Role of RFX4 in Brain Development and Function

该项目的启动是由于观察到在六个心脏特异性过表达人CYP 2 J2的转基因品系中的一个中，大部分小鼠表现出头部肿胀，随后迅速神经功能恶化并在成年早期死亡。我们假设转基因中断了一个重要基因的编码或调控区。我们鉴定了与单个转基因插入位点相邻的5 ′和3 ′基因组序列，发现它们与含有RFX 4基因座的人类12号染色体序列高度相关。将转基因插入RFX 4基因的内含子中，这种插入阻止了导致先天性脑积水发展的新变体转录物（称为RFX4_v3）的表达。我们发现野生型（+/+）和转基因中断的等位基因（一个中断的等位基因为+/-，两个中断的等位基因为-/-）可以通过Southern印迹和PCR轻易区分。来自半合子（+/-）小鼠的脑表达约50%的正常水平的RFX4_v3转录物，并表现出与连合下器官（SCO）形成失败相关的侧脑室和第三脑室的严重先天性脑积水，导致Sylvius导水管狭窄。与此相反，纯合子无效（-/-）小鼠端脑形成有严重缺陷，导致产前脑畸形和围产期死亡。事实上，在E12.5时对-/-小鼠的研究表明，它们失去了关键的中线结构，包括半球间裂，导致形成单个中央脑室而不是两个侧脑室。尽管脊髓也畸形，但-/-小鼠的面部结构、视网膜、嗅上皮和所有其他检查的非脑组织均未受影响。克隆了人和小鼠RFX4_v3 cDNA，发现其96%相同，表明该变体转录物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RFX 4转录本共享的外显子组成。RFX4_v3转录物仅在脑中表达，并且最初出现在E7.5和E9.5之间。原位杂交结果表明，RFX4_v3的表达是高度动态的大脑发育过程中。重要的是，在E14.5时，在尾部间脑中发育中的SCO区域中发现RFX4_v3的丰富表达。总之，这些数据表明RFX4_v3对正常的大脑发育至关重要。此外，SCO在+/-小鼠中形成的失败表明，这种不寻常的脑器官对RFX4_v3的正常发育表达极其敏感。为了鉴定RFX4_v3野生型和突变型小鼠脑中差异表达的基因，从胚胎第10.5天（E10.5）的头部分离RNA，并用Agilent小鼠寡核苷酸微阵列进行杂交。在本研究中使用四对单独的RNA和一对合并的RNA，并且每对样品与两个寡核苷酸微阵列杂交，每个RNA样品用每个荧光团标记以解释荧光团掺入偏倚。在5对样本中至少有4对样本中，34个基因表达上调，75个基因表达下调。基于i）通过微阵列分析的相对大的倍数变化，和ii）在脑发育期间的潜在重要功能，选择离群值列表中的24个基因用于通过实时PCR进行验证。实时PCR结果表明，24个基因中的22个被证实差异表达，与微阵列数据相比，具有相似或更大的倍数变化。一些已验证的基因是众所周知的神经元发育调节因子。这些潜在的下游基因的检查可能会导致更好地了解RFX4_v3在大脑发育中的功能的分子机制。微阵列数据和实时PCR结果之间的高度相关性证明了使用微阵列在脑形态发生过程中识别转录因子的潜在下游靶基因的实用性。

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. To identify the differentially expressed genes in the brains of RFX4_v3 wild-type and mutant mice, RNAs were isolated from the embryonic day 10.5 (E10.5) heads and hybridizations were performed with the Agilent mouse oligo microarrays. Four pairs of individual RNAs and one pair of pooled RNAs were used in the present studies and each pair of samples was hybridized to two oligonucleotide microarrays with each RNA sample labeled with each fluorophore to account for fluorophore incorporation bias. Thirty-four genes were up-regulated and 75 genes were down-regulated in at least 4 out of 5 pairs of samples. Twenty-four genes in the outlier lists were chosen for validation by real-time PCR based on i) relatively large fold changes by microarray analysis, and ii) potentially important functions during brain development. Real-time PCR results indicated that 22 out of 24 genes were confirmed to express differentially with similar or larger fold changes compared to microarray data. Some of the validated genes are well-known regulators for neuronal development. Examination of these potential downstream genes may lead to better understanding of the molecular mechanisms underlying the function of RFX4_v3 in brain development. The high correlation between microarray data and real-time PCR results demonstrates the utility of using microarrays in identifying potential downstream target genes of transcription factors during brain morphogenesis.