Hox genes encode transcription factors that have been implicated in embryonic, adult and disease processes. The earliest developmental program known to be directed by Hox genes is the timing of ingression of presumptive axial mesoderm during gastrulation. We previously used morpholino (MO)‐based knockdown to implicate the zebrafish hoxd4a gene in the specification of the hemangioblast, an event occurring at pre‐gastrulation stages, well before the earliest known Hox gene function. The precise time at which hoxd4a function is required for this specification is not defined. We therefore fused the hoxd4a coding region to the human estrogen receptor (hER^T2). Following co‐injection of anti‐hoxd4a MO with mRNA encoding the Hoxd4a‐ER^T2 fusion protein, hemangioblast specification was fully rescued when embryos were exposed to the estrogen analog 4‐hydroxy‐tamoxifen (4‐OHT) at 4 hr post‐fertilization (hpf), but only poorly at 6 hpf and not at all at 8 hpf, thereby defining a pre‐gastrulation role for Hoxd4a, the earliest developmental function of a vertebrate Hox gene so far described. Both DNA binding and interaction with cofactor Pbx were further shown to be required for rescue of the morphant phenotype. Confirmation of the morphant phenotype was sought via the generation of hoxd4a null mutants using CRISPR/Cas9 technology. Null mutants of hoxd4a up to the third generation (F₃) failed to recapitulate the morphant phenotype, and were largely refractory to the effects of injected anti‐hoxd4a MO suggesting the action of genetic compensation.

中文翻译：

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斑马鱼的hox基因在胃化之前起作用，以指定成血管细胞。

Hox基因编码与胚胎，成年和疾病过程有关的转录因子。已知由Hox基因指导的最早的发育程序是推定胚性中胚层进入胃的时间。我们以前使用基于吗啉代（MO）的敲低将斑马鱼hoxd4a基因包含在成血成血管细胞的规格中，该事件发生在孕前阶段，早于已知的最早的Hox基因功能。没有定义此规范要求hoxd4a函数的确切时间。因此，我们将hoxd4a编码区与人类雌激素受体（hER ^T2）融合在一起。共注射抗hoxd4a后MO带有编码Hoxd4a-ER ^T2融合蛋白的mRNA ，当胚胎在受精后4小时（hpf）暴露于雌激素类似物4-羟基-他莫昔芬（4-OHT），但在6岁时却很差，可以完全拯救成血管细胞规范hpf而根本没有达到8 hpf，从而定义了Hoxd4a的预妊娠作用，Hoxd4a是迄今为止描述的脊椎动物Hox基因的最早发育功能。DNA结合和与辅因子Pbx的相互作用都进一步证明了挽救形态表型是必需的。通过使用CRISPR / Cas9技术生成hoxd4a无效突变体来寻求对表型的确认。hoxd4a的无效突变体直至第三代（F ₃）未能概括出吗啡表型，并且对所注射的抗hoxd4a MO的作用有很大的抵抗力，提示了遗传补偿的作用。