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.

Figure 1

ATRX expression increases in postmitotic neurons. (A–E) Fixed tissue sections were treated with a polyclonal antibody (H300) detecting the full-length ATRX protein. (A) Coronal section through the cortical region at E13.5 reveals highest expression in the preplate (PP). (B) Sagittal section through the cortex at P0.5 shows high levels of ATRX staining in postmitotic neurons of the marginal zone (MZ), cortical plate (CP), and subplate (SP), but lower levels of staining in the proliferative subventricular (SVZ) and ventricular zones (VZ). (C) Sagittal section at P0.5 through the hippocampus demonstrates high ATRX levels in CA1, CA3, and dentate gyrus (DG) subregions. (D and E) Saggital section at P31 through the brain demonstrates that ATRX expression is maintained in the adult throughout the cortex (CX), the hippocampus (CA1 and CA3), and the dentate gyrus subregions. (F–K) Cortical progenitor cells dissected from E12.5 telencephalon were cultured for 6 days and were costained for ATRX (F) and for MAP2 (G), a marker of neuronal differentiation. The merged image (H) reveals coexpression of ATRX and MAP2 in the differentiated cells. In contrast, progenitors costained for ATRX (I) and proliferating cells as measured by BrdU incorporation (J) show low levels of ATRX in the BrdU-positive cells (K, merged image). Higher magnification (×20, K, bottom-right inset) image shows that BrdU and ATRX staining does not significantly overlap. Magnification, ×20 (A, C–K), ×10 (B).

Figure 2

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 Neo^r 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 (Atrxlox^P) 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.

Figure 3

Cre-mediated conditional deletion of Atrx in the forebrain. (A and B) Cre in situ detection in sagittal sections of control (A) and AtrxFoxg1Cre (B) E13.5 embryos demonstrates that Cre expression is restricted to the telencephalon and anterior retina. (C–F) Sections of the cortex at P0.5 immunostained with antibodies specific for ATRX (C and D) and Cre recombinase (E and F). ATRX protein is undetectable in AtrxFoxg1Cre male cortex, and loss of expression is correlated with the presence of Cre recombinase. (G and H) Merged image of ATRX expression (red) and Cre expression (green) demonstrating the loss of ATRX protein in the cortex but not in neighboring brain tissues and the presence of rare ATRX-positive cells in the cortex (arrow, H). (H) Higher magnification of the image in G. (I) Reduced size of P0.5 AtrxFoxg1Cre pup and lack of milk in stomach (indicated by arrows). (J) Graph depicting decreased weight of P0.5 AtrxFoxg1Cre males compared with that of wild-type and heterozygous Cre⁺ female mice (P < 0.001). (K) Reduced size of AtrxFoxg1Cre forebrain compared with that of a littermate control, with greater reduction in caudal-medial area. Magnification, ×10 (C–G) and ×40 (H).

Figure 4

Cortical and hippocampal size reduction and hypocellularity. (A–L) Control (A, C, E, G, I, and K) and knockout (AtrxFoxg1Cre; B, D, F, H, J, and L) brain sections from P0.5 pups were stained with hematoxylin and eosin. Coronal views of rostral (A and B) and caudal (C and D) forebrain demonstrate the size reduction of the cortex and hippocampus in the conditional knockout animals. There is dysgenesis of the hippocampus (E and F) and lack of a dentate gyrus (asterisk in F) in AtrxFoxg1Cre males, as well as reduced cell density in the CA1 hippocampal field (G and H). There is decreased cortical plate thickness in E15.5 AtrxFoxg1Cre cortex (I and J). There is reduced cell density in rostral cortex at P0.5 in the AtrxFoxg1Cre cortex (K and L). Insets show higher magnification. II–IV, V, and VI represent cortical layers present at birth. (M) Graph depicting cell numbers in the cortical plate at E13.5 and E15.5 and rostral (R) and caudal (C) sections at P0.5. Magnification, ×40 (A–L).

Figure 5

AtrxNestinCre mice have a phenotype similar to that of AtrxFoxg1Cre animals. (A and B) In situ whole-mount analysis of NestinCre-transgenic mice (E13.5) from line 2472 on a β-geo reporter strain background shows high Cre activity in the forebrain and spinal cord. Atrxlox^P mice were mated to NestinCre-transgenic mouse line 2472 to achieve selective recombination of the Atrx gene in neuronal progenitors. ATRX (C) and DAPI (D) staining of forebrains isolated from AtrxNestinCr^e animals at birth demonstrate ATRX expression in early-born neurons of the marginal zone and the subplate, plus variegated expression throughout the cortex and hippocampus. Hematoxylin and eosin staining of cortex isolated from P0.5 mice of wild-type (E) and AtrxNestinCre animals (F) shows reduced cellularity. Similarly, neonatal hippocampal tissue sections from AtrxNestinCre (H) and a control littermate (G) also demonstrate hypocellularity in all hippocampal fields. Magnification, ×20 (C–H).

Figure 6

Many AtrxFoxg1Cre progenitor cells born at E15.5 never reach the superficial layers of the cortex and the dentate gyrus. Female mice homozygous for the Atrx floxed allele were bred to Foxg1Cre-heterozygous males and were injected with BrdU at E15.5, and newborn pups were sacrificed at P0.5. (A) Graph depicting BrdU staining of coronal sections of rostral and caudal forebrain. Fewer labeled cells migrate to the superficial layers of the cortex (rostral, n = 3, P < 0.001; caudal, n = 3, P < 0.001). Control was set as 100% labeling. (B and C) BrdU labeling in the dentate gyrus of cells at E15.5. Fewer labeled cells have migrated to the dentate gyrus in the ATRX-deficient hippocampus (C). Magnification, ×40 (B and C).

Figure 7

Normal proliferation in AtrxFoxg1Cre cortex during embryogenesis. Timed matings were set up between homozygous Atrx-floxed female mice and Foxg1Cre-heterozygous males. Pregnant female mice were subjected to a 1-hour BrdU pulse prior to sacrifice. Embryos were fixed and frozen, and sections were processed for BrdU immunostaining at E11.5 (A and B), E13.5 (C and D), E15.5 (E and F). Normal controls (A, C, and E) are compared with AtrxFoxg1Cre mice (B, D, and F). (G) BrdU-positive cells were counted in an identically sized area and data are expressed as a percentage of the total number of cells (DAPI-positive) at E11.5, E13.5, and E15.5. No statistically significant differences in proliferation were observed at any of the time points examined (E11.5, n = 4, P = 1.000; E13.5, n = 6, P = 0.181; E15.5, n = 5, P = 0.482). Magnification, ×40 (A and B), ×20 (E and F), and ×10 (C and D).

Figure 8

Increased number of pyknotic cell clusters during corticogenesis. (A–F) Cortical histology was examined at E11.5 (A and B) and E13.5 (C–F) by hematoxylin and eosin staining. Control littermates are depicted in A, C, and D, and AtrxFoxg1Cre in B, E, and F. Large numbers of darkly stained pyknotic clusters (arrows) indicative of DNA condensation and apoptosis are observed in the cortex of E11.5 (B) and E13.5 (E and F) ATRX-deficient embryos. Greater numbers of these clusters are also present in the hippocampal area at E13.5 of AtrxFoxg1Cre embryos (E and F) compared with controls (C and D). Magnification, ×40 (A and B), ×20 (D and F), and ×10 (C and E).

Figure 9

Increased apoptosis in AtrxFoxg1Cr^e embryonic cortex and postnatal hippocampus. (A–I) The TUNEL assay was performed on embryos at E11.5 (A and B) and E13.5 (C and D) and at P0.5 in the hippocampus (H and I). AtrxFoxg1Cre sections (B, D, G, and I) are compared with littermate controls (A, C, F, and H). A substantial increase in the number of TUNEL-positive cells is observed in AtrxFoxg1Cre E11.5 neuroepithelial layer (compare A with B). This increase persists in the cortex of E13.5 embryos (C and D) and is not restricted to the proliferative ventricular layer. (E) Graph depicting the increased number of apoptotic cells in ATRX-deficient embryos detected by TUNEL assay. TUNEL-positive cells were counted and data expressed as a percentage of total number of cells (DAPI-positive; E11.5, n = 3, P = 0.045; E13.5, n = 4, P < 0.001). At P0.5, increased levels of TUNEL-positive cells are detected in the subventricular zone of the CA1 field (F and G) and in the developing dentate gyrus (H and I). PL, pial layer; H, hippocampus; MS, migratory stream. Missing DG in I is indicated by an asterisk. Magnification, ×20 (A, B, and F–I) and ×10 (C and D).

Figure 10

Increased apoptosis but normal proliferation in ATRX-deficient primary cortical cultures. (A–I) Primary cultures of cortical progenitor cells were established from E12.5 telencephalon of AtrxFoxg1Cre mice (B, D, F, and H), control Cre^– littermates (A, C, E, G), or heterozygote (Het) Cre⁺ littermates (I), and were grown for 6 days in culture. (A and B) BrdU staining for 16 hours of control (A) and knockout (B) colonies demonstrates that the proliferative capacity of the cultured cells is not diminished in the absence of ATRX protein expression. (C and D) Control progenitors show a high level of ATRX protein (red) in differentiating MAP2-positive cells (green). AtrxFoxg1Cre progenitors that are ATRX deficient (lack of red staining in D) still express high levels of MAP2. (E and F) Corresponding ATRX staining of clones in G and H demonstrates the absence of ATRX protein in AtrxFoxg1Cre progenitors. (G and H) Merged images of TUNEL-positive cells (green) and DAPI staining (blue). Note the increased level of TUNEL staining in AtrxFoxg1Cre cell colonies after 6 days in culture. (I) Heterozygous Cre⁺ progenitor cell clone stained for ATRX protein (red) and TUNEL (green), clearly showing that ATRX-deficient areas of the clone display higher levels of apoptosis. Magnification, ×20 (A–I).