Cell fate choices by Tbx1 in forming the mammalian heart

Tbx1 在形成哺乳动物心脏过程中的细胞命运选择

• 批准号: 10451598
• 负责人: BERNICE E MORROW
• 金额: $ 60.56万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-20 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451598
• 关键词: 22q11; 22q11.2; 3-Dimensional; APLN gene; ATAC-seq; Affect; Alleles; Anterior; Aorta; Binding; Binding Proteins; Binding Sites; Biological Process; Cardiac; Cardiac development; Cell Differentiation process; Cell model; Cells; ChIP-seq; Chromatin; Chromosomes; Data; Data Set; Defect; DiGeorge Syndrome; Ectopic Expression; Embryo; Embryonic Development; Face; Gene Expression Profile; Gene Proteins; General Population; Genes; Genetic; Genetic Transcription; Heart; Heart Abnormalities; Human Genetics; Immunofluorescence Immunologic; Individual; Lung; Mammals; Maps; Mesenchyme; Mesoderm; Mesoderm Cell; Molecular; Mus; Muscle; Muscle Fibers; Mutant Strains Mice; Mutation; Myocardium; Neck; Neonatal; Pathway interactions; Patients; Penetrance; Persistent Truncus Arteriosus; Pharyngeal Apparatus; Phenocopy; Population; Population Sizes; Process; Regulatory Element; Risk Factors; Shprintzen syndrome; Testing; Time; Tissues; base; cell behavior; cell type; conditional mutant; congenital heart disorder; embryo tissue; experimental study; functional genomics; gastrulation; gene network; interest; mouse model; mutant; precursor cell; progenitor; programs; receptor; single-cell RNA sequencing; spatiotemporal; stem cells; transcription factor; transcriptomics

TBX1 encodes a T-box transcription factor required for cardiac development. This gene maps to the chromosome 22q11.2 region that is deleted in patients with DiGeorge syndrome/velo-cardio- facial syndrome or 22q11.2 deletion syndrome (22q11.2DS). Approximately 60% of 22q11.2DS patients have congenital heart disease that mostly affects the cardiac outflow tract. A subset of individuals with a mutation of the TBX1 gene but not a deletion has been identified and they partially phenocopy patients with 22q11.2DS. Inactivation of one allele of Tbx1 in mice results in mild defects, but inactivation of both alleles results in neonatal lethality with a persistent truncus arteriosus, in which the aorta and pulmonary trunk fail to separate. This defect also occurs in 5- 10% of 22q11.2DS patients with congenital heart disease. To understand the function of Tbx1 in mammals, we performed single cell RNA-sequencing (scRNA-seq) of cardiopharyngeal mesoderm progenitor cells within the pharyngeal apparatus. We discovered a multilineage progenitor (MLP) population that expresses genes important for forming the cardiac outflow tract as well as branchiomeric muscles of the face and neck. The MLP population expands at the expense of more differentiated populations when Tbx1 is inactivated in the cardiopharyngeal mesoderm. Our main hypothesis is that Tbx1 is required in the MLP population for progression towards more differentiated states needed for cardiac development. We propose three specific aims to test this hypothesis. In the first aim, we will perform additional scRNA-seq experiments and analyze the complete dataset to understand how the progression of MLP cells are altered in Tbx1 conditional and global mutant mouse embryos. In Aim 2, we will determine where the MLP cells are localized in the embryo. Preliminary data suggests that these cells are localized to the nascent mesenchyme of the elongating pharyngeal apparatus. We will also inactivate Tbx1 specifically within the MLP cells to determine its particular functions. In Aim 3, we will turn to functional genomic studies and will identify open and accessible chromatin for which harbors TBX1 protein binding sites using ATAC-seq and ChIP-seq from embryo tissue. Preliminary data suggests that we are able to identify direct transcriptional target genes. By these three aims, we will understand the molecular functions of TBX1 in the progression of progenitor cells to more differentiated states to build the cardiac outflow tract.

TBX 1编码心脏发育所需的T-box转录因子。这个基因定位于 在DiGeorge综合征/velo-cardio患者中缺失的染色体22q11.2区域， 面部综合征或22q11.2缺失综合征（22q11.2DS）。约60%的22q11.2DS 患者患有先天性心脏病，主要影响心脏流出道。的子集 已经鉴定出具有TBX 1基因突变但没有缺失的个体， 22q11.2DS的部分表型复制患者。在小鼠中Tbx 1的一个等位基因的失活导致 轻度缺陷，但两个等位基因的失活导致新生儿死亡，并伴有持续性干 动脉，其中主动脉和肺动脉干未能分离。这一缺陷也发生在5- 10%的22q11.2DS患者患有先天性心脏病。为了了解Tbx 1在 在哺乳动物中，我们进行了心咽部的单细胞RNA测序（scRNA-seq）， 咽器内的中胚层祖细胞。我们发现了一个多血统的 表达对形成心脏流出道重要的基因的祖细胞（MLP）群体 以及面部和颈部的鳃节肌肉。MLP人口在 当Tbx 1在心咽中失活时， 中胚层我们的主要假设是，MLP人群中需要Tbx 1进行进展 朝着心脏发育所需的更分化的状态发展。我们提出三个具体的 旨在验证这一假设。在第一个目标中，我们将进行额外的scRNA-seq实验， 并分析完整的数据集，以了解MLP细胞的进展是如何改变的， tbx 1条件和整体突变小鼠胚胎。在目标2中，我们将确定MLP 细胞位于胚胎中。初步数据表明，这些细胞定位于 伸长的咽器的新生间充质。我们还将提供Tbx 1 特别是在MLP细胞内，以确定其特定功能。在目标3中，我们将转向 功能基因组研究，并将确定开放和可访问的染色质，其中港口 使用来自胚胎组织的ATAC-seq和ChIP-seq的TBX 1蛋白结合位点。初步数据 表明我们能够识别直接转录靶基因。通过这三个目标，我们 将了解TBX 1在祖细胞向更多细胞分化过程中的分子功能， 分化状态，以建立心脏流出道。