Role of RFX4 in Brain Development and Function

该项目的启动是由于观察到，在6个心脏特异性过表达人类CYP2J2的转基因系中，有很大比例的小鼠在成年早期表现出头部肿胀，随后出现快速的神经退化和死亡。我们假设转基因中断了一个重要基因的编码区或调控区。我们鉴定了单个转基因插入位点附近的5 ‘和3 ’基因组序列，发现它们与包含RFX4位点的人类12号染色体序列高度相关。该转基因被插入到RFX4基因的内含子中，该插入阻止了一种新的变异转录物（称为RFX4_v3）的表达，从而导致先天性脑积水的发展。我们发现野生型（+/+）和转基因中断等位基因（+/-表示一个中断等位基因，-/-表示两个中断等位基因）可以很容易地通过Southern blotting和PCR区分。半合子（+/-）小鼠的大脑表达RFX4_v3转录本的水平约为正常水平的50%，并表现出严重的先天性侧脑室和第三脑室脑积水，伴有合骨下器官（SCO）形成失败，导致Sylvius输水管道狭窄。相比之下，纯合子null（-/-）小鼠在端脑形成方面存在严重缺陷，导致产前脑畸形和围产期死亡。事实上，对E12.5的-/-小鼠的研究表明，它们已经失去了关键的中线结构，包括半球间裂缝，导致形成一个单一的中央脑室而不是两个侧脑室。虽然脊髓也畸形，但面部结构、视网膜、嗅上皮和所有其他非脑组织检查在-/-小鼠中未受影响。克隆了人和小鼠的RFX4_v3 cdna，发现96%相同，表明该变异转录本在这两个物种之间高度保守。基因组序列分析显示，小鼠RFX4_v3转录本由独特的外显子和与其他RFX4转录本共享的外显子组成。RFX4_v3转录本仅在脑中表达，最初出现在E7.5 ~ E9.5之间。原位杂交显示RFX4_v3的表达在大脑发育过程中是高度动态的。重要的是，在E14.5时，在尾间脑发育中的SCO区域发现了大量RFX4_v3的表达。总之，这些数据表明RFX4_v3对正常的大脑发育至关重要。此外，在+/-小鼠中SCO的形成失败表明，这种不寻常的大脑器官对RFX4_v3的正常发育表达极其敏感。

This project was initiated by the observation that a large percentage of mice in one of six transgenic lines with cardiac-specific overexpression of human CYP2J2 exhibited head swelling followed by rapid neurological deterioration and death in young adulthood. We hypothesized that the transgene had interrupted the coding or regulatory region of an important gene. We identified the 5 prime and 3 prime genomic sequences adjacent to the single transgene insertional site and found them to be highly related to a human chromosome 12 sequence that contained the RFX4 locus. The transgene was inserted into an intron of the RFX4 gene and this insertion prevented expression of a novel variant transcript (termed RFX4_v3) which led to the development of congenital hydrocephalus. We found that wild type (+/+) and transgene-interrupted alleles (+/- for one interrupted allele and -/- for two interrupted alleles) could be readily distinguished by Southern blotting and PCR. Brains from hemizygous (+/-) mice expressed approximately 50% of normal levels of the RFX4_v3 transcript and exhibited severe congenital hydrocephalus of the lateral and third ventricles associated with failure of formation of the subcommissural organ (SCO), leading to stenosis of the aqueduct of Sylvius. In contrast, the homozygous null (-/-) mice had a severe defect in telencephalon formation that led to gross prenatal brain malformations and death in the perinatal period. Indeed, investigation of -/- mice at E12.5 showed that they had lost critical midline structures including the interhemispheric fissure resulting in the formation of a single central ventricle instead of two lateral ventricles. Although the spinal cords were also malformed, facial structures, retinas, olfactory epithelium and all other non-brain tissues examined were unaffected in the -/- mice. The human and mouse RFX4_v3 cDNAs were cloned and found to be 96% identical, indicating that this variant transcript was highly conserved between these two species. Analysis of genomic sequences revealed that the mouse RFX4_v3 transcript was composed of both unique and shared exons with other RFX4 transcripts. The RFX4_v3 transcript was expressed only in brain and initially appeared between E7.5 and E9.5. In situ hybrization revealed that RFX4_v3 expression was highly dynamic during brain development. Importantly, abundant expression of RFX4_v3 was found in the region of the developing SCO in the caudal diencephalon at E14.5. Together, these data indicate that RFX4_v3 is critical for normal brain development. Moreover, the failure of formation of the SCO in +/- mice suggests that this unusual brain organ is extremely sensitive to normal developmental expression of RFX4_v3.