Role of the novel regulator lbh in neural crest and craniofacial development

描述（由申请人提供）：颅面畸形是最常见的人类出生缺陷之一，影响1/700的出生。虽然许多导致这些缺陷的基因是已知的，但许多其他基因的身份和功能尚不清楚。本提案的总体目标是评估一个很大程度上尚未表征的基因，肢体芽和心脏同源基因（lbh）在斑马鱼颅面发育过程中的作用。该基因可能介导CHARGE综合征，一种神经嵴细胞（NCCs）疾病，包括颅面缺陷，如小颌畸形。此外，我们还将该基因与慈鲷颌的进化联系起来，这种进化模式在颅面形态上表现出广泛的多样性，其中许多模仿人类面部畸形。综合这些数据，我们假设lbh是NCC和颅面发育的一种新的调节因子，我们将通过以下实验目的来验证这一假设。目的1：考虑到lbh信息的缺乏，我们将首先利用全安装和切片原位杂交，并与已知颅面标记物进行比较，表征lbh在斑马鱼面部发育过程中的表达模式和细胞特异性。目的2：我们将评估调节lbh表达（morpholino敲低或mRNA过表达）对颅面骨骼的影响。初步实验表明，Lbh的消耗导致颅面骨骼ncc和相应缺陷的减少。具体来说，我们将确定这种缺陷的细胞机制是由于诱导失败、迁移失败、存活率降低还是NCCs过早分化，使用原位杂交、报告斑马鱼品系和细胞测定相结合。目的3：我们将利用从不同下颌骨长度的稚鱼中分离的lbh的生物学相关等位基因来分析lbh的功能保护和进化。总之，这些研究将阐明lbh在颅面发育、疾病和进化中的作用。

DESCRIPTION (provided by applicant): Craniofacial malformations are among the most common human birth defects, affecting 1/700 births. While many of the genes that underlie these defects are known, the identity and functions of many others are not. The overarching goal of this proposal is evaluate the role of a largely uncharacterized gene, limb bud and heart homolog (lbh), during zebrafish craniofacial development. This gene may mediate CHARGE syndrome, a disease of neural crest cells (NCCs) that includes craniofacial defects such as micrognathia. Additionally, we have associated this gene with the evolution of jaws in cichlids, an evolutionary model that exhibits extensive diversity in craniofacial form, many of which mimic human facial malformations. Combined, these data led to the hypothesis that lbh is a novel regulator of NCC and craniofacial development, which will be tested through the following experimental aims. Aim 1: Given the paucity of information about lbh, we will first characterize the expression pattern and cellular specificity of lbh throughout zebrafish facial development using whole-mount and sectioned in situ hybridization and comparison with known craniofacial markers. Aim 2: We will evaluate the effects of modulating lbh expression (morpholino knockdown or mRNA overexpression) on the craniofacial skeleton. Preliminary experiments indicate that depletion of Lbh results in reduction of NCCs and commensurate defects in the craniofacial skeleton. Specifically, we will determine if the cellular mechanism of this defect is due to a failure of induction, failure of migration, decreased survival, or premature differentiatin of NCCs using a combination of in situ hybridization, reporter zebrafish strains, and cellular assays. Aim 3: We will analyze the functional conservation and evolution of lbh using biologically relevant alleles of lbh isolated from cichlids with differing mandible lengths. Overal, these studies will illuminate the role of lbh in craniofacial development, disease, and evolution.