Genetic and developmental mechanisms of vertebrate craniofacial variation

项目总结 颅面形态发生是一种高度协调和进化保守的发育 进程。在某些情况下，脊椎动物正常头面部变异背后的基因变化 会导致人体的解剖缺陷。因此，全面了解遗传和 颅面多样性的发育机制将为人类先天性的研究提供重要的见解 颅面部疾病占所有出生缺陷的三分之一以上，通常会导致听力损失， 言语障碍和智力残疾。 在家鸽品种中，单一物种内喙形态的根本差异 是几千年来人工选择的结果。初步的基因组分析支持经典的结果 繁殖实验表明，鸽子喙的形态是由多个遗传位点控制的， 包括至少一个与性有关的因素。通过比较中小喙鸽子的基因组，我得到了 确定了两个候选基因ROR2和SMAD6，它们编码非典型性Wnt和 BMP信号通路。在人类中，这些基因的突变会导致先天性颅面 疾病，包括罗宾诺综合征和颅缝早闭。这个项目利用了跨学科的 理解特定基因组合功能的方法和互补脊椎动物模型 以及脊椎动物头面部骨骼图案化过程中的信号通路。此外，这个项目 利用家鸽喙形态上的显著差异来鉴定基因 颅面形态特定维度的结构，包括单个骨骼的大小和形状 头面部骨骼的特征。 这个项目将追求两个截然不同的目标。目标一号将测试单个和整合的分子 ROR2和Smad6在脊椎动物颅面形态发生中的作用我将使用以下组合 原位杂交(ISH)和RNA测序(RNA-SEQ)技术 ROR2和/或SMAD6及其各自的信号通路与喙的变异有关 禽类胚胎的形态。此外，我将使用遗传、发育和活体成像方法来 检测ROR2和Smad6在鸡和斑马鱼颅面形态发生中的功能作用。目标2 将识别与个体骨骼大小和形状变化相关的特定基因 脊椎动物的头面部骨骼。我将对两只鸽子F2进行数量性状基因座(QTL)定位 杂交以确定导致鸽子喙形态变异的基因组基因座。为了补充 关于基因作图实验，我将利用夏皮罗实验室的广泛的“鸽子”工具包来精细作图 每个QTL中与喙形态尺寸相关的特定基因或DNA序列。 这些实验将广泛地定义鸽子头面部变异的遗传结构 发现特定的候选基因，以供进一步研究。 这些互补的遗传、基因组和发育方法将定义 脊椎动物头面部形态发生中的两个基因并将确定令人惊讶的分子基础 创新模型系统中的头面部变异。总而言之，该项目的目标将开辟新的途径 了解特定基因在脊椎动物正常和疾病变异中的作用。

PROJECT SUMMARY Craniofacial morphogenesis is a highly orchestrated and evolutionarily conserved developmental process. In some cases, changes to the genes that underlie normal craniofacial variation across vertebrates can cause anatomical defects in humans. Thus, a comprehensive understanding of the genetic and developmental mechanisms of craniofacial diversity will provide important insights into human congenital craniofacial disorders, which account for more than one third of all birth defects and often cause hearing loss, speech impediment and intellectual disability. Among breeds of domestic pigeon, radical variation in beak morphology within a single species has resulted from millennia of artificial selection. Preliminary genomic analyses support the results of classical breeding experiments, which suggest that pigeon beak morphology is controlled by multiple genetic loci, including at least one sex-linked factor. By comparing the genomes of small and medium beak pigeons, I have identified two candidate genes, ROR2 and SMAD6, which encode members of the non-canonical Wnt and BMP signaling pathways, respectively. In humans, mutations in these genes cause congenital craniofacial disorders, including Robinow syndrome and Craniosynostosis. This project utilizes cross-disciplinary approaches and complementary vertebrate models to understand the combinatorial function of specific genes and signaling pathways during patterning of the vertebrate craniofacial skeleton. Furthermore, this project takes advantage of the striking variation in beak morphology within domestic pigeons to identify the genetic architecture of specific dimensions of craniofacial morphology, including the size and shape of individual bones of the craniofacial skeleton. This project will pursue two distinct Aims. Aim 1 will test the individual and integrated molecular functions of ROR2 and SMAD6 during vertebrate craniofacial morphogenesis. I will use a combination of in situ hybridization (ISH) and RNA sequencing (RNA-seq) techniques to determine if differential expression of ROR2 and/or SMAD6 and their respective signaling pathways are associated with variation in beak morphology in avian embryos. In addition, I will employ genetic, developmental and live imaging approaches to test the functional roles of ROR2 and SMAD6 during chick and zebrafish craniofacial morphogenesis. Aim 2 will identify specific genes associated with variation in the size and shape of individual bones of the vertebrate craniofacial skeleton. I will perform quantitative trait locus (QTL) mapping in two pigeon F2 intercrosses to identify genomic loci that underlie variation in pigeon beak morphology. To complement the genetic mapping experiments, I will leverage the Shapiro lab's extensive “pigeonomic” toolkit to fine map the specific genes or DNA sequences within each QTL that are associated with dimensions of beak morphology. These experiments will broadly define the genetic architecture of craniofacial variation in pigeons and will implicate specific candidate genes for further investigation. These complementary genetic, genomic, and developmental approaches will define the precise roles of two genes in vertebrate craniofacial morphogenesis and will identify the molecular basis of astonishing craniofacial variation in an innovative model system. Together, the Aims of this project will open new avenues to understand the roles of specific genes in normal and disease variation among vertebrates in general.