Department of Anatomy and Embryology, Maastricht University Medical Center, University of Maastricht, PO Box 616, 6200 MD, Maastricht, The Netherlands.
Hum Mol Genet. 2011 Apr 15;20(8):1536-46. doi: 10.1093/hmg/ddr031. Epub 2011 Jan 24.
Cranial neural tube defects (NTDs) occur in mice carrying mutant alleles of many different genes, whereas isolated spinal NTDs (spina bifida) occur in fewer models, despite being common human birth defects. Spina bifida occurs at high frequency in the Axial defects (Axd) mouse mutant but the causative gene is not known. In the current study, the Axd mutation was mapped by linkage analysis. Within the critical genomic region, sequencing did not reveal a coding mutation whereas expression analysis demonstrated significant up-regulation of grainyhead-like 2 (Grhl2) in Axd mutant embryos. Expression of other candidate genes did not differ between genotypes. In order to test the hypothesis that over-expression of Grhl2 causes Axd NTDs, we performed a genetic cross to reduce Grhl2 function in Axd heterozygotes. Grhl2 loss of function mutant mice were generated and displayed both cranial and spinal NTDs. Compound heterozygotes carrying both loss (Grhl2 null) and putative gain of function (Axd) alleles exhibited normalization of spinal neural tube closure compared with Axd/+ littermates, which exhibit delayed closure. Grhl2 is expressed in the surface ectoderm and hindgut endoderm in the spinal region, overlapping with grainyhead-like 3 (Grhl3). Axd mutants display delayed eyelid closure, as reported in Grhl3 null embryos. Moreover, Axd mutant embryos exhibited increased ventral curvature of the spinal region and reduced proliferation in the hindgut, reminiscent of curly tail embryos, which carry a hypomorphic allele of Grhl3. Overall, our data suggest that defects in Axd mutant embryos result from over-expression of Grhl2.
颅神经管缺陷（NTDs）发生在携带许多不同基因突变体的小鼠中，而孤立的脊柱 NTDs（脊柱裂）发生在较少的模型中，尽管这是常见的人类出生缺陷。Axial defects（Axd）小鼠突变体中脊柱裂的发生率很高，但致病基因尚不清楚。在本研究中，通过连锁分析对 Axd 突变进行了定位。在关键基因组区域内，测序未发现编码突变，而表达分析表明 Axd 突变体胚胎中 grainyhead-like 2（Grhl2）的表达显著上调。基因型之间其他候选基因的表达没有差异。为了验证 Grhl2 过表达导致 Axd NTDs 的假设，我们进行了遗传杂交以降低 Axd 杂合子中的 Grhl2 功能。生成了 Grhl2 功能丧失突变小鼠，并显示出颅神经和脊柱 NTDs。携带 Grhl2 缺失（Grhl2 null）和假定功能获得（Axd）等位基因的复合杂合子与 Axd/+同窝仔鼠相比，脊柱神经管闭合正常，而 Axd/+同窝仔鼠的神经管闭合延迟。Grhl2 在脊柱区域的表面外胚层和后肠内胚层中表达，与 grainyhead-like 3（Grhl3）重叠。Axd 突变体显示出延迟的眼睑闭合，正如在 Grhl3 缺失胚胎中报道的那样。此外，Axd 突变体胚胎表现出脊柱区域的腹侧曲率增加和后肠增殖减少，类似于携带 Grhl3 功能降低等位基因的卷曲尾巴胚胎。总的来说，我们的数据表明，Axd 突变体胚胎的缺陷是由于 Grhl2 的过表达所致。
