ABSTRACT
A set of genes in the posterior end of developing mouse embryos shows oscillatory expression, thereby regulating periodic somite segmentation. Although the mechanism for generating oscillation has extensively been clarified, what regulates the oscillation period is still unclear. We attempted to elongate the oscillation period by increasing the time to transcribe Hes7 in this research. We generated knock-in mice, in which a large intron was inserted into Hes7 3'UTR. The exogenous intron was unexpectedly not properly spliced out and the transcripts were prematurely terminated. Consequently, Hes7 mRNA lost its 3'UTR, thereby reducing the amount of Hes7 protein. Oscillation was damped in the knock-in embryos and periodic somite segmentation does not occur properly. Thus, we demonstrated that Hes7 3'UTR is essential to accumulate adequate amounts of Hes7 protein for the somite segmentation clock that orchestrates periodic somite formation.
Subject(s)
3' Untranslated Regions/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Body Patterning/genetics , Embryo, Mammalian/cytology , Somites/embryology , Somites/metabolism , Animals , Base Sequence , Cells, Cultured , Embryo, Mammalian/metabolism , Embryonic Development , In Situ Hybridization , Mice , Molecular Sequence Data , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Somites/cytologyABSTRACT
Somite segmentation, a prominent periodic event in the development of vertebrates, is instructed by cyclic expression of several genes, including Hes7 and Lunatic fringe (Lfng). Transcriptional regulation accounts for the cyclic expression. In addition, because the expression patterns vary in a cycle, rapid turnover of mRNAs should be involved in the cyclic expression, although its contribution remains unclear. Here, we demonstrate that 3'-UTR-dependent rapid turnover of Lfng and Hes7 plays a critical role in their dynamic expression patterns. The regions active in the transcription of Lfng and Hes7 are wholly overlapped in the posterior presomitic mesoderm (PSM) of the mouse embryo. However, their distribution patterns are slightly different; Hes7 mRNA shows a broader distribution pattern than Lfng mRNA in the posterior PSM. Lfng mRNA is less stable than Hes7 mRNA, where their 3'-UTRs are responsible for the different stability. Using transgenic mice expressing Venus under the control of the Hes7 promoter, which leads to cyclic transcription in the PSM, we reveal that the Lfng 3'-UTR provides the narrow distribution pattern of Lfng mRNA, whereas the Hes7 3'-UTR contributes the relatively broad distribution pattern of Hes7 mRNA. Thus, we conclude that 3'-UTR-dependent mRNA stability accounts for the differential distribution patterns of Lfng and Hes7 mRNA. Our findings suggest that 3'-UTR-dependent regulation of mRNA turnover plays a crucial role in the diverse patterns of mRNA distribution during development.
Subject(s)
3' Untranslated Regions/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , Gene Expression Regulation, Developmental , Glycosyltransferases/physiology , RNA, Messenger/metabolism , Animals , Blotting, Western , Body Patterning , Female , HEK293 Cells , Humans , In Situ Hybridization , Male , Mesoderm/cytology , Mesoderm/metabolism , Mice , Mice, Transgenic , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Somites/cytology , Somites/metabolismABSTRACT
During vertebrate development, the primary body axis elongates towards the posterior and is periodically divided into somites, which give rise to the vertebrae, skeletal muscles and dermis. Somites form periodically from anterior to posterior, and the anterior somites form in a more rapid cycle than the posterior somites. However, how this anteroposterior (AP) difference in somitogenesis is generated and how it contributes to the vertebrate body plan remain unclear. Here, we show that the AP difference in zebrafish somitogenesis originates from a variable overlapping segmentation period between one somite and the next. The AP difference is attributable to spatiotemporal inhibition of the clock gene her1 via retinoic acid (RA) regulation of the transcriptional repressor ripply1. RA depletion thus disrupts timely somite formation at the transition, eventually leading to the loss of one somite and the resultant cervical vertebra. Overall, our results indicate that RA regulation of the AP difference is crucial for proper linkage between the head and trunk in the vertebrate body plan.
