Expression profiling by high throughput sequencing
Summary
Purpose: The transcription factor p53 classically regulates the cellular response to DNA damage. Inappropriate activation of p53 during embryonic development can lead to a range of developmental defects such as microcephaly. Here, we studied the cellular and molecular mechanisms underlying microcephaly induced by acute irradiation of mouse fetuses at the onset of neurogenesis. This leads to transient DNA damage followed by apoptosis and premature neuronal differentiation, both of which are p53-dependent as they are reduced in dorsal forebrain-specific Trp53 knockout mice. A functional genomics screen shows that radiation induces the p53-dependent activation of both apoptosis- and differentiation-associated genes. Irradiation furthermore induces an epithelial-to-mesenchymal transition-like process characterized by a reduction of epithelial markers and a disruption of the apical adherens junction belt. Altogether, we have identified a novel function of p53 as an activator of neuronal differentiation in response to acute embryonic DNA damage further demonstrating its importance as a regulator of cell fate. Methods: Pregnant mouse dams were (sham-)irradiated at gestational day 11 after which they were sacrificed for dissection of fetuses after 2 h, 6 h and 12 h. Cerebral cortices were removed for transcriptomic analysis via RNA-seq.
Overall design
Mice were coupled during a 2-h time period and the morning of coupling was counted as embryonic day (E) 0. Pregnant dams were (sham-)irradiated at E11 after which they were sacrificed for dissection of fetuses after 2 h, 6 h and 12 h. Cerebral cortices were subsequently removed for transcriptomic analysis. Both wild-type (WT) mice and Emx1-cre;Trp53-LoxP mice (cKO) were used, and (sham)irradiated as described before. For each experimental condition 3 individual fetuses were used from at least 2 different mothers except for cKO_1 Gy_6 h for which all three fetuses were littermates. Fetal cortices were dissected and stored in RLT Plus lysis buffer (Qiagen) at -80°C until further processing.
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