Tbx1 regulates a cardiopharyngeal mesodermal niche in forming the heart and branchiomeric muscles [scRNA-Seq 4]

The heart and branchiomeric muscles are formed from the cardiopharyngeal mesoderm (CPM) during mammalian embryogenesis. The molecular mechanisms for lineage progression in the CPM in mammals remain elusive. Here, we have used single cell RNA-seq and lineage analysis and identified a cardiopharyngeal niche containing multilineage primed cells termed multilineage progenitors (MLPs), which is maintained throughout maturation of the pharyngeal apparatus. We found that MLP function is dependent on Tbx1, encoding a T-box transcription factor and the gene for 22q11.2 deletion syndrome. TBX1 positively regulates novel MLP enriched genes such as Aplnr and Nrg1, as well as known CPM genes related to both BrM and cardiac muscle cell development. Further, loss of Tbx1 results in ectopic expression of neuronal and other non-mesodermal specification genes such as Bdnf and Pax8, respectively, indicating that normal developmental regulation is disrupted. Integration of the multi-omic data generated a TBX1 gene regulatory network, including Isl1, Pitx2, Foxc2, Six1/2 and Tcf21, that regulates CPM lineage progression from MLPs. Our finding suggests that TBX1 is a one of the key regulators in MLP to maintain CPM property and promote differentiation toward both BrM and cardiac muscle cells. Mesp1 gene expresses in early cardiac mesodermal cells. To capture mesodermal cell lineages, sorted GFP positive cell from Mesp1-Cre/+;GFPflox/+ embryos at E8.0 to E10.5 embryos were used for scRNA-seq analysis.在哺乳动物胚胎发生过程中，心脏与鳃弓肌由心咽中胚层（cardiopharyngeal mesoderm, CPM）发育形成。目前，哺乳动物心咽中胚层内谱系进展的分子机制仍不甚明晰。本研究借助单细胞RNA测序（single cell RNA-seq）与谱系分析技术，鉴定出一个心咽微环境，其中包含被命名为多谱系祖细胞（multilineage progenitors, MLPs）的多谱系预启动细胞，该微环境在咽器官的整个成熟过程中得以维持。研究发现，MLP的功能依赖于Tbx1基因——该基因编码T-box转录因子（T-box transcription factor），同时是22q11.2缺失综合征（22q11.2 deletion syndrome）的致病基因。TBX1正向调控一系列在MLP中富集的新基因（如Aplnr与Nrg1），以及与鳃弓肌（branchiomeric muscles, BrM）和心肌细胞发育相关的已知CPM基因。进一步研究显示，Tbx1的缺失会导致神经元及其他非中胚层定向基因的异位表达，其中分别对应神经元的Bdnf与非中胚层定向的Pax8，表明正常的发育调控程序遭到扰乱。整合本次研究获得的多组学数据，我们构建了TBX1基因调控网络（gene regulatory network），该网络包含Isl1、Pitx2、Foxc2、Six1/2及Tcf21等调控因子，可调控心咽中胚层从MLP出发的谱系进展。本研究结果表明，TBX1是维持MLP心咽中胚层特性，并促进其向鳃弓肌与心肌细胞分化的关键调控因子之一。Mesp1基因在早期心脏中胚层细胞中表达。为捕获中胚层细胞谱系，我们选取了胚胎发育第8.0天至第10.5天的Mesp1-Cre/+;GFPflox/+胚胎中分选得到的GFP阳性细胞，用于单细胞RNA测序分析。