Gene Regulatory Networks that Establish Mandible and Maxilla Patterning

建立下颌骨和上颌骨模式的基因调控网络

• 批准号: 10454286
• 负责人: David E. Clouthier
• 金额: $ 59.85万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454286
• 关键词: Address; Affect; Alleles; Area; Automobile Driving; BHLH Protein; Bone structure; Branchial arch structure; CRISPR/Cas technology; Cells; Congenital Abnormality; Craniofacial Abnormalities; Data; Defect; Deformity; Development; Developmental Process; Distant; Dlx protein; Dorsal; Elements; Environment; Etiology; Event; Face; First Pharyngeal Arch; Future; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic Transcription; Genetic study; Goals; Hand functions; Health; Helix-Turn-Helix Motifs; Human; Jaw; Knowledge; Live Birth; Location; Maintenance; Mandible; Mandibular Prominence; Maps; Maxillary Prominence; Mediating; Mediator of activation protein; Mission; Modeling; Molecular; Morphogenesis; Mus; Mutant Strains Mice; Mutate; Neural Crest Cell; Operative Surgical Procedures; Patients; Pattern; Pierre Robin Syndrome; Play; Process; Public Health; Quality of life; Research; Research Proposals; Role; Signal Transduction; Smad Proteins; Structural Genes; Structure; Syndrome; Temporomandibular Joint; Testing; Transcriptional Regulation; United States National Institutes of Health; Zygomatic Arch; craniofacial complex; craniofacial development; craniofacial structure; dimer; gain of function; gene network; gene regulatory network; insight; loss of function; mandible/maxilla; middle ear; network models; neural patterning; novel; novel strategies; overexpression; reconstruction; single-cell RNA sequencing; spatiotemporal; synergism; transcriptome

Abstract Craniofacial abnormalities affecting the mandible, maxilla and jaw joint are commonly encountered birth defects, most of which require surgical correction to establish quality of life and in some cases survival. While one or more organizing centers within the developing pharyngeal arches, from which the face arises, may hold the key to understanding the etiologies of these defects, the existence of such centers has never been proven, leading to a poor understanding of how gene regulatory networks are regulated and integrated during facial development. This limiting knowledge stifles new approaches to efficaciously treat these deformities. Endothelin1 (through DLX proteins) and BMP (through SMAD proteins) signaling establishes positional and structural identity of neural crest cells within the ventral mandibular arch. This is primarily achieved by induction of the basic bHLH transcription factor HAND2. HAND2 and BMP synergy is required for induction of the bHLH factor HAND1 within the ventral-most “cap” of the mandibular arch. Interestingly, altering HAND1 dimer partner choice (thus altering HAND1-mediated signaling) results in pronounced mid-facial clefting, even though Hand1 is not expressed in the mid-face structures. Our data shows that loss of either BMP or HAND2 activity disrupts the establishment of the ventral cap. These findings establish our hypothesis that the intersection of BMP and HAND2 activity establishes a ventral cap-signaling center, which acts in both cell and non-cell autonomous manners to drive upper and lower jaw development. Additionally, we hypothesize that DLX activity antagonizes BMP/HAND2 synergy. This proposal takes advantage of the craniofacial development expertise of Dr. David Clouthier, the bHLH signaling expertise of Dr. Anthony Firulli and a number of novel mutant mouse alleles to test these hypotheses in two Aims. In Aim 1, we will use single cell (sc) RNA-seq to define the gene regulatory networks that are initiated by the coordinated action of both HAND2 and BMP that act either in an autonomous (ventral cap) or non-cell autonomously (more dorsal first arch areas) manner. Following analysis of scRNA-seq data, the top HAND2/BMP effector candidates will be functionally evaluated in loss-of-function and genetic studies. In Aim 2, the role of DLX proteins in confining ventral cap size will be examined using a novel gain-of- function Dlx5 mouse allelefollowed by functional testing of DLX action by creating a Hand1 mouse mutant lacking DLX cis-element inputs. Together, these novel approaches will provide the first direct evidence that the mandibular arch ventral cap is a signaling center required for facial development. Relevance: Craniofacial abnormalities are common and require intensive reconstructive surgical corrections. HAND2 and BMPs play key roles in patterning the neural crest cells that form the face. Gaining insight into the molecular mechanism of this understudied developmental process could have great potential for initial development of non-surgical treatments for congenital craniofacial defects in patients.

摘要 颅面畸形影响下颌骨，上颌骨和颌关节是常见的出生 缺陷，其中大多数需要手术矫正，以建立生活质量，在某些情况下生存。而 在发育中的咽弓内的一个或多个组织中心，面部从其产生， 理解这些缺陷的病因的关键，这些中心的存在从未被证明， 导致对基因调控网络在面部发育过程中如何调控和整合的理解不足。 发展这种有限的知识扼杀了有效治疗这些畸形的新方法。 内皮素1（通过DLX蛋白）和BMP（通过SMAD蛋白）信号传导建立了位置和功能。 下颌弓腹侧神经嵴细胞的结构特征。这主要是通过诱导 基本bHLH转录因子HAND 2。诱导bHLH需要HAND 2和BMP协同作用 HAND 1因子在下颌弓的最腹侧“帽”内。有趣的是，改变HAND 1二聚体伴侣 选择（从而改变HAND 1介导的信号）导致明显的面中部裂，即使HAND 1 在面中部结构中没有表现出来。我们的数据显示，BMP或HAND 2活性的丧失破坏了 腹帽的形成。这些发现确立了我们的假设，即BMP和 HAND 2活动建立了一个腹侧帽信号中心，它在细胞和非细胞自主活动中起作用。 方式来驱动上下颌的发展。此外，我们假设DLX活性拮抗 BMP/HAND 2协同作用。这项建议利用了大卫博士的颅面发育专业知识 Clouthier，Anthony Firulli博士的bHLH信号传导专业知识和一些新的突变小鼠等位基因， 在两个目标中测试这些假设。在目标1中，我们将使用单细胞（SC）RNA-seq来定义基因调控 由HAND 2和BMP的协调动作发起的网络， （腹帽）或非细胞自主（更多的背侧第一弓区）的方式。scRNA-seq分析 数据，顶级HAND 2/BMP效应子候选者将在功能丧失和遗传学方面进行功能评估。 问题研究在目标2中，DLX蛋白在限制腹帽大小中的作用将使用一种新的获得性蛋白来检查。 功能Dlx 5小鼠等位基因，然后通过产生Hand 1小鼠突变体进行DLX作用的功能测试 缺乏DLX顺式元件输入。总之，这些新方法将提供第一个直接证据， 下颌弓腹帽是面部发育所需的信号中枢。 相关性：颅面畸形很常见，需要密集的重建手术矫正。 HAND 2和BMP在形成面部的神经嵴细胞的图案中起着关键作用。了解了 这种未充分研究的发育过程的分子机制可能具有很大的潜力， 发展非手术治疗先天性颅面缺损的患者。