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: 10210382
• 负责人: David E. Clouthier
• 金额: $ 64.89万
• 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/10210382
• 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; Regulator Genes; 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; 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 影响下颌、上颌和颌骨关节的颅面畸形是出生时常见的疾病。 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。而当 在发育中的咽弓内的一个或多个组织中心，面部从那里凸起，可以保持 了解这些缺陷的原因的关键是，这些中心的存在从未得到证实， 导致对面部过程中基因调控网络是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。 Endothelin1(通过DLX蛋白)和BMP(通过SMAD蛋白)信号建立位置和 下颌骨腹侧弓内神经脊细胞的结构同一性。这主要是通过归纳来实现的。 碱性bHLH转录因子HAND2。HAND2和BMP的协同作用是诱导bHLH所必需的 下颌弓腹侧最“帽”内的HAND1因子。有趣的是，改变HAND1二聚体伙伴 选择(从而改变HAND1介导的信号传递)会导致明显的面中部裂伤，即使Hand1 在面中部结构中不表达。我们的数据显示，BMP或HAND2活动的丢失都会扰乱 腹侧盖的建立。这些发现建立了我们的假设，即BMP和BMP的交集 HAND2活动建立了腹侧帽信号中心，它既在细胞内发挥作用，也在非细胞内发挥作用 引导上下颌骨发育的方式。此外，我们假设DLX活性具有拮抗作用 BMP/HAND2的协同作用。这项建议利用了大卫博士的头面部发育专业知识。 Clouthier，Anthony Firulli博士和一些新的突变小鼠等位基因的bHLH信号专业知识 在两个目标上检验这些假设。在目标1中，我们将使用单细胞(Sc)rna-seq来定义基因调控。 由HAND2和BMP的协调操作启动的网络 (腹帽)或非细胞自主(更多背侧第一弓区)方式。对scRNA-seq序列进行后续分析 数据，前HAND2/BMP效应者将在功能丧失和遗传方面进行评估 学习。在目标2中，DLX蛋白在限制腹帽大小方面的作用将通过一种新的增益- 通过创建一个Hand1小鼠突变体，对Dlx5小鼠进行功能测试 缺少DLX顺式元素输入。总之，这些新颖的方法将提供第一个直接证据，证明 下颌弓腹帽是面部发育所必需的信号中枢。 相关性：颅面畸形很常见，需要进行密集的重建手术矫正。 HAND2和BMPs在形成面部神经脊细胞的模式方面起着关键作用。深入了解 这一未被研究的发育过程的分子机制可能具有巨大的潜力 先天性颅面骨缺损的非手术治疗进展。