Roles of Cell Polarity and Cilia in Cartilage Patterning in the Craniofacial Skeleton

项目摘要/摘要 从未受精卵到成虫特化器官，细胞和细胞内结构的极化 是动物发育的一个基本方面。在多细胞组织中，平面细胞极性(PCP) 在果蝇的上皮细胞中进行了广泛的研究，但对细胞的作用知之甚少 脊椎动物间充质组织发育中的极性，如软骨和骨。软骨由以下部分组成 极化的软骨细胞堆积导致软骨内骨化的极化进展。这些 堆叠是通过协调的插层延伸和骨骼凝结内的分化形成的。 之前，我们已经证明了两个PCP信号通路，Fat-Dachsous和Wnt-Fz，是 斑马鱼颌骨发育中软骨细胞的极化堆积和分化。的一个显著特点 在Fat3或Wnt5缺陷的胚胎中，这些被破坏的极化堆栈是微管的位置- 每个细胞内的组织中心，它标志着初级纤毛的位置。这表明， 初级纤毛在软骨形成过程中可能起到一定的作用(S)。两只小鼠的研究 而Chick进一步将PCP和初级纤毛用于确定长骨中的软骨极性。此外， Wnt-FZ和Fat-Dachsous信号的缺陷会导致人类疾病。Wnt5A或ROR2突变导致 Robinow综合征和FAT4突变导致Van Maldergem综合征，这两种综合征都包括骨骼 缺陷。同样，由纤毛基因突变引起的疾病，统称为“纤毛病”，通常也会出现。 有骨骼缺陷，主要影响面部。来自老鼠和小鸡的这些和其他数据表明 细胞极性的缺陷是骨骼异常的基础，包括那些影响头面部骨骼的异常。 我们的初步数据表明，Hedgehog(HH)信号和初级纤毛在软骨细胞极性和 对于生长带(GZ)形成中的极性，它相当于长骨生长板，并形成 在极性反转部位的软骨内骨化过程中。我们假设PCP和初级纤毛 共同作用在软骨发育中极化软骨细胞以建立软骨内生长部位 和骨化。在目标1中，我们将测试对初级纤毛和软骨中HH信号的新要求 斑马鱼的极性：1)分析Fat-PCP和Wnt-PCP信号对破坏纤毛和HH的反应 信号，2)利用纤毛缺失的Bbs/ofd1突变体和HH通过细胞移植创建遗传马赛克 信号缺陷动物探索纤毛和HH对软骨图案形成的细胞非自主作用，以及 3)确定Wnt-PCP和Fat-PCP在初级纤毛信号下游的作用。在目标2中，我们将确定 极性在斑马鱼软骨形态发生和GZ形成中的作用 极性和初级纤毛模式GZ，2)分析极性和初级纤毛如何影响软骨反应 3)研究BBS9/OFD1在软骨极性中可能的纤毛非依赖性作用， 包括有丝分裂纺锤体极定向和胞质分裂。

Project Summary/Abstract From the unfertilized egg to specialized organs in the adult, the polarization of cells and intracellular structures is a fundamental aspect of animal development. In multicellular tissues, planar cell polarity (PCP) has been extensively studied in epithelia in Drosophila, however, comparatively little is known about the roles of cell polarity in developing mesenchymal tissues in vertebrates such as cartilage and bone. Cartilages composed of polarized stacks of chondrocytes give rise to a polarized progression of endochondral ossification. These stacks form through coordinated intercalation elongation, and differentiation within a skeletal condensation. Previously, we have shown that two PCP signaling pathways, Fat-Dachsous and Wnt-Fz, are required for the polarized stacking and differentiation of chondrocytes in the developing zebrafish jaw. A prominent feature of these polarized stacks that is disrupted in Fat3- or Wnt5-deficient embryos is the position of the microtubule- organizing center within each cell, which marks the location of the primary cilium. This suggests that the primary cilium may serve some specific function(s) during the formation of cartilages. Studies from both mouse and chick further implicate PCP and primary cilia in determining cartilage polarity in long bones. Moreover, defects in Wnt-Fz and Fat-Dachsous signaling cause human diseases. WNT5A or ROR2 mutations cause Robinow syndrome and FAT4 mutations cause Van Maldergem syndrome, both of which include skeletal defects. Similarly, diseases caused by mutations in cilia genes, collectively termed “ciliopathies,” often present with skeletal defects, primarily affecting the face. These and other data from both mouse and chick suggest that defects in cell polarity underlie skeletal abnormalities including those affecting the craniofacial skeleton. Our preliminary data suggest a role for Hedgehog (Hh) signaling and primary cilia in chondrocyte polarity and for polarity in the formation of “growth zones” (GZs), which are equivalent to long bone growth plates, and form during endochondral ossification at the sites of polarity reversals. We hypothesize that PCP and primary cilia function together to polarize chondrocytes in developing cartilages to establish sites of endochondral growth and ossification. In Aim 1 we will test novel requirements for the primary cilium and Hh signaling in cartilage polarity in zebrafish by 1) analyzing Fat-PCP and Wnt-PCP signaling responses to disrupting cilia and Hh signaling, 2) creating genetic mosaics with cell transplantation using cilia-deficient bbs/ofd1 mutants and Hh signaling-defective animals to explore cell non-autonomous effects of cilia and Hh on cartilage patterning, and 3) determining roles for Wnt- and Fat-PCP downstream of primary cilia signaling. In Aim 2 we will determine the roles of polarity in cartilage morphogenesis and GZ formation in zebrafish by 1) testing how cartilage polarity and primary cilia pattern GZs, 2) analyzing how polarity and primary cilia influence cartilage responses to Hh, and 3) studying potential cilia-independent roles for BBS9/OFD1 which may affect in cartilage polarity, including mitotic spindle pole orientation and cytokinesis in GZs.