Mechanisms of Sonic hedgehog mediated skeletal patterning in zebrafish fin appendages

声波刺猬介导斑马鱼鳍附属物骨骼模式的机制

基本信息

批准号：
10645527
负责人：
Amy Robbins
金额：
$ 1.9万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10645527
关键词：
Advocacy Attenuated Automobile Driving Binding Sites Bone Diseases CRISPR/Cas technology Cell Count Cells Cellular biology Confocal Microscopy Data Defect Detection Development Developmental Biology Digit structure Disease Distal Doctor of Philosophy Epidermis Fellowship Fishes Genetic Genetic Transcription Genomic approach Growth Human Imaging Device Immature Bone Individual Introns Lateral Ligands Limb structure Mediating Mentorship Messenger RNA Modeling Monitor Morphogenesis Mosaicism Mutagenesis Natural regeneration Nucleic Acid Regulatory Sequences Orthologous Gene Osteoblasts Pattern Population Proteins Publishing Regenerative Medicine Regulatory Element Reporter Reporting Research SHH gene Shapes Signal Pathway Signal Transduction Site Skeleton Sonic Hedgehog Pathway Source Testing Tetrapoda Therapeutic Training Transcription Coactivator Transcriptional Activation Transgenic Organisms Trauma Variant WNT Signaling Pathway Work Zebrafish appendage beta catenin bone bone cell comparative genomics functional genomics in vivo insight interest molecular imaging mutant novel prevent progenitor regenerative repaired response skeletal smoothened signaling pathway

项目摘要

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.

项目摘要/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的骨骼模式在开发和再生期间。申请人还将追求专业发展，Mentalship，和奖学金期间的倡导活动。斑马鱼鳍与骨射线的分支骨骼，可能类似于四足动物的数字。射线分叉是由祖细胞成骨细胞（POB）池的分裂，逐渐产生骨骼射线。声音刺猬信号是在开发和再生过程中特别需要的射线分支。在这两个中案例，shha是由在不同的山地表皮域上唯一表达的超越鳍的方面。基础表皮细胞不断分开迁移，仅在进入POB定义的生长区。重要的是，射线分支形态发生似乎是由 Shha+ - 表皮域的侧向分裂，然后是SHH响应性POB。因此，钥匙尚未解决射线分支的上游步骤是基本表皮SHHA的转录激活。该建议旨在通过研究如何在pob-neighboring中诱导SHHA来发现骨骼形态发生的机制基础表皮。先前的研究和申请人的初步数据表明了典型的Wnt/b-catenin，很好已知的鳍再生生长所需的是基本表皮SHHA的上游。此外，申请人已经确定了为规范wnt效应子lef1配置的候选绑定位点功能上重要的SHHA调节区域。总的来说，这些结果和其他结果支持申请人的假设POB生产的Wnt激活附近基本表皮中的规范Wnt/B-catenin信号传导细胞诱导SHHA表达。申请人将以两个具体的目标探讨这一假设。目标1意志确定Wnt信号如何使用可诱导在体内操纵典型的Wnt活性和CRISPR/CAS9诱变的典型POB表达 Wnt。 AIM 2将探索SHHA基因座中候选LEF1结合位点的功能要求。此外，申请人将使用比较基因组学和诱变的转基因报告来识别顺式调节元件专门驱动远端鳍片表皮中的Shha表达。这些目标将在一起提供详细的了解如何相交的信号通路直接直接骨骼图案附属物。拟议的研究将发现许多潜在相关性的SHH信号传导机制生物医学环境和支持骨损伤和疾病的新型再生医学方法。