Hair follicle morphogenesis depends on Wnt, Shh, Notch, BMP and other signaling pathways interplay between epithelial and mesenchymal cells. The Wnt pathway plays an essential role during hair follicle induction, Shh is involved in morphogenesis and late stage differentiation, Notch signaling determines stem cell fate while BMP is involved in cellular differentiation. The Wnt pathway is considered to be the master regulator during hair follicle morphogenesis. Wnt signaling proceeds through EDA/EDAR/NF-κB signaling. NF-κB regulates the Wnt pathway and acts as a signal mediator by upregulating the expression of Shh ligand. Signal crosstalk between epithelial and mesenchymal cells takes place mainly through primary cilia. Primary cilia formation is initiated with epithelial laminin-511 interaction with dermal β-1 integrin, which also upregulates expression of downstream effectors of Shh pathway in dermal lineage. PDGF signal transduction essential for crosstalk is mediated through epithelial PDGF-A and PDGFRα expressed on the primary cilia. Dermal Shh and PDGF signaling up-regulates dermal noggin expression; noggin is a potent inhibitor of BMP signaling which helps in counteracting BMP mediated β-catenin inhibition. This interplay of signaling between the epithelial and dermal lineage helps in epithelial Shh signal amplification. The dermal Wnt pathway helps in upregulation of epithelial Notch expression. Dysregulation of these pathways leads to certain abnormalities and in some cases even tumor outgrowth.

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.

The craniofacial disorder mandibulofacial dysostosis Guion-Almeida type is caused by haploinsufficiency of the U5 snRNP gene EFTUD2/SNU114. However, it is unclear how reduced expression of this core pre-mRNA splicing factor leads to craniofacial defects. Here we use a CRISPR-Cas9 nickase strategy to generate a human EFTUD2-knockdown cell line and show that reduced expression of EFTUD2 leads to diminished proliferative ability of these cells, increased sensitivity to endoplasmic reticulum (ER) stress and the mis-expression of several genes involved in the ER stress response. RNA-Seq analysis of the EFTUD2-knockdown cell line revealed transcriptome-wide changes in gene expression, with an enrichment for genes associated with processes involved in craniofacial development. Additionally, our RNA-Seq data identified widespread mis-splicing in EFTUD2-knockdown cells. Analysis of the functional and physical characteristics of mis-spliced pre-mRNAs highlighted conserved properties, including length and splice site strengths, of retained introns and skipped exons in our disease model. We also identified enriched processes associated with the affected genes, including cell death, cell and organ morphology and embryonic development. Together, these data support a model in which EFTUD2 haploinsufficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on gene expression, including altering the expression of ER stress response genes and genes involved in the development of the craniofacial region. The increased burden of unfolded proteins in the ER resulting from mis-splicing would exceed the capacity of the defective ER stress response, inducing apoptosis in cranial neural crest cells that would result in craniofacial abnormalities during development.

The coronary vasculature is an essential vessel network providing the blood supply to the heart. Disruptions in coronary blood flow contribute to cardiac disease, a major cause of premature death worldwide. The generation of treatments for cardiovascular disease will be aided by a deeper understanding of the developmental processes that underpin coronary vessel formation. From an ENU mutagenesis screen, we have isolated a mouse mutant displaying embryonic hydrocephalus and cardiac defects (EHC). Positional cloning and candidate gene analysis revealed that the EHC phenotype results from a point mutation in a splice donor site of the Myh10 gene, which encodes NMHC IIB. Complementation testing confirmed that the Myh10 mutation causes the EHC phenotype. Characterisation of the EHC cardiac defects revealed abnormalities in myocardial development, consistent with observations from previously generated NMHC IIB null mouse lines. Analysis of the EHC mutant hearts also identified defects in the formation of the coronary vasculature. We attribute the coronary vessel abnormalities to defective epicardial cell function, as the EHC epicardium displays an abnormal cell morphology, reduced capacity to undergo epithelial-mesenchymal transition (EMT), and impaired migration of epicardial-derived cells (EPDCs) into the myocardium. Our studies on the EHC mutant demonstrate a requirement for NMHC IIB in epicardial function and coronary vessel formation, highlighting the importance of this protein in cardiac development and ultimately, embryonic survival.