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

Abstract

Background In vertebrate embryos, a "segmentation clock" times somitogenesis. Clock-linked genes, including Lunatic fringe (Lfng), exhibit cyclic expression in the presomitic mesoderm (PSM), with a period matching the rate of somite formation. The clock period varies widely across species, but the mechanisms that underlie this variability are not clear. The half-lives of clock components are proposed to influence the rate of clock oscillations, and are tightly regulated in the PSM. Interactions between Lfng and mir-125a-5p in the embryonic chicken PSM promote Lfng transcript instability, but the conservation of this mechanism in other vertebrates has not been tested. Here, we examine whether this interaction affects clock activity in a mammalian species. Results Mutation of mir-125 binding sites in the Lfng 3'UTR leads to persistent, non-oscillatory reporter transcript expression in the caudal-most mouse PSM, though dynamic transcript expression recovers in the central PSM. Despite this, expression of endogenous mir-125a-5p is dispensable for mouse somitogenesis. Conclusions These results suggest that mir-125a sites in the Lfng 3'UTR influence transcript turnover in both mouse and chicken embryos, and support the existence of position-dependent regulatory mechanisms in the PSM. They further suggest the existence of compensatory mechanisms that can rescue the loss of mir-125a-5p in mice.

Keeping development on time: Insights into post‐transcriptional mechanisms driving oscillatory gene expression during vertebrate segmentation

“…Results from these experiments revealed that the hairy2a 3′UTR was necessary to reconstitute the endogenous expression pattern and drive the rapid decay of reporter transcripts. Similar findings have been reported in zebrafish, mouse, and chick embryos (Kawamura et al, 2016 ; Riley et al, 2013 ; Tietz et al, 2020 ; Wahi et al, 2017 ), and more recent studies have identified specific cis‐regulatory elements within segmentation clock gene mRNA 3′UTRs that influence transcript stability (Riley et al, 2013 ; Tietz et al, 2020 ; Wahi et al, 2017 ). These studies have motivated future experiments aimed at assessing the role of specific mRNA regulatory factors on segmentation clock mRNA decay and translation, and thus, the tempo of genetic oscillations.…”

“…Reporter‐based studies of segmentation clock transcript 3′UTRs have been conducted to analyze the expression dynamics of segmentation clock mRNAs in several vertebrate genetic model systems (Davis et al, 2001 ; Gallagher et al, 2017 ; Hilgers et al, 2005 ; Nitanda et al, 2013 ; Riley et al, 2013 ; Tietz et al, 2020 ; Wahi et al, 2017 ). This was first demonstrated in Xenopus laevis embryos for the segmentation clock gene hairy2a , whereby the expression patterns produced from reporter constructs containing different regions of the hairy2a gene were analyzed to identify the minimal regions required to recapitulate the endogenous striped hairy2 expression pattern (Davis et al, 2001 ).…”

“…miRNA‐mediated regulation has been implicated in a broad range of developmental processes, including the maternal to zygotic transition in zebrafish and Xenopus embryos (Giraldez et al, 2006 ; Lund et al, 2009 ), muscle differentiation (Goljanek‐Whysall et al, 2011 ), and development of multiple organ systems (Ason et al, 2006 ; Bhaskaran et al, 2009 ; Zhao et al, 2007 ). Previous studies have investigated the role of miRNA‐mediated segmentation clock regulation by identifying miRNAs expressed in the PSM of developing embryos and analyzing expression of reporter genes containing segmentation clock gene 3′UTRs in the presence and absence of specific miRNAs (Riley et al, 2013 ; Wahi et al, 2017 ). miR‐125a‐5p is expressed in the PSM of chick and mouse embryos, and binding sites for the miR‐125a‐5p seed sequence are present in the 3′UTR of the oscillating gene Lunatic fringe ( Lfng ; Riley et al, 2013 ; Wahi et al, 2017 ).…”

“…Previous studies have investigated the role of miRNA‐mediated segmentation clock regulation by identifying miRNAs expressed in the PSM of developing embryos and analyzing expression of reporter genes containing segmentation clock gene 3′UTRs in the presence and absence of specific miRNAs (Riley et al, 2013 ; Wahi et al, 2017 ). miR‐125a‐5p is expressed in the PSM of chick and mouse embryos, and binding sites for the miR‐125a‐5p seed sequence are present in the 3′UTR of the oscillating gene Lunatic fringe ( Lfng ; Riley et al, 2013 ; Wahi et al, 2017 ). Expression of a reporter gene containing either the chick or mouse Lfng 3′UTR was strongly downregulated following exogenous overexpression of miR‐125a‐5p , and this effect was abrogated upon mutation of the putative miR‐125 binding sites in both 3′UTRs (Riley et al, 2013 ).…”

The vertebrate Embryo Clock: Common players dancing to a different beat

“…Moreover, Lfng and hairy1 lost their oscillatory expression pattern, further evidencing that miRNA-mediated regulation is necessary for EC gene expression oscillations (Riley et al, 2013). A regulatory action of miR-125a-5p on Lfng mRNA degradation and expression dynamics was also documented in the mouse embryo (Wahi et al, 2017). Mathematical modelling performed by Jing et al (2015) provided important insights regarding miRNA role in the segmentation clock.…”