The basis underlying microcephaly caused by defects in replication or DNA repair.

从灵长类动物到人类的进化导致了相对大脑大小的增加，这就需要胚胎神经干细胞进行快速分裂以扩大干细胞库。这个过程需要一个及时的转换，从分裂产生两个子干细胞/祖细胞到分裂产生一个子干细胞和一个分化的细胞，最终成为一个神经元，这被描述为从对称到不对称的细胞分裂的变化。不能适当地调节这种开关会严重影响神经元的形成。有效的神经发生需要大量促进快速复制的蛋白质，精确调节细胞分裂，以及细胞间的沟通以实现及时的转换。DNA损伤反应蛋白也需要修复由于快速复制和/或高代谢活性而引起的DNA损伤。这些突起的缺陷会导致头围小，这种情况被称为小头畸形。此外，胚胎大脑对DNA损伤剂极其敏感。小头性原始侏儒症（MPD）是一种头围小并伴有明显生长迟缓的疾病。最近，我们在MPD患者中发现了ORC1L突变。ORC1L编码Orc1，一种开始DNA复制所需的蛋白质。对Orc1缺陷患者细胞的分析表明，Orc1活性降低会损害维持快速增殖的能力。然而，最近的研究结果也表明，orc1缺乏会影响细胞通讯和/或细胞分裂的准确性。该建议的目的之一是了解orc1缺陷患者小头畸形的基础。LIG4综合征是另一种导致MPD的人类疾病，由编码DNA连接酶IV的基因LigIV突变引起，DNA连接酶IV是修复DNA双链断裂（DSBs）所需的蛋白质，DNA双链断裂是DNA损伤的重要形式。使用LIG4综合征的小鼠模型（LigIVY288C），我们已经证明在LigIVY288C胚胎的神经干和早期分化室中存在细胞死亡（称为凋亡）增加。同时，我们观察到更多的DSB，可能是由于DSB修复缺陷。损伤水平比其他组织更明显，这表明快速增殖的干细胞产生了高水平的dsb。然而，我们不知道细胞死亡的增加是否会影响下游的发育步骤，包括从对称细胞分裂到不对称细胞分裂的转变。我们还观察到胚胎神经干细胞在低剂量辐射照射后对细胞死亡（凋亡）异常敏感。此外，我们的目标是继续对liivy288c小鼠的神经发生和低水平辐射的影响进行分析。我们将利用在orc1缺陷患者中观察到的突变变化来创建敲入小鼠模型。利用我们在LigIVY288C胚胎研究中已经开发的程序，以及新的方法，我们将研究DNA连接酶IV， Orc1或辐射暴露的缺陷如何影响胚胎干细胞的增殖能力，从对称到不对称细胞分裂转换的时间以及分化细胞的迁移。我们的工作将为有效神经发生所需的关键过程提供见解。鉴于胚胎神经细胞对辐射的高度敏感性，我们的工作将为优化怀孕期间胚胎的护理提供信息。最后，胚胎神经干细胞室是研究干细胞生物学的理想系统，因为干细胞、祖细胞和分化细胞可以通过位置和/或标记进行区分。因此，我们的工作将为有效的干细胞复制和编程到分化状态所需的因素提供更广泛的见解。

Evolution from primates to humans has resulted in increased relative brain size, necessitating that the embryonic neuronal stem cells undergo rapid division to expand the stem cell pool. This process necessitates a timely switch from divisions generating two daughter stem/progenitor cells to divisions generating a daughter stem cell and a differentiated cells that finally becomes a neuron, described as a change from symmetric to asymmetric cell division. Failure to appropriately regulate this switch can severely impact upon neuron formation. Efficient neurogenesis requires a plethora of proteins promoting rapid replication, precision in regulating cell division, as well as communication between cells to effect the timely switches. DNA damage response proteins are also required to repair DNA damage that arises as a consequence of rapid replication and/or high metabolic activity. Defects in these processes can cause small head circumference, a condition described as microcephaly. Further, the embryonic brain is extremely sensitive to DNA damaging agents. Microcephalic primordial dwarfism (MPD) is a disorder in which small head circumference is accompanied by marked growth delay. Recently, we identified mutations in ORC1L in patients with MPD. ORC1L encodes Orc1, a protein required for the commencement of DNA replication. Analysis of cells from Orc1-deficient patients suggested that reduced Orc1 activity impairs the ability to sustain rapid proliferation. However, recent findings also suggest that Orc1-deficiency can impact upon cell communication and/or the precision of cell division. One aim of this proposal is to understand the basis underlying microcephaly in Orc1-deficient patients. LIG4 Sydrome is another human disorder conferring MPD, which arises from mutations in LigIV, a gene encoding DNA ligase IV, a protein required for the repair of DNA double strand breaks (DSBs), an important form of DNA damage. Using a mouse model (LigIVY288C) for LIG4 Syndrome, we have shown that there is increased cell death (termed apoptotis) in the neuronal stem and early differentiated compartments of LigIVY288C embryos. Concomitantly, we observed more DSBs, likely due to the DSB repair defect. The level of damage is more marked than in other tissues, suggesting that the rapidly proliferating stem cells incur high levels of DSBs. However, we do not know whether the elevated cell death impacts upon downstream developmental steps including the switch from symmetric to asymmetric cell division. We have also observed that the embryonic neuronal stem cells are hypersensitive to cell death (apoptosis) after low dose radiation exposure. We aim additionally to pursue our analysis of neurogenesis in the LigIVY288C mouse and the impact of low levels of radiation. We will exploit the mutational changes observed in Orc1-deficient patients to create a knock-in mouse model. Using procedures already developed from our study of LigIVY288C embryos, together with new approaches, we will examine how deficiency in DNA ligase IV, Orc1, or radiation exposure affects the proliferation capacity of the embryonic stem cells, the timing of the switch from symmetric to asymmetric cell division and the migration of the differentiated cells. Our work will provide insight into critical processes required for efficient neurogenesis. Given the high sensitivity of the embryonic neuronal cells to radiation, our work will provide information to optimise the care of embryos during pregnancy. Finally, the embryonic neuronal stem cell compartment is an ideal system for studying stem cell biology since the stem, progenitor and differentiated cells can be distinguished by position and/or markers. Thus, our work will provide broader insight into factors required for efficient stem cell replication and programming to a differentiated state.