Neural crest cell derived DKK1 modulates Wnt signalling in the second heart field to orchestrate cardiac outflow tract development.

Heart morphogenesis is highly complex, and depends on the generation of diverse cell types which interact with each other in an orchestrated manner to remodel the primitive heart tube into a functional organ. Cardiac outflow tract formation critically depends on continued contribution of cardiac progenitor cells from the anterior second heart field to ensure proper growth of the outflow tract. Prior to entering the outflow tract, neural crest cells migrate in close apposition to the second heart field and may play important roles in regulating second heart field growth dynamics, however the molecular mechanisms by which neural crest cells interact with the second heart field have remained elusive. Here, we discover that neural crest cells are a primary source of Dickkopf1 (DKK1), a secreted Wnt signalling inhibitor, which modulates Wnt signalling activity in the second heart field to impose a balance between progenitor maintenance and differentiation. Further, we identify the ubiquitin ligase NEDD4 as a critical regulator of DKK1 levels, with disruption of Nedd4 activity leading to outflow tract defects. In the context of disease pathogenicity, we show a novel human congenital heart disease variant of NEDD4 has lost the ability to ubiquitinate DKK1, and is associated with heart defects in a mouse model of the genetic variant. Our findings point to an unexpected role for neural crest cells acting as a rheostat of Wnt signalling activity in cardiac progenitors, identifying a new molecular pathway underpinning correct outflow tract morphogenesis, and a new causative factor of congenital heart disease. Overall design: Tissue sections from embryonic day E9.5 mouse embryos were acquired and subjected to laser capture microdissection. A region containing the anterior second heart field and associated neural crest and pharyngeal endoderm tissue was laser dissected, and total RNA prepared. This was done from 3 independent wildtype and Wnt1-Cre;Nedd4fl/fl embryos. Prepared RNA was subjected to bulk RNAseq.

心脏形态发生（Heart morphogenesis）过程极为复杂，依赖于多种细胞类型的产生：这些细胞以协同有序的方式相互作用，将原始心管重塑为具备正常功能的器官。心脏流出道（cardiac outflow tract）的形成，关键依赖于前第二心场（anterior second heart field）来源的心脏祖细胞的持续贡献，以保障流出道的正常生长。在进入流出道之前，神经嵴细胞（neural crest cells）会紧贴第二心场迁移，并可能在调控第二心场的生长动态中发挥重要作用，然而神经嵴细胞与第二心场相互作用的分子机制仍不明确。本研究发现，神经嵴细胞是Dickkopf1（DKK1）的主要来源——该蛋白是一种分泌型Wnt信号通路抑制剂（Wnt signalling inhibitor），可通过调控第二心场内的Wnt信号活性，在祖细胞维持与分化之间建立平衡。进一步研究证实，泛素连接酶NEDD4（ubiquitin ligase NEDD4）是DKK1蛋白水平的关键调控因子，Nedd4活性的缺失会导致流出道发育缺陷。在疾病致病性层面，我们发现一种全新的人类NEDD4先天性心脏病变异体，其丧失了泛素化DKK1的能力，且在携带该遗传变异的小鼠模型中可引发心脏发育缺陷。本研究揭示了神经嵴细胞作为心脏祖细胞中Wnt信号活性调控"变阻器"的意外功能，明确了一条支撑正常流出道形态发生的全新分子通路，同时鉴定出一种新的先天性心脏病致病因素。整体实验设计：获取胚胎发育第9.5天（embryonic day E9.5）的小鼠胚胎组织切片，进行激光捕获显微切割（laser capture microdissection）。对包含前第二心场、相关神经嵴及咽内胚层的组织区域进行激光显微切割，并提取总RNA。该实验分别取材自3组独立的野生型胚胎，以及3组Wnt1-Cre;Nedd4fl/fl胚胎。所提取的总RNA均进行了批量RNA测序（bulk RNAseq）。