Number of vertebrae is fine-tuned by Notch signaling via control of period of the somite segmentation clock

The number of vertebrae is strictly defined for any given species1, and depends on the number of somites, which are periodically formed as perfectly matched cell masses during mid-embryogenesis, where the somite segmentation clock prescribes the timing. The tempo of the clock is affected by surrounding condition, despite which the same number of somites is formed, suggesting that the clock may be tunable to adapt to the environmental condition. Here, we demonstrate a tunability of the segmentation clock in the period depending on the level of Notch signaling that senses the surrounding information to adjust the number of somites and vertebrae precisely, in which we propose a mechanism of the clock with a feedback loop of Notch signaling by Notch-regulated ankyrin repeat protein (Nrarp). Disruption of Nrarp in mouse resulted in the loss of two vertebrae, due to 4-min extension of the period of the clock elicited by the up-regulation of Notch activity, whereas pharmacological diminishment of Notch activity shortens it by a few min. The Notch inhibitor rescues the phenotype of Nrarp knockout mice in the period. These results are comprehended by mathematical analyses, in which the period of the clock is fine-tuned by Notch activity that Nrarp adjusts. Overall, our results are the first to provide molecular evidence of fine-tuning of the segmentation clock that preserves the number of somites and vertebrae. Nrarp mutant mouse which have LacZ gene instead of Nrarp coding region was generated by homologous recombinant. Pregnant female heterozygous mutant mice, mated with heterozygous mutant male, were dissected at embryonic day 10.5 (E10.5). 12 PSMs were collected in each genotype to extract total RNA.对于任一特定物种而言，椎骨数量有着严格的固定定义[1]，其取决于体节（somites）的数量。体节是胚胎发育中期周期性形成的高度规整的细胞团块，体节分节钟（somite segmentation clock）调控着体节分节的时序。尽管该分节钟的节律会受周围环境条件的影响，但最终形成的体节总数始终保持一致，这提示分节钟可通过调整自身节律以适应环境变化。本研究证实了分节钟的周期可根据感知周围环境信息的Notch信号通路水平进行精准调控，从而微调体节与椎骨的数量；我们还提出了一种由Notch调控的锚蛋白重复蛋白（Notch-regulated ankyrin repeat protein，Nrarp）介导的Notch信号反馈环路的分节钟调控机制。小鼠体内Nrarp的敲除会导致椎骨缺失两块，这是由于Notch活性上调使得分节钟周期延长了4分钟；而通过药理学手段降低Notch活性，则可使周期缩短数分钟。Notch抑制剂能够挽救Nrarp敲除小鼠的分节钟周期异常表型。上述研究结果可通过数学分析得到合理解释：Nrarp通过调控Notch活性，对分节钟的周期进行精细微调。总体而言，本研究首次为分节钟的精细调控提供了分子生物学证据，该调控可维持体节与椎骨的数量恒定。本研究通过同源重组技术构建了编码区被LacZ基因替换的Nrarp突变小鼠。将杂合突变型雄性小鼠与杂合突变型雌性小鼠交配后，于胚胎发育第10.5天（E10.5）对孕鼠实施解剖，收集各基因型小鼠的12个体节前中胚层（presomitic mesoderm，PSM）组织以提取总RNA。