Identification of the factors underlying tooth field size and competency

项目摘要/摘要 成年脊椎动物的整体牙齿形态是由特定的牙场开始运动的。 身体中的各个区域在早期发育期间进行规划。此后，乳牙按顺序形成，每个乳牙都展开 牙场，因为在牙场边缘添加了新的牙齿。基因和信号通路的基础研究 在这些过程的基础上，将揭示哪些细胞类型和遗传特征与牙野有关 启动和扩展，从而为未来创造和成功植入活牙的尝试提供信息。通过 确定哪些细胞类型能够转化为牙科命运，为 实验室制造的牙齿器官的创造将被揭晓。此外，识别相关联的遗传签名 牙齿区域的扩大和停滞将提供有关牙弓 促进了扩张。本研究将使用一组新开发的稳定的转基因刺鱼 和斑马鱼，以确定细胞类型，转录谱和信号事件，这是基础的过程 牙齿区域的启动、扩展和停止。这些模式鱼提供了一个了解牙齿的独特机会 牙齿的形态发生，因为牙齿的位置和大小可以用基因工具来操纵。作为回应 为了过度表达EDA，两个物种在头部或头部高度一致的位置形成了异位牙区， 同时也扩大了内源性牙田。相比之下，刺鱼中Dkk2的过度表达会减少牙齿 通过抑制牙野扩张来调节牙野大小。目标1试图了解基因表达的动力学 通过评估编码肿瘤坏死基因的精细空间表达模式来切换到牙齿器官的命运 因子受体(TNFR)，可能对EDA产生反应的候选受体，并执行单细胞 从EDA过度表达和WT刺鱼中显微解剖的牙齿功能区域的RNA测序。 该项目的目标2将通过重复成像FISH来纵向观察异位牙的形成过程 当他们长出异位面部牙齿时，利用谱系追踪技术、报告基因表达和活性染料 标记以了解哪些细胞对异位牙齿有贡献。目标3将阐明哪些发育途径 与牙野大小和牙齿行数有关的基因表达谱 解剖的牙野边缘源于扩大(EDA过表达)、停滞(Dkk2过表达)和 正常的刺背牙区。总体而言，这些实验将提供有关如何指定牙齿的信息 在发育中(目标1和2)，以及哪些基因表达反应伴随着有利的或 牙野扩大的不利条件(目标3)。这项工作的第一部分将在加州大学伯克利分校进行 (K99阶段)，由Square博士的咨询委员会、机构和目前的实验室提供帮助。这项工程的完成 工作和相关的职业发展活动将支持Square博士实现他成为校长的目标 研究牙齿和颅面发育界面的调查者。

Project Summary/Abstract The overall dental morphology of an adult vertebrate is set in motion by the initiation of tooth fields at specific regions in the body plan during early development. Thereafter, primary teeth form sequentially, expanding each tooth field as new teeth are added at the tooth field margin. Basic research into the genes and signaling pathways underlying these processes will reveal which cell types and genetic signatures are associated with tooth field initiation and expansion, thus informing future attempts to create and successfully implant live teeth. By determining which cell types are capable of undergoing transformation to a dental fate, new avenues for the creation of lab-made tooth organs will be revealed. Additionally, identifying the genetic signatures associated with tooth field expansion and arrest will provide information about the greater context under which dental arcade expansion is facilitated. The present study will use a newly developed set of stable transgenic stickleback fish and zebrafish to identify the cell types, transcript profiles, and signaling events that underlie the processes of tooth field initiation, expansion, and arrest. These model fishes present a unique opportunity to understand tooth field morphogenesis, because tooth field position and size can be manipulated using genetic tools. In response to Eda overexpression, both species form ectopic tooth fields in highly consistent locations in or on the head, while also expanding endogenous tooth fields. By contrast, Dkk2 overexpression in stickleback reduces tooth field size by inhibiting tooth field expansion. Aim 1 seeks to understand the gene expression dynamics of the switch to a tooth organ fate by assessing the fine spatial expression patterns of genes encoding Tumor Necrosis Factor Receptors (TNFRs), candidate receptors that may confer the response to Eda, and performing single-cell RNA sequencing on tooth-competent regions microdissected from Eda overexpressing and WT sticklebacks. Aim 2 of this project will longitudinally observe the process of ectopic tooth formation by repeatedly imaging fish as they grow ectopic facial teeth, utilizing lineage tracing techniques, reporter gene expression, and vital dye labeling to understand which cells contribute to ectopic teeth. Aim 3 will elucidate which developmental pathways are associated with tooth field size and tooth row number by comparing the gene expression profiles of the dissected tooth field margins derived from expanded (Eda overexpression), arrested (Dkk2 overexpression), and normal stickleback tooth fields. Overall, these experiments will yield information on how teeth can be specified in development (Aims 1 and 2), and which gene expression responses are concomitant with favorable or unfavorable conditions for tooth field expansion (Aim 3). The first portion of this work will occur at UC-Berkeley (K99 phase), facilitated by Dr. Square’s advisory committee, institution, and current lab. The completion of this work and the associated career development activities will support Dr. Square in his goal to become a principal investigator studying the interface of tooth and craniofacial development.