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

Abstract

Vertebrate somitogenesis is regulated by a segmentation clock. Clock-linked genes exhibit cyclic expression, with a periodicity matching the rate of somite production. In mice, lunatic fringe (Lfng) expression oscillates, and LFNG protein contributes to periodic repression of Notch signaling. We hypothesized that rapid LFNG turnover could be regulated by protein processing and secretion. Here, we describe a novel Lfng allele (Lfng RLFNG ), replacing the N-terminal sequences of LFNG, which allow for protein processing and secretion, with the N-terminus of radical fringe (a Golgi-resident protein). This allele is predicted to prevent protein secretion without altering the activity of LFNG, thus increasing the intracellular half-life of the protein. This allele causes dominant skeletal and somite abnormalities that are distinct from those seen in Lfng loss-of-function embryos. Expression of clock-linked genes is perturbed and mature Hes7 transcripts are stabilized in the presomitic mesoderm of mutant mice, suggesting that both transcriptional and post-transcriptional regulation of clock components are perturbed by RLFNG expression. Contrasting phenotypes in the segmentation clock and somite patterning of mutant mice suggest that LFNG protein may have context-dependent effects on Notch activity.

Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

“…Analysing the potential oscillation of Notch target genes showed that NRARP and LFNG peaked in G1, similar to HES7 ( Fig 5L ). These genes act as a negative feedback loop to repress Notch gene expression and constitute a transcriptional circuit that plays a fundamental role in development during, for example, somitogenesis [ 46 – 49 ]. When exploring our data for receptors other than Notch, members of several classes of receptors were found to oscillate in HeLa and U2OS cells (Panel B in S4 Fig ).…”

“…Activated NOTCH2-ICD protein expression peaked in G2/M and was followed by expression of Notch target genes in G1 in HeLa cells. In somitogenesis, Notch interacts with the WNT/Frizzled and FGFR signalling pathways (reviewed in [ 46 , 63 , 64 ]) through a critical negative feedback loop mediated by HES1 and HES7 [ 49 , 65 ]. This feedback function appeared to be present in the HeLa and U2OS cell cycles as well, as HES1 and HES7 oscillated in our data and peaked in G1, where NOTCH2 expression itself was downregulated.…”

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.…”

“…Mathematical models predict that the maintenance of synchronized clock gene oscillations requires regulation at both transcriptional and post transcriptional levels (Lewis, 2003;Feng and Navaratna, 2007;Gonzalez and Kageyama, 2009). 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., Lewis, 2003;Goldbeter and Pourquie, 2008;Herrgen et al, 2010;Schroter et al, 2012;Wiedermann et al, 2015).…”

Pnrc2 regulates 3’UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation

“…More recently, studies have also investigated post-transcriptional mechanisms regulating transcript processing and clearance (Cibois et al, 2010; Fujimuro et al, 2014; Hanisch et al, 2013; Nitanda et al, 2014). Notable are studies that indicate splicing is a critical parameter (Harima et al, 2013; Takashima et al, 2011), mRNA export is a rate-limiting step (Hoyle and Ish-Horowicz, 2013), translational delays contribute to traveling waves of expression (Ay et al, 2014), oscillatory protein turnover is required for transcriptional and post-transcriptional clock function (Williams et al, 2016), cyclic transcript 3′UTRs can promote decay (Delaune et al, 2012, Fujimuro et al, 2014; Giudicelli et al, 2007), and miRNAs regulate decay of some cyclic transcripts (Bonev et al, 2012; Riley et al, 2013; Tan et al, 2012; Wong et al, 2015). Rapid clearance of cyclic transcripts likely occurs using mRNA decay machinery that promotes deadenylation, 5′ cap removal, and/or exonucleolytic cleavage of natural, non-aberrant transcripts (Garneau et al, 2007; Ghosh and Jacobson, 2010; Houseley and Tollervey, 2009; Lykke-Andersen and Jensen, 2015; Schoenberg and Maquat, 2012), though how cyclic transcripts are efficiently targeted and cleared remains largely unknown.…”