Progressive Tooth Pattern Changes in Cilk1-Deficient mice Depending on Hedgehog Signaling

Primary cilia function as critical sensory organelles that mediate multiple signaling pathways, including the Hedgehog (Hh) pathway, which is essential for organ patterning and morphogenesis. Disruptions in Hh signaling have been implicated in supernumerary tooth formation and molar fusion in mutant mice. Cilk1, a highly conserved serine/threonine-protein kinase localized within primary cilia, plays a critical role in ciliary transport. Loss of Cilk1 results in severe ciliopathy phenotypes, including polydactyly, edema, and cleft palate. However, the role of Cilk1 in tooth development remains unexplored. In this study, we investigated the role of Cilk1 in tooth development. Cilk1 deficiency resulted in downregulation of Hh target genes, leading to the formation of supernumerary teeth. This study reveals a previously unrecognized role of Cilk1 in controlling tooth morphology via Hh signaling. Cilk1 (Ciliogenesis associated kinase1) knockout (Cilk1-/-) mice were generated by crossing Cilk1fl/fl mice with EIIa-Cre transgenic mice for Cre-mediated deletion of Cilk1 exon 6. Cilk1fl/fl mice were previously described (https://doi.org/10.7554/eLife.56551). Cilk1-/-;PCS1-MRCS1delta/delta mice were generated by crossing Cilk1-/- and PCS1-MRCS1delta/delta mice. PCS1-MRCS1delta/delta mice with a deletion of a 70 kb region from PCS1 to MRCS1 were previously described (https://doi.org/10.1177/0022034518758642). All animal procedures were conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University. RNA was extracted from the maxillary molar tooth germs of Cilk1-/-, Cilk1-/-;PCS1-MRCS1delta/delta and Cilk1+/+ mice at embryonic day 14.5, followed by bulk RNA sequencing. Comparative gene expression profiling was then performed on the RNA-seq data.初级纤毛（Primary cilia）作为关键的感觉细胞器，介导包括刺猬（Hedgehog, Hh）信号通路在内的多条信号转导途径，而该通路对于器官模式形成与形态发生至关重要。Hh信号通路异常已被证实与突变小鼠的多生牙（supernumerary teeth）形成及磨牙融合相关。Cilk1是一种定位于初级纤毛内的高度保守丝氨酸/苏氨酸蛋白激酶（serine/threonine-protein kinase），在纤毛运输过程中发挥关键作用。Cilk1缺失会引发严重的纤毛病（ciliopathy）表型，包括多指（趾）畸形、水肿及腭裂。然而，Cilk1在牙齿发育中的功能仍未被探明。本研究探究了Cilk1在牙齿发育中的作用，研究发现Cilk1缺陷会导致Hh靶基因表达下调，进而引发多生牙形成。本研究揭示了Cilk1此前未被认知的功能——通过Hh信号通路调控牙齿形态。 Cilk1（纤毛发生相关激酶1, Ciliogenesis associated kinase1）敲除（Cilk1-/-）小鼠通过将Cilk1fl/fl小鼠与EIIa-Cre转基因小鼠杂交，经Cre介导敲除Cilk1第6外显子构建获得；其中Cilk1fl/fl小鼠的构建方法此前已有报道（https://doi.org/10.7554/eLife.56551）。Cilk1-/-;PCS1-MRCS1delta/delta双突变小鼠通过将Cilk1-/-小鼠与PCS1-MRCS1delta/delta小鼠杂交构建获得；而PCS1-MRCS1delta/delta小鼠（缺失从PCS1至MRCS1的70kb片段）的构建方法此前也已有报道（https://doi.org/10.1177/0022034518758642）。所有动物实验操作均严格遵循机构相关指南，并获得延世大学实验动物伦理审查委员会（Institutional Animal Care and Use Committee, IACUC）的批准。 本研究提取了胚胎第14.5天的Cilk1-/-、Cilk1-/-;PCS1-MRCS1delta/delta及Cilk1+/+小鼠上颌磨牙牙胚（maxillary molar tooth germs）的总RNA，随后进行批量RNA测序（bulk RNA sequencing）。基于RNA测序数据，本研究开展了比较基因表达谱分析（comparative gene expression profiling）。