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The platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases consists of two receptors, PDGFRα and PDGFRβ, that homodimerize and heterodimerize upon ligand binding. Here, we tested the hypothesis that differential internalization and trafficking dynamics of the various PDGFR dimers underlie differences in downstream intracellular signaling and cellular behavior. Using a bimolecular fluorescence complementation approach, we demonstrated that PDGFRα/β heterodimers are rapidly internalized into early endosomes. We showed that PDGFRα/β heterodimer activation does not induce downstream phosphorylation of ERK1/2 and significantly inhibits cell proliferation. Further, we identified MYO1D as a protein that preferentially binds PDGFRα/β heterodimers and demonstrated that knockdown of MYO1D leads to retention of PDGFRα/β heterodimers at the plasma membrane, increased phosphorylation of ERK1/2 and increased cell proliferation. Collectively, our findings impart valuable insight into the molecular mechanisms by which specificity is introduced downstream of PDGFR activation to differentially propagate signaling and generate distinct cellular responses. Here they show that heterodimerization of the receptor tyrosine kinases PDGFRa and PDGFRb negatively affects downstream ERK1/2 signaling and cellular proliferation, due in part to rapid internalization mediated by binding to the unconventional myosin protein MYO1D.

Palatogenesis is a complex morphogenetic process, disruptions in which result in highly prevalent birth defects in humans. In recent decades, the use of model systems such as genetically-modified mice, mouse palatal organ cultures and primary mouse embryonic palatal mesenchyme (MEPM) cultures has provided significant insight into the molecular and cellular defects underlying cleft palate. However, drawbacks in each of these systems have prevented high-throughput, large-scale studies of palatogenesis in vitro. Here, we report the generation of an immortalized MEPM cell line that maintains the morphology, migration ability, transcript expression and responsiveness to exogenous growth factors of primary MEPM cells, with increased proliferative potential over primary cultures. The immortalization method described in this study will facilitate the generation of palatal mesenchyme cells with an unlimited capacity for expansion from a single genetically-modified mouse embryo and enable mechanistic studies of palatogenesis that have not been possible using primary culture.

Hereditary hypertrichoses are a group of hair overgrowth syndromes that are extremely rare in humans. We have previously demonstrated that a position effect on TRPS1 is associated with hypertrichosis in humans and mice. To gain insight into the functional role of Trps1, we analyzed the late morphogenesis vibrissae phenotype of Trps1(Δgt) mutant mice, which is characterized by follicle degeneration after peg downgrowth has been initiated. We found that Trps1 directly represses expression of the hair follicle stem cell regulator Sox9 to control proliferation of the follicle epithelium. Furthermore, we identified a copy number variation upstream of SOX9 in a family with hypertrichosis that significantly decreases expression of the gene in the hair follicle, providing new insights into the long-range regulation of SOX9. Our findings uncover a novel transcriptional hierarchy that regulates epithelial proliferation in the developing hair follicle and contributes to the pathology of hypertrichosis.

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during mouse skeletal development. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting and widespread alternative RNA splicing (AS) changes. Here, we demonstrated via enhanced UV-crosslinking and immunoprecipitation of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. We found that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole-exome sequencing. We identified a single base pair substitution in the 5' donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles. The homozygous recessive disruption of ABCA5 leads to reduced lysosome function, which results in an accumulation of autophagosomes, autophagosomal cargos as well as increased endolysosomal cholesterol in CGHT keratinocytes. In an unrelated sporadic case of CGHT, we identified a 1.3 Mb cryptic deletion of chr17q24.2-q24.3 encompassing ABCA5 and found that ABCA5 levels are dramatically reduced throughout patient hair follicles. Collectively, our findings support ABCA5 as a gene underlying the CGHT phenotype and suggest a novel, previously unrecognized role for this gene in regulating hair growth.

Cranial neural crest (NC) cells delaminate from the neural folds in the forebrain to the hindbrain during mammalian embryogenesis and migrate into the frontonasal prominence and pharyngeal arches. These cells generate the bone and cartilage of the frontonasal skeleton, among other diverse derivatives. RNA-binding proteins (RBPs) have emerged as critical regulators of NC and craniofacial development in mammals. Conventional RBPs bind to specific sequence and/or structural motifs in a target RNA via one or more RNA-binding domains to regulate multiple aspects of RNA metabolism and ultimately affect gene expression. In this review, we discuss the roles of RBPs other than core spliceosome components during human and mouse NC and craniofacial development. Where applicable, we review data on these same RBPs from additional vertebrate species, including chicken, Xenopus and zebrafish models. Knockdown or ablation of several RBPs discussed here results in altered expression of transcripts encoding components of developmental signaling pathways, as well as reduced cell proliferation and/or increased cell death, indicating that these are common mechanisms contributing to the observed phenotypes. The study of these proteins offers a relatively untapped opportunity to provide significant insight into the mechanisms underlying gene expression regulation during craniofacial morphogenesis.

Down syndrome (DS), the genetic condition caused by trisomy 21, is characterized by variable cognitive impairment, immune dysregulation, dysmorphogenesis and increased prevalence of diverse co-occurring conditions. The mechanisms by which trisomy 21 causes these effects remain largely unknown. We demonstrate that triplication of the interferon receptor ( IFNR ) gene cluster on chromosome 21 is necessary for multiple phenotypes in a mouse model of DS. Whole-blood transcriptome analysis demonstrated that IFNR overexpression associates with chronic interferon hyperactivity and inflammation in people with DS. To define the contribution of this locus to DS phenotypes, we used genome editing to correct its copy number in a mouse model of DS, which normalized antiviral responses, prevented heart malformations, ameliorated developmental delays, improved cognition and attenuated craniofacial anomalies. Triplication of the Ifnr locus modulates hallmarks of DS in mice, suggesting that trisomy 21 elicits an interferonopathy potentially amenable to therapeutic intervention. A mouse model of Down syndrome (DS) highlights the importance of triplication of the IFNR gene cluster for a variety of DS-associated traits. Copy number correction resulted in amelioration of multiple phenotypes associated with the condition.

Recently, we reported that mutations in the R-spondin 4 (RSPO4) gene underlie inherited anonychia/hyponychia. Here, we studied five consanguineous Pakistani families with recessive inheritance of a combination of anonychia and hyponychia. Homozygous mutations were identified in the RSPO4 gene in all five families. Three families had a splice site mutation at the exon 2–intron 2 boundary. One family had a 26bp deletion encompassing the start codon, and the final family had a missense mutation changing the initiating methionine to isoleucine. We demonstrated by in situ hybridization that Rspo4 is exclusively expressed in the mesenchyme underlying the digit tip epithelium in the mouse at embryonic day 14.5 (e14.5). These findings expand our understanding of the role of RSPO4 in nail development and disease.

X-linked congenital generalized hypertrichosis (Online Mendelian Inheritance in Man 307150) is an extremely rare condition of hair overgrowth on different body sites. We previously reported linkage in a large Mexican family with X-linked congenital generalized hypertrichosis cosegregating with deafness and with dental and palate anomalies to Xq24-27. Using SNP oligonucleotide microarray analysis and whole-genome sequencing, we identified a 389-kb interchromosomal insertion at an extragenic palindrome site at Xq27.1 that completely cosegregates with the disease. Among the genes surrounding the insertion, we found that Fibroblast Growth Factor 13 (FGF13) mRNA levels were significantly reduced in affected individuals, and immunofluorescence staining revealed a striking decrease in FGF13 localization throughout the outer root sheath of affected hair follicles. Taken together, our findings suggest a role for FGF13 in hair follicle growth and in the hair cycle.

Activity of the receptor tyrosine kinase PDGFRα and the tyrosine phosphatase SHP2 are critical for vertebrate craniofacial development. We sought to determine the effect of SHP2 binding to PDGFRα via phenotypic and biochemical analyses of an allelic series of mouse embryos with combined loss of both proteins in the neural crest lineage. We demonstrated that SHP2 preferentially binds PDGFRα/α homodimers among the three PDGFR dimers. Analysis of allelic series mutant embryos revealed increased cell death in the lateral nasal and maxillary processes at E10.5, variably penetrant facial blebbing, facial hemorrhaging, midline clefting and loss of the mandibular region at E13.5, and widespread craniofacial bone and cartilage defects at birth. Further, we showed that loss of SHP2 leads to increased phosphorylation of PDGFRα and the downstream effector Erk1/2 in E10.5 allelic series mutant embryo lysates. Together, our findings demonstrate additive effects on craniofacial development upon conditional ablation of PDGFRα and SHP2 in the mouse neural crest lineage and indicate that SHP2 may negatively and positively regulate PDGFRα signaling through distinct mechanisms.