Mechanisms of Sonic hedgehog mediated skeletal patterning in zebrafish fin appendages

项目总结/摘要： 该项目将为申请人提供博士学位。细胞和发育生物学培训。论文研究将 进一步了解细胞信号网络如何调节斑马鱼鳍附属物的骨骼模式 在发育和再生过程中。申请人还将追求专业发展，导师， 研究金期间的宣传活动。斑马鱼鳍的发育和再生具有很强的 骨条的分支骨骼，可能类似于四足动物的趾。光线分叉发生在 祖细胞成骨细胞（pOb）池分裂，逐渐产生延伸的骨骼射线。声波 hedgehog信号是发育和再生过程中射线分支所特别需要的。无论是 在一些病例中，shha由位于远端的pOb池上的小基底表皮结构域唯一表达。 长出鳍的方面。基底表皮细胞不断向远端迁移，只有当 移动到pOB定义的生长区。重要的是，射线分支形态发生似乎是由 shha+-表皮结构域的横向分裂，随后是Shh-响应性pOb。因此，一个未解决的关键 射线分支的上游步骤是基底表皮shha的转录激活。这项建议旨在 通过研究shha是如何在pob-邻近的细胞中诱导的，揭示骨骼形态发生的机制。 基部表皮以前的研究和申请人的初步数据表明，典型的Wnt/b-连环蛋白，以及 已知是指再生生长所必需的，是基底表皮shha的上游。此夕h 申请人已经鉴定了典型Wnt效应子Lef 1的候选保守结合位点， 功能上重要的shha调节区。总的来说，这些和其他结果支持申请人的 pOB产生的Wnt激活附近基底表皮中典型Wnt/β-连环蛋白信号传导的假设 细胞以诱导SHHA表达。申请人将以两个具体目标探讨这一假设。目标1将 确定Wnt信号传导如何作用于Shh信号传导的上游，用于射线分支形态发生， 典型pOb表达的Wnt基因的典型Wnt活性和CRISPR/Cas9诱变的体内操作 Wnt.目的2将探讨候选Lef 1结合位点的shha基因座内的功能要求。 此外，申请人将使用比较基因组学和诱变转基因报告来鉴定顺式- 在远鳍基底表皮中特异性驱动shha表达的调节元件。总之，这些目标将 提供了一个详细的了解如何交叉信号通路直接骨骼模式的鳍 附肢拟议的研究将揭示Shh信号机制的潜在相关性， 生物医学背景和支持新的再生医学方法的骨损伤和疾病。

PROJECT SUMMARY/ ABSTRACT: This project will provide the applicant with Ph.D. training in cell and developmental biology. Thesis research will further our understanding how cell signaling networks regulate skeletal pattering of zebrafish fin appendages during development and regeneration. The applicant also will pursue professional development, mentorship, and advocacy activities during the fellowship period. Zebrafish fins robustly develop and regenerate with a branched skeleton of bony rays, which are likely analogous to tetrapod digits. Ray bifurcation occurs by the splitting of progenitor osteoblast (pOb) pools that progressively produce extending skeletal rays. Sonic hedgehog signaling is specifically required for ray branching during development and regeneration. In both cases, shha is uniquely expressed by small basal epidermis domains overlying pOb pools found at the distal aspect of outgrowing fins. Basal epidermal cells constantly migrate distally, upregulating shha only when moving into the pOb-defined growth zone. Importantly, ray branching morphogenesis appears to initiate by the lateral splitting of the shha+-epidermal domain followed by Shh-responsive pObs. Therefore, a key unresolved upstream step in ray branching is the transcriptional activation of basal epidermal shha. This proposal aims to uncover mechanisms of skeletal morphogenesis by investigating how shha is induced in pOb-neighboring basal epidermis. Previous studies and the applicant’s preliminary data suggest canonical Wnt/b-catenin, well known to be required for fin regenerative outgrowth, is upstream of basal epidermal shha. Further, the applicant has identified candidate conserved binding sites for the canonical Wnt effector, Lef1, within a functionally important shha regulatory region. Collectively, these and other results support the applicant’s hypothesis that a pOb-produced Wnt activates canonical Wnt/b-catenin signaling in nearby basal epidermal cells to induce shha expression. The applicant will explore this hypothesis with two specific aims. Aim 1 will determine how Wnt signaling acts upstream of Shh signaling for ray branching morphogenesis using inducible in vivo manipulations of canonical Wnt activity and CRISPR/Cas9 mutagenesis of a canonical pOb-expressed Wnt. Aim 2 will explore functional requirements of the candidate Lef1 binding sites within the shha locus. Further, the applicant will use comparative genomics and mutagenized transgenic reports to identify cis- regulatory elements specifically driving shha expression in distal fin basal epidermis. Together, these aims will provide a detailed understanding how intersecting signaling pathways direct skeletal patterning of fin appendages. The proposed research will uncover Shh signaling mechanisms of potential relevance in many biomedical contexts and support novel regenerative medicine approaches for bone damage and disease.