Differential regulation of cranial and cardiac neural crest by serum response factor and its cofactors
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Evaluation Summary:
This carefully executed study suggests new mechanisms by which Serum Response Factor (Srf) regulates transcription. The manuscript reports the effects that loss of Srf function has on different neural crest lineages in the mouse. The authors conclude that within neural crest, the main function of Srf is in the cardiac neural crest lineage where it regulates cytoskeletal genes.
(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
Serum response factor (SRF) is an essential transcription factor that influences many cellular processes including cell proliferation, migration, and differentiation. SRF directly regulates and is required for immediate early gene (IEG) and actin cytoskeleton-related gene expression. SRF coordinates these competing transcription programs through discrete sets of cofactors, the ternary complex factors (TCFs) and myocardin-related transcription factors (MRTFs). The relative contribution of these two programs to in vivo SRF activity and mutant phenotypes is not fully understood. To study how SRF utilizes its cofactors during development, we generated a knock-in Srf aI allele in mice harboring point mutations that disrupt SRF-MRTF-DNA complex formation but leave SRF-TCF activity unaffected. Homozygous Srf aI/aI mutants die at E10.5 with notable cardiovascular phenotypes, and neural crest conditional mutants succumb at birth to defects of the cardiac outflow tract but display none of the craniofacial phenotypes associated with complete loss of SRF in that lineage. Our studies further support an important role for MRTF mediating SRF function in cardiac neural crest and suggest new mechanisms by which SRF regulates transcription during development.
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Author Response:
Reviewer #2:
The SRF transcription factor regulates gene transcription through associating with ERK- and actin-regulated cofactors belonging to the TCF and Myocardin families. Each family has multiple members, which exhibit differing expression profiles, and which are to varying extents functionally redundant, which has complicated their functional analysis. Thus, while inactivation of SRF itself leads to failure of gastrulation, inactivation of individual TCF and myocardin-family genes results in much later developmental defects, or barely affects development. Nevertheless studies of this sort have established that myocardin is limiting for VSM development at e10.5, while MRTF-B and myocardin function become limiting in neural crest cells at e14.
Vasudevan and Soriano previously presented evidence for a PDGF-SRF …
Author Response:
Reviewer #2:
The SRF transcription factor regulates gene transcription through associating with ERK- and actin-regulated cofactors belonging to the TCF and Myocardin families. Each family has multiple members, which exhibit differing expression profiles, and which are to varying extents functionally redundant, which has complicated their functional analysis. Thus, while inactivation of SRF itself leads to failure of gastrulation, inactivation of individual TCF and myocardin-family genes results in much later developmental defects, or barely affects development. Nevertheless studies of this sort have established that myocardin is limiting for VSM development at e10.5, while MRTF-B and myocardin function become limiting in neural crest cells at e14.
Vasudevan and Soriano previously presented evidence for a PDGF-SRF axis operating in neural crest cells during craniofacial development: NC-specific SRF inactivation caused facial clefting, and in embryonic palatal mesenchymal cells, PDGF signalling to SRF cytoskeletal target genes that are controlled through MRTF in fibroblasts was impaired. These results pointed to a role for MRTF signalling to SRF in craniofacial development.
"Differential regulation…." by Dinsmore and Soriano revisits these findings. They take a novel approach to assessment of SRF cofactor function by analysing an SRF variant, SRF-alpha1. This SRF derivative was previously shown to be defective in recruitment of MRTF-A (and by extension other myocardin family members) but remained competent to recruit the TCFs. They show that:
• Homozygous SRF-1 mutant mouse embryos survive to e10.5, when they succumb to defects similar to the myocd knockout.
• Anterior mesoderm (Mesp1-cre) SRF-alpha1 embryos last to e10.5, similar to the null, and phenocopy global Myocd mutants.
• Surprisingly, (Wnt1-cre) SRF-alpha1 embryos do not show facial clefting, and there is no genetic interaction with PDGFR, although their palatal MEPM cells are selectively defective in MRTF-SRF target gene expression
• Instead, (Wnt1-cre) SRF-alpha1animals survive to birth, and succumb to cyanosis resulting from highly penetrant cardiac outflow defects reminiscent of those seen in two other models: a hypomorphic MRTF-B genetrap mutation, and an NC-specific (Wnt1-cre) Myocd mutant.
The results raise two main questions:
- What is the basis of the gastrulation defect seen in SRF-null embryos? The results suggest that in cannot not reflect a deficient MRTF signalling, but triple TCF-deficient embryos live beyond this point, so why is there a defect?
- What is the basis for the craniofacial defects in wnt1-Cre SRF-null embryos? The previous proposal that they result from defective PDGF-MRTF-SRF signalling was based on correlation with defective MRTF gene expression, but the SRF-alpha1 result suggests this is not in fact correct.
The authors cannot answer these questions, but propose three possible explanations for their findings: (i) that the SRF-alpha1 allele is a hypomorph not a null for MRTF interaction; (ii) that the TCF cofactors execute some SRF pathways; and (iii) that other undefined SRF cofactors may be involved.
I found this paper enjoyable to read, but hard to review, because it is a clever experiment that raises more questions than it answers. It is an interesting study for a specialist in the SRF field, but less conclusive in terms of clarifying SRF's biological roles.
Unfortunately the paper does not directly answer the questions it raises - it does not directly test the role of MRTFs (and/or myocardin) in the processes analysed, and does not assess the requirement for SRF cofactors per se using appropriate SRF mutants. For example, global MRTF-A/B double knockouts (or A/Mycd, BMycd), which could give direct insights into potential early myocardin-family functions, have not been reported. In the view of this reviewer, however, to do such experiments as part of this study would be inappropriate, and the paper would be of value as a spur to the field.
However, I do have concerns as to the way the data are presented and discussed. To a more general reader in development or transcription, the discussion does not pose the issues clearly, and would benefit from reworking. It would be clearer if the alternative explanations for variance from the simple MRTF-null view of SRF-alpha1 should be posed briefly, and then the basis for each of the phenotypes observed considered in turn.
This is an excellent suggestion to better orient the non-SRF cognoscenti and we have updated the Discussion accordingly, as well as incorporating the reviewer’s extremely insightful ideas that follow. We briefly address them point by point below.
In addition, the authors leave some issues unaddressed in their discussion. For example, they do not consider:
- That the gastrulation defect of SRF-nulls may reflect a cofactor-independent SRF activity. This is plausible, since SRF does have a constitutive transcription activation function. One possible way to test it would be to introduce a mutation such as SRF V194E that blocks both TCF and myocardin-family interactions with SRF (Ling et al 1998 JBiolChem). This mutant phenocopies an SRF-null in immune cells (Mylona et al, 2011 MCB), and should it bypass the gastrulation defect, TCF/MRTF-independent SRF function would be highly likely.
This is an important possibility that we now discuss along with the possibility of alternate cofactors.
- That the SRF-alpha1 allele is a hypomorph for MRTF interaction but a null (or stronger hypomorph) for Myocd interaction. On this model the SRF-alpha1 phenotypes might reflect Myocd recruitment - the lack of craniofacial phenotype might reflect residual MRTF-B interaction, but the later cardiac outflow phenotype would arise from limiting Mycd interaction.
This interpretation is totally consistent with our results and we now include it along with the discussion of tissue-specific thresholds.
- That for some functions the TCF and Myocardin families act through SRF in a functionally redundant manner, so inactivating one family would not impair function.
We meant to suggest this with our first interpretation, “TCF factors … can somehow compensate for loss of SRF-MRTF activity. It is true that some SRF targets can be bound and regulated by both MRTF and TCF factors.” However, we now more strongly invoke their possible redundancy to highlight this point.
- That the MRTF-A and MRTF-B proteins act functionally redundantly with myocardin.
This is an important consideration in tissues where Myocd is expressed and we now address this.
- Their previous paper identified Mrtfa as the only MRTF expressed above background level in MEPM cells. However, the MRTFa knockout mouse develops normally. Thus, if MRTFs are involved in the clefting phenotype, a substantial decrease in MRTF activity can be tolerated before the phenotype becomes manifest. The nonclefting phenotype of the SRF-alpha1 mutant would not be unexpected if this were the case.
The reviewer’s interpretation is correct as stated, but we believe this is an instance where the shortcomings of cell culture complicate matters. Mrtfa is expressed roughly 5x higher than Mrtfb at the mRNA level in passage 2 MEPMs, based on RNA-Seq data from Vasudevan et al., 2014 and Vasudevan et al., 2015. However, in whole E13.5 palate (from which MEPMs are derived) the expression levels are nearly equal. We find in our bulk RNA-Seq that Mrtfa and Mrtfb are expressed at equal levels. More strikingly, in NC cells sorted from E10.5 and E11.5 facial prominences, another study (Minoux et al., 2017) found that Mrtfb is expressed roughly 3x higher than Mrtfa. Rather than being well-tolerated like the loss of Mrtfa, Mrtfb null mutant mice have much stronger developmental phenotypes and the hypomorphic Mrtfb gene trap allele has NC-specific defects. All that stated, it is true that neither Mrtfa nor Mrtfb have been associated with facial clefts in the mouse. It would be interesting to know whether Mrtfa+/-; Mrtfb-/- embryos, Mrtfa-/-; Mrtfb-/- embryos (if they survive long enough), or Mrtfa-/-; Mrtfbflox/flox; Wnt1-CreTg/+ embryos would have clefts. We now include the gene expression data as a supplemental figure (Figure 1, Supplement 2E). Given the expression data from the Minoux study, we are cautious about interpreting the lack of a phenotype in Mrtfa mutants as strong evidence that NC can tolerate substantial depletion of Mrtf genes without clefting.
- The TCFs and MRTFs seem to compete to some extent at most SRF targets - for example, loss of TCFs potentiates cytoskeletal contractility. Thus the effectiveness of the SRF-alpha1 mutation in blocking MRTF-SRF activation in a given setting will be dependent not only on MRTF level but also on TCF level.
This is an interesting point that we had not considered. We mentioned TCF-MRTF competition but now add this intriguing possibility as a further interpretation.
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Evaluation Summary:
This carefully executed study suggests new mechanisms by which Serum Response Factor (Srf) regulates transcription. The manuscript reports the effects that loss of Srf function has on different neural crest lineages in the mouse. The authors conclude that within neural crest, the main function of Srf is in the cardiac neural crest lineage where it regulates cytoskeletal genes.
(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 reports the effects that loss of Srf function has on different neural crest lineages in the mouse. SRF is an important transcription factor that regulates proliferation, migration and differentiation via discrete sets of co factors, such as TCFs and MRTFs. The authors generated a null mouse, as well as a mutant that inhibits Srf interactions with MRTF factors specifically. The manuscript describes a detailed and beautifully illustrated phenotypic analysis and comparison of the resulting phenotypes together with bulk RNASeq analyses. The authors find that Srf mice with point mutations that disrupt Srf interactions with MRTFs show the same gene expression changes as Srf flox/flox mice, but they lack the craniofacial defects. Therefore, they conclude that within neural crest, the main function of …
Reviewer #1 (Public Review):
The manuscript reports the effects that loss of Srf function has on different neural crest lineages in the mouse. SRF is an important transcription factor that regulates proliferation, migration and differentiation via discrete sets of co factors, such as TCFs and MRTFs. The authors generated a null mouse, as well as a mutant that inhibits Srf interactions with MRTF factors specifically. The manuscript describes a detailed and beautifully illustrated phenotypic analysis and comparison of the resulting phenotypes together with bulk RNASeq analyses. The authors find that Srf mice with point mutations that disrupt Srf interactions with MRTFs show the same gene expression changes as Srf flox/flox mice, but they lack the craniofacial defects. Therefore, they conclude that within neural crest, the main function of Srf is in the cardiac neural crest lineage where it regulates cytoskeletal genes. The study does not provide mechanistic insight into why the Srfα allele mostly affects the cardiac neural crest lineage and not the early embryo or other crest lineages, but the possible mechanisms are sufficiently discussed. The carefully executed study suggests new mechanisms by which Srf regulates transcription.
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Reviewer #2 (Public Review):
The SRF transcription factor regulates gene transcription through associating with ERK- and actin-regulated cofactors belonging to the TCF and Myocardin families. Each family has multiple members, which exhibit differing expression profiles, and which are to varying extents functionally redundant, which has complicated their functional analysis. Thus, while inactivation of SRF itself leads to failure of gastrulation, inactivation of individual TCF and myocardin-family genes results in much later developmental defects, or barely affects development. Nevertheless studies of this sort have established that myocardin is limiting for VSM development at e10.5, while MRTF-B and myocardin function become limiting in neural crest cells at e14.
Vasudevan and Soriano previously presented evidence for a PDGF-SRF axis …
Reviewer #2 (Public Review):
The SRF transcription factor regulates gene transcription through associating with ERK- and actin-regulated cofactors belonging to the TCF and Myocardin families. Each family has multiple members, which exhibit differing expression profiles, and which are to varying extents functionally redundant, which has complicated their functional analysis. Thus, while inactivation of SRF itself leads to failure of gastrulation, inactivation of individual TCF and myocardin-family genes results in much later developmental defects, or barely affects development. Nevertheless studies of this sort have established that myocardin is limiting for VSM development at e10.5, while MRTF-B and myocardin function become limiting in neural crest cells at e14.
Vasudevan and Soriano previously presented evidence for a PDGF-SRF axis operating in neural crest cells during craniofacial development: NC-specific SRF inactivation caused facial clefting, and in embryonic palatal mesenchymal cells, PDGF signalling to SRF cytoskeletal target genes that are controlled through MRTF in fibroblasts was impaired. These results pointed to a role for MRTF signalling to SRF in craniofacial development.
"Differential regulation...." by Dinsmore and Soriano revisits these findings. They take a novel approach to assessment of SRF cofactor function by analysing an SRF variant, SRF-alpha1. This SRF derivative was previously shown to be defective in recruitment of MRTF-A (and by extension other myocardin family members) but remained competent to recruit the TCFs. They show that:
• Homozygous SRF-1 mutant mouse embryos survive to e10.5, when they succumb to defects similar to the myocd knockout.
• Anterior mesoderm (Mesp1-cre) SRF-alpha1 embryos last to e10.5, similar to the null, and phenocopy global Myocd mutants.
• Surprisingly, (Wnt1-cre) SRF-alpha1 embryos do not show facial clefting, and there is no genetic interaction with PDGFR, although their palatal MEPM cells are selectively defective in MRTF-SRF target gene expression
• Instead, (Wnt1-cre) SRF-alpha1animals survive to birth, and succumb to cyanosis resulting from highly penetrant cardiac outflow defects reminiscent of those seen in two other models: a hypomorphic MRTF-B genetrap mutation, and an NC-specific (Wnt1-cre) Myocd mutant.
The results raise two main questions:
What is the basis of the gastrulation defect seen in SRF-null embryos? The results suggest that in cannot not reflect a deficient MRTF signalling, but triple TCF-deficient embryos live beyond this point, so why is there a defect?
What is the basis for the craniofacial defects in wnt1-Cre SRF-null embryos? The previous proposal that they result from defective PDGF-MRTF-SRF signalling was based on correlation with defective MRTF gene expression, but the SRF-alpha1 result suggests this is not in fact correct.
The authors cannot answer these questions, but propose three possible explanations for their findings: (i) that the SRF-alpha1 allele is a hypomorph not a null for MRTF interaction; (ii) that the TCF cofactors execute some SRF pathways; and (iii) that other undefined SRF cofactors may be involved.
I found this paper enjoyable to read, but hard to review, because it is a clever experiment that raises more questions than it answers. It is an interesting study for a specialist in the SRF field, but less conclusive in terms of clarifying SRF's biological roles.
Unfortunately the paper does not directly answer the questions it raises - it does not directly test the role of MRTFs (and/or myocardin) in the processes analysed, and does not assess the requirement for SRF cofactors per se using appropriate SRF mutants. For example, global MRTF-A/B double knockouts (or A/Mycd, BMycd), which could give direct insights into potential early myocardin-family functions, have not been reported. In the view of this reviewer, however, to do such experiments as part of this study would be inappropriate, and the paper would be of value as a spur to the field.
However, I do have concerns as to the way the data are presented and discussed. To a more general reader in development or transcription, the discussion does not pose the issues clearly, and would benefit from reworking. It would be clearer if the alternative explanations for variance from the simple MRTF-null view of SRF-alpha1 should be posed briefly, and then the basis for each of the phenotypes observed considered in turn.
In addition, the authors leave some issues unaddressed in their discussion. For example, they do not consider:
That the gastrulation defect of SRF-nulls may reflect a cofactor-independent SRF activity. This is plausible, since SRF does have a constitutive transcription activation function. One possible way to test it would be to introduce a mutation such as SRF V194E that blocks both TCF and myocardin-family interactions with SRF (Ling et al 1998 JBiolChem). This mutant phenocopies an SRF-null in immune cells (Mylona et al, 2011 MCB), and should it bypass the gastrulation defect, TCF/MRTF-independent SRF function would be highly likely.
That the SRF-alpha1 allele is a hypomorph for MRTF interaction but a null (or stronger hypomorph) for Myocd interaction. On this model the SRF-alpha1 phenotypes might reflect Myocd recruitment - the lack of craniofacial phenotype might reflect residual MRTF-B interaction, but the later cardiac outflow phenotype would arise from limiting Mycd interaction.
That for some functions the TCF and Myocardin families act through SRF in a functionally redundant manner, so inactivating one family would not impair function.
That the MRTF-A and MRTF-B proteins act functionally redundantly with myocardin.
Their previous paper identified Mrtfa as the only MRTF expressed above background level in MEPM cells. However, the MRTFa knockout mouse develops normally. Thus, if MRTFs are involved in the clefting phenotype, a substantial decrease in MRTF activity can be tolerated before the phenotype becomes manifest. The nonclefting phenotype of the SRF-alpha1 mutant would not be unexpected if this were the case.
The TCFs and MRTFs seem to compete to some extent at most SRF targets - for example, loss of TCFs potentiates cytoskeletal contractility. Thus the effectiveness of the SRF-alpha1 mutation in blocking MRTF-SRF activation in a given setting will be dependent not only on MRTF level but also on TCF level.
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Reviewer #3 (Public Review):
The manuscript "Differential regulation of cranial and cardiac neural crest by Serum Response Factor" aims to illuminate the differential functional mechanism of Srf during cranial and cardiac neural crest development, especially with respect to the SRF-TCF and SRF-MRTF complexes. The early embryonic lethality of Wnt1-Cre;Srffl/fl mice suggests that Srf has a critical role during neural crest development. By in vivo analysis of marker genes involved in cranial neural crest patterning, the authors found that craniofacial patterning was largely normal in these mice. Further bulk-RNAseq analysis showed that the most differentially expressed genes in Wnt1-Cre;Srffl/fl compared to controls are those targeted by the SRF-MRTF complex, which led the authors to hypothesize that this complex may play a critical role …
Reviewer #3 (Public Review):
The manuscript "Differential regulation of cranial and cardiac neural crest by Serum Response Factor" aims to illuminate the differential functional mechanism of Srf during cranial and cardiac neural crest development, especially with respect to the SRF-TCF and SRF-MRTF complexes. The early embryonic lethality of Wnt1-Cre;Srffl/fl mice suggests that Srf has a critical role during neural crest development. By in vivo analysis of marker genes involved in cranial neural crest patterning, the authors found that craniofacial patterning was largely normal in these mice. Further bulk-RNAseq analysis showed that the most differentially expressed genes in Wnt1-Cre;Srffl/fl compared to controls are those targeted by the SRF-MRTF complex, which led the authors to hypothesize that this complex may play a critical role in midfacial development. To test this hypothesis, the authors generated point mutation mice (SrfαI/αI mice) in which SRF-MRTF-DNA formation is disrupted, while the SRF-TCF-DNA complex is unaffected. SrfαI/αI mice were found to die at the early embryonic stage and morphological defects were observed at E9.5, including turning defects, delayed neural tube closure, missing/hypoplastic second pharyngeal arch, abnormal hematopoiesis, and more. Further in vivo analysis showed reduction in the F-actin level, the number of CD31-positive cells, and cell proliferation, along with significantly increased cell death. The authors then investigated the function of Srf in the anterior mesodermal lineage using Srfflox/flox;Mesp1Cre/+ and SrfαI/flox;Mesp1Cre/+ and found that the defects observed in these two models were similar. Surprisingly, no craniofacial defects were observed in SrfαI/flox; Wnt1-CreTg/+ mice, although the trends of gene and protein expression were very similar between SrfαI/flox;Wnt1-CreTg/+ and Srfflox/flox;Wnt1-CreTg/+ mice. Meanwhile, SrfαI/flox;Wnt1-CreTg/+ mice died at newborn stage and exhibited outflow tract defects.
This study provides an excellent model to investigate the function of Srf in cardiovascular development. While the discovery of a differential response to the same regulator within different neural crest populations is novel and interesting, the detailed regulatory mechanism will need additional clarification.
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