Modifying transcript lengths of cycling mouse segmentation genes

Abstract

Regular production of somites, precursors of the axial skeleton and attached muscles is controlled by a molecular oscillator, the segmentation clock, which drives cyclic transcription of target genes in the unsegmented presomitic mesoderm (PSM). The clock is based on a negative feedback loop which generates pulses of transcription that oscillate with the same periodicity as somite formation. Mutants in several oscillating genes including the Notch pathway gene Lunatic fringe (Lfng) and the Notch target Hes7, result in defective somitogenesis and disorganised axial skeletons. Both genes encode negative regulators of Notch signalling output, but it is not yet clear if they are just secondary clock targets or if they encode components of a primary, pacemaker oscillator. In this paper, we try to identify components in the primary oscillator by manipulating delays in the feedback circuitry. We characterise recombinant mice in which Lfng and Hes7 introns are lengthened in order to delay mRNA production. Lengthening the third Hes7 intron by 10 or 20 kb disrupts accurate RNA splicing and inactivates the gene. Lfng expression and activity is normal in mice whose Lfng is lengthened by 10 kb, but no effects on segmentation are evident. We discuss these results in terms of the relative contributions of transcriptional and post-transcriptional delays towards defining the pace of segmentation, and of alternative strategies for manipulating the period of the clock.