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

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.

We aim to understand the basis underlying microcephaly in humans. We will study neurogenesis in mouse embryos since this represents a good model system. We will examine neurogenesis in mice deficient in Orc1, a condition recently found to cause microcephaly in patients. We will also examine mice deficient in DNA ligase IV and following exposure to ionising radiation.
Mouse embryos at E13.5 to E17 will be obtained from mice and subjected to PCR analysis to assess their genotype. Studies will be performed on heterozygous crosses, allowing homozygous deficient and heterozygous mice from identical litters to be examined in parallel. Our studies with LigIVY288C mice have shown that embryonic neuronal development is similar in wild type and heterozygous mice. This will be established for Orc1-deficient mice. The embryonic neuronal tissues will be snap frozen or prefixed (depending on the analysis to be undertaken), sliced and processed for immunostaining using neuronal markers and/or antibodies to DNA damage response proteins or markers of replication. We have established that p-histone H3 staining, which marks the apical and basal mitotic layers, provides a suitable marker to separate the VZ/SVZ from the IZ. Neuronal markers will include Tbr2, Tuj1 and Pax6. Apoptosis will be assessed by TUNEL staining and DNA double strand breakage identified by gammaH2AX or 53BP1 foci analysis. Where necessary BrdU will be given to the mice by peritoneal injection. Where necessaryembryos mice will be irradiated in utero with low doses using an X-ray source and maintained as necessary prior to sacrifice. These procedures have predominantly been described previously REF.
Mouse embryo fibroblasts (MEFs) will be obtained from Orc-1 deficient mice using standard procedures and examined using assays described in our recent publication (Bicknell et al., Nat Genet. 43: 350-355, 2011).
Generation of an Orc1-deficient knock-in mice will be undertaken using a standard procedure as described.

1) One class of person benefitting from the research will be patients (and their families) with microcephaly or microcephalic primordial dwarfism.
Although the work is unlikely to identify a means to reverse either their growth delay or microcephaly, the work should
a) Provide a clinical diagnosis and greater understanding of the basis underlying microcephaly. From past experience, the parents of such patients find this beneficial.
b) Enhance our ability to carry out prenatal diagnosis.
c) Enhance the care of such patients eg by assessing whether exposure to diagnostic tools such as CT scanning/X-rays causes greater cell death and/or other phenotypes in such patients.
d) Enhance our ability to identify further candidate genetic defects causing microcephaly.

2) Regulators evaluating the impact of exposure to radiation and/or other DNA damaging agents could also benefit from the work. Gaining further insight into how radiation exposure affects neurogenesis is the most important aspect here. Additionally, chemicals can cause replication fork arrest and the work could be of significance in that context.

3) Scientists and/or the commercial sector interested in optimising stem cell growth. The exploitation of stem cell biology is a currently advancing area of research with potential enormous benefit both for repairing tissue damage and for other medical procedures. Further understanding factors required for stem cell proliferation may be exploitable to prevent cancer stem cell growth. Glioblastoma represent a neurological stem cell tumour that is particularly hard to treat. Any procedure that might help restrict the growth of the glioblastoma stem cell has the potential to be exploitable.