Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways

Abstract

SUMMARYThe isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.

Grhl2 is required in nonneural tissues for neural progenitor survival and forebrain development

“…Grh is also downstream of erk signaling in Drosophila wound healing. In zebrafish, subphenotypic concentrations of grhl2b and fgf8 morpholino knockdown lead to MHB patterning defects (Dworkin et al ., ). These links to FGF signaling are consistent with the finding FGF is required for neuronal survival in the mouse forebrain (Paek et al ., ).…”

“…Grhl2 has been liked to the control of cell proliferation in a number of different contexts. Morpholino knockdown of grhl1b in fish leads to increased apoptosis in the CNS (Dworkin et al, 2012). Grh also regulates neuroblast proliferation in Drosophila (Brody and Odenwald, 2000;Cenci and Gould, 2005;Maurange et al, 2008).…”

Diverse expression patterns of the EMT suppressor grainyhead‐like 2 (GRHL2) in normal and tumour tissues

“…Although the results of our immunohistochemical and GRHL2 mRNA expression analyses clearly point towards a preferential expression of GRHL2 in epithelial tumours, for the first time evidence also for an expression of GRHL2 in tumours of non‐epithelial origin is provided. Developmental studies already revealed that GRHL2 represents a key regulator of neural patterning and morphogenesis . Grhl2 mutant mice among many developmental defects present with general defects in heart and embryonal neural tube formation .…”

Development of the cerebellum: simple steps to make a ‘little brain’

“…been shown that distinct pathways govern the morphological and molecular features of the MHB downstream of the common transcription factor grainyhead-like 2 (Grhl2), with engrailed 2 acting downstream of Grhl2 to promote cell survival and formation of the molecular boundary (Dworkin et al, 2012). In addition to the co-repressive actions of Otx2 and Gbx2 at the boundary, Fgf8, Gbx2 and Notch signalling (Sunmonu et al, 2011;Tossell et al, 2011) promote cell sorting and, hence, lineage restriction at the boundary.…”