mir-125a-5p-Mediated Regulation of Lfng Is Essential for the Avian Segmentation Clock

Abstract

Summary Somites are embryonic precursors of the axial skeleton and skeletal muscles, and establish the segmental vertebrate body plan. Somitogenesis is controlled in part by a segmentation clock that requires oscillatory expression of genes including Lunatic fringe (Lfng). Oscillatory genes must be tightly regulated both at the transcriptional and post-transcriptional levels for proper clock function. Here we demonstrate that microRNA-mediated regulation of Lfng is essential for proper segmentation during chick somitogenesis. We find that mir-125a-5p targets evolutionarily conserved sequences in the Lfng 3′UTR, and that preventing interactions between mir-125a-5p and Lfng transcripts in vivo causes abnormal segmentation and perturbs clock activity. This provides strong evidence that miRNAs function in the post-transcriptional regulation of oscillatory genes in the segmentation clock. Further, this demonstrates that the relatively subtle effects of miRNAs on target genes can have broad effects in developmental situations that have critical requirements for tight post-transcriptional regulation.

Putative binding sites for mir‐125 family miRNAs in the mouse Lfng 3′UTR affect transcript expression in the segmentation clock, but mir‐125a‐5p is dispensable for normal somitogenesis

“…Large changes in component half-lives generally disrupt segmentation clock function (Hirata et al, 2004, Riley et al, 2013, Williams et al, 2016). However, mathematical models and biological data suggest that, within a narrow window, changes in half-lives of clock components may influence the period of oscillations, which could contribute to species-specific clock periods (e.g.…”

A novel targeted Lunatic fringe allele predicted to reduce protein secretion is dominant and disrupts somitogenesis

“…We cannot formally exclude the possibility that the Lfng RLFNG allele might have some effect on transcript stability, which could exacerbate the reduced RNA expression we observe. There is no evidence that the transcript regions altered in our mutant mice harbor any RNA stability signals, and the Lfng 3′UTR, which we and others have suggested influences transcript stability (Chen et al, 2005;Nitanda et al, 2014;Riley et al, 2013), is intact in the Lfng RLFNG allele. Further, our qRT-PCR analyses indicate that both alleles are expressed to similar levels in heterozygous embryos, and reductions in transcript stability could not explain the dominant nature of the phenotypes.…”

The many roles of Notch signaling during vertebrate somitogenesis

“…When interactions between the Lfng 3 UTR and miR-125a were disrupted in chicken embryos, Lfng transcripts were initially stabilized in the PSM, resulting in the formation of misshapen and poorly patterned somites. These results suggest that rapid turnover of Lfng transcripts is essential for somitogenesis and is regulated by mir-125a during chick somitogenesis [81].…”

“…Consistent with these findings, mathematical models predict that mRNA and protein half lives need to be quite short to allow rapid oscillations during somitogenesis [20,79]. The 3 untranslated regions (3 UTR) of cyclic genes such as Hes7 and Lfng are essential for rapid RNA turnover in the clock [51,80,81]. The 3 UTR of Lfng has evolutionarily conserved regions that destabilize the mRNA [51], and contains conserved binding sites for miR-125a-5p [81].…”

“…The 3 untranslated regions (3 UTR) of cyclic genes such as Hes7 and Lfng are essential for rapid RNA turnover in the clock [51,80,81]. The 3 UTR of Lfng has evolutionarily conserved regions that destabilize the mRNA [51], and contains conserved binding sites for miR-125a-5p [81]. When interactions between the Lfng 3 UTR and miR-125a were disrupted in chicken embryos, Lfng transcripts were initially stabilized in the PSM, resulting in the formation of misshapen and poorly patterned somites.…”