Plexins promote Hedgehog signaling through their cytoplasmic GAP activity
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Curated by eLife
Evaluation Summary:
Prompted by previous findings that Neuropilins can stimulate activity of the Hh signalling pathway, the authors have investigated the possible involvement of Plexins in this process. Using NIH3T3 cells as an assay system, they present evidence that multiple Plexins are able to enhance the transcriptional response to SHH ligand and that this requires activity of the C-terminal intracellular domain of the Plexin protein. They present in vivo evidence that Plexins can stimulate and are required for HH-dependent processes in the CNS. The data are compelling and add further insights into the workings of this important signalling pathway.
(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 #3 agreed to share their name with the authors.)
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Abstract
Hedgehog signaling controls tissue patterning during embryonic and postnatal development and continues to play important roles throughout life. Characterizing the full complement of Hedgehog pathway components is essential to understanding its wide-ranging functions. Previous work has identified neuropilins, established semaphorin receptors, as positive regulators of Hedgehog signaling. Neuropilins require plexin co-receptors to mediate semaphorin signaling, but the role of plexins in Hedgehog signaling has not yet been explored. Here, we provide evidence that multiple plexins promote Hedgehog signaling in NIH/3T3 mouse fibroblasts and that plexin loss of function in these cells results in significantly reduced Hedgehog pathway activity. Catalytic activity of the plexin GTPase-activating protein (GAP) domain is required for Hedgehog signal promotion, and constitutive activation of the GAP domain further amplifies Hedgehog signaling. Additionally, we demonstrate that plexins promote Hedgehog signaling at the level of GLI transcription factors and that this promotion requires intact primary cilia. Finally, we find that plexin loss of function significantly reduces the response to Hedgehog pathway activation in the mouse dentate gyrus. Together, these data identify plexins as novel components of the Hedgehog pathway and provide insight into their mechanism of action.
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Author Response
Reviewer #1 (Public Review):
The authors use both in vitro signaling assays, knockdown in chick neural tube patterning assays and some limited use of Plexin mutant mice. The in vitro work convincingly demonstrates that misexpression of several Plexins is sufficient to enhance HH signaling in a way that depends on the Plexin GAP domain.
We thank the reviewer for the positive evaluation of our work.
Not addressed is how the GAP activity promotes HH signaling.
The reviewer raises an interesting point that we hope to address in future mechanistic studies.
The in vivo data are extremely interesting. However, alternative interpretations of the data are not assessed and need to be before the conclusions favored by the authors can be asserted.
We agree with the reviewer.
Reviewer #2 (Public Review):
This is …
Author Response
Reviewer #1 (Public Review):
The authors use both in vitro signaling assays, knockdown in chick neural tube patterning assays and some limited use of Plexin mutant mice. The in vitro work convincingly demonstrates that misexpression of several Plexins is sufficient to enhance HH signaling in a way that depends on the Plexin GAP domain.
We thank the reviewer for the positive evaluation of our work.
Not addressed is how the GAP activity promotes HH signaling.
The reviewer raises an interesting point that we hope to address in future mechanistic studies.
The in vivo data are extremely interesting. However, alternative interpretations of the data are not assessed and need to be before the conclusions favored by the authors can be asserted.
We agree with the reviewer.
Reviewer #2 (Public Review):
This is interesting work that expands our knowledge of Hedgehog signaling. The work is well-done, well-written, and the figures are clear. I have comments that would help strengthen some of the experiments and improve the manuscript. In particular, the in vivo loss of function experiments could be measured in additional ways (using additional endpoints) to provide a convincing case of the role that Plexins play in Hh signaling in vivo.
We thank the reviewer for their favorable assessment and appreciate their recommendations to add additional in vivo loss of function experiments, which are addressed in the response to Essential Revisions.
- The authors show that the effect of SmoM2 or Gli1 overexpression on Hh pathway activity can be potentiated by Plexins. They then conclude that "These data suggest that PLXNs function downstream of HH ligand at the level of GLI regulation...". It is unclear to me how this experiment allows them to conclude this, as the effect of Plexins could be downstream of Gli1, through the regulation of the transcription machinery, for example.
See response to Essential Revisions.
- Are primary cilia formed normally and present at normal frequency in cells with loss or over-expression of Plexins? This could help understand better how Plexins act to modulate the Hh pathway.
See response to Essential Revisions.
- Are Gli1 protein levels affected by Plexins?
We have not directly examined GLI1 protein levels. Future studies will investigate the consequence of PLXNs on levels, processing and localization of all GLI proteins based on the findings from this study.
- In order to provide a convincing case for the role that Plexins play in Hh signaling in vivo, the in vivo Plexin loss of function experiments should be assessed in additional ways to Gli1-lacZ (Figure 6). Also, proliferation should be measured (as previously shown to be Hh-dependent).
See response to Essential Revisions.
- Data showing whether Plexins bind Shh (or not) should be presented.
The reviewer raises an interesting point. However, the data with the Plxna1∆ECD construct, which lacks the entire extracellular domain suggests that PLXN binding to SHH is not required for HH pathway promotion (see Figure 3). Instead, our experiments suggest that PLXN functions downstream of HH ligand (see Figure 3).
- The authors show that increased Plexin activity in chick neural tubes increases cell migration into the neural tube lumen. Is this effect of Plexins Gli-dependent?
See response to Essential Revisions.
- In the chick neural tube experiments, how can the authors conclude that Plexin promotes Gli-dependent cellular responses since their data show that Plexin is not significantly affecting the fate (NKX6.1 and PAX7) of the cells? I was confused by this. The image shows a change, but the quantification does not.
See response to Essential Revisions.
- Could loss of function experiments in chick neural tube using RNAi against multiple Plexins be performed? This would provide a very convincing case of the requirement of Plexins for Shh signaling.
While we appreciate the reviewer’s suggestion, this experiment would be technically very challenging, given that several PLXNs are expressed in the chicken neural tube (Mauti et al. 2006), and we would likely need to achieve robust knockdown of multiple Plxns to reveal a phenotype. Instead, we have relied on knockdown approaches in cell culture and genetic deletion in mice to assess the consequences of PLXN loss-of-function on HH signaling.
- Figure 1 panels H-I need a negative control for siRNAs.
As noted in the methods (lines 571-573) and in the results (lines 128-131), negative controls for siRNAs were included in each experiment.
- Figure 3B needs to control for Plxn1ΔECD expression levels (by western). Can higher activation of the pathway be explained by higher Plexin protein expression?
While higher PLXNDECD protein levels is one possible explanation for the increase in HH pathway activity, the subsequent data with the GAP domain and FYN kinase mutants (in the context of PLXNDECD, would argue that this is not simply a matter of protein expression, but instead is due to the previously demonstrated increase in GAP activity caused by deletion of the PLXN extracellular domain.
Reviewer #3 (Public Review):
The main strengths of this study are the compelling data derived from the use of well-established cell-based assays of Hedgehog signalling and novelty of the finding that Plexins can modulate the response of cells to Hedgehog. The experiments are well designed and carefully controlled.
We thank the reviewer for their favorable assessment.
The main weaknesses are as follows:
- Plxna2 is expressed at levels lower than a3, b2 and d1, but it is not explained why this gene was knocked out in cell lines in preference to the other three.
We initially generated Plxna1-/-;Plxna2-/- MEFs simply due to the availability of these animals (i.e., we do not have Plxnb2 or Plxnd1 mutant mice in our colony). We then utilized siRNA to achieve a further loss of Plxna3, Plxnb2, and Plxnd1.
- Most of the analysis and the main conclusions of the study are based on the 3T3 experiments. The data supporting the in vivo significance of these findings are less strong:
First, using electroporation of the chick neural tube, they revealed that constitutive Plexin activity can replicate only a subset of the effects of Gli over-expression. It would be relevant to know if ectopic cell migration can be caused by levels of Gli activity lower than those sufficient to induce Nkx6.1 expression - I am not sure if this is already known.
See response to Essential Revisions above.
Second, the authors investigate the consequences of loss of plexin function in the hippocampus, using mouse Plxna1 and Plxna2 mutants. This is a bit puzzling given that their own cell-based assays show that loss of either or both of these proteins has no impact on the response of 3T3 cells to ligand.
We agree with the reviewer that these were surprising results. However, the profile of PLXN expression in the hippocampus is distinct from that of NIH/3T3 cells, and the relative abundance of PLXNs also differs in these sites. Therefore, it is difficult to assess the relative importance of individual PLXNS in the hippocampus, other than analyzing individual Plxn mutant animals. Future studies investigating the individual and combined contributions of PLXNs to HH-dependent embryogenesis will be of high significance.
Moreover, a previous study cited by the authors (Cheng et al 2001) reported that Plxna3 shows the highest and most widespread expression in the CNS and in the hippocampus in particular. Plxna2, by contrast is expressed at much lower levels whilst Plxna1 was detected principally in mature pyramidal cells. It is not clear why the authors chose to focus on these particular Plexins and to what extent the requirements for Plexin function have been rigorously tested.
We agree that investigating the role of other PLXNs in the CNS will be of great value. However, as noted above, we only had access to Plxna1 and Plxna2 mutants at the time of this study.
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Evaluation Summary:
Prompted by previous findings that Neuropilins can stimulate activity of the Hh signalling pathway, the authors have investigated the possible involvement of Plexins in this process. Using NIH3T3 cells as an assay system, they present evidence that multiple Plexins are able to enhance the transcriptional response to SHH ligand and that this requires activity of the C-terminal intracellular domain of the Plexin protein. They present in vivo evidence that Plexins can stimulate and are required for HH-dependent processes in the CNS. The data are compelling and add further insights into the workings of this important signalling pathway.
(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. …
Evaluation Summary:
Prompted by previous findings that Neuropilins can stimulate activity of the Hh signalling pathway, the authors have investigated the possible involvement of Plexins in this process. Using NIH3T3 cells as an assay system, they present evidence that multiple Plexins are able to enhance the transcriptional response to SHH ligand and that this requires activity of the C-terminal intracellular domain of the Plexin protein. They present in vivo evidence that Plexins can stimulate and are required for HH-dependent processes in the CNS. The data are compelling and add further insights into the workings of this important signalling pathway.
(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 #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors use both in vitro signaling assays, knockdown in chick neural tube patterning assays and some limited use of Plexin mutant mice. The in vitro work convincingly demonstrates that misexpression of several Plexins is sufficient to enhance HH signaling in a way that depends on the Plexin GAP domain. Not addressed is how the GAP activity promotes HH signaling. The in vivo data are extremely interesting. However, alternative interpretations of the data are not assessed and need to be before the conclusions favored by the authors can be asserted.
-
Reviewer #2 (Public Review):
This is interesting work that expands our knowledge of Hedgehog signaling. The work is well-done, well-written, and the figures are clear. I have comments that would help strengthen some of the experiments and improve the manuscript. In particular, the in vivo loss of function experiments could be measured in additional ways (using additional endpoints) to provide a convincing case of the role that Plexins play in Hh signaling in vivo.
Specific comments, in no particular order:
1- The authors show that the effect of SmoM2 or Gli1 overexpression on Hh pathway activity can be potentiated by Plexins. They then conclude that "These data suggest that PLXNs function downstream of HH ligand at the level of GLI regulation...". It is unclear to me how this experiment allows them to conclude this, as the effect of …
Reviewer #2 (Public Review):
This is interesting work that expands our knowledge of Hedgehog signaling. The work is well-done, well-written, and the figures are clear. I have comments that would help strengthen some of the experiments and improve the manuscript. In particular, the in vivo loss of function experiments could be measured in additional ways (using additional endpoints) to provide a convincing case of the role that Plexins play in Hh signaling in vivo.
Specific comments, in no particular order:
1- The authors show that the effect of SmoM2 or Gli1 overexpression on Hh pathway activity can be potentiated by Plexins. They then conclude that "These data suggest that PLXNs function downstream of HH ligand at the level of GLI regulation...". It is unclear to me how this experiment allows them to conclude this, as the effect of Plexins could be downstream of Gli1, through the regulation of the transcription machinery, for example.
2- Are primary cilia formed normally and present at normal frequency in cells with loss or over-expression of Plexins? This could help understand better how Plexins act to modulate the Hh pathway.
3- Are Gli1 protein levels affected by Plexins?
4- In order to provide a convincing case for the role that Plexins play in Hh signaling in vivo, the in vivo Plexin loss of function experiments should be assessed in additional ways to Gli1-lacZ (Figure 6). Also, proliferation should be measured (as previously shown to be Hh-dependent).
5- Data showing whether Plexins bind Shh (or not) should be presented.
6- The authors show that increased Plexin activity in chick neural tubes increases cell migration into the neural tube lumen. Is this effect of Plexins Gli-dependent?
7- In the chick neural tube experiments, how can the authors conclude that Plexin promotes Gli-dependent cellular responses since their data show that Plexin is not significantly affecting the fate (NKX6.1 and PAX7) of the cells? I was confused by this. The image shows a change, but the quantification does not.
8- Could loss of function experiments in chick neural tube using RNAi against multiple Plexins be performed? This would provide a very convincing case of the requirement of Plexins for Shh signaling.
Minor points:
9- Figure 1 panels H-I need a negative control for siRNAs.
10- Figure 3B needs to control for Plxn1ΔECD expression levels (by western). Can higher activation of the pathway be explained by higher Plexin protein expression?
-
Reviewer #3 (Public Review):
The main strengths of this study are the compelling data derived from the use of well-established cell-based assays of Hedgehog signalling and novelty of the finding that Plexins can modulate the response of cells to Hedgehog. The experiments are well designed and carefully controlled.
The main weaknesses are as follows:
1. Plxna2 is expressed at levels lower than a3, b2 and d1, but it is not explained why this gene was knocked out in cell lines in preference to the other three.
2. Most of the analysis and the main conclusions of the study are based on the 3T3 experiments. The data supporting the in vivo significance of these findings are less strong:
First, using electroporation of the chick neural tube, they revealed that constitutive Plexin activity can replicate only a subset of the effects of Gli …Reviewer #3 (Public Review):
The main strengths of this study are the compelling data derived from the use of well-established cell-based assays of Hedgehog signalling and novelty of the finding that Plexins can modulate the response of cells to Hedgehog. The experiments are well designed and carefully controlled.
The main weaknesses are as follows:
1. Plxna2 is expressed at levels lower than a3, b2 and d1, but it is not explained why this gene was knocked out in cell lines in preference to the other three.
2. Most of the analysis and the main conclusions of the study are based on the 3T3 experiments. The data supporting the in vivo significance of these findings are less strong:
First, using electroporation of the chick neural tube, they revealed that constitutive Plexin activity can replicate only a subset of the effects of Gli over-expression. It would be relevant to know if ectopic cell migration can be caused by levels of Gli activity lower than those sufficient to induce Nkx6.1 expression - I am not sure if this is already known.
Second, the authors investigate the consequences of loss of plexin function in the hippocampus, using mouse Plxna1 and Plxna2 mutants. This is a bit puzzling given that their own cell-based assays show that loss of either or both of these proteins has no impact on the response of 3T3 cells to ligand. Moreover, a previous study cited by the authors (Cheng et al 2001) reported that Plxna3 shows the highest and most widespread expression in the CNS and in the hippocampus in particular. Plxna2, by contrast is expressed at much lower levels whilst Plxna1 was detected principally in mature pyramidal cells. It is not clear why the authors chose to focus on these particular Plexins and to what extent the requirements for Plexin function have been rigorously tested. -
