Dynamic acetylation profile during mammalian neurulation

Abstract

Background: Neural tube defects (NTDs) result from failure of neural tube closure during embryogenesis. These severe birth defects of the central nervous system include anencephaly and spina bifida, and affect 0.5-2 per 1,000 pregnancies worldwide in humans. It has been demonstrated that acetylation plays a pivotal role during neural tube closure, as animal models for defective histone acetyltransferase proteins display NTDs. Acetylation represents an important component of the complex network of posttranslational regulatory interactions, suggesting a possible fundamental role during primary neurulation events. This study aimed to assess protein acetylation contribution to early patterning of the central nervous system both in human and murine specimens.

Methods: We used both human and mouse (Cited2 ^-/- ) samples to analyze the dynamic acetylation of proteins during embryo development through immunohistochemistry, western blot analysis and quantitative polymerase chain reaction.

Results: We report the dynamic profile of histone and protein acetylation status during neural tube closure. We also report a rescue effect in an animal model by chemical p53 inhibition.

Conclusions: Our data suggest that the p53-acetylation equilibrium may play a role in primary neurulation in mammals.

Keywords: Cited2; acetylation profile; neural tube defects; neurodevelopment; p53.

Figure 1

Acetylation status is dynamic during human and mouse neural tube development. (a) Cells staining for acetylated‐histone H4 antibody are present in the normal developing human neural tube (Control), both at 9 and 20 WG. Note positive‐staining cells also in the spinal cord of a 21 WG individual with spina bifida (Case). Bar represents 250 μm. The orange line represent the level of histological section (lumbar for all analyzed fetuses). (b) Quantification of acetylated‐histone H4 staining: the median value is shown as a horizontal line inside the boxes; upper and lower limits of the boxes represent upper and lower quartiles, respectively; whiskers represent the maximum and minimum values. Among normal fetuses (controls), an early stage of gestation (9 WG) has a higher number of acetylated‐histone H4 positive cells per unit area than stages from 12 WG onward. The fetus with spina bifida (case) shows a similar density of positive cells as unaffected individuals of a comparable stage. (c) Western blot demonstrates the dynamic acetylation profile of wild‐type mouse embryos at different developmental stages. Note the band at 50 KDa which has a peak of expression intensity at E9.5. (d) The acetylation profile of wild‐type and Cited2 ^−/− embryos without (closed) and with (open) exencephaly. A 50 KDa band is upregulated in Cited2 ^−/− embryos compared with wild‐type, with particularly strong acetylation in mutants with open neural tube

Figure 2

p53 acetylation contribution to the Cited2 ^−/− NTD phenotype. (a) Quantification of acetylated p53, normalized to actin expression, shows an increase in affected Cited2 ^−/− embryos (three embryos per pool per genotype were used. Three replicates were performed). Bars show standard deviation. (b) Relative western blot analysis of acetylated p53. (c) In contrast, total p53 gene expression (qPCR) does not differ between Cited2 genotypes at E9.5. (d) Chemical inhibition of p53 activity using pifithrin‐α results in a significant decrease in the frequency of NTDs in Cited2 ^−/− embryos (Fisher's exact test: *p = 0.023)