Trps1 and Its Target Gene Sox9 Regulate Epithelial Proliferation in the Developing Hair Follicle and Are Associated with Hypertrichosis

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 Trps1Δgt 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

Mutations in the Cholesterol Transporter Gene ABCA5 Are Associated with Excessive Hair Overgrowth

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

Generation of an immortalized mouse embryonic palatal mesenchyme cell line.

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

There and Back Again: Hair Follicle Stem Cell Dynamics

Recently in Cell, Hsu et al. (2011) defined the relationship between stem cells and differentiated progeny within a hair follicle lineage. Their work reveals that stem cell descendants that have migrated out of the bulge can return to this niche and actively contribute to its function

Immortalized MEPM cells maintain responsiveness to growth factor stimulation.

<p>(A) Biochemical analysis of PDGFR activation and phosphorylation of downstream signaling molecules in passage 3 primary (left) and passage 12 immortalized (right) MEPM cells following treatment with PDGF-AA, PDGF-BB or PDGF-DD ligand. In both cell types, PDGF-AA treatment led to autophosphorylation of PDGFRα, while stimulation with PDGF-BB and PDGF-DD led to autophosphorylation of PDGFRα and PDGFRβ. PDGF-AA treatment led to modest increases in phospho-Akt and phospho-Erk1/2 levels whereas stimulation with PDGF-BB and PDGF-DD generated robust increases in the levels of each phosphoprotein in both primary and immortalized MEPM cells. (B) Biochemical analysis of PDGF-AA-dependent Akt target phosphorylation in passage 9 immortalized MEPM cells. IP, immunoprecipitation; WCL, whole cell lysate; WB, Western blot.</p

Expression of palatal shelf transcripts is similar between primary and immortalized MEPM cells.

<p>Bar graph depicting qRT-PCR values revealing reduced expression of <i>Msx1</i>, <i>Meox2</i> and <i>Alx3</i> in passage 13 immortalized MEPM cells as compared to their passage 1 primary counterparts and similar expression of <i>Osr2</i>, <i>Pdgfra</i>, <i>Shox2</i>, <i>Efnb1</i>, <i>Mn1</i>, <i>Tbx22</i> and <i>Shh</i> across the two cell types. qRT-PCR values are normalized to <i>B2m</i>. qRT-PCR data are presented as mean ± SEM. n.s., not significant. *, p<0.05. E13.5 primary MEPM RNA-sequencing FPKM (fragments per kilobase of transcript per million mapped reads) values [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179078#pone.0179078.ref012" target="_blank">12</a>] are normalized to <i>B2m</i> and E13.5 palatal shelf RNA-sequencing RPKM (reads per kilobase of transcript per million mapped reads) values (<a href="http://www.facebase.org/" target="_blank">www.facebase.org</a>, accession FB00000278) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179078#pone.0179078.ref033" target="_blank">33</a>] are represented as an average of normalization to <i>Actb</i>, <i>Hsp90ab1</i> and <i>Ppia</i>.</p

Primers used in qRT-PCR analysis.

<p>Primers used in qRT-PCR analysis.</p

Immortalized MEPM cells maintain migration ability.

<p>(A-B’) Primary (A,A’) and immortalized (B,B’) MEPM cells migrate comparable distances following scratching on a fibronectin substrate. Dashed lines indicate the scratch boundary at 0 hours. Black arrowheads denote farthest migration on either side of the scratch. Solid rectangles indicate the frame of the following panel. (C-D”) Expression of phalloidin (red; C,D) and paxillin (green; C’,D’) as assessed by immunofluorescence analyses in passage 1 primary (C-C”) and passage 14 immortalized (D-D”) MEPM cells. Both primary and immortalized MEPM cells at the leading edge display filamentous actin arcs and focal adhesion formation. Nuclei were stained with DAPI (blue; C”,D”). (E-F) Crystal violet staining of primary (E) and immortalized (F) MEPM cells following migration through a porous membrane. Scale bars, 100 μm. (G) Bar graph depicting integrated densities revealing comparable migration of passage 2 primary and passage 23 immortalized MEPM cells in Transwell assays. Data are presented as mean ± SEM. n.s., not significant.</p

The Role of RNA-Binding Proteins in Vertebrate Neural Crest and Craniofacial Development

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