Grhl3 modulates epithelial structure formation of the circumvallate papilla during mouse development

Abstract

Grainyhead-like 3 (Grhl3) is a transcription factor involved in epithelial morphogenesis. In the present study, we evaluated the developmental role of Grhl3 in structural formation of the circumvallate papilla (CVP), which undergoes dynamic morphological changes during organogenesis. The specific expression pattern of Grhl3 was examined in the CVP-forming region, specifically in the apex and epithelial stalk from E13.5 to E15.5 using in situ hybridization. To determine the role of Grhl3 in epithelial morphogenesis of the CVP, we employed an in vitro tongue culture method, wherein E13.5 tongue were isolated and cultured for 2 days after knocking down of Grhl3. Knockdown of Grhl3 resulted in significant changes to the epithelial structure of the CVP, such that the apical region of the CVP was smaller in size, and the epithelial stalks were more deeply invaginated. To define the mechanisms underlying these morphological alterations, we examined cell migration, proliferation, and apoptosis using phalloidin staining, immunohistochemistry against Ki67, ROCK1, and E-cadherin, and a TUNEL assay, respectively. These results revealed an increase in proliferation, a reduction in apoptosis, and an altered pattern of cytoskeletal formation in the CVP-forming epithelium, following Grhl3 knockdown. In addition, there were changes in the specific expression patterns of signaling and apoptosis-related molecules such as Axin2, Bak1, Bcl2, Casp3, Casp8, Ctnnb1, Cnnd1, Gli3, Lef1, Ptch1, Rock1, Shh, and Wnt11, which could explain the altered cellular and morphological events. Based on these results, we propose that developmental stage-specific Grhl3 plays a significant role in CVP morphogenesis not by just disruption of epithelial integrity but by regulating epithelial cell proliferation, apoptosis, and migration via Shh, Wnt, and apoptosis signaling during mouse embryogenesis.

Neural tube closure depends on expression of Grainyhead-like 3 in multiple tissues

“…However, studies in other systems implicate Grhl3 as a negative influence on cell proliferation. For example, in developing skin, Grhl3 mutations cause hyper-proliferation ( Ting et al, 2005 ), while in normal tongue papillae, loss of Grhl3 results in enhanced cell proliferation ( Adhikari et al, 2017 ). Hence, the specific requirement for Grhl3 in hindgut cell proliferation remains to be determined.…”

Mis-expression of grainyhead-like transcription factors in zebrafish leads to defects in enveloping layer (EVL) integrity, cellular morphogenesis and axial extension

“…Severely affected zebrafish mutants lacking e-cadherin exhibit a highly-similar FTEA phenotype 57 to our grhl3 over-expressing fish, and it is a possibility that defective ECM scaffolding through repressed e-cadherin may contribute to failed cellular migration and subsequent tail extension. Furthermore, recent studies indicate that in addition to activating E-Cadherin , or serving to alter E-Cadherin localisation 58 , Grhl3 may also act as an E-cadherin suppressor 59 , indicative of a dichotomous relationship between these two factors, which together may regulate convergence-extension mediated movements during embryogenesis.…”

Structural basis of gene regulation by the Grainyhead/CP2 transcription factor family

“…In animals, Grhl proteins are predominantly expressed in epithelial tissues and are essential regulators of epithelial development and extracellular barrier repair after tissue damage ( 4 , 6 , 7 ). GRHL2 or GRHL3 null mutations in mice lead to embryonic lethality with defects in dorsal/ventral closure ( 8 , 9 ), while mice lacking GRHL1 exhibit delayed hair coat growth, defective hair anchoring and palmoplantar keratoderma. Many studies have been devoted to identifying Grhl target genes.…”