural crest migration in the trunk. Rekler and Kalcheim choose to focused on a distinct function of RA at a later timepoint. The main contribution of the present study is the demonstration that - at later stages - RA produced by the neural tube has the opposite effect, acting to inhibit the BMP/Wnt cascade and halt neural crest production. Thus, RA would be a major regulator of the timing of neural crest production, acting to both trigger and repress neural crest migration.
The study's strengths lie in an experimental strategy that allows the authors to manipulate RA function in a stagespecific manner and therefore uncover a later role for the signaling system in neural crest production. The authors also show that RA inhibition results in an incomplete fate switch and results in the generation of cells that share regulatory features of neural crest and roof plate cells. A significant limitation of the study is that the molecular mechanisms that endow RA signaling with stage-specific functions remain unknown. This is of particularly important since the early vs. late RA seem to have opposing effects, acting to either promote or terminate neural crest production.
We thank this referee for her/his positive comments on our manuscript. We agree with the referee that a key question is understanding how RA signaling is differentially interpreted over time given its multistage activity in dorsal NT development.
This is based on the following findings: Years ago, we uncovered that the balance between activities of BMP/Wnt and noggin in the dorsal NT trigger the onset of NC EMT. Martinez-Morales et al. strengthened our findings by reporting that a balance between somitic RA and FGF works on the reported BMP/Wnt modules to initiate the process. This group found that at gastrulation stages, RA is required for NC specification, as revealed by analysis of VAD quail embryos. Next, during somite formation, somitic RA is necessary for the onset of emigration of specified NC progenitors but at advanced somite stages it is dispensable for the subsequent maintenance of cell emigration. Presently, we find that RP-derived RA ends NC production. Together, this highlights a dynamic behavior of RA at 4 sequential stages of NC ontogeny. Clearly enough, the two first effects are mediated by an influence of RA dorsoventral patterning of the early NT, as distribution of ventral NT markers was strongly affected. In our case, RA from the nascent RP has no such effects suggesting that RP-derived RA acts at a post-patterning phase to specifically affect the dorsal NT.
All things considered, we think that the problem is not simply a binary question of “opposing functions of RA signaling in starting or terminating NC production”. Instead, it is the understanding of a differential interpretation to the same morphogen by progenitor cells with changing states and at sequential stages.
To the referee’s request, we begun addressing the question of how does RA inhibit BMP signaling close to the RP stage. To this end, we decided first to examine the temporal regulation of Raldh2 expression that is restricted to the RP stage, and is therefore a prerequisite for the late activity of RA. Whereas repressing RA activity extends the NC phase including the continuous transcription of Foxd3, Sox9 and Snail2 (Fig.3), we now found that extending the activity of each of these transcription factors close to the RP stage represses the onset of Raldh2 transcription in the nascent RP (new Fig. 9). We interpret these results to mean that as long as NC genes are active in the dorsal NT (NC stage), local Raldh2 and consequent RA synthesis in the NT does not take place, so Raldh2 in RP is repressed by NC-specific traits. The significance of these data is twofold: first, they explain the late onset of Raldh2 production at the RP stage. Second, since we also report the reciprocal result, that RA represses NC genes (Fig.3), we conclude that a cross repressive interaction exists between NC and RP-specific genes downstream of RA, being an emerging temporal property of the network. These data further indicate that the changing roles of RA throughout development of the dorsal neural primordium, largely depend on a different interpretation of the signal mediated by changing and mutually repressive codes.
We have now presented these data in Fig.9. To clarify our thoughts further, we now provide a working model summarizing the effects of RA in NC to RP transition (Fig.10B).
Our article uncovers for the first time and thoroughly documents, a role of local RA activity on the end of NC production and ensuing RP architecture. We believe that a comprehensive elucidation of the molecular mechanism responsible for inhibition of BMP signaling by local RA is the next obligatory step. We show in this study the selective activation of BMP inhibitors by endogenous RA and previously found that one of them, Hes/hairy, indeed inhibits BMP signaling and NC EMT (Nitzan et al, 2016). Therefore we propose that upregulation of BMP inhibitors by RA is a possible mechanism. However, we also predict that this is not the only one, and a deeper understanding of this problem is beyond the scope of the present study.
Additional possibilities that fit with our data were now discussed: RA expression in somites vs. RP can be regulated by different enhancers and thus have distinct functions. For example, a specific enhancer driving expression of Raldh2 was found to be activated only at the definitive RP stage (Castillo et al., 2010). This enhancer contains Tcf binding sites and thus may be activated by Wnt signaling. In turn, as we show, RP-derived Raldh2 and resulting RA could negatively feed-back on Wnt signaling in the formed RP either directly or through BMP acting upstream of Wnt (now presented in Fig. 10B).
Another possible scenario is that RA represses BMP signaling by inactivating Smad proteins via ubiquitination, as shown to be the case in selected cell lines (Sheng et al., 2010). These possibilities were discussed and await to be systematically explored.
Comments:
Previous studies have demonstrated that early RA production (presumably from the mesoderm) is necessary for the expression of early dorsal neural tube / neural crest genes like Pax7, Msx, Wnt1, and even BMP ligands. This is in contrast to the local source of RA, which presumably would be silencing these genes. Thus, mesoderm-derived RA would have the opposite effect in these progenitors than the RA synthesized in the neural tube. The study does not provide a mechanism that explains these stage-specific effects of the morphogen.
As elaborated in our reply above to the general comment, we believe that RA whether emanating from somites or nascent RP, provides an initial signal that is later relayed upon target factors unique to each stage. It is possible that the precise source of factor plays a role; along this line we showed that somitic RA is dispensable for late events, reciprocally, there is no RA synthesis in the early NT that could affect NC cells. Having said that, there is RA activity in the NT at both stages and the output is still different. Hence, there should be more to this: In the revised version, we report that NC and RP-specific genes stand in a mutually repressive interaction downstream of RA, and this may contribute to the stage-specific effects of the morphogen.
The effects of RA manipulation are often examined with non-quantitative techniques, like in situ hybridization (Fig. 2, 3). The incorporation of quantitative approaches (e.g., qPCR) would allow for the precise characterization of phenotypes (and better estimation of penetrance, etc.). Furthermore, the study lacks molecular/biochemical strategies to define the regulatory linkages between genes and pathways. This is a considerable limitation of the study since it prevents the establishment of a regulatory axis that would directly connect RA signaling to the BMP pathway.
As the referee may notice, most genes examined are not restricted solely to the dorsal NT/RP domains. Since it is technically not accurate to isolate only the regions of interest for qPCR analysis, collecting entire NTs following unilateral or bilateral electroporations for qPCR would be highly inaccurate. In situ hybridization and immunohistochemistry provide a precise tool to assess the spatial localization of the transcripts/proteins of interest. To note is that in all cases examined, development of the color reactions was for the same length of time for control and experimental cases and photography was performed under identical conditions. Furthermore, in most cases, effects between treatments were dramatic, readily apparent at a qualitative level and easily quantifiable from ISH or fluorescent images.
As to regulatory linkages between genes and pathways, the referee is correct; we do not demonstrate direct molecular interactions between the different players at the biochemical level. The present study provides a wealth of novel data connecting morphogens such as RA with BMP and Wnt activities, and those with a variety of downstream genes specific for either NC or RP stages. The next step will be to ask about the precise nature of the linkages between specific molecules/pathways.
The function (and the regulation) of RALDH2 at the dorsal neural tube has been studied thoroughly, and RA is a known player in the dorsal-ventral patterning of the CNS. It is not clear to what extent the phenotypes observed by the authors are due to the disruption of a neural crest-intrinsic mechanism or if they are secondary to the overall changes in the cellular organization of the neural tube caused by loss of RA.
This is a good point as RA is known to have multiple effects on NT development whose nature changes with stage. Available data emanating from young caudal neural plate explants and from VAD embryos that lack RA, showed that early RA signaling from developing somites is required for ventral patterning of the neural tube (motoneurons and V1, V2 interneurons) and for neuronal differentiation (Diez del Corral et al, 2003, Sockanathan and Jessel, 1998, Liu et al, 2001, Maden et al, 1996). These effects were shown to depend, at least partly, on antagonistic activities of RA and FGF in mesoderm which affect ventral, but not dorsal NT patterning (Diez del Corral et al, 2003). Our study focuses on a later stage when D-V neural tube patterning is already established.
To address the referee’s comment, we now examined the effects of RA attenuation on expression of Pax7, a dorsal factor, and Hb9, a motoneuron-specific protein. We found that RARa403 does not affect the localization and/or extent of expression of Hb9, and causes only a mild 12% increase in the area of expression of Pax7. Consistent with these results, we also show in several figures that in the absence of RA signaling pSmad and Wnt activities, Foxd3, Snai2 and Sox9 expression patterns are prolonged in time but not in D-V extent.
These data corroborate that the effects documented are directed to the dorsal NT and do not result from overall changes in D-V patterning. The data were now added as Fig.7 Supplementary 1.
The authors rely solely upon overexpression constructs to manipulate the activity of the RA signaling pathway, which may be prone to artifacts. Furthermore, both overexpression constructs aim at inhibiting RA activity. This limits the impact of the work since there is no demonstration that RA is sufficient to activate BMP inhibitors and halt neural crest production.
The tools we used to repress RA signaling consist of RARa403 that acts as a pan-dominant negative construct to abrogate receptor activity, and Cyp26A1, an enzyme that degrades RA. To activate RA signaling in a ligand- independent manner, we now implemented VP16-RAR-alpha in the revised version of this manuscript. All these tools are extensively and routinely employed in the literature in a variety of animal species and were shown to act in vivo as expected both by others and further confirmed by us in the present study. Having said that, we are currently optimizing the CRISPR-Cas9 method for gene editing of RA-specific genes and hope to succeed in the near future.
We have now performed experiments to address the sufficiency of RA. Data were now added as Fig.5 Supplem 2 and 3 and Fig.6 Supp.2 .
As we expected, gain of RA function at NC stages is not sufficient to prematurely activate BMP inhibitors like BAMBI, to end prematurely BMP signaling (pSMAD) or NC EMT, to alter the dynamics of expression of NC-specific genes, or to cause an earlier appearance of RP-specific traits. This is fully consistent with RA being active at NC stages when BMP/Wnt signaling, NC EMT, etc are operational. The fact that RA is necessary but not sufficient for these processes further suggest that the key is how NC cells at various stages of their ontogeny and then RP cells, differentially interpret the signal given the profound changes in cellular and molecular landscapes apparent between these stages.
Reviewer #2 (Public Review):
The manuscript presents a novel role for RA signaling during development as the mediator of the switch that occurs in the dorsal neural tube after the neural crest cells have migrated and the roof plate forms. The finding is interesting and novel as the events that take place at the end of neural crest stage are poorly understood. The strengths of the manuscript are that the study is well planned and executed to show the interesting phenotype of delayed/disturbed roof plate formation accompanied with prolonged neural crest stage caused by inhibition of RA signaling in the dorsal neural tube. The results also show that RA signaling marks the RP territory and inhibits the DI1 interneurons from invading the region. The results bring novel information to the field. The original finding of the involvement of RA in the process was revealed in a RNAseq screen comparison between the neural crest and the roof plate (which was recently published by the same lab). However, the current study doesn't use any new technology such as high throughput screens or high resolution or live imaging etc., but rather relies mainly on "old fashioned" techniques: electroporation to induce transient inhibition of RA signaling in the dorsal neural tube followed by analysis of the phenotype by using chromogenic in situ hybridization. The chosen techniques are sufficient to convincingly show the point the authors want to make and the study serves as a reminder that fancy new techniques are not necessarily a requirement for creating a solid story. The manuscript is also well written and easy to follow.
We thank this referee for a very positive feedback on our study. Although we are always motivated by the implementation of new techniques, we agree that the primary goal is to answer a biologically meaningful question with suitable means.
Finally, the manuscript links the activation of RA signaling to the decline of BMP signaling and specifically the upregulation of BMP inhibitors in the dorsal neural tube at the end of the NC stage, but in its current form the proof of this proposed link remains weak.
Our article uncovers for the first time and thoroughly documents, a role of local RA activity on the end of NC production and ensuing RP architecture. We believe that a comprehensive elucidation of the molecular mechanism responsible for inhibition of BMP signaling by local RA is the next obligatory step. We show in this study the selective activation of BMP inhibitors by endogenous RA and previously found that one of them, Hes/hairy, indeed inhibits BMP signaling and NC EMT (Nitzan et al, 2016). Therefore we propose that upregulation of BMP inhibitors by RA is a possible mechanism. However, we also predict that this is not the only one, and a deeper understanding of this problem is beyond the scope of the present study.
Additional possibilities that fit with our data were now discussed: RA expression in somites vs. RP can be regulated by different enhancers and thus have distinct functions. For example, a specific enhancer driving expression of Raldh2 was found to be activated only at the definitive RP stage (Castillo et al., 2010). This enhancer contains Tcf binding sites and thus may be activated by Wnt signaling. In turn, as we show, RP-derived Raldh2 and resulting RA could negatively feed-back on Wnt signaling in the formed RP either directly or through BMP acting upstream of Wnt (this was now presented in a working model in Fig. 10B).
Another possible scenario is that RA represses BMP signaling by inactivating Smad proteins via ubiquitination, as shown to be the case in selected cell lines (Sheng et al., 2010). These possibilities were discussed and await to be explored systematically.
Similarly, the manuscript does not address the consequences of exposure of RA to the dorsal neural tube during NC stage and it thus remains unknown whether RA signaling is sufficient to end the NC stage and activate roof plate formation prematurely. Additional experiments of this kind would help clarify the role of RA in the dorsal neural tube and the reciprocal roles of the two signaling pathways (RA and BMP).
We have now performed experiments to address the sufficiency of RA. Data were now added as Fig.5 Supp.2 and Supp.3, and Fig.6 Supp.2, and discussed.
As we expected, gain of RA function at NC stages is not sufficient to prematurely activate BMP inhibitors, to end prematurely BMP signaling (pSMAD) or NC EMT, to alter the dynamics of expression of NC-specific genes, or to cause an earlier appearance of RP-specific traits.
This result is totally understandable in light of RA being anyway active (but not produced) in NT at NC stages (original Fig.1) when BMP/Wnt signaling, a NC-specific gene network, and NC EMT are operational.
The fact that RA is necessary but not sufficient for these processes further suggests that the key is in the following, perhaps complementary mechanisms: 1) a different interpretation of the same signal by NC progenitors at sequential stages of their ontogeny and then by RP cells, accounted for by the profound changes in cellular and molecular landscapes apparent between these stages. 2) the possibility that somite-derived versus RP-derived RA are differentially interpreted by the dorsal NT cells owing, for example, to a distinctive mode of ligand presentation (e.g; by CRABP1 expressed in RP but not NC, etc).
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Evaluation Summary:
This manuscript examines how retinoid acid signaling controls the timing of neural crest production in avian embryos. The authors propose that local production of retinoid acid signaling activates the expression of BMP inhibitors in the dorsal neural tube. Disruption of BMP signaling results in the termination of neural crest migration and the establishment of the definite neural plate.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Rekler and Kalcheim examines the role of neural tube-derived retinoic acid (RA) in neural crest development. They observe that the onset of expression of the RA-synthesizing enzyme RALDH2 in the dorsal neural tube coincides with the end of neural crest production. The authors propose that this local source of RA is essential to activate the transcription of Bambi other BMP inhibitors, leading to the disruption of BMP signaling. Loss of BMP activity at the dorsal neural tube would halt neural crest production, leading to the establishment of the definite roof plate. Thus, precise temporal regulation of RALDH2 in the dorsal neural tube would dictate the timing of neural crest production and the segregation of PNS and CNS progenitors.
Previous studies have already identified a role for RA in …
Reviewer #1 (Public Review):
The manuscript by Rekler and Kalcheim examines the role of neural tube-derived retinoic acid (RA) in neural crest development. They observe that the onset of expression of the RA-synthesizing enzyme RALDH2 in the dorsal neural tube coincides with the end of neural crest production. The authors propose that this local source of RA is essential to activate the transcription of Bambi other BMP inhibitors, leading to the disruption of BMP signaling. Loss of BMP activity at the dorsal neural tube would halt neural crest production, leading to the establishment of the definite roof plate. Thus, precise temporal regulation of RALDH2 in the dorsal neural tube would dictate the timing of neural crest production and the segregation of PNS and CNS progenitors.
Previous studies have already identified a role for RA in the control of the timing of neural crest production. Martinez-Morales et al (JCB 2011) have shown that during early trunk development, mesoderm-derived RA works with FGF signaling to jumpstart the BMP/Wnt cascade that drives neural crest migration in the trunk. Rekler and Kalcheim choose to focused on a distinct function of RA at a later timepoint. The main contribution of the present study is the demonstration that - at later stages - RA produced by the neural tube has the opposite effect, acting to inhibit the BMP/Wnt cascade and halt neural crest production. Thus, RA would be a major regulator of the timing of neural crest production, acting to both trigger and repress neural crest migration.
The study's strengths lie in an experimental strategy that allows the authors to manipulate RA function in a stage-specific manner and therefore uncover a later role for the signaling system in neural crest production. The authors also show that RA inhibition results in an incomplete fate switch and results in the generation of cells that share regulatory features of neural crest and roof plate cells. A significant limitation of the study is that the molecular mechanisms that endow RA signaling with stage-specific functions remain unknown. This is of particularly important since the early vs. late RA seem to have opposing effects, acting to either promote or terminate neural crest production.
Comments:
Previous studies have demonstrated that early RA production (presumably from the mesoderm) is necessary for the expression of early dorsal neural tube / neural crest genes like Pax7, Msx, Wnt1, and even BMP ligands. This is in contrast to the local source of RA, which presumably would be silencing these genes. Thus, mesoderm-derived RA would have the opposite effect in these progenitors than the RA synthesized in the neural tube. The study does not provide a mechanism that explains these stage-specific effects of the morphogen.
The effects of RA manipulation are often examined with non-quantitative techniques, like in situ hybridization (Fig. 2, 3). The incorporation of quantitative approaches (e.g., qPCR) would allow for the precise characterization of phenotypes (and better estimation of penetrance, etc.). Furthermore, the study lacks molecular/biochemical strategies to define the regulatory linkages between genes and pathways. This is a considerable limitation of the study since it prevents the establishment of a regulatory axis that would directly connect RA signaling to the BMP pathway.
The function (and the regulation) of RALDH2 at the dorsal neural tube has been studied thoroughly, and RA is a known player in the dorsal-ventral patterning of the CNS. It is not clear to what extent the phenotypes observed by the authors are due to the disruption of a neural crest-intrinsic mechanism or if they are secondary to the overall changes in the cellular organization of the neural tube caused by loss of RA.
The authors rely solely upon overexpression constructs to manipulate the activity of the RA signaling pathway, which may be prone to artifacts. Furthermore, both overexpression constructs aim at inhibiting RA activity. This limits the impact of the work since there is no demonstration that RA is sufficient to activate BMP inhibitors and halt neural crest production.
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Reviewer #2 (Public Review):
The manuscript presents a novel role for RA signaling during development as the mediator of the switch that occurs in the dorsal neural tube after the neural crest cells have migrated and the roof plate forms. The finding is interesting and novel as the events that take place at the end of neural crest stage are poorly understood. The strengths of the manuscript are that the study is well planned and executed to show the interesting phenotype of delayed/disturbed roof plate formation accompanied with prolonged neural crest stage caused by inhibition of RA signaling in the dorsal neural tube. The results also show that RA signaling marks the RP territory and inhibits the DI1 interneurons from invading the region. The results bring novel information to the field. The original finding of the involvement of RA …
Reviewer #2 (Public Review):
The manuscript presents a novel role for RA signaling during development as the mediator of the switch that occurs in the dorsal neural tube after the neural crest cells have migrated and the roof plate forms. The finding is interesting and novel as the events that take place at the end of neural crest stage are poorly understood. The strengths of the manuscript are that the study is well planned and executed to show the interesting phenotype of delayed/disturbed roof plate formation accompanied with prolonged neural crest stage caused by inhibition of RA signaling in the dorsal neural tube. The results also show that RA signaling marks the RP territory and inhibits the DI1 interneurons from invading the region. The results bring novel information to the field. The original finding of the involvement of RA in the process was revealed in a RNAseq screen comparison between the neural crest and the roof plate (which was recently published by the same lab). However, the current study doesn't use any new technology such as high throughput screens or high resolution or live imaging etc., but rather relies mainly on "old fashioned" techniques: electroporation to induce transient inhibition of RA signaling in the dorsal neural tube followed by analysis of the phenotype by using chromogenic in situ hybridization. The chosen techniques are sufficient to convincingly show the point the authors want to make and the study serves as a reminder that fancy new techniques are not necessarily a requirement for creating a solid story. The manuscript is also well written and easy to follow.
Finally, the manuscript links the activation of RA signaling to the decline of BMP signaling and specifically the upregulation of BMP inhibitors in the dorsal neural tube at the end of the NC stage, but in its current form the proof of this proposed link remains weak. Similarly, the manuscript does not address the consequences of exposure of RA to the dorsal neural tube during NC stage and it thus remains unknown whether RA signaling is sufficient to end the NC stage and activate roof plate formation prematurely. Additional experiments of this kind would help clarify the role of RA in the dorsal neural tube and the reciprocal roles of the two signaling pathways (RA and BMP).
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