Functional analysis of ZFHX1 family transcription factors in embryonic development

ZFHX 1家族是一个高度保守的转录因子家族，包括δ-晶体蛋白增强因子1（δ-crystallin enhancer factor 1，δ EF 1）和Smad-interacting protein 1（SIP1）。在小鼠胚胎中，这些蛋白质基因的表达在一些组织中广泛重叠（例如，神经板和颅神经节）并且通常也发生在同一组织的相邻区域（例如，颅间充质和神经管）。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、颅神经嵴迁移停滞和体节发生缺陷;而δ EF1-/-纯合子胚胎仅在妊娠中期后出现轻度缺陷。δ EF 1-/--; SIP1-/-双纯合子胚胎在前神经板中表现出比SIP1-/-纯合子胚胎更明显的表型，即神经板较薄，与非神经外胚层没有明显的形态边界。这种表型的严重程度与Sox 2表达水平的降低高度相关。由于SIP1-/-纯合胚胎在E9后死亡，在δ EF1 - 1-; SIP1 +/-的条件下检查ZFHXI蛋白的复合缺陷的影响。在这种条件下，在δ EF 1-/-纯合胚胎中没有观察到的各种形态缺陷（例如，脑室发育紊乱，尾部神经管开放）或SEF1-/-纯合胚胎中较轻度缺陷的强化形式（例如，颅面异常）。复合突变体的这些表型表明δ EF1和SIP1在其表达重叠处主要具有冗余功能。

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 embryos 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 ZFHXI proteins was examined under the condition of δEF1-1-;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 SEF1-/- 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.