The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.

斑马鱼是一种出色的模型动物，适合于遗传学方法的发展。为了揭示脊椎动物中未知的发育调控机制，我们进行了化学诱变筛选，并鉴定了一个新的突变kanazutsi（kzt）。这种突变是隐性的，其纯合子具有胚胎致死性。突变的胚胎遭受各种形态学缺陷，例如头部扁平，心包水肿，循环缺陷，黑素细胞分布模式破坏，眼睛矮小，下颌缺损以及头部广泛的细胞凋亡，这表明主要受影响的组织是衍生的来自神经rest细胞（NCC）。组织特异性标志物在kzt中的表达突变体表明，NCC的早期规格是正常的，但其后期分化受到严重影响。通过连锁分析将突变定位到3号染色体，靠近细胞球蛋白1（cygb1），其产物是球蛋白家族呼吸蛋白。尽管基因产物具有正常的初级结构，但在野生型胚胎的体节和颅中NCC中，cygb1的表达被激活，但在突变型胚胎中却被显着下调。所述KZT突变通过表型模拟cygb1敲低与低剂量吗啉代寡聚物和通过部分地救出cygb1过表达。cygb1的严重敲低和无效突变通过CRISPR / Cas9技术建立的cDNA在早期导致了严重得多的缺陷。因此，很有可能cygb1的下调是导致kzt突变的许多（即使不是全部）表型的原因。这些结果揭示了脊椎动物胚胎中对球蛋白家族蛋白的需求，特别是在NCC的分化和后续发育中。