An Alternative Splicing Program for Mouse Craniofacial Development

Abstract

Alternative splicing acts as a fundamental mechanism to increase the number of functional transcripts that can be derived from the genome-and its appropriate regulation is required to direct normal development, differentiation, and physiology, in many species. Recent studies have highlighted that mutation of splicing factors, resulting in the disruption of alternative splicing, can have profound consequences for mammalian craniofacial development. However, there has been no systematic analysis of the dynamics of differential splicing during the critical period of face formation with respect to age, tissue layer, or prominence. Here we used deep RNA sequencing to profile transcripts expressed in the developing mouse face for both ectodermal and mesenchymal tissues from the three facial prominences at critical ages for facial development, embryonic days 10.5, 11.5, and 12.5. We also derived separate expression data from the nasal pit relating to the differentiation of the olfactory epithelium for a total of 60 independent datasets. Analysis of these datasets reveals the differential expression of multiple genes, but we find a similar number of genes are regulated only via differential splicing, indicating that alternative splicing is a major source of transcript diversity during facial development. Notably, splicing changes between tissue layers and over time are more prevalent than between prominences, with exon skipping the most common event. We next examined how the variation in splicing correlated with the expression of RNA binding proteins across the various datasets. Further, we assessed how binding sites for splicing regulatory molecules mapped with respect to intron exon boundaries. Overall these studies help define an alternative splicing regulatory program that has important consequences for facial development.

AP-2α and AP-2β cooperatively function in the craniofacial surface ectoderm to regulate chromatin and gene expression dynamics during facial development

“…Next, we employed the corresponding E11.5 gene expression profiles of the mouse craniofacial ectoderm and mesenchyme (Hooper et al, 2020) and correlated the relative expression between these two tissue layers with the list of E11.5 genomic elements and associated genes identified using ATACseq. Genes from the expression analysis were first binned into groups (Table S1) based upon whether they had: no associated peaks; peaks associated only with C1 (tissue generic), C2 (ectoderm favored), or C3 (ectoderm unique); or peaks in multiple categories (e.g., C1+C2).…”

“…Association of gene expression and ATAC-seq peaks: First, gene expression for the craniofacial ectoderm and mesenchyme, at E11.5, was calculated using our publicly available datasets profiling the facial ectoderm and mesenchyme from E10.5 through E12.5 (Hooper et al, 2020) (available through the Facebase Consortium website, www.facebase.org, under the accession number FB00000867).…”

Srsf3 mediates alternative RNA splicing downstream of PDGFRα signaling

“…The relatively low extent of overlap between identified transcripts is not surprising given that these RNA-seq experiments were performed in different, but related, tissues across a 48-hour timeframe. In fact, AS changes in the developing murine face from E10.5-E12.5 are more significant across age than across facial prominence location (Hooper et al, 2020).…”

Species-specific deployment of Runx2 isoforms and differential regulation of target genes during avian jaw development and evolution

“…Increased levels and complexity of alternative splicing especially in higher vertebrates can underlie phenotypic change (Kim et al, 2007;Nilsen and Graveley, 2010;Chen et al, 2012c) and drive rapid adaptive radiation such as in the jaws of cichlid fish (Singh et al, 2017). Alternative splicing is critical for normal development of the craniofacial complex and dysregulation of splicing factors can disrupt patterning (Bélanger et al, 2018;Hooper et al, 2020;Lee et al, 2020;Wood et al, 2020). Multiple craniofacial disorders have spliceosomopathies as their primary etiology (Bernier et al, 2012;Lehalle et al, 2015;Merkuri and Fish, 2019).…”