Elucidating Ciliopathies: The Role of Empty Spiracles Homeobox 2 and Cannabinoid Signaling in Cilia Development across Embryonic Tissues

Cilia are exquisite organelles with essential roles in ontogeny and physiology, and ciliogenesis defects underline a range of diseases ranging from rare genetic, chronic to highly infectious diseases. In our work, we elucidated the role of empty spiracles homeobox 2 (emx2) and cannabinoid receptor 1 (cnr1) in zebrafish embryonic cilia development across tissues, highlighting their roles in regulating renal MCCs. We report that emx2 is essential for cilia development across the ear, neuromasts and Kupffer’s vesicle (KV). emx2 deficient embryos manifest altered fluid homeostasis and kidney defects including decreased multiciliated cells (MCCs), determining that emx2 is essential to properly establish several renal lineages. Further, emx2 deficiency disrupted renal monociliated cells, MCCs and led to aberrant basal positioning. We reported that emx2 regulates prostaglandin biosynthesis in ciliogenesis and renal fate changes through key factors including ppargc1a, ptgs1 and PGE2. Our findings reveal essential roles of emx2 in tissue cilia development and identify emx2 as a critical regulator of prostaglandin biosynthesis during renal development and ciliogenesis. In our second study, we report that Cnr1 is essential for cilia development across tissues and regulates renal MCCs via cyclic AMP (cAMP) signaling. We found that the loss of function, agonism and antagonism of cnr1 all lead to reduced mature renal MCC populations. cnr1 deficiency also led to reduced cilia development across tissues, including the pronephros, ear, KV, and nasal placode. Interestingly, treatment with the cAMP activator Forskolin (FSK) restored renal MCC defects in agonist-treated embryos, suggesting cnr1 mediates cAMP signaling in renal MCC development. Meanwhile, treatment with the cAMP inhibitor SQ-22536 alone or with cnr1 deficiency both led to reduced MCC populations, suggesting cnr1 mediates renal MCC development independently of cAMP signaling. Our findings indicate that cnr1 has a critical role in controlling renal MCC development both via cAMP signaling and independent pathway.

纤毛（Cilia）是一类精巧的细胞器，在个体发育（ontogeny）与生理稳态中发挥核心功能，而纤毛发生（ciliogenesis）缺陷可引发一系列疾病，涵盖罕见遗传病、慢性疾病乃至高传染性疾病。本研究阐明了空位同源盒2（empty spiracles homeobox 2, emx2）与大麻素受体1（cannabinoid receptor 1, cnr1）在斑马鱼（zebrafish）多组织胚胎纤毛发育中的作用，并重点揭示了二者对肾多纤毛细胞（multiciliated cells, MCCs）的调控功能。我们证实，emx2对耳、神经丘（neuromasts）及库氏泡（Kupffer’s vesicle, KV）的纤毛发育至关重要。emx2功能缺失的胚胎会出现流体稳态紊乱及肾脏发育缺陷，包括多纤毛细胞数量减少，由此确定emx2是建立多种肾系细胞谱系的必要因子。进一步研究发现，emx2功能缺失会破坏肾脏单纤毛细胞与多纤毛细胞的稳态，并导致基底定位异常。本研究还表明，emx2可通过ppargc1a、ptgs1及PGE2等关键因子调控纤毛发生过程中的前列腺素生物合成（prostaglandin biosynthesis）与肾系命运转变。本研究结果揭示了emx2在组织纤毛发育中的核心功能，并确定emx2是肾脏发育与纤毛发生过程中前列腺素生物合成的关键调控因子。在第二项研究中，我们报道了cnr1对多组织纤毛发育的必需作用，并揭示其通过环腺苷酸（cyclic AMP, cAMP）信号通路调控肾多纤毛细胞的机制。我们发现，cnr1的功能缺失、激动剂处理与拮抗剂处理均会导致成熟肾多纤毛细胞群体数量减少。cnr1功能缺失还会造成多组织（包括原肾管、耳、库氏泡及鼻基板）的纤毛发育受损。有趣的是，使用环腺苷酸激活剂福司柯林（Forskolin, FSK）处理可挽救激动剂处理胚胎的肾多纤毛细胞缺陷，提示cnr1可通过cAMP信号通路参与肾多纤毛细胞的发育。与此同时，单独使用cAMP抑制剂SQ-22536，或联合cnr1功能缺失处理，均会导致多纤毛细胞数量减少，这表明cnr1还可通过不依赖cAMP的信号通路调控肾多纤毛细胞发育。综上，本研究结果表明，cnr1可同时通过cAMP信号通路与非依赖cAMP的通路，在肾多纤毛细胞发育过程中发挥关键调控作用。