Transcription profile of zebrafish hand2 (hanS6 allele) mutant embryos at 19hpf

Heart formation requires the fusion of bilateral cardiomyocyte populations as they move toward the embryonic midline. The bHLH transcription factor Hand2 is essential for cardiac fusion; however, the effector genes that execute this function of Hand2 are unknown. Here, we provide the first evidence for a downstream component of the Hand2 pathway that mediates cardiac morphogenesis. Although hand2 is expressed in cardiomyocytes, mosaic analysis demonstrates that hand2 plays a non-autonomous role in regulating cardiomyocyte movement. Gene expression profiles reveal heightened expression of fibronectin 1 (fn1) in hand2 mutant embryos. Reciprocally, overexpression of hand2 leads to decreased Fibronectin levels. Furthermore, reduction of fn1 function enables rescue of cardiac fusion in hand2 mutants: bilateral cardiomyocyte populations merge and exhibit improved tissue architecture, albeit without major changes in apicobasal polarity. Together, our data provide a novel example of a tissue creating a favorable environment for its morphogenesis: the Hand2 pathway establishes an appropriate environment for cardiac fusion through negative modulation of Fn1 levels. Embryos from three independent hand2 mutant (hanS6 allele) heterozyogous crosses were collected. Examination of Tg(myl7:egfp) expression allow sorting of hand2 mutant embryos from their wild-type siblings.

心脏形成过程中，双侧心肌细胞群向胚胎中线迁移时需要发生融合。碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）转录因子Hand2对于心脏融合至关重要，但介导Hand2发挥该功能的效应基因至今仍未明确。本研究首次报道了介导心脏形态发生的Hand2信号通路下游组分。尽管hand2基因在心肌细胞中表达，但嵌合分析（mosaic analysis）结果显示，hand2通过非细胞自主方式调控心肌细胞的迁移。基因表达谱分析显示，hand2突变体胚胎中纤连蛋白1（fibronectin 1, fn1）的表达水平显著升高。反之，过表达hand2则会降低纤连蛋白的表达水平。进一步实验发现，降低fn1的功能可挽救hand2突变体的心脏融合缺陷：双侧心肌细胞群能够发生融合，且组织架构得到改善，尽管细胞顶基极性（apicobasal polarity）未出现显著变化。综上，本研究数据揭示了一种组织为自身形态发生构建有利微环境的全新范式：Hand2信号通路通过负调控纤连蛋白1的表达水平，为心脏融合建立适宜的环境。本研究收集了3个独立的hand2突变体（hanS6等位基因）杂合交配产生的胚胎。通过检测Tg(myl7:egfp)的表达，可将hand2突变体胚胎与其野生型同胞胚胎区分开来。