Functional characterization of D630023F18Rik in mouse brain development

Radiation may cause tissue and/or organ damage, such as damage to DNA. The brain is the most sensitive organ for irradiation, leading for example, to microcephaly (“small head”). DNA damage is a constantly occurring event and cells must be able to deal with it to maintain viability, and to prevent that cells harboring damaged genetic material undergo replication. Upon radiation-induced DNA damage cell cycle arrest occurs, which is regulated by p53, a tumor suppressor gene. P53 is a transcription factor involved in many different processes, among which, cell cycling, DNA repair, apoptosis and senescence. DNA damages activates p53 by for example disrupting its interaction with Mdm2. In turn, p53 is able to affect gene transcription. In the past, multiple p53 target genes have already been identified and it seems that after radiation exposure, p53 preferentially targets developmentally regulated genes. In 2015, researchers at SCK CEN identified novel p53 targets that are activated in the embryonic mice brain after irradiation. One of the most activated genes was D630023F18Rik. D630023F18Rik is characterized by the expression of several long and short transcript isoforms. Induction of p53 expression upon irradiation results in alternative transcription of D630023F18Rik due to p53 binding to an alternative promotor. This leads to the expression of the radiation responsive gene part of D630023F18Rik (short-transcript variants). It was previously found that expression of long transcripts is strongly induced during in vivo brain development and in vitro neuronal differentiation, suggesting that D630023F18Rik might be relevant in brain development and neuronal maturation. It is hypothesized that the short isoforms may be involved in p53-dependent premature differentiation of neuronal progenitors after irradiation. In this thesis, I contributed to the characterization of this promising gene D630023F18Rik. We investigated the effectivity/specificity of different D630023F18Rik antibodies on both wild-type (WT) and knock-out (KO) mouse brains and neuron progenitor cells. The tested antibodies were found to be non-specific for D630023F18Rik. To enhance antibody specificity and to limit background signals both on western blots and immunohistochemistry (IHC) assays, a switch from polyclonal antibodies to monoclonal antibodies can be considered. For future work, it can be investigated what exactly is recognized by these antibodies, for example by mass spectrometry. QPCR analysis of D630023F18Rik showed the lack of D630023F18Rik Exon3-4 expression in the KO and 50% reduction in heterozygote expression levels, which confirmed successful D630023F18Rik knock-out. Furthermore, this analysis revealed an increase of D630023F18Rik short transcripts expression in response to irradiation. Finally, to investigate whether the absence of D630023F18Rik long transcript variants affects overall brain morphology, cortical layering or interneuron dispersion, immunohistochemistry assays were performed with different markers for cortical layers at two distinct developmental time points, embryonic day 18 (E18) and post-natal day 30 (P30). D630023F18Rik KO mice did not display major abnormalities in cortical lamination. A more detailed analysis of D630023F18Rik expressing neuronal subtypes can be further assessed to account for more subtle differences that were not detected in this study. Furthermore, behavioral assays can be conducted to investigate possible impairments in brain function as a consequence of D630023F18Rik knock-out.