Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways.
The 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.
Department of Medicine, Monash University Central Clinical School, Prahran VIC 3181, Australia.
Sebastian
S
Dworkin
Charbel
C
Darido
Smitha R
SR
Georgy
Tomasz
T
Wilanowski
Seema
S
Srivastava
Felix
F
Ellett
Luke
L
Pase
Yanchao
Y
Han
Anming
A
Meng
Joan K
JK
Heath
Graham J
GJ
Lieschke
Stephen M
SM
Jane
D000818:Animals; D017209:Apoptosis; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D008636:Mesencephalon / Q000254:growth & development* / Q000378:metabolism; D009024:Morphogenesis; D060172:Morpholinos / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D010802:Phylogeny; D012249:Rhombencephalon / Q000254:growth & development* / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*; D000099266:Engrailed 2 Protein
D002352:Carrier Proteins; D018398:Homeodomain Proteins; D060172:Morpholinos; D009419:Nerve Tissue Proteins; D014157:Transcription Factors; D029961:Zebrafish Proteins; C570455:Cdc42se1 protein, zebrafish; C079428:en2a protein, zebrafish; D000099266:Engrailed 2 Protein; C570454:grhl2b protein, zebrafish