Minor class splicing shapes the zebrafish transcriptome during development.

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

Complex architecture and regulated expression of the Sox2ot locus during vertebrate development.

The Sox2 gene is a key regulator of pluripotency embedded within an intron of a long noncoding RNA (ncRNA), termed Sox2 overlapping transcript (Sox2ot), which is transcribed in the same orientation. However, this ncRNA remains uncharacterized. Here we show that Sox2ot has multiple transcription start sites associated with genomic features that indicate regulated expression, including highly conserved elements (HCEs) and chromatin marks characteristic of gene promoters. To identify biological processes in which Sox2ot may be involved, we analyzed its expression in several developmental systems, compared to expression of Sox2. We show that Sox2ot is a stable transcript expressed in mouse embryonic stem cells, which, like Sox2, is down-regulated upon induction of embryoid body (EB) differentiation. However, in contrast to Sox2, Sox2ot is up-regulated during EB mesoderm-lineage differentiation. In adult mouse, Sox2ot isoforms were detected in tissues where Sox2 is expressed, as well as in different tissues, supporting independent regulation of expression of the ncRNA. Sox2dot, an isoform of Sox2ot transcribed from a distal HCE located >500 kb upstream of Sox2, was detected exclusively in the mouse brain, with enrichment in regions of adult neurogenesis. In addition, Sox2ot isoforms are transcribed from HCEs upstream of Sox2 in other vertebrates, including in several regions of the human brain. We also show that Sox2ot is dynamically regulated during chicken and zebrafish embryogenesis, consistently associated with central nervous system structures. These observations provide insight into the structure and regulation of the Sox2ot gene, and suggest conserved roles for Sox2ot orthologs during vertebrate development.

Mtx2 directs zebrafish morphogenetic movements during epiboly by regulating microfilament formation.

The homeobox transcription factor Mtx2 is essential for epiboly, the first morphogenetic movement of gastrulation in zebrafish. Morpholino knockdown of Mtx2 results in stalling of epiboly and lysis due to yolk rupture. However, the mechanism of Mtx2 action is unknown. The role of mtx2 is surprising as most mix/bix family genes are thought to have roles in mesendoderm specification. Using a transgenic sox17-promoter driven EGFP line, we show that Mtx2 is not required for endoderm specification but is required for correct morphogenetic movements of endoderm and axial mesoderm. During normal zebrafish development, mtx2 is expressed at both the blastoderm margin and in the zebrafish equivalent of visceral endoderm, the extra-embryonic yolk syncytial layer (YSL). We show that formation of the YSL is not Mtx2 dependent, but that Mtx2 directs spatial arrangement of YSL nuclei. Furthermore, we demonstrate that Mtx2 knockdown results in loss of the YSL F-actin ring, a microfilament structure previously shown to be necessary for epiboly progression. In summary, we propose that Mtx2 acts within the YSL to regulate morphogenetic movements of both embryonic and extra-embryonic tissues, independently of cell fate specification.

Prescribing patterns of oral antiplatelets in Wales: evolving trends from 2005 to 2016.

AIM: Antiplatelets have been used for decades to prevent atherothrombotic disease, but there is limited 'real-life' prescribing data. We hereby report the prescribing patterns for oral antiplatelets in Wales, UK. METHODS/RESULTS: Retrospective analysis of anonymized data in Wales from 2005 to 2016 revealed differences in prescribing patterns of oral antiplatelets. Aspirin and dipyridamole use declined with a corresponding increase in clopidogrel prescription. Costs declined with a sharp decrease coinciding with clopidogrel coming off patent. Prasugrel and ticagrelor have shown significant cost contribution (29% of total) despite only forming 1% of total items prescribed in 2016. CONCLUSION: This first-look analysis of real-life antiplatelet data demonstrates a decrease in the overall prescribing costs with varying patterns. This may aid policy-makers in reviewing funding strategies.

Evolution of gene function and regulatory control after whole-genome duplication: comparative analyses in vertebrates.

The significance of whole-genome duplications (WGD) for vertebrate evolution remains controversial, in part because the mechanisms by which WGD contributed to functional evolution or speciation are still incompletely characterized. Fish genomes provide an ideal context in which to examine the consequences of WGD, because the teleost lineage experienced an additional WGD soon after divergence from tetrapods and because five teleost genomes are available for comparative analysis. Here we present an integrated approach to characterize these post-duplication genomes based on genome-scale synteny, phylogenetic, temporal, and spatial gene expression and on protein sequence data. A minimum of 3%-4% of protein-coding loci have been retained in two copies in each of the five fish genomes, and many of these duplicates are key developmental genes that function as transcription factors or signaling molecules. Almost all duplicate gene pairs we examined have diverged in spatial and/or temporal expression during embryogenesis. A quarter of duplicate pairs have diverged in function via the acquisition of novel protein domains or via changes in the subcellular localization of their encoded proteins. We compared the spatial expression and protein domain architecture of zebrafish WGD-duplicates to those of their single mouse ortholog and found many examples supporting a model of neofunctionalization. WGD-duplicates have acquired novel protein domains more often than have single-copy genes. Post-WGD changes at the gene regulatory level were more common than changes at the protein level. We conclude that the most significant consequence of WGD for vertebrate evolution has been to enable more-specialized regulatory control of development via the acquisition of novel spatiotemporal expression domains. We find limited evidence that reciprocal gene loss led to reproductive isolation and speciation in this lineage.