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, 45 (9), 577-87

Transcription Factor TEAD2 Is Involved in Neural Tube Closure

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Transcription Factor TEAD2 Is Involved in Neural Tube Closure

Kotaro J Kaneko et al. Genesis.

Abstract

TEAD2, one of the first transcription factors expressed at the beginning of mammalian development, appears to be required during neural development. For example, Tead2 expression is greatest in the dorsal neural crest where it appears to regulate expression of Pax3, a gene essential for brain development. Consistent with this hypothesis, we found that inactivation of the Tead2 gene in mice significantly increased the risk of exencephaly (a defect in neural tube closure). However, none of the embryos exhibited spina bifida, the major phenotype of Pax3 nullizygous embryos, and expression of Pax3 in E11.5 Tead2 nullizygous embryos was normal. Thus, Tead2 plays a role in neural tube closure that is independent of its putative role in Pax3 regulation. In addition, the risk of exencephaly was greatest with Tead2 nullizygous females, and could be suppressed either by folic acid or pifithrin-alpha. These results reveal a maternal genetic contribution to neural tube closure, and suggest that Tead2-deficient mice provide a model for anencephaly, a common human birth defect that can be prevented by folic acid.

Figures

FIG. 1
FIG. 1
Construction of a Tead2 conditional KO mouse. The mouse Tead2 gene is located at 23 cM on chromosome 7 in ~30 kb region that includes the Dkkl1 and CD37 genes. (A) Schematic depiction of the features of the Tead2 gene that are relevant for construction of the knockout allele. Locations of cleavage sites for EcoRV (RV), Nhe I (N), and Xho I (X) restriction endonucleases are indicated. Locations of the 5′ and 3′-sequence specific DNA probes used in Southern blotting-hybridization analysis are indicated. Locations of primers A, B, and C used for multiplex genomic DNA PCR assays are indicated. Primer sequences are in Table 4. A putative Tead2 gene enhancer (“E”) is located within intron 1. (B) The targeting vector spanned the region from just upstream of the Tead2 promoter through part of intron 7. LoxP sites (closed triangles) were inserted within introns 1 and 3. A neomycin resistance gene (Neo) driven by the phosphoglycerol kinase (PGK) gene promoter, flanked by Flip recombinase target (FRT) sites (closed ovals), was inserted into intron 3 just upstream of 3′-loxP site. Newly created restriction sites are indicated with an asterisk. (C) Exposure of the targeted allele to Cre recombinase results in excision of the region between the two loxP sites. The deleted allele was screened by muliplex genomic DNA PCR (primer A/C) as well as by Southern blotting-hybridization analysis after digesting with EcoRV enzyme and probing with KO probe.
FIG. 2
FIG. 2
Genomic DNA analysis confirmed the presence of the Tead2 mutant allele. (A) Genomic DNA was extracted from the tails of pups born from matings between chimeric males and C57BL/6J females. DNA was digested with the indicted restriction endonuclease, subjected to Southern blotting-hybridization, and then detected with the indicated probe (Fig. 1A, B). Lanes 2–4 contain DNA from agouti pups. Lane 1 contains DNA from a black littermate. XhoI-5′ probe gives rise to a 19.9 kb DNA fragment from the wild type (Wt) allele, and a 4.6 kb DNA fragment from the mutant allele (Mt). NheI-3′ probe gives rise to 8.4 kb fragment from the Wt allele, and a 9.5 kb fragment from the mutant allele. Genomic DNA from WT ES cells was included as a positive control. (B) Mice containing the mutant allele were mated to EllaCre+ mice, and resulting pups were assayed for the deleted/KO allele. Genomic DNA was digested with EcoRV restriction endonuclease, subjected to Southern blotting-hybridization, and the DNA detected with “KO probe” (Fig. 1C). The Wt allele generates a 7.2 kb DNA fragment (Wt). The knockout allele generates a 8.5 kb DNA fragment (KO). Result shown is an example from such matings (“parents”). These Tead2 heterozygous parents (male and female) were then mated to each other and their litters of five pups were similarly assayed for the presence of KO allele. (C) The DNA samples (“litter”) in panel B were also subjected to multiplex genomic DNA PCR analysis. Primers A and B (Fig. 1B) amplified a 165 bp fragment from the Wt allele. Primers A and C (Fig. 1C) amplified a 210 bp fragment from the KO allele.
FIG. 3
FIG. 3
Full-length Tead2 mRNA was absent in Tead2 nullizygous mice. Tead2 heterozygous mice were mated, and DNA extracted from the tails of littermates was genotyped. (A) Total RNA (15 μg) was prepared from adult forebrain (lane 1) and fetal brain (E13.5; lane 2) from Tead2 WT mouse, and fetal brain (E13.5) from a Tead2 nullizygous mouse (lane 3), fractionated by agarose gel electrophoresis, transferred to nylon membrane, and hybridized with radiolabeled Tead2 cDNA (Kaneko et al., 1997). Similarly, total RNA (15 μg) was prepared from the lungs of a Tead2 homozygous (lane 5), a heterozygous (lane 6) and a nullizygous (lane 7) littermate. Samples were subjected to Northern blotting-hybridization analysis. Total RNA (15 μg) isolated from WT CCE ES cells (lane 4), which abundantly express Tead2 (Kaneko et al., 2004), was used as a positive control. Ethidibum bromide stained 28S rRNA was used as loading control. 0.24–9.5 kb RNA ladder from Invitrogen was used as marker. (B, C) Tead2 KO mice produce no detectable level of Tead2 mRNA, but still express the Fgfr4 and Tead4 genes. Total RNA from lung (B) or ovary (C) tissue was isolated from mice that were WT, heterozygous, or nullizygous at Tead2 allele and then used as a template for cDNA synthesis. PCR was performed for the indicated number of cycles using Tead2 primers that amplified the region from exons 2 to 6 (Kaneko et al., 1997). The same aliquot of RNA was used to amplify Fgfr4-specific and Tead4-specific RNA. DNA marker used was 1 kb ladder from Invitrogen. Gapdh control primers confirmed that relatively equivalent RNA samples were amplified (data not shown).
FIG. 4
FIG. 4
Exencephalic embryos were detected as early as E11.5, although Pax3 expression was not altered by the absence of TEAD2. (A) Total RNA was prepared from 11 embryos at E11.5 that were obtained from a single heterozygous female mated to a heterozygous male. This RNA was assayed for Pax3 sequences using RT-PCR. RNA from E13.5 fetal brain served as a positive control, while RNA from ES cells served as a negative control. One embryo that exhibited exencephaly (asterisk) is shown in panel C. (B) Multiplex PCR analysis was used to determine the genotype of each embryo. (C) One normal (Tead2+/−) and one exencephalic (Tead2−/−) embryo from the same litter at E11.5 are shown using bright-field illumination. Arrows indicate sites where neural tube failed to close.
FIG. 5
FIG. 5
Exencephalic embryos were clearly evident by E13.5. Tead2 nullizygous females were mated to a heterozygous male, and embryos were harvested at day E13.5. Two exencephalic and one normal embryo from the same litter are shown using dark-field illumination.
FIG. 6
FIG. 6
By E16.5, some exencephalic embryos are alive while others are being resorbed. Tead2 nullizygous females were mated to a heterozygous male, and embryos were harvested at day E16.5. (A) Tead2−/− exencephalic embryo. (B) Tead2−/− exencephalic embryo undergoing resorption. (C) Tead2+/− littermate.

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