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Developmental Cell
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SRF Regulates Craniofacial Development through Selective Recruitment of MRTF Cofactors by PDGF Signaling

      Highlights

      • Neural crest conditional Srf mouse mutants exhibit facial clefting
      • Pdgfra mutants, but not Fgfr1 mutants, interact genetically with Srf mutants
      • Both PDGF and FGF responsive gene promoters overlap with SRF ChIP-seq targets
      • PDGF selectively regulates MRTF-SRF dependent cytoskeletal gene expression

      Summary

      Receptor tyrosine kinase signaling is critical for mammalian craniofacial development, but the key downstream transcriptional effectors remain unknown. We demonstrate that serum response factor (SRF) is induced by both platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signaling in mouse embryonic palatal mesenchyme cells and that Srf neural crest conditional mutants exhibit facial clefting accompanied by proliferation and migration defects. Srf and Pdgfra mutants interact genetically in craniofacial development, but Srf and Fgfr1 mutants do not. This signal specificity is recapitulated at the level of cofactor activation: while both PDGF and FGF target gene promoters show enriched genome-wide overlap with SRF ChIP-seq peaks, PDGF selectively activates a network of MRTF-dependent cytoskeletal genes. Collectively, our results identify a role for SRF in proliferation and migration during craniofacial development and delineate a mechanism of receptor tyrosine kinase specificity mediated through differential cofactor usage, leading to a PDGF-responsive SRF-driven transcriptional program in the midface.

      Graphical Abstract

      Introduction

      Receptor tyrosine kinases (RTKs) engage shared signaling effectors, such as extracellular signal-related kinase (ERK) and phosphatidylinositol 3-kinase (PI3K), but the in vivo phenotypes associated with different RTK mutants can be quite distinct (
      • Lemmon M.A.
      • Schlessinger J.
      Cell signaling by receptor tyrosine kinases.
      ). A central question revolves around how signal specificity arises from a seemingly general set of transduction pathways. At a transcriptional level, RTK signaling classically modulates the expression of immediate early genes (IEGs) (
      • Cochran B.H.
      • Zullo J.
      • Verma I.M.
      • Stiles C.D.
      Expression of the c-fos gene and of an fos-related gene is stimulated by platelet-derived growth factor.
      ,
      • Lau L.F.
      • Nathans D.
      Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc.
      ). While different RTK pathways, such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signaling, induce similar sets of IEGs in cultured cells (
      • Fambrough D.
      • McClure K.
      • Kazlauskas A.
      • Lander E.S.
      Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes.
      ), genetic experiments in mice suggest a degree of IEG specificity downstream of PDGF signaling (
      • Schmahl J.
      • Raymond C.S.
      • Soriano P.
      PDGF signaling specificity is mediated through multiple immediate early genes.
      ). Thus, a major goal remains to characterize the key transcriptional mediators regulated by RTK signaling and determine their specificity downstream of different receptors.
      Development of the mammalian face comprises derivatives from all three germ layers, including a unique contribution from the neural crest. Many components of RTK signaling are linked to craniofacial syndromes and phenotypes in both mice and humans (
      • Newbern J.
      • Zhong J.
      • Wickramasinghe R.S.
      • Li X.
      • Wu Y.
      • Samuels I.
      • Cherosky N.
      • Karlo J.C.
      • O’Loughlin B.
      • Wikenheiser J.
      • et al.
      Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.
      ,
      • Bentires-Alj M.
      • Kontaridis M.I.
      • Neel B.G.
      Stops along the RAS pathway in human genetic disease.
      ). Mice harboring neural crest cell (NCC) conditional loss of PDGF receptor α (PDGFRα) using the Wnt1-Cre transgene exhibit cleft face and palate (
      • Tallquist M.D.
      • Soriano P.
      Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells.
      ). Combined loss of both PDGFRα-specific ligands, PDGFA and PDGFC, results in facial clefting (
      • Ding H.
      • Wu X.
      • Boström H.
      • Kim I.
      • Wong N.
      • Tsoi B.
      • O’Rourke M.
      • Koh G.Y.
      • Soriano P.
      • Betsholtz C.
      • et al.
      A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling.
      ). In humans, mutations in and around PDGFC (
      • Choi S.J.
      • Marazita M.L.
      • Hart P.S.
      • Sulima P.P.
      • Field L.L.
      • McHenry T.G.
      • Govil M.
      • Cooper M.E.
      • Letra A.
      • Menezes R.
      • et al.
      The PDGF-C regulatory region SNP rs28999109 decreases promoter transcriptional activity and is associated with CL/P.
      ,
      • Calcia A.
      • Gai G.
      • Di Gregorio E.
      • Talarico F.
      • Naretto V.G.
      • Migone N.
      • Pepe E.
      • Grosso E.
      • Brusco A.
      Bilaterally cleft lip and bilateral thumb polydactyly with triphalangeal component in a patient with two de novo deletions of HSA 4q32 and 4q34 involving PDGFC, GRIA2, and FBXO8 genes.
      ) and PDGFRα (
      • Rattanasopha S.
      • Tongkobpetch S.
      • Srichomthong C.
      • Siriwan P.
      • Suphapeetiporn K.
      • Shotelersuk V.
      PDGFRa mutations in humans with isolated cleft palate.
      ) have been associated with cleft lip and palate, reflecting a conserved role for PDGF signaling in mammalian midface development. It is interesting that NCC conditional loss of FGF receptor 1 (FGFR1) also results in craniofacial defects (
      • Trokovic N.
      • Trokovic R.
      • Mai P.
      • Partanen J.
      Fgfr1 regulates patterning of the pharyngeal region.
      ,
      • Wang C.
      • Chang J.Y.F.
      • Yang C.
      • Huang Y.
      • Liu J.
      • You P.
      • McKeehan W.L.
      • Wang F.
      • Li X.
      Type 1 fibroblast growth factor receptor in cranial neural crest cell-derived mesenchyme is required for palatogenesis.
      ), indicating a requirement for both PDGF and FGF signaling in NCCs for craniofacial morphogenesis.
      Serum response factor (SRF) is a transcription factor critical for coupling actin dynamics and signaling pathways to gene expression (
      • Posern G.
      • Treisman R.
      Actin’ together: serum response factor, its cofactors and the link to signal transduction.
      ,
      • Olson E.N.
      • Nordheim A.
      Linking actin dynamics and gene transcription to drive cellular motile functions.
      ). SRF was identified as a regulator of the serum response in fibroblasts (
      • Treisman R.
      Identification and purification of a polypeptide that binds to the c-fos serum response element.
      ), and more recent work has focused on understanding the mechanisms of SRF specificity at the transcriptional level (
      • Gineitis D.
      • Treisman R.
      Differential usage of signal transduction pathways defines two types of serum response factor target gene.
      ), particularly in regard to interactions with its two major cofactor families: ternary complex factors (TCFs) and myocardin-related transcription factors (MRTFs) (
      • Esnault C.
      • Stewart A.
      • Gualdrini F.
      • East P.
      • Horswell S.
      • Matthews N.
      • Treisman R.
      Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts.
      ). SRF can be activated in response to many extracellular stimuli, including PDGF and FGF (
      • Treisman R.
      Regulation of transcription by MAP kinase cascades.
      ,
      • Wang Z.
      • Wang D.Z.
      • Hockemeyer D.
      • McAnally J.
      • Nordheim A.
      • Olson E.N.
      Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression.
      ). However, the specificity of SRF activation at a receptor level is unclear, and a direct comparison of SRF function downstream of multiple RTKs has not been carried out.
      SRF is essential across many developmental and physiological contexts, including mesoderm formation (
      • Arsenian S.
      • Weinhold B.
      • Oelgeschläger M.
      • Rüther U.
      • Nordheim A.
      Serum response factor is essential for mesoderm formation during mouse embryogenesis.
      ), cardiac development (
      • Parlakian A.
      • Tuil D.
      • Hamard G.
      • Tavernier G.
      • Hentzen D.
      • Concordet J.P.
      • Paulin D.
      • Li Z.
      • Daegelen D.
      Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.
      ), angiogenesis (
      • Franco C.A.
      • Mericskay M.
      • Parlakian A.
      • Gary-Bobo G.
      • Gao-Li J.
      • Paulin D.
      • Gustafsson E.
      • Li Z.
      Serum response factor is required for sprouting angiogenesis and vascular integrity.
      ), oligodendrocyte differentiation (
      • Stritt C.
      • Stern S.
      • Harting K.
      • Manke T.
      • Sinske D.
      • Schwarz H.
      • Vingron M.
      • Nordheim A.
      • Knöll B.
      Paracrine control of oligodendrocyte differentiation by SRF-directed neuronal gene expression.
      ), neuronal migration (
      • Alberti S.
      • Krause S.M.
      • Kretz O.
      • Philippar U.
      • Lemberger T.
      • Casanova E.
      • Wiebel F.F.
      • Schwarz H.
      • Frotscher M.
      • Schütz G.
      • Nordheim A.
      Neuronal migration in the murine rostral migratory stream requires serum response factor.
      ), and circadian regulation (
      • Gerber A.
      • Esnault C.
      • Aubert G.
      • Treisman R.
      • Pralong F.
      • Schibler U.
      Blood-borne circadian signal stimulates daily oscillations in actin dynamics and SRF activity.
      ). SRF was first implicated in neural crest development through an in situ hybridization screen (
      • Adams M.S.
      • Gammill L.S.
      • Bronner-Fraser M.
      Discovery of transcription factors and other candidate regulators of neural crest development.
      ), and neural crest conditional Srf mouse mutants show defects in dorsal root ganglion (DRG) formation (
      • Wickramasinghe S.R.
      • Alvania R.S.
      • Ramanan N.
      • Wood J.N.
      • Mandai K.
      • Ginty D.D.
      Serum response factor mediates NGF-dependent target innervation by embryonic DRG sensory neurons.
      ), cardiac outflow tract development, and mandible formation (
      • Newbern J.
      • Zhong J.
      • Wickramasinghe R.S.
      • Li X.
      • Wu Y.
      • Samuels I.
      • Cherosky N.
      • Karlo J.C.
      • O’Loughlin B.
      • Wikenheiser J.
      • et al.
      Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.
      ). No facial clefting phenotypes have been previously reported, and the role of SRF in midface development remains unknown.
      In the present study, we report that SRF is required for craniofacial development and responds differentially to PDGF and FGF signaling through selective interactions with MRTF and TCF cofactors. Wnt1-Cre; Srffl/fl mutants exhibit overt facial clefting as well as proliferation and migration deficits in the cranial neural crest and its derivatives. We find that Srf and Pdgfra double mutants (Wnt1-Cre; Srf+/fl; Pdgfra+/fl) display varying degrees of craniofacial defects, but Srf and Fgfr1 (Wnt1-Cre; Srf+/fl; Fgfr1+/fl) do not interact genetically, indicating that SRF function downstream of these two RTKs is not identical. We demonstrate that this specificity is encoded at the level of MRTF-SRF activation and recapitulated in the genome-wide binding profile of SRF and MRTF at the promoters of PDGF target genes, particularly those involved in cytoskeletal organization. Taken together, our studies illustrate a role for SRF in controlling proliferation and migration during craniofacial development and uncover an example of RTK specificity mediated by a common transcription factor through differential cofactor usage and unique output gene expression signatures.

      Results

      PDGF Activates SRF in MEPMs, and PDGFRα and SRF Are Coexpressed during Craniofacial Development

      To identify transcriptional targets of PDGF signaling in the midface, we carried out RNA sequencing (RNA-seq) in embryonic day (E)13.5 mouse embryonic palatal mesenchyme (MEPM) cells treated with PDGFA (which specifically activates PDGFRα), identifying Srf as a PDGF target gene (Table S2 available online; Gene Expression Omnibus [GEO] accession number GSE61755). MEPMs express many palatal mesenchyme markers, including Pdgfra, and have been used to study PDGF (
      • Fantauzzo K.A.
      • Soriano P.
      PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways.
      ) and Ephrin signaling (
      • Bush J.O.
      • Soriano P.
      Ephrin-B1 forward signaling regulates craniofacial morphogenesis by controlling cell proliferation across Eph-ephrin boundaries.
      ). A quantitative PCR (qPCR) time course revealed the peak of Srf mRNA induction to occur at 60 min following PDGF treatment (Figure 1A), and western blot confirmed this increase at the protein level (Figure 1B). The increase in SRF protein prior to Srf mRNA is likely due in part to post-transcriptional regulation of IEG induction (
      • Avraham R.
      • Yarden Y.
      Feedback regulation of EGFR signalling: decision making by early and delayed loops.
      ). We observed the appearance of a shifted band following PDGF treatment; indeed, SRF is phosphorylated at multiple residues in response to growth factor treatment, and previous work has shown that these modifications can affect SRF activity in vitro (
      • Rivera V.M.
      • Miranti C.K.
      • Misra R.P.
      • Ginty D.D.
      • Chen R.H.
      • Blenis J.
      • Greenberg M.E.
      A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.
      ,
      • Iyer D.
      • Chang D.
      • Marx J.
      • Wei L.
      • Olson E.N.
      • Parmacek M.S.
      • Balasubramanyam A.
      • Schwartz R.J.
      Serum response factor MADS box serine-162 phosphorylation switches proliferation and myogenic gene programs.
      ). Thus, we treated PDGF-stimulated MEPM lysates with calf intestinal phosphatase, which resulted in loss of the upper band (Figure S1A), indicating that PDGF treatment promotes SRF phosphorylation. To determine the signaling dependence of SRF induction, we performed western blots following PDGF treatment in the presence of PD325901 (MEK inhibitor), LY294002 (PI3K inhibitor), latrunculin B (MRTF inhibitor), and cytochalasin D (MRTF activator). We found that PDGF-mediated SRF induction requires both PI3K and ERK signaling as well as MRTF activity (Figure 1C).
      Figure thumbnail gr1
      Figure 1SRF Is a Target of PDGF Signaling in Craniofacial Development
      (A and B) In E13.5 MEPMs, PDGF stimulation increases (A) Srf mRNA (2-fold peak induction) and (B) protein (7-fold peak induction) (n = 3). Data plotted as mean ± SEM. p < 0.05.
      (C) SRF induction following PDGF stimulation requires ERK, PI3K, and MRTF activity, as evidenced by inhibition of these pathways. Cells treated with 30 ng/ml PDGFAA for desired duration. PD, PD325901; LY, LY294002; LB, latrunculin B; CD, cytochalasin D.
      (D) At E11.5, Pdgfra and Srf mRNA are coexpressed in the developing MNP and less robustly in the LNP.
      See also and .
      Next, we analyzed the expression pattern of Srf and Pdgfra during craniofacial development. Whole-mount in situ hybridization (WISH) revealed that both genes are expressed in the E11.5 medial nasal process (MNP) (Figure 1D), and we confirmed protein coexpression in the developing MNP and maxillary process (MxP) with anti-SRF immunofluorescence on Pdgfra+/GFP reporter embryos (
      • Hamilton T.G.
      • Klinghoffer R.A.
      • Corrin P.D.
      • Soriano P.
      Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.
      ) (Figure S1B). At E13.5, both Srf and Pdgfra were present broadly in the craniofacial region, with expression noted in the anterior palate at both the mRNA and protein levels (Figures S1C and S1D). These experiments show that PDGF induces SRF in an ERK-, PI3K-, and MRTF-dependent manner and that SRF is coexpressed with PDGFRα in the midface.

      Srf Conditional Mutants Exhibit Overt Facial Clefting and Interact Genetically with Pdgfra Mutants but Not Fgfr1 Mutants

      While cardiac, neuronal, and mandibular defects have been observed in NCC conditional Srf mutants (
      • Newbern J.
      • Zhong J.
      • Wickramasinghe R.S.
      • Li X.
      • Wu Y.
      • Samuels I.
      • Cherosky N.
      • Karlo J.C.
      • O’Loughlin B.
      • Wikenheiser J.
      • et al.
      Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.
      ,
      • Wickramasinghe S.R.
      • Alvania R.S.
      • Ramanan N.
      • Wood J.N.
      • Mandai K.
      • Ginty D.D.
      Serum response factor mediates NGF-dependent target innervation by embryonic DRG sensory neurons.
      ), detailed analysis of the craniofacial phenotypes in these mice has not been carried out. Therefore, we conditionally disrupted Srf in NCCs using the Wnt1-Cre driver (
      • Danielian P.S.
      • Muccino D.
      • Rowitch D.H.
      • Michael S.K.
      • McMahon A.P.
      Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.
      ). We found fully penetrant facial clefting in Wnt1-Cre; Srffl/fl mutants compared to heterozygous Wnt1-Cre; Srf+/fl controls, which appear grossly normal (Figures 2A–2B’ ). Furthermore, Wnt1-Cre; Srf+/fl; Pdgfra+/fl double heterozygotes exhibit partially penetrant facial clefting (Figures 2A’’ and 2B’’), and infrequently recovered Wnt1-Cre; Srffl/fl; Pdgfra+/fl mutant embryos display even more severe phenotypes, characterized by gross midline hemorrhage and blistering in the cephalic region (Figures 2A’’’ and 2B’’’) (Table S1). The defects observed in Pdgfra/Srf double mutants are reminiscent of Pdgfra−/− knockouts, which also exhibit facial clefting, hemorrhaging, and blisters (
      • Soriano P.
      The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites.
      ). We next performed hematoxylin and eosin staining, confirming that the facial clefting extends through the midline in both Wnt1-Cre; Srffl/fl and Wnt1-Cre; Srf+/fl; Pdgfra+/fl mutants at E11.5 (Figure 2C). We carried out morphometric analysis to quantify the relative severity of clefting across genotypes. We found significantly increased distances between the nasal pits (Figure 2D), as well as reduced MxP length (Figure 2D’), in Srf mutants beginning as early as E10.5. No differences in mandibular morphogenesis were detected at these stages (data not shown). Thus, SRF function is required in NCCs for craniofacial development, and Srf/Pdgfra double heterozygotes exhibit phenotypes despite the grossly normal appearance of Srf or Pdgfra heterozygotes, suggesting that these two genes may function within a common network.
      Figure thumbnail gr2
      Figure 2Wnt1-Cre; Srffl/fl Mutants Exhibit Facial Clefting and Interact Genetically with Pdgfra
      (A–B’’’) NCC conditional deletion of Srf results in fully penetrant facial clefting at both (A’) E11.5 and (B’) E13.5. (A’’ and B’’) Embryos heterozygous for both Srf and Pdgfra display a partially penetrant clefting phenotype while (A’’’and B’’’) Srf homozygous mutants missing one copy of Pdgfra display exacerbated phenotypes, including severe midline hemorrhage and blebbing.
      (C) Frontal sections in E11.5 embryos show clefting and forebrain expansion in both the Wnt1-Cre; Srffl/fl and Wnt1-Cre; Srf+/fl; Pdgfra+/fl mutants as well as midline hemorrhage in the double heterozygous condition.
      (D) Morphometry reveals differences in the distance between nasal pits and (D’) MxP length in Wnt1-Cre; Srffl/fl mutants (n = 5 at E10.5, n = 11 at E11.5) and Wnt1-Cre; Srf+/fl; Pdgfra+/fl embryos (n = 4 at E10.5, n = 9 at E11.5) compared to controls (n = 10 at E10.5, n = 10 at E11.5). p < 0.05; ∗∗p < 0.01. Scale bars, 200 μm. All data plotted as mean ± SEM.
      See also and .
      Given the activation of SRF in response to many extracellular signals (including FGF), we hypothesized that SRF may also function downstream of FGF signaling during craniofacial development. Indeed, FGF stimulation induces Srf mRNA in E13.5 MEPMs; however, in contrast to PDGF signaling, FGF-mediated SRF induction required ERK signaling but not PI3K or MRTF activity (Figures S2A and S2B). Furthermore, no interaction between Fgfr1 and Srf conditional mutants (Wnt1-Cre; Srf+/fl; Fgfr1+/fl) was observed (Figure S2C), despite the fact that FGFR1 is the primary FGF receptor in the neural crest and craniofacial mesenchyme (
      • Trokovic N.
      • Trokovic R.
      • Mai P.
      • Partanen J.
      Fgfr1 regulates patterning of the pharyngeal region.
      ,
      • Park E.J.
      • Watanabe Y.
      • Smyth G.
      • Miyagawa-Tomita S.
      • Meyers E.
      • Klingensmith J.
      • Camenisch T.
      • Buckingham M.
      • Moon A.M.
      An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart.
      ). These results suggest that activation of SRF by PDGF and FGF signaling is fundamentally different and, more broadly, that these two receptors perform at least a subset of nonoverlapping functions.
      We recently showed that the original Wnt1-Cre results in Wnt1 overexpression and enlargement of the midbrain and, therefore, generated a Wnt1-Cre2 transgenic line as an alternative without these caveats (
      • Lewis A.E.
      • Vasudevan H.N.
      • O’Neill A.K.
      • Soriano P.
      • Bush J.O.
      The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling.
      ). Facial clefting phenotypes obtained with this Cre driver were similar to those observed with the original Wnt1-Cre (Figure S3A).

      SRF Mutants Display Cell Proliferation and Migration Deficits during Craniofacial Development

      SRF is known to control a diverse range of cellular outcomes, including cell proliferation, migration, survival, and differentiation. Thus, we examined each of these processes in Wnt1-Cre; Srffl/fl mutants to determine the basis for the observed clefting phenotypes. We found reduced proliferation in the MNP of Srf mutants (Figures 3A–3B’’ ) at both E10.5 and E11.5. Similarly, we found fewer cells specifically in the MNP of E11.5 Wnt1-Cre; Srffl/fl mutant embryos (Figure 3C), although no such reduction in total cell number was observed at E10.5 (data not shown). This spatiotemporally specific proliferation defect in Wnt1-Cre; Srffl/fl mutants is consistent with previous work showing that Wnt1-Cre; Pdgfrafl/fl mutants also exhibit reduced MNP proliferation (
      • He F.
      • Soriano P.
      A critical role for PDGFRα signaling in medial nasal process development.
      ). We did not find any difference in apoptosis between control and Srf mutant embryos in the MNP or lateral nasal process (LNP), and we did not detect any change in the expression of MNP marker genes such as Alx3 (data not shown).
      Figure thumbnail gr3
      Figure 3Wnt1-Cre; Srffl/fl Mutants Display Proliferation and Lineage Tracing Defects In Vivo
      (A–B’’) Here, (A and B) Srf mutants exhibit decreased proliferation specifically in the MNP at (A and A’’) E10.5 (n = 5) and (B and B’’) E11.5 (n = 7). ∗∗p < 0.001.
      (C) The MNP of Srf conditional mutants is hypocellular, with significantly fewer cells at E11.5 (n = 7). Cell counts were normalized to number of cells in littermate control. p < 0.005.
      (D–E’) Here, (D and E) lineage tracing using the ROSA26 reporter (R26R) reveals reduced contribution of NCCs to (D and D’) the first and second branchial arches (BA1, BA2) and (E and E’) FNP at E9.5 (somite number indicated). Blue (lacZ positive) cells are generated by Wnt1Cre-mediated recombination and thus label the neural crest and its derivatives.
      Scale bars, 100 μm. All data plotted as mean ± SEM.
      Classic studies have shown that many craniofacial structures are predominantly derived from the neural crest (
      • Couly G.F.
      • Coltey P.M.
      • Le Douarin N.M.
      The triple origin of skull in higher vertebrates: a study in quail-chick chimeras.
      ,
      • Chai Y.
      • Jiang X.
      • Ito Y.
      • Bringas Jr., P.
      • Han J.
      • Rowitch D.H.
      • Soriano P.
      • McMahon A.P.
      • Sucov H.M.
      Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis.
      ). To visualize defects in migration and population of the craniofacial mesenchyme by NCCs, we crossed Srf conditional mutants to R26R-lacZ mice (
      • Soriano P.
      Generalized lacZ expression with the ROSA26 Cre reporter strain.
      ). At E9.5, Wnt1-Cre; Srffl/fl; R26R+/− mutant embryos showed impaired neural crest contribution to the frontonasal prominence (FNP), first branchial arch (BA1), and second branchial arch (BA2) (Figures 3D and 3E). We often observed BA1 and BA2 defects in early E9.5 embryos (<18 somites, seven of nine mutant embryos with phenotype) and FNP and BA2 defects in late E9.5 embryos (≥18 somites, six of nine mutant embryos with phenotype). The combination of lineage tracing and proliferation defects reflects the requirement of SRF activity in the neural crest to both fully populate the craniofacial mesenchyme and respond to proliferative signals.

      SRF Is Required for Cellular Responses to RTK Signaling in FPCs

      Since Wnt1-Cre; Srffl/fl embryos are recovered below Mendelian ratios at E13.5, we turned to an earlier stage of craniofacial development to further investigate SRF function. Given the in vivo defects observed in the E11.5 midface, we established facial prominence cells (FPCs) from E11.5 embryos as a primary cell culture model to study the effects of SRF loss; control FPCs show robust expression of Srf, Pdgfra, and Fgfr1, and Wnt1-Cre2; Srffl/fl mutant FPCs express almost no Srf mRNA (Figure S3B) or protein (Figure S3C).
      SRF is critical for maintaining proper cytoskeletal morphology (
      • Schratt G.
      • Philippar U.
      • Berger J.
      • Schwarz H.
      • Heidenreich O.
      • Nordheim A.
      Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells.
      ). Thus, we stained Srf mutant FPCs for F-actin and β-tubulin and observed gross defects in actin stress fiber formation and microtubule organization (Figure 4A). Next, we tested the proliferative response of FPCs to PDGF and FGF stimulation. Surprisingly, only PDGF induced proliferation in control FPCs, while Srf mutant FPCs fail to proliferate following PDGF treatment, reflecting the requirement of SRF function for PDGF-dependent cell proliferation (Figure 4B). The selective response of FPCs to PDGF may partially explain the phenotypic interactions observed between Srf and Pdgfra conditional mutants but not Srf and Fgfr1 conditional mutants. Finally, we performed scratch assays to compare the response of control and Srf mutant FPCs. In Wnt1-Cre2; Srf+/fl FPCs, 10% fetal bovine serum (FBS), PDGF, and FGF all induced significant wound closure, but this response was abrogated under all conditions in Wnt1-Cre2, Srffl/fl mutant FPCs, reflecting an intrinsic defect in Srf mutant cells (Figure 4C; Figure S3D). Knockdown of SRF is crucial for both cell motility and directional persistence (
      • Medjkane S.
      • Perez-Sanchez C.
      • Gaggioli C.
      • Sahai E.
      • Treisman R.
      Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis.
      ); therefore, we performed time-lapse microscopy and single cell tracking to better understand this deficit. These experiments revealed that, although a subset of Wnt1-Cre2; Srffl/fl mutant FPCs do move efficiently (Figure 4D; Figure S3E), Srf mutant FPCs, overall, are significantly slower and exhibit decreased directional persistence compared to control cells (Figure 4E). In summary, Wnt1-Cre2; Srffl/fl FPCs display proliferation and motility defects in response to growth factor stimulation, linking the observed in vivo proliferation and lineage tracing defects in Srf mutants to RTK signaling and demonstrating the functional relationship between these pathways in the midface.
      Figure thumbnail gr4
      Figure 4Srf Mutant E11.5 FPCs Do Not Proliferate in Response to PDGF and Exhibit Defective Wound Healing
      (A) Srf mutant FPCs lack actin stress fibers, show decreased total F-actin staining, and display gross microtubule disorganization (n > 100 cells per condition).
      (B) Control FPCs exhibit a modest proliferative response to PDGF stimulation, but Srf mutant FPCs fail to proliferate in response to PDGF (n = 4). BrdU, bromodeoxyuridine.
      (C) Although control FPCs show significant wound healing when treated with 10% FBS, PDGF, or FGF compared to 0.1% FBS, Srf mutant FPCs fail to show significant closure when compared to 0.1% FBS starved cells (n = 3). Furthermore, Srf mutant FPCs show significant decreases in wound healing across all growth factor conditions when compared to control FPCs.
      (D) Representative trajectories from ten cells tracked during wound healing in response to either 10% FBS or PDGF. Srf mutant FPCs show both decreased directionality and total distance traveled, although some mutant cells move relatively efficiently (blue trajectory in 10% FBS condition). The heterogeneous migration properties were not due to incomplete loss of SRF (B and S3C).
      (E) Srf mutant cells show decreased speed and persistence in response to both 10% FBS and PDGF when compared to control cells (quartile plot with whiskers spanning 5%–95%).
      Scale bars, 25 μm. Data in (A)–(C) plotted as mean ± SEM. p < 0.05; ∗∗p < 0.001; ˆp < 0.001. Cells were treated with either 30 ng/ml PDGFAA or 50 ng/ml FGF1 + 1 μg/ml heparin.
      See also .

      PDGF Mediates MRTFA-SRF Complex Formation and Activates a Set of MRTF-SRF-Associated Cytoskeletal Genes

      In addition to direct transcriptional induction, many mechanisms have been described to regulate SRF function, including alternative splicing (
      • Belaguli N.S.
      • Zhou W.
      • Trinh T.H.
      • Majesky M.W.
      • Schwartz R.J.
      Dominant negative murine serum response factor: alternative splicing within the activation domain inhibits transactivation of serum response factor binding targets.
      ), direct nuclear translocation (
      • Camoretti-Mercado B.
      • Liu H.W.
      • Halayko A.J.
      • Forsythe S.M.
      • Kyle J.W.
      • Li B.
      • Fu Y.
      • McConville J.
      • Kogut P.
      • Vieira J.E.
      • et al.
      Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor.
      ), and differential cofactor usage (
      • Posern G.
      • Treisman R.
      Actin’ together: serum response factor, its cofactors and the link to signal transduction.
      ). Therefore, we investigated how these parameters are modulated by RTK activation and whether signal specificity was encoded through these mechanisms. We did not detect alternative splicing of SRF (Figures S4A–S4C), and PDGF treatment did not significantly alter the cellular localization of SRF in MEPMs (Figure S4D). Similarly, we did not observe changes in SRF splicing or localization following FGF treatment (data not shown), suggesting these mechanisms are not utilized by either RTK in this context.
      The two major cofactor families utilized by SRF are the TCFs (Elk1, Elk3/Net, and Elk4/Sap1) and MRTFs (MRTFA/Mkl1 and MRTFB/Mkl2). MRTF-dependent SRF activity occurs downstream of changes in actin concentration predominantly mediated by Rho-family small GTPases (
      • Miralles F.
      • Posern G.
      • Zaromytidou A.I.
      • Treisman R.
      Actin dynamics control SRF activity by regulation of its coactivator MAL.
      ,
      • Vartiainen M.K.
      • Guettler S.
      • Larijani B.
      • Treisman R.
      Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL.
      ). In contrast, TCF-dependent SRF activation lies downstream of ERK signaling (
      • Posern G.
      • Treisman R.
      Actin’ together: serum response factor, its cofactors and the link to signal transduction.
      ). The distinction between the two mechanisms of SRF activation has a functional consequence, as unique SRF-regulated gene sets are controlled through each of these pathways (
      • Gineitis D.
      • Treisman R.
      Differential usage of signal transduction pathways defines two types of serum response factor target gene.
      ). Although RTK signaling is traditionally associated with robust activation of ERK, PDGF and FGF have also been shown to modulate small GTPase function in many contexts, including the midface (
      • He F.
      • Soriano P.
      A critical role for PDGFRα signaling in medial nasal process development.
      ). We began by screening the expression of SRF cofactor genes in MEPM RNA-seq and published E13.5 palate RNA-seq data (http://www.facebase.org; FaceBase accession number FB00000278.2); only Elk1, Elk3, and Mrtfa are expressed above a threshold of ten fragments per kilobase of transcript per million mapped reads (FPKM) in both data sets (Figure 5A). We next performed WISH in E11.5 embryos to determine the expression pattern of these cofactors and compared them to Srf and Pdgfra. Both Elk1 and Elk3 show strong expression in the MNP, but Mrtfa is also expressed throughout the craniofacial region, albeit more diffusely (Figure 5B). Thus, based on expression pattern alone, any of these cofactors may synergize with SRF in the midface.
      Figure thumbnail gr5
      Figure 5Both MRTF and TCF Cofactors Play Roles Downstream of RTK Signaling in Craniofacial Development
      (A) Of the five major TCF and MRTF cofactor family members, only Elk1, Elk3, and Mrtfa are expressed above a threshold of ten FPKM in both E13.5 MEPMs and E13.5 palate.
      (B) WISH reveals that Elk1 and Elk3 mRNA are enriched in the E11.5 MNP. Mrtfa mRNA expression in the midface is more diffuse but shares expression domains with Pdgfra and Srf.
      (C) PDGF modestly increases SRF-MRTFA association while FGF reduces SRF-MRTFA complex formation. MRTFA levels are not modulated by PDGF or FGF treatment, thus serving as an additional loading control.
      (D) MRTFA-SRF association following 30 min PDGF stimulation requires PI3K activity. All biochemistry performed in E13.5 MEPMs. CD, cytochalasin D; PD, PD325901; LY, LY294002; U, untreated cells; P, 30 min 30 ng/ml PDGFAA; F, 30 min 50 ng/ml FGF1 + 1 μg/ml heparin.
      (E) MRTFA immunofluorescence shows greater nuclear accumulation of MRTFA in response to PDGF compared to FGF, although a significant number of FGF-treated cells contain nuclear MRTFA. Red arrowheads mark cells counted as containing nuclear MRTFA. Cytochalasin D and latrunculin B were used as a positive and a negative control, respectively. ∗∗p < 0.05.
      Data plotted as mean ± SEM. See also .
      In order to directly test SRF-cofactor complex formation, we treated E13.5 MEPMs with either PDGF or FGF, performed immunoprecipitation (IP) for either Elk1 or MRTFA, and then western blotted for SRF. Although both PDGF and FGF promoted formation of an SRF-Elk1 complex, only PDGF treatment resulted in SRF-MRTFA association; conversely, FGF stimulation reduced the amount of SRF-MRTFA complex (Figure 5C; Figure S4E). This PDGF-mediated SRF-MRTFA association required PI3K (Figure 5D) activity, while SRF-Elk1 association required both ERK and PI3K signaling (Figure S4E), consistent with previous work implicating PI3K as the key effector of PDGFRα signaling during craniofacial development (
      • Klinghoffer R.A.
      • Hamilton T.G.
      • Hoch R.
      • Soriano P.
      An allelic series at the PDGFalphaR locus indicates unequal contributions of distinct signaling pathways during development.
      ,
      • Fantauzzo K.A.
      • Soriano P.
      PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways.
      ). Since MRTF activation results in shuttling of the protein from the cytoplasm to the nucleus (
      • Miralles F.
      • Posern G.
      • Zaromytidou A.I.
      • Treisman R.
      Actin dynamics control SRF activity by regulation of its coactivator MAL.
      ), we next performed MRTFA immunofluorescence in MEPMs (Figure 5E). While MRTFA is predominantly cytoplasmic in starved cells, PDGF stimulation increases nuclear MRTFA. FGF also induces MRTFA translocation, but to a lesser extent than PDGF. Indeed, PDGF-induced MRTFA shuttling occurs at ratios comparable to those of cytochalasin-D-treated MEPMs, consistent with a full MRTFA response to PDGF. The observed heterogeneity of the MRTFA response in MEPMs may be, in part, a result of our primary cell culture system, and quantitatively similar changes in nuclear MRTFA have been observed in other studies (
      • Ho C.Y.
      • Jaalouk D.E.
      • Vartiainen M.K.
      • Lammerding J.
      Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics.
      ).
      These results raised the possibility that PDGF signaling preferentially drives MRTFA-dependent SRF activity, while both PDGF and FGF activate Elk1-mediated SRF function. In order to gain insight toward the genome-wide role of SRF downstream of these pathways, we integrated our MEPM RNA-seq data with SRF chromatin immunoprecipitation sequencing (ChIP-seq) data from mouse C2C12 (
      ENCODE Project Consortium
      An integrated encyclopedia of DNA elements in the human genome.
      ; GEO accession number GSM915168) and 3T3 (
      • Esnault C.
      • Stewart A.
      • Gualdrini F.
      • East P.
      • Horswell S.
      • Matthews N.
      • Treisman R.
      Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts.
      ) cells. Although SRF binding events are unlikely to be fully conserved across different contexts, a previous study estimated that ∼60% of SRF binding events in the proximal promoter are shared between cell types (
      • Sullivan A.L.
      • Benner C.
      • Heinz S.
      • Xie L.
      • Miano J.M.
      • Glass C.K.
      • Huang W.
      Serum response factor utilizes distinct mechanisms to regulate cytoskeletal gene expression in macrophages serum response factor utilizes distinct promoter- and enhancer-based mechanisms to regulate cytoskeletal gene expression.
      ), supporting our correlative approach. We also recently generated an analogous RNA-seq data set for FGF-treated MEPMs, allowing comparison of the transcriptional outputs from both PDGF and FGF signaling (Table S2). First, we plotted the distribution of SRF ChIP-seq peaks from the transcriptional start site (TSS) of all genes upregulated at 1 hr by either PDGF (125 significant genes) or FGF (135 significant genes) (Figures 6A and 6B ). We found enrichment of SRF ChIP-seq peaks upstream from the TSS of RTK regulated genes, suggesting that SRF-mediated transcription plays a key role in the genome-wide response to both PDGF and FGF. A full list of these peaks and genes is provided (Table S3). No such enrichment was observed in randomly selected, expression-matched control genes (Figures 6A and 6B, black lines) unresponsive to growth factor treatment or when plotting the peak distribution from ChIP-seq data for Jun (induced on PDGF treatment in MEPMs), Pax5 (not expressed in MEPMs), or p300 (a transcriptional coactivator sampled in the E13.5 palate) (Figures S5A–S5C).
      Figure thumbnail gr6
      Figure 6Both PDGF- and FGF-Responsive Genes Correlate with SRF Binding Genome-wide, but Only PDGF Target Gene Promoters Are Enriched for MRTF
      (A and B) Both PDGF (red) and FGF (blue) responsive genes show enrichment for SRF binding events from C2C12 (
      ENCODE Project Consortium
      An integrated encyclopedia of DNA elements in the human genome.
      ) and 3T3 (
      • Esnault C.
      • Stewart A.
      • Gualdrini F.
      • East P.
      • Horswell S.
      • Matthews N.
      • Treisman R.
      Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts.
      ) SRF ChIP-seq data. Randomly sampled expression matched control genes (black, n = 3 random sets) show no such enrichment. A total of 67 PDGF responsive genes have an SRF ChIP-seq peak within 10 kb of the TSS, while 52 FGF-responsive genes have an SRF ChIP-seq peak within 10 kb of the TSS. p < 0.1; ∗∗p < 0.05; ∗∗∗p < 0.01.
      (C) Closer inspection of SRF target genes downstream of each RTK reveals 56% overlap, with many classic IEGs (Fos, Jun, Egr) activated jointly by both PDGF and FGF.
      (D) PDGF-SRF targets show enrichment for actin cytoskeletal elements, while FGF-SRF targets show minimal functional organization.
      (E) PDGF-SRF targets show significantly increased MRTF scores compared to FGF-SRF targets, consistent with PDGF-specific activation of an MRTF-associated transcriptional program.
      (F) SRF and MRTFA ChIP-qPCR in E13.5 MEPMs reveals increased binding of these factors at the promoters of cytoskeletal genes in response to PDGF (red) in contrast to FGF (blue). Inhibition of PI3K signaling (patterned bars) reduces PDGF-stimulated SRF binding, although a significant response is still observed at some promoters. p < 0.1; ∗∗p < 0.05, compared to serum starved. (n = 3).
      Cells were treated with 30 ng/ml PDGFAA or 50 ng/ml FGF1 + 1 μg/ml heparin. See also and .
      A total of 94 PDGF and 95 FGF target genes contain an SRF ChIP-seq peak within 70 kb of the TSS in either C2C12 or 3T3 cells; over half of these genes are induced by both pathways (56% shared) (Figure 6C). Many classic IEGs (such as Fos, Jun, and Egr) fall into the group of 53 genes with SRF binding events that are jointly induced by both PDGF and FGF signaling. Notably, a high percentage of genes (56%–75%) was conserved in both ChIP-seq data sets (Table S3). Gene ontology analysis revealed that PDGF-responsive SRF targets show overrepresentation of genes associated with the actin cytoskeleton, while FGF-mediated SRF targets show no such relationship (Figure 6D). To visualize these target genes at the level of cofactor specificity, we next integrated our data sets with recently published MRTF and TCF ChIP-seq data sets in 3T3 cells that assigned a score for each target gene reflecting the relative binding of these cofactors (
      • Esnault C.
      • Stewart A.
      • Gualdrini F.
      • East P.
      • Horswell S.
      • Matthews N.
      • Treisman R.
      Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts.
      ) (Table S4). We found that many shared RTK targets possess high TCF scores, but a subset of PDGF-specific genes show high MRTF scores (Figures S5D and S5E). Indeed, MRTF scores for PDGF-SRF target genes are significantly increased compared to MRTF scores or FGF-SRF target genes (Figure 6E), but no such difference is observed for TCF scores (Figure S5F). This correlation between PDGF target genes and SRF-MRTF binding genome-wide may reflect a PDGF-MRTF-SRF circuit not regulated by FGF signaling.

      SRF Regulates the Expression of a Cytoskeletal Network Critical for Craniofacial Development

      Our genomic analyses suggest that PDGF-mediated SRF activation results in activation of a key cytoskeletal transcriptional program; however, given that the ChIP-seq and RNA-seq data were generated from different cell types, our correlative approach alone does not delineate whether these binding events and gene expression changes are functional. Thus, we probed this network in more detail, using a candidate-based approach. We selected eight genes for further study based on published mouse craniofacial phenotypes and reported disease associations in humans (Figure S6A). Many of these genes show high MRTF scores and, in the case of Acta1 and Myh9, have been previously identified as MRTF target genes (
      • Sun Y.
      • Boyd K.
      • Xu W.
      • Ma J.
      • Jackson C.W.
      • Fu A.
      • Shillingford J.M.
      • Robinson G.W.
      • Hennighausen L.
      • Hitzler J.K.
      • et al.
      Acute myeloid leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function.
      ,
      • Medjkane S.
      • Perez-Sanchez C.
      • Gaggioli C.
      • Sahai E.
      • Treisman R.
      Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis.
      ). We performed endogenous SRF ChIP in E13.5 MEPMs at previously identified SRF binding sites in these eight target promoters, finding six (Vcl, Acta2, Myh9, Actb, Tgln, Flna) to show SRF binding (Figure S6B). Five of these six promoters (Acta2, Vcl, Myh9, Actb, Flna) exhibit increased SRF binding following PDGF stimulation; comparing the two RTKs, all six targets show significantly greater SRF binding in response to PDGF, while only one promoter (Actb) exhibits increased binding following FGF stimulation (Figure 6F). In contrast, SRF binding at the promoter of the shared target Fos is induced by both PDGF and FGF signaling, and low occupancy was observed in the promoter of Arid5b, an IEG not bound by SRF in either ChIP-seq data set (Figure S6C).
      Given the importance of PI3K signaling downstream of PDGF in the midface, we assayed the effect of PI3K inhibition on SRF binding. We found decreased PDGF-mediated SRF binding at all six targets following pretreatment with LY294002; however, two target promoters (Acta2 and Actb) still showed significant responses, indicating that PI3K signaling is not always required for SRF binding (Figure 6F). In addition, PI3K inhibition significantly decreased SRF binding at the Tagln promoter across all conditions, suggesting that PI3K promotes SRF maintenance at this locus. Next, we carried out endogenous MRTFA ChIP and demonstrate significantly increased MRTFA binding at four loci (Acta2, Vcl, Myh9, Actb) in response to PDGF treatment (Figure 6F). In contrast, FGF induced significant MRTFA binding at only the Actb promoter, consistent with the increased SRF binding at this region. No MRTFA binding was observed at either the Flna or the Fos (Figure S6C) promoter. Collectively, these results indicate that PDGF and FGF differentially modulate SRF and MRTFA binding at target gene promoters, in part through PI3K signaling.
      To determine the MRTF dependence of these genes, we stimulated MEPMs with PDGF or FGF in the presence of latrunculin B (Figure 7A). All six genes are selectively induced by PDGF. Furthermore, latrunculin B inhibits the PDGF-mediated expression of five genes (Acta2, Vcl, Myh9, Actb, Tagln), confirming that these targets are indeed MRTF dependent. While MRTFA nuclear accumulation is observed at 30 min following PDGF treatment, both the repressive effect of latrunculin B and observed MRTFA binding at target gene promoters is more pronounced at 4 hr, suggesting that MRTF-mediated changes in gene expression may be a delayed response. The induction of Fos by both PDGF and FGF is not affected by latrunculin B (Figure S6D). We then measured the expression of these six genes in E11.5 facial prominences (MNP, LNP, and MxP) dissected from Srf, Pdgfra, and Fgfr1 conditional mutants. As expected, the expression of Srf, Pdgfra, and Fgfr1 were decreased in the corresponding mutants; furthermore, we observed downregulation of all six targets in Srf mutant facial prominences (Figure 7B). Finally, we found significantly decreased expression of four PDGF-SRF targets (Vcl, Myh9, Actb, and Flna) in Pdgfra mutants, but not Fgfr1 mutants (Figure 7B). It is interesting that a modest increase in Pdgfra expression was observed in Srf mutants, possibly indicating a compensatory feedback mechanism. These results reflect the perturbation of a PDGF-responsive, MRTF-dependent cytoskeletal circuit specifically in Pdgfra and Srf mutants.
      Figure thumbnail gr7
      Figure 7A PDGF-MRTF-SRF Axis Controls Expression of Key Cytoskeletal Regulators in Craniofacial Development
      (A) PDGF (red bars) robustly activates expression of cytoskeletal target genes in MEPMs, while FGF (blue bars) does not. Latrunculin B treatment (patterned bars) inhibits PDGF-mediated gene expression, indicating that induction of these shared PDGF-SRF targets is MRTF dependent.
      (B) The expression of all six cytoskeletal SRF target genes is reduced in E11.5 Wnt1-Cre; Srffl/fl mutant facial prominences (gray bars). While none of these genes show reduced expression in Wnt1-Cre; Fgfr1fl/fl mutant facial prominences (blue bars), four of six genes (Vcl, Myh9, Actb, and Flna) show downregulation in Wnt1-Cre; Pdgfrafl/fl mutant facial prominences (red bars).
      (C) PPI network constructed from PDGF-regulated SRF target genes (red) and FGF-regulated SRF target genes (purple) recapitulates unique SRF functions downstream of PDGF signaling. Furthermore, the PDGF specific SRF network contains an enrichment of MRTF target genes (squares) compared to the shared network, which has equal TCF (triangle), MRTF (square), and nonspecific (circles) genes. All six genes with altered expression in SRF mutants (bold) fall under the PDGF-specific network, and many PDGF-SRF target genes have known roles in craniofacial development (sources: http://www.informatics.jax.org; http://www.omim.org).
      Data are plotted as mean ± SEM. p < 0.1; ∗∗p < 0.05, compared to serum starved or wild-type control; ˆˆp < 0.05, compared to latrunculin B treatment (n = 3).
      See also and .
      Our data suggest the following model: PDGF mediates MRTFA-SRF association and binding at select target gene promoters to drive MRTF-dependent expression of key actomyosin cytoskeleton elements (such as Vcl, Actb, Acta2, and Myh9). In contrast, both PDGF and FGF signaling increase Elk1-SRF complex formation to modulate the classic IEG signature (including Fos, Fosb, and Junb) observed downstream of these pathways. By building a protein-protein interaction (PPI) network of the targets downstream of PDGF, FGF, and SRF (
      • Chen E.Y.
      • Xu H.
      • Gordonov S.
      • Lim M.P.
      • Perkins M.H.
      • Ma’ayan A.
      Expression2Kinases: mRNA profiling linked to multiple upstream regulatory layers.
      ), we can better visualize this specificity at both the RTK and cofactor levels (Figure 7C). Consistent with our framework, the PDGF-specific PPI network shows strong correlation with MRTF target genes. Our studies imply that loss of this PDGF-MRTF-SRF axis explains, in part, both the craniofacial phenotypes of Pdgfra and Srf conditional mutants and the observed phenotypic interaction between these two genes.

      Discussion

      SRF is a classic regulator of the transcriptional response to growth factor signaling. In the present study, we find that neural crest conditional loss of SRF results in facial clefting accompanied by proliferation and migration defects. By analyzing SRF activation downstream of both PDGF and FGF signaling, we uncover a PDGF-MRTF-SRF circuit critical for cytoskeletal gene expression in the midface. We conclude that SRF is required for craniofacial development and that RTK signaling encodes the specificity of SRF-mediated gene expression at the level of cofactor recruitment in this developmental context.
      Many phenotypes have been reported in SRF neural crest conditional mutants (
      • Newbern J.
      • Zhong J.
      • Wickramasinghe R.S.
      • Li X.
      • Wu Y.
      • Samuels I.
      • Cherosky N.
      • Karlo J.C.
      • O’Loughlin B.
      • Wikenheiser J.
      • et al.
      Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.
      ,
      • Wickramasinghe S.R.
      • Alvania R.S.
      • Ramanan N.
      • Wood J.N.
      • Mandai K.
      • Ginty D.D.
      Serum response factor mediates NGF-dependent target innervation by embryonic DRG sensory neurons.
      ), and we now describe gross facial clefting in these mutants, which had not been previously appreciated. We further demonstrate that SRF drives proliferation of the NCC-derived MNP mesenchyme. MNP cell proliferation is also decreased in Wnt1-Cre; Pdgfrafl/fl mice (
      • He F.
      • Soriano P.
      A critical role for PDGFRα signaling in medial nasal process development.
      ), suggesting a common mechanism underlying both PDGF and SRF activity. Consistent with this notion, SRF mutant FPCs fail to proliferate in response to PDGF stimulation. Thus, PDGF signaling acts through SRF to drive a functional proliferation program in the midface. In the epidermis, loss of Srf leads to cell proliferation defects due to abnormalities in the actomyosin network (
      • Luxenburg C.
      • Pasolli H.A.
      • Williams S.E.
      • Fuchs E.
      Developmental roles for Srf, cortical cytoskeleton and cell shape in epidermal spindle orientation.
      ); many targets of the PDGF-MRTF-SRF axis elucidated in our study (such as Actb, Myh9, Flna, and Actg1) were also implicated in the proliferation defects observed in these epidermis-specific Srf mutants, reflecting the importance of these genes. We further show that Wnt1-Cre; Srffl/fl embryos exhibit lineage tracing defects characterized by decreased infiltration of NCCs into the FNP and first two branchial arches, raising the possibility that the facial clefting may be, in part, the result of an insufficient number of neural-crest-derived progenitors populating the craniofacial mesenchyme. To understand the basis for these deficits, we analyzed SRF-deficient primary cells isolated from the cranial NCC-derived facial prominences and demonstrated decreased speed and directional persistence in wound healing assays. On delamination from the dorsal neural tube, NCCs migrate in stereotypic streams throughout the embryo to populate a diverse range of derivatives, responding to guidance cues as they migrate collectively to their final destinations (
      • Theveneau E.
      • Mayor R.
      Neural crest delamination and migration: from epithelium-to-mesenchyme transition to collective cell migration.
      ). Many PDGF-regulated SRF target genes in our study are involved in force generation (Myh9, Myl6, Myl12b), focal adhesion formation (Vcl, Flna, Zyx), and cytoskeletal organization (Actb, Actg1, Acta2), key processes for cell migration. In addition, MRTFs have been shown to mediate cell motility and directionality in other contexts (
      • Medjkane S.
      • Perez-Sanchez C.
      • Gaggioli C.
      • Sahai E.
      • Treisman R.
      Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis.
      ), suggesting that the observed defects may be MRTF dependent.
      On activation, RTKs become phosphorylated and engage downstream effectors, which, in turn, activate many shared intracellular pathways. One outcome of this signaling cascade is the transcription of IEGs. Many RTKs induce overlapping sets of IEGs, leaving open the question of how transcriptional specificity is generated. We demonstrate that PDGF, but not FGF, selectively promotes recruitment of SRF and MRTFA to target gene promoters, leading to induction of a unique gene expression program. This PDGF-MRTF-SRF axis is enriched for cytoskeletal regulators, many of which exhibit MRTF-dependent induction and decreased expression in Srf and Pdgfra mutants. On the other hand, both PDGF and FGF increase Elk1-SRF complex formation, in line with our observation that over half of the identified SRF target genes are jointly activated by both PDGF and FGF. The targets downstream of this shared RTK-Elk1-SRF axis, such as Fos, Jun, and Egr, constitute a classic set of IEGs activated by multiple pathways (
      • Fambrough D.
      • McClure K.
      • Kazlauskas A.
      • Lander E.S.
      Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes.
      ), suggesting that other stimuli can compensate for loss of Elk1-SRF-mediated transcription. Consistent with this notion, Srf null embryos express residual amounts of Fos and Egr1 but show complete loss of Acta1 expression (
      • Arsenian S.
      • Weinhold B.
      • Oelgeschläger M.
      • Rüther U.
      • Nordheim A.
      Serum response factor is essential for mesoderm formation during mouse embryogenesis.
      ). We propose that the PDGF-MRTF-SRF axis has unique roles in the developing midface, an assertion supported by our epistasis results and gene expression studies.
      Two critical points merit further discussion. First, what are the key signaling parameters encoding differential cofactor activation and SRF-mediated gene expression downstream of PDGF and FGF signaling? A potential answer lies in the importance of PI3K activity in the formation of an MRTFA-SRF complex and in driving maximal SRF binding at target gene promoters in response to PDGF stimulation. Previous studies have shown PI3K to be the primary effector downstream of PDGF signaling during midface development, closely mirroring the craniofacial phenotypes observed in PDGFRα mutants (
      • Klinghoffer R.A.
      • Hamilton T.G.
      • Hoch R.
      • Soriano P.
      An allelic series at the PDGFalphaR locus indicates unequal contributions of distinct signaling pathways during development.
      ,
      • Fantauzzo K.A.
      • Soriano P.
      PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways.
      ). Thus, one explanation is that PDGF-activated PI3K signaling selectively promotes MRTFA-SRF association, perhaps through Rho-family small GTPases, which modulate actin dynamics and MRTF activity in other contexts (
      • Pipes G.C.T.
      • Creemers E.E.
      • Olson E.N.
      The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis.
      ). Both Rac1 and Cdc42 are required in the neural crest for craniofacial development (
      • Thomas P.S.
      • Kim J.
      • Nunez S.
      • Glogauer M.
      • Kaartinen V.
      Neural crest cell-specific deletion of Rac1 results in defective cell-matrix interactions and severe craniofacial and cardiovascular malformations.
      ,
      • Fuchs S.
      • Herzog D.
      • Sumara G.
      • Büchmann-Møller S.
      • Civenni G.
      • Wu X.
      • Chrostek-Grashoff A.
      • Suter U.
      • Ricci R.
      • Relvas J.B.
      • et al.
      Stage-specific control of neural crest stem cell proliferation by the small rho GTPases Cdc42 and Rac1.
      ) and have been implicated downstream of PDGF signaling in MEPMs (
      • He F.
      • Soriano P.
      A critical role for PDGFRα signaling in medial nasal process development.
      ,
      • Fantauzzo K.A.
      • Soriano P.
      PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways.
      ). Although FGF signaling can modulate both Rac1 and Cdc42 in other systems (
      • Fera E.
      • O’Neil C.
      • Lee W.
      • Li S.
      • Pickering J.G.
      Fibroblast growth factor-2 and remodeled type I collagen control membrane protrusion in human vascular smooth muscle cells: biphasic activation of Rac1.
      ,
      • Clark I.B.N.
      • Muha V.
      • Klingseisen A.
      • Leptin M.
      • Müller H.A.J.
      Fibroblast growth factor signalling controls successive cell behaviours during mesoderm layer formation in Drosophila.
      ), it is unclear whether this relationship is conserved in the midface. Alternatively, while both PDGF and FGF signal through common kinase cascades, the magnitude and duration of this induction can be quite different. Indeed, PDGF- and FGF-mediated pERK activation patterns are markedly different in MEPMs, with PDGF stimulation resulting in a transient pERK pulse but FGF treatment driving sustained pERK activation (unpublished data). Consistent with this observation, a recent study showed that sporadic pERK pulses drive SRF-mediated transcription more efficiently than sustained pERK activity (
      • Aoki K.
      • Kumagai Y.
      • Sakurai A.
      • Komatsu N.
      • Fujita Y.
      • Shionyu C.
      • Matsuda M.
      Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation.
      ), reflecting yet another layer of control. In summary, a combination of both qualitative and quantitative differences in signaling parameters likely accounts for the observed SRF specificity.
      Second, is this specificity of SRF activation “hard wired” into the PDGF and FGF signaling networks, or is it context dependent? The answer is almost certainly the latter, as the magnitude and kinetics of activation downstream of even the same RTK can vary depending on many parameters, including expression level (
      • Traverse S.
      • Seedorf K.
      • Paterson H.
      • Marshall C.J.
      • Cohen P.
      • Ullrich A.
      EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor.
      ). The expression pattern of many signaling components is restricted over the course of development, necessitating diverse, context-specific control systems. The neural crest itself is a multipotent population with many sublineages, all expressing different combinations and amounts of receptors and signaling effectors. In the case of PDGFRα and SRF, we describe a PDGF-SRF circuit in the midface. However, many of the observed hemorrhaging and blistering phenotypes in these mutants may be due, in part, to requirements for PDGF signaling and SRF activity in neural-crest-derived vascular components, such as smooth muscle cells and pericytes (
      • Etchevers H.C.
      • Vincent C.
      • Le Douarin N.M.
      • Couly G.F.
      The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain.
      ). The PDGF-SRF signaling axis may be wired differently in these cells, particularly at the levels of receptor activation, effector requirements, and cofactor recruitment. In DRG sensory neurons, SRF activity downstream of nerve growth factor is dependent on ERK-mediated MRTFA activity, but not on TCF-SRF complex formation (
      • Wickramasinghe S.R.
      • Alvania R.S.
      • Ramanan N.
      • Wood J.N.
      • Mandai K.
      • Ginty D.D.
      Serum response factor mediates NGF-dependent target innervation by embryonic DRG sensory neurons.
      ). Thus, SRF function can be controlled by different RTKs in divergent neural crest lineages through a common set of cofactors and signaling effectors. The degree of sophistication and stimulus-dependent utilization of SRF regulatory mechanisms is remarkable, and determining the exact rules governing SRF activity following receptor activation across these diverse developmental contexts will be rewarding. Our studies provide one such example of the intricate control systems in place to encode transcriptional specificity downstream of two different RTKs at the level of SRF cofactor recruitment.

      Experimental Procedures

      Mouse Strains

      All animal experiments were approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai. Srftm1Rmn mice (
      • Ramanan N.
      • Shen Y.
      • Sarsfield S.
      • Lemberger T.
      • Schütz G.
      • Linden D.J.
      • Ginty D.D.
      SRF mediates activity-induced gene expression and synaptic plasticity but not neuronal viability.
      ), referred to as Srffl/fl in the text, were a gift from Dr. Xin Sun, University of Wisconsin-Madison, and were backcrossed a minimum of three generations to 129S4 mice prior to all experiments included in this study. Tg(Wnt1-Cre)2Sor mice (
      • Lewis A.E.
      • Vasudevan H.N.
      • O’Neill A.K.
      • Soriano P.
      • Bush J.O.
      The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling.
      ), referred to as Wnt1-Cre2 in the text, were backcrossed to 129S4 mice for four generations prior to all experiments in this study. Pdgfratm11(EGFP)Sor (
      • Hamilton T.G.
      • Klinghoffer R.A.
      • Corrin P.D.
      • Soriano P.
      Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.
      ), referred to as PdgfraGFP/+ in the text, and Gt(ROSA)26Sortm1Sor mice (
      • Soriano P.
      Generalized lacZ expression with the ROSA26 Cre reporter strain.
      ), referred to as R26R in the text, were maintained on a C57BL/6 background. PDGFRαtm8Sor mice (
      • Tallquist M.D.
      • Soriano P.
      Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells.
      ), referred to as Pdgfrafl/fl, in the text, FGFR1tm5.1Sor mice (
      • Hoch R.V.
      • Soriano P.
      Context-specific requirements for Fgfr1 signaling through Frs2 and Frs3 during mouse development.
      ), referred to as Fgfr1fl/fl in the text, and Tg(Wnt1-Cre)11Rth mice (
      • Danielian P.S.
      • Muccino D.
      • Rowitch D.H.
      • Michael S.K.
      • McMahon A.P.
      Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.
      ), referred to as Wnt1-Cre in the text, were all maintained on a 129S4 genetic background.

      Tissue Culture and Proliferation/Scratch Assays

      Primary MEPM cells were isolated from E13.5 secondary palatal shelves (day of plug: E0.5) as previously described (
      • Fantauzzo K.A.
      • Soriano P.
      PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways.
      ). Primary mouse FPCs were generated from E11.5 mouse facial prominences (Figure S3B), but we were unable to passage these FPCs in culture. Therefore, MEPMs were used for further experiments investigating RTK-mediated control of SRF function, such as western blots and ChIP. For proliferation assays, cells were starved overnight and then incubated with 10 μm bromodeoxyuridine for 4 hr (
      • Bush J.O.
      • Soriano P.
      Ephrin-B1 forward signaling regulates craniofacial morphogenesis by controlling cell proliferation across Eph-ephrin boundaries.
      ) with either 30 ng/ml PDGFAA or 50 ng/ml FGF1 + 1 μg/ml heparin. For wound healing assays, cells were grown to confluence, starved overnight, and then scratched. Details are available in the Supplemental Experimental Procedures.

      Histology, In Situ Hybridization, and Immunofluorescence

      Embryos were dissected and embedded in either paraffin or optimal cutting temperature compound for sectioning. In situ hybridization and immunofluorescence were performed according to standard protocols. See the Supplemental Experimental Procedures for further details.

      qPCR

      For analysis of SRF induction, E13.5 MEPMs were starved overnight and then treated with PDGF or FGF for the desired time duration. For analysis of SRF target gene expression in mutant embryos, E11.5 facial prominence lysates were harvested, and RNA was extracted directly from tissue. All experiments were conducted on litters from three independent biological replicates. Statistical analysis was performed using a two-tailed, paired Student’s t test for MEPM time courses, in which cells from the same embryo were considered paired. For comparison of expression between facial prominence lysates from different genotypes, a two-tailed, unpaired Student’s t test was used. Further details are available in the Supplemental Experimental Procedures.

      Time-Lapse Imaging

      FPCs were prepared and scratch assays performed as described earlier. Live cell imaging was carried out on an Olympus IX-70 wide-field epifluorescence system with a stage-top incubation chamber to maintain cell viability. Images were taken with a 10× lens every 5 min across 250 min, for a total of 50 images per field of view. Four fields of view per condition per embryo were imaged, and two independently dissected control and mutant embryos were analyzed. Ten cells were randomly selected in each field of view for tracking and calculation of migration parameters; thus, a total of 40 cells per condition per embryo were analyzed. Image analysis was performed in ImageJ (v 1.47; NIH) using the Manual Tracking plugin. Calculation of trajectories, speed, and persistence were implemented through custom code in R (http://www.R-project.org/) (
      R Core Team
      R: A Language and Environment for Statistical Computing.
      ).

      IP and Western Blot

      E13.5 MEPMs were serum starved for 24 hr in 0.1% FBS and stimulated with PDGF or FGF for desired duration. When applicable, cells were pretreated for 1 hr with 10 μM of desired inhibitor. IPs and western blots were performed according to standard protocols using horseradish-peroxidase-conjugated secondary antibodies and quantified in ImageJ (v 1.47; NIH). A minimum of two biological replicates were performed for each set of IP experiments. Further details are provided in the Supplemental Experimental Procedures.

      ChIP

      E13.5 MEPMs were isolated and stimulated with PDGF or FGF as described earlier. ChIP was performed as previously described (
      • Fantauzzo K.A.
      • Soriano P.
      PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways.
      ) to test occupancy in input, immunoglobulin G, and antibody (anti-SRF or anti-MRTFA)-precipitated samples. qPCR was carried out as described earlier, and statistics were performed using a two-tailed, paired Student’s t test, in which cells from the same embryo were considered paired. Data presented are from three independent biological replicates. Further details are provided in the Supplemental Experimental Procedures.

      ChIP-Seq and RNA-Seq Data Integration

      C2C12 SRF ChIP-seq peak data were downloaded directly from the ENCODE Project Consortium (http://www.encodeproject.org; GEO accession number: GSM915168) in the “narrowPeaks” format, which lists significant peaks identified by the ENCODE Project Consortium. 3T3 SRF ChIP-seq data were similarly obtained (
      • Esnault C.
      • Stewart A.
      • Gualdrini F.
      • East P.
      • Horswell S.
      • Matthews N.
      • Treisman R.
      Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts.
      ). Chromosomal coordinates for each of these peaks were then compared to the TSS for each gene regulated by PDGF or FGF signaling at 1 hr, and a frequency distribution was generated by counting the number of peaks within successive 10 kb bins of the TSS. Strategy was implemented through custom code in R. Similar distributions for control gene sets (randomly selected, expression-matched genes that are not regulated by RTK activation) were generated to assess baseline ChIP-seq peak enrichment. A similar approach was implemented for the following control ChIP-seq data sets:

      Acknowledgments

      We are very grateful to Fenglei He for performing the initial RNA-seq experiment. We thank Jason Newbern at the University of North Carolina for discussion on SRF mutant phenotypes. Srffl/fl mice were a gift from Dr. Xin Sun at the University of Wisconsin-Madison. Excellent genotyping and technical support were provided by Tony Chen, Aryel Heller, and Anne Levine. Live imaging was performed in the Microscopy Shared Resource Facility at the Icahn School of Medicine at Mount Sinai, and we thank Rumana Huq and Lauren O’Rourke for assistance. RNA-seq experiments were performed at the Mount Sinai Genomics Core, and we thank Omar Jabado, Yumi Kasai, Milind Mahajan, and Avi Ma’ayan for advice and discussions. We are grateful to the Developmental Studies Hybridoma Bank for reagents. ENCODE ChIP-seq data sets were downloaded for SRF (Wold lab), Jun (Snyder lab) and Pax5 (Hardison lab). FaceBase data sets were downloaded for E13.5 palate p300 ChIP-seq and E13.5 palate RNA-seq (Visel lab). We thank Robert Krauss, Susan Parkhurst, and the members of the P.S. laboratory for helpful discussion and critical comments on this manuscript. This work was supported by National Institute of Dental and Craniofacial Research (NIDCR) grants R01DE022363 and R01DE022778 to P.S. and NIDCR Ruth L. Kirschstein NRSA Individual Predoctoral Fellowship F31DE023456 to H.N.V.

      Accession Numbers

      The GEO accession number for the MEPM RNA-seq data reported in this paper is GSE61755.

      Supplemental Information

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