Understanding the role of minor intron splicing in cortical development

小头畸形是一种毁灭性的发育缺陷，可由遗传突变引起，如 寨卡病毒感染的症状有哪些？为了更好地理解 潜在的分子和细胞缺陷，导致小头畸形，我们必须首先了解分子和 正常发育过程中的细胞变化。目前的建议扩展了我们的发现，失活的 在我们的U11条件性敲除（cKO）小鼠中，与Emx 1-Cre杂交的小剪接体导致： 出生时观察到的小头畸形。我们发现，小头畸形的主要原因是自我放大功能的丧失， 放射状胶质细胞（RGC）和中间祖细胞（IPC）的延迟死亡，而没有相应的 皮质发育早期神经元的丧失。尽管在E14中NPC完全消失， 突变的皮质设法产生通常在E14/之后出生的第IV层神经元。我们发现这 神经元产生的变化与通过EdU脉冲/追踪测量的神经发生增加有关 实验基于这些复杂的表型，我们提出了三个目标来检验假设， 小剪接体以细胞类型特异性方式起作用，以告知细胞周期调节、NPC能力和 神经元生成目的1a中提出的实验旨在阐明细胞周期的精确调控 以及这两种细胞类型是否正在进行自我扩增或神经源性细胞 分裂在目标1b中，我们探索了U11无效RGC和IPC的变化如何影响神经元的产生。在aim中 目的是了解U11丢失的细胞类型特异性效应的分子基础。所述一 RGC的唯一标识符是它们与IPC相比分裂迅速，因此目的中提出的实验 2a检验细胞周期速度赋予对U11丢失的敏感性的假设。另一种可能是 目标2b中提出的实验是每种细胞类型都具有独特的含小内含子基因的标记 （MIGs）可能使细胞对U1丢失具有抗性/易感性。最后，RNAseq数据显示，P53- 介导的凋亡途径和细胞周期缺陷的U11-null组织。目标3a中提出的实验 测试挽救细胞周期缺陷是否会阻止P53介导的凋亡或P53介导的凋亡 与细胞周期缺陷无关。目标3b测试了这样的想法，如果P53是消融，它会拯救细胞周期还是 细胞死亡进而挽救小头畸形。总之，我们将发现这种新的基因调控形式在以下方面的作用： 皮质发育，反过来又提供了对在疾病中观察到的小头畸形的见解， 1型小头骨发育不良性原始侏儒症（MOPD 1）和Roifman综合征， 由有缺陷的小剪接体引起的。

Microcephaly is a devastating developmental defect that can be caused by inherited mutations such as those that inactivate the minor spliceosome or more recently Zika virus infections. In order to better understand the underlying molecular and cellular defects that cause microcephaly, we must first understand the molecular and cellular changes during normal development. The current proposal extends our finding that inactivation of the minor spliceosome in our U11 conditional knockout (cKO) mouse crossed with Emx1-Cre results in microcephaly observed at birth. We found that the primary cause of the microcephaly is loss of self-amplifying radial glial cells (RGCs) and delayed death of intermediate progenitor cells (IPCs) without the corresponding loss of neurons during early cortical development. Despite the complete loss of NPCs by E14, the developing mutant cortex managed to produce layer IV neurons that are normally born at/after E14. We found that this shift in neuron production is in conjunction with increased neurogenesis measured by EdU pulse/chase experiments. Based on these complex phenotypes, we have proposed three aims to test the hypothesis that minor spliceosome acts in a cell-type specific manner to inform cell cycle regulation, NPC competence, and neuron production. Experiments proposed in aim 1a are designed to elucidate the precise cell-cycle regulation of RGCs and IPCs and whether these two cell-types are undergoing self-amplifying or neurogenic cell divisions. In aim 1b, we explore how the changes in U11-null RGCs and IPCs impact neuron production. In aim 2, the objective is to understand the molecular underpinning of the cell-type specific effect of U11 loss. The one unique identifier of RGCs is that they divide rapidly compared to the IPCs, so the experiments proposed in aim 2a test the hypothesis that cell cycle speed confers susceptibility to loss of U11. Another possibility that the experiments proposed in aim 2b is that each cell-type has a unique signature of minor intron-containing genes (MIGs) that might make cells resistant/susceptible to U1 loss. Finally, RNAseq data showed activation of P53- medated apoptosis pathway and cell cycle defect in the U11-null tissue. The experiments proposed in aim 3a test whether rescuing cell cycle defect would prevent P53-mediated apoptosis or is P53-mediated apoptosis independent of the cell cycle defect. Aim 3b tests the idea that if P53 is ablation, would it rescue cell cycle or cell death and in turn rescue microcephaly. In all, we will find the role of this novel form of gene regulation in cortical development and in turn provide insights into microcephaly observed in diseases such as microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1) and Roifman syndrome that are both caused by defective minor spliceosome.