Microarray analysis of alternative splicing in PTBP2 knockout mouse brain

The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation. The splicing regulatory proteins PTBP1 and PTBP2 show distinct temporal expression profiles in the developing brain. Neuronal progenitor cells predominantly express PTBP1, whereas developing neurons express high levels of PTBP2, which are subsequently reduced late in neuronal maturation. We show here that PTBP2 and the program of splicing it controls are essential to proper neuronal maturation and survival. To investigate its in vivo function, we generated conditional PTBP2 null alleles in mice. Loss of PTBP2 in neuronal progenitor cells leads to neonatal death without gross defects in brain architecture. Mice with specific depletion of PTBP2 in the cortex and forebrain are viable. However over the first three postnatal weeks, when the normal cortex expands and develops mature circuits, the PTBP2 null cortices degenerate. We find that PTBP2-/- neurons cultured from embryonic brain show the same initial viability as wild type cells with proper early marker expression and neurite outgrowth. Strikingly, between 10 and 20 days in culture PTBP2 null neurons undergo a catastrophic failure to mature and die. To assess the target transcripts leading to these phenotypes, we examined the genomewide splicing changes in the PTBP2 null brains. This identified a large number of mis-regulated exons that share a temporal pattern of regulation; in the absence of PTBP2 many isoforms normally found in adults are precociously expressed in the developing brain. Transcripts following this pattern encode essential neuronal proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program essential for neuronal survival and maturation, where PTBP2 acts to temporarily repress expression of protein isoforms until the final maturation of the neuron. Mice carrying a conditional floxed PTBP2 allele of PTBP2 were crossed to mice carrying Cre recombinase driven by the nestin promoter. The resulting knockout mutant mouse brains were analyzed for changes in gene expression and alternative splicing. Knockout mice were compared to wildtype littermates. Whole mouse brain polyA plus RNA was isolated from three Nestin-cre knockout embryos at embryonic day 18 and compared to three wildtype littermates. RNA was converted to cDNA and used to probe Affymetrix MJAY splicing sensitive microarrays and analysed by Omniviewer to identify changes in splicing.剪接调控因子PTBP2调控神经元成熟所需的胚胎剪接程序。剪接调控蛋白PTBP1与PTBP2在发育中的大脑内呈现截然不同的时序表达模式：神经元祖细胞主要表达PTBP1，而发育中的神经元则高表达PTBP2，该蛋白的表达水平随后会在神经元成熟后期降低。本研究证实，PTBP2及其调控的剪接程序对于神经元的正常成熟与存活至关重要。为探究PTBP2的体内功能，我们在小鼠中构建了条件性PTBP2敲除等位基因。在神经元祖细胞中敲除PTBP2会导致新生小鼠死亡，但大脑结构并无明显异常。在大脑皮层与前脑特异性敲除PTBP2的小鼠可存活。然而在出生后的前三周（此时正常大脑皮层会扩张并形成成熟神经环路），PTBP2敲除的皮层会发生退行性病变。我们发现，从胚胎大脑中分离培养的PTBP2基因敲除（PTBP2-/-）神经元，其初始存活能力与野生型细胞一致，且早期标志物表达正常、神经突向外生长良好。值得注意的是，在培养至10至20天时，PTBP2敲除神经元会出现成熟障碍并发生灾难性死亡。为探究导致上述表型的靶标转录本，我们分析了PTBP2敲除大脑中的全基因组剪接变化。该分析鉴定出大量调控异常的外显子，这些外显子具有共同的时序调控模式：在PTBP2缺失的情况下，许多正常情况下仅在成年脑中表达的剪接异构体，会在发育中的大脑中提前表达。遵循该调控模式的转录本，其编码的关键神经元蛋白可影响神经突生长、突触前后组装以及突触传递。本研究结果确立了一套对神经元存活与成熟至关重要的全新遗传调控程序，其中PTBP2的作用是暂时抑制蛋白质异构体的表达，直至神经元最终成熟。将携带条件性floxed PTBP2等位基因的小鼠，与携带巢蛋白启动子驱动的Cre重组酶的小鼠进行杂交。对所得的敲除突变小鼠大脑进行分析，以检测基因表达与可变剪接的变化。将敲除小鼠与其野生型同窝仔鼠进行对照比较。我们从3只胚胎第18天的Nestin-cre敲除胚胎中分离全脑polyA+ RNA，并与3只野生型同窝仔鼠的样本进行对照。将RNA反转录为cDNA后，用于Affymetrix MJAY剪接敏感型微阵列的杂交检测，并通过Omniviewer软件分析以鉴定剪接变化。