Strain-specific modifier genes of Cecr2-associated exencephaly in mice: genetic analysis and identification of differentially expressed candidate genes.

Although neural tube defects (NTDs) are common in humans, little is known about their multifactorial genetic causes. While most mouse models involve NTDs caused by a single mutated gene, we have previously described a multigenic system involving susceptibility to NTDs. In mice with a mutation in Cecr2, the cranial NTD exencephaly shows strain-specific differences in penetrance, with 74% penetrance in BALB/cCrl and 0% penetrance in FVB/N. Whole genome linkage analysis showed that a region of chromosome 19 was partially responsible for this difference in penetrance. We now reveal by genetic analysis of three subinterval congenic lines that the chromosome 19 region contains more than one modifier gene. Analysis of embryos showed that although a Cecr2 mutation causes wider neural tubes in both strains, FVB/N embryos overcome this abnormality and close. A microarray analysis comparing neurulating female embryos from both strains identified differentially expressed genes within the chromosome 19 region, including Arhgap19, which is expressed at a lower level in BALB/cCrl due to a stop codon specific to that substrain. Modifier genes in this region are of particular interest because a large portion of this region is syntenic to human chromosome 10q25, the site of a human susceptibility locus.

CECR2 is involved in spermatogenesis and forms a complex with SNF2H in the testis.

The regulation of nucleosome positioning and composition by ATP-dependent chromatin remodeling enzymes and their associated binding partners plays important biological roles in mammals. CECR2 is a binding partner to the ISWI (imitation switch) ATPase SNF2L/SMARCA1 and is involved in neural tube closure and inner ear development; however, its functions in adult tissues have not been examined. Here, we report that CECR2 contributes to spermatogenesis and forms a complex that includes the other ISWI ATPase SNF2H/SMARCA5 in the testis. Cecr2 mutant males non-penetrant for neural tube defects sired smaller litters than wild-type males. Strikingly, while we found that Cecr2 mutants have normal seminiferous epithelium morphology, sperm count, motility, and morphology, the mutant spermatozoa were compromised in their ability to fertilize oocytes. Investigation of CECR2/ISWI complexes in the testis showed that SNF2H interacted with CECR2, and this interaction was also observed in embryonic stem cells, suggesting that CECR2 may interact with SNF2H or SNF2L depending on the cell type. Finally, we found that Cecr2 mutants exhibit misregulation of the homeobox transcription factor Dlx5 in the testis, suggesting that CECR2 complexes may regulate gene expression during spermatogenesis. Taken together, our results demonstrate a novel role of CECR2-containing complexes in spermatogenesis and show that CECR2 interacts predominantly with SNF2H instead of SNF2L in the testis.

Role of chromatin remodeling gene Cecr2 in neurulation and inner ear development.

The loss of Cecr2, which encodes a chromatin remodeling protein, has been associated with the neural tube defect (NTD) exencephaly and open eyelids in mice. Here, we show that two independent mutations of Cecr2 are also associated with specific inner ear defects. Homozygous mutant 18.5 days post coitus (dpc) fetuses exhibited smaller cochleae as well as rotational defects of sensory cells and extra cell rows in the inner ear reminiscent of planar cell polarity (PCP) mutants. Cecr2 was expressed in the neuroepithelium, head mesenchyme, and the cochlear floor. Although limited genetic interaction for NTDs was seen with Vangl2, a microarray analysis of PCP genes did not reveal a direct connection to this pathway. The mechanism of Cecr2 action in neurogenesis and inner ear development is likely complex.

Cecr2 mutations causing exencephaly trigger misregulation of mesenchymal/ectodermal transcription factors.

BACKGROUND: Over 200 mouse genes are associated with neural tube defects (NTDs), including Cecr2, the bromodomain-containing subunit of the CERF chromatin remodeling complex. METHODS: Gene-trap mutation Cecr2(Gt45Bic) results in 74% exencephaly (equivalent of human anencephaly) on the BALB/c strain. Gene expression altered during cranial neural tube closure by the Cecr2 mutation was identified through microarray analysis of 11-14 somites stage Cecr2(Gt45Bic)embryos. RESULTS: Analysis of Affymetrix Mouse 430 2.0 chips detected 60 transcripts up-regulated and 54 transcripts down-regulated in the Cecr2(Gt45Bic) embryos (fold > 1.5, p < 0.05). The Cecr2 transcript was reduced only approximately 7- to 14-fold from normal levels, suggesting the Cecr2(Gt45Bic) is a hypomorphic mutation. We therefore generated a novel Cecr2 null allele (Cecr2 (tm1.1Hemc)). Resulting mutants displayed a stronger penetrance of exencephaly than Cecr2(Gt45Bic) in both BALB/c and FVB/N strains, in addition to midline facial clefts and forebrain encephalocele in the FVB/N strain. The Cecr2 transcript is reduced 260-fold in the Cecr2(tm1.1Hemc) line. Subsequent qRT-PCR using Cecr2 (tm1.1Hemc) mutant heads confirmed downregulation of transcription factors Alx1/Cart1, Dlx5, Eya1, and Six1. CONCLUSIONS: As both Alx1/Cart1 and Dlx5 mouse mutations result in exencephaly, we hypothesize that changes in expression of these mesenchymal/ectodermal transcription factors may contribute to NTDs associated with Cecr2.