The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis
Nathalie G. Bérubé, … , Ruth S. Slack, David J. Picketts
Nathalie G. Bérubé, … , Ruth S. Slack, David J. Picketts
Published February 1, 2005
Citation Information: J Clin Invest. 2005;115(2):258-267. https://doi.org/10.1172/JCI22329.
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Article Neuroscience

The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis

Abstract

Mutations in genes encoding chromatin-remodeling proteins, such as the ATRX gene, underlie a number of genetic disorders including several X-linked mental retardation syndromes; however, the role of these proteins in normal CNS development is unknown. Here, we used a conditional gene-targeting approach to inactivate Atrx, specifically in the forebrain of mice. Loss of ATRX protein caused widespread hypocellularity in the neocortex and hippocampus and a pronounced reduction in forebrain size. Neuronal “birthdating” confirmed that fewer neurons reached the superficial cortical layers, despite normal progenitor cell proliferation. The loss of cortical mass resulted from a 12-fold increase in neuronal apoptosis during early stages of corticogenesis in the mutant animals. Moreover, cortical progenitors isolated from Atrx-null mice undergo enhanced apoptosis upon differentiation. Taken together, our results indicate that ATRX is a critical mediator of cell survival during early neuronal differentiation. Thus, increased neuronal loss may contribute to the severe mental retardation observed in human patients.

Authors

Nathalie G. Bérubé, Marie Mangelsdorf, Magdalena Jagla, Jackie Vanderluit, David Garrick, Richard J. Gibbons, Douglas R. Higgs, Ruth S. Slack, David J. Picketts

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Generation of Atrx gene-targeted mice. (A) A 6.2-kb SstI fragment of the...
Generation of Atrx gene-targeted mice. (A) A 6.2-kb SstI fragment of the Atrx gene was used to engineer a targeting construct containing a floxed Neor cassette within intron 17 and loxP sites flanking exon 18. Two herpes simplex virus thymidine kinase (HSV TK) genes were included to allow for positive and negative selection of recombinant ES cell clones. Female mice homozygous for the floxed Atrx allele (AtrxloxP) were bred to heterozygous Foxg1Cre male mice to generate KO male mice, referred to as AtrxFoxg1Cre. (B) Southern blot analysis of genomic DNA isolated from the cortices of newborn pups of different genotypes. F/Y, floxed-ATRX X chromosome; F/WT, wild-type X chromosome; Cre+, Cre-positive; Cre–, Cre-negative. Right margin: F, floxed allele; R, recombined allele. (C) RT-PCR analysis of RNA isolated from newborn forebrains of AtrxFoxg1Cre males (F/Y, Cre +), control littermates (F/Y, Cre –), or heterozygous females (F/WT) with or without Cre. Fragments were amplified with Atrx primers 17F and 20R, as indicated in A, or Gapdh primers (lower band) in control reactions. As expected, the amplified product was shorter when Cre was present due to recombination of loxP sites. (D) Western blot analysis of proteins isolated from newborn forebrains of wild-type, heterozygous, or knockout mice using an antibody that recognizes both full-length ATRX protein and the truncated isoform ATRXt (39f) or a C-terminal antibody (H300) that detects only the full-length isoform. Loss of full-length ATRX expression is observed in knockout animals, while ATRXt expression is not affected. Tubulin was used as a loading control.