Increased expression of Grainyhead-like-3 rescues spina bifida in a folate-resistant mouse model

Abstract

Neural tube defects (NTDs), such as spina bifida, are common and severe birth defects in humans but the underlying causes are poorly understood. The pathogenesis and etiology of spina bifida in the curly tail mouse closely resemble defects in humans, providing a well-characterized model of NTDs. Grainyhead-like-3 (Grhl3), which encodes a transcription factor, was recently identified as a candidate gene for curly tail based on chromosomal location and the occurrence of spina bifida in Grhl3 null mice. However, the causative curly tail mutation has not been established, while the relationship between Grhl3 gene expression and the known cellular defect leading to NTDs in curly tail is unknown. Spina bifida in curly tail results from a cell type-specific proliferation defect in the hindgut endoderm, and we find that Grhl3 is expressed specifically in this tissue during the final stages of spinal neural tube closure in wild type embryos. Moreover, Grhl3 expression is diminished in the spinal region of neurulation-stage curly tail embryos. Curly tail mice do not carry a coding region mutation in Grhl3, however, we found a putative regulatory mutation upstream of the Grhl3 gene, which may be responsible for the expression deficit. In order to test the hypothesis that spina bifida in curly tail mice results from insufficient expression of Grhl3, we generated Grhl3-expressing curly tail mice by bacterial artificial chromosome-mediated transgenesis and demonstrated complete rescue of spina bifida. This study provides evidence for a critical role of diminished Grhl3 expression in causing spinal NTDs in the curly tail mouse model.

Mechanisms of tissue fusion during development

“…Axd mutants exhibit increased Grhl2 expression, and NT closure can be restored by decreasing the level of Grhl2 (Brouns et al, 2011). Ct mutants show decreased expression of Grhl3, and NT closure can be rescued by Grhl3 overexpression (Gustavsson et al, 2007). Thus, these studies suggest that Grhl activity must be tightly controlled during NT closure.…”

“…However, closure point 1 occurs in the absence of both Grhl2 and Grhl3 (Rifat et al, 2010). A role for Grhl3 in hindgut endoderm has been suggested in spinal NT closure, as loss of Grhl3 expression in the hindgut endoderm leads to decreased proliferation, which causes increased ventral curvature and hence pulls the neural folds apart so that fusion cannot take place (Ting et al, 2003;Gustavsson et al, 2007). Two well-characterized mouse NTD models that arose from spontaneous mutations are axial defects (Axd) and curly tail (Ct).…”

Apoptosis is not required for mammalian neural tube closure

“…Sections were counterstained with methyl green. The numbers of positive cells were counted as described previously (32,33).…”

Insights into prevention of human neural tube defects by folic acid arising from consideration of mouse mutants

“…For at least some of these genes, it is possible that hypomorphic mutants with partial gene function could respond to folic acid. This situation appears to be demonstrated by the NTD-preventative response to inositol in the curly tail strain (in which the major risk allele, ct, is a putative hypomorph at the Grhl3 locus) and the lack of preventative response in the Grhl3-null embryos (Gustavsson et al, 2007;Gustavsson et al, 2008;Ting et al, 2003).…”