Mechanisms of Sonic hedgehog mediated skeletal patterning in zebrafish fin appendages

项目摘要/摘要： 该项目将为申请者提供细胞和发育生物学方面的博士培训。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肢的骨骼图案 在发育和再生过程中。申请者还将追求职业发展、指导、 并在研究金期间开展宣传活动。斑马鱼的鳍通过一种 骨射线的分支骨骼，很可能类似于四足动物的脚趾。光线分叉通过以下方式发生 祖细胞成骨细胞(POB)池分裂，逐渐产生延伸的骨骼射线。音速 Hedgehog信号对于发育和再生过程中的射线分枝是特别需要的。在这两个地方 病例中，shha的独特表达是在远端的POB池上发现的小的基表皮结构域 鱼鳍生长不足的一个方面。基表细胞不断向远端迁移，仅在以下情况下上调shha 进入POB定义的增长区域。重要的是，射线分枝形态发生似乎是由 SHHA-ESPECT域的横向分裂，随后是SHH反应的POBS。因此，一个尚未解决的关键问题 射线分枝的上游步骤是基表皮shha的转录激活。这项建议旨在 通过研究POB邻近的SHHA是如何诱导的来揭示骨骼形态发生的机制 基生表皮。以前的研究和申请人的初步数据表明，Wnt/b-catenin是典型的 已知是鳍再生生长所必需的，位于基表皮毛的上游。此外， 申请人已经确定了规范的Wnt效应器Lef1的候选保守结合位点 具有重要功能的SHHA监管区域。总体而言，这些结果和其他结果支持申请人的 假设POB产生的Wnt激活附近基表中典型的Wnt/b-catenin信号 诱导shha表达的细胞。申请者将带着两个特定的目标来探索这一假设。目标1将 用可诱导的方法确定Wnt信号如何在Shh信号的上游作用于射线分支形态发生 规范Wnt活性的体内操作及规范表达的POB基因的CRISPR/Cas9突变 WNT。目标2将探索SHHA基因座内候选Lef1结合位点的功能需求。 此外，申请者将使用比较基因组学和诱变转基因报告来鉴定顺式- 调控元件特异性地驱动shha在远端鳍基表皮中的表达。这些目标加在一起，将 详细了解交叉信号通路如何引导鳍的骨架模式 附属物。拟议中的研究将揭示Shh信号机制在许多 生物医学背景和支持骨损伤和疾病的新的再生医学方法。

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.