Functional analysis of ZFHX1 family transcription factors in embryonic development

ZFHX1转录因子家族在高等脊椎动物中由δ-晶体蛋白增强因子1 （δ-crystallin enhancer factor 1）和SIP1 （Smad-interacting protein 1）组成，包含高度保守的锌指簇和一个同源结构域。在小鼠胚胎中，这些蛋白基因的表达在某些组织（如神经板和颅神经节）中广泛重叠，也经常发生在同一组织的邻近区域（如颅间质和神经管）。为了研究这两个因素之间的功能关系，我们产生了复合敲除小鼠胚胎并研究了由此产生的表型。SIP1-/-纯合子胚胎在E8后出现形态异常，前神经板闭合缺陷，颅神经嵴迁移停止，体发育缺陷；而δEF1-/-纯合子胚胎仅在妊娠中期后出现轻微缺陷。δEF1 - / -;SIP1-/-双纯合子胚胎在前神经板上的表型比SIP1-/-纯合子胚胎更严重，即神经板薄，与非神经外胚层没有明确的形态界限。该表型的严重程度与Sox2表达水平的降低高度相关。由于SIP1-/-纯合子胚胎在E9后死亡，在δEF1-/-条件下检测了ZFHX1蛋白复合缺乏的影响；SIP1 + / -。在这种情况下，δEF1-/-纯合子胚胎出现了多种形态缺陷（如脑室发育紊乱、尾部神经管打开），δEF1-/-纯合子胚胎出现了多种形态缺陷（如颅面畸形）。复合突变体的这些表型表明，δEF1和SIP1在其表达重叠的地方具有主要的冗余功能。

ZFHX1 family of transcription factors, containing highly conserved two-partite zinc finger clusters and a homeodomain, comprises δEF1 (δ-crystallin enhancer factor 1) and SIP1 (Smad-interacting protein 1) in higher vertebrates. In mouse embryo, expression of these protein genes overlap extensively in some tissues (e.g., neural plate and cranial ganglia) and often also take place in adjacent domains of the same tissue (e.g., cranial mesenchyme and neural tube). To investigate the functional relationships between these two factors, we generated compound knockout mouse embryos and investigated the resulting phenotype. SIP1-/- homozygous embryos develop morphological abnormality after E8, defect in anterior neural plate closure, arrest of migration of cranial neural crest, and defective somitogenesis ; by contrast, δEF1-/- homozygous embryos display mild defects only after mid-gestation period. δEF1-/- ; SIP1-/- double homozygous einbryos show severer phenotype than SIP1-/- homozygous embryo in the anterior neural plate, namely thin neural plate without a clear morphological boundary to the non-neural ectoderm. The severity of this phenotype is highly correlated with decrease of Sox2 expression level. As SIP1-/- homozygous embryos die after E9, effect of compounded deficiency of ZFHX1 proteins was examined under the condition of δEF1-/- ; SIP1+/-. Under this condition, various morphological defects either not observed in δEF1-/- homozygous embryos (e.g., disorganized development of brain ventricles, and open neural tube in the tail) or in strengthened form of milder defects in δEF1-/- homozygous embryos (e.g., craniofacial abnormality). These phenotypes of the compound mutants indicate that δEF1 and SIP1 have primarily redundant functions where their expression overlaps.