title: Grainyhead-like 3 regulation of endothelin-1 in the pharyngeal endoderm is critical for growth and development of the craniofacial skeleton.
abstract: Craniofacial development is a highly conserved process that requires complex interactions between neural crest cells (NCCs) and pharyngeal tissues derived from all three germ layers. Signals emanating from the pharyngeal endoderm drive differentiation of NCCs into craniofacial cartilage, and disruption of this process underpins several human craniofacial defects (CFD). Here, we demonstrate that morpholino (MO)-mediated knockdown in zebrafish of the highly conserved transcription factor grainyhead-like 3 (grhl3), which is selectively expressed in the pharyngeal endoderm, leads to severe hypoplasia of the lower jaw cartilages. Phylogenetic analysis of conserved grhl-binding sites in gene regulatory regions identified endothelin-1 (edn1) as a putative direct grhl3 target gene, and this was confirmed by chromatin precipitation (ChIP) assays in zebrafish embryos. Injection of sub-phenotypic concentrations of MOs targeting both grhl3 and edn1 induced jaw abnormalities, and injection of edn1 mRNA into grhl3-morphants rescued both pharyngeal expression of the downstream effectors of edn1, and jaw cartilage formation. This study sheds new light on the role of endodermal endothelin-1 in vertebrate jaw development, and highlights potential new genetic defects that could underpin human CFD.
authors.affiliation: Department of Medicine, Monash University Central Clinical School, Prahran, VIC 3181, Australia. Electronic address: sebastian.dworkin@monash.edu.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Dworkin
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia.
authors.forename: Johanna
authors.initials: J
authors.lastname: Simkin
authors.affiliation: Department of Medicine, Monash University Central Clinical School, Prahran, VIC 3181, Australia.
authors.forename: Charbel
authors.initials: C
authors.lastname: Darido
authors.affiliation: Department of Medicine, Monash University Central Clinical School, Prahran, VIC 3181, Australia.
authors.forename: Darren D
authors.initials: DD
authors.lastname: Partridge
authors.affiliation: Department of Medicine, Monash University Central Clinical School, Prahran, VIC 3181, Australia.
authors.forename: Smitha R
authors.initials: SR
authors.lastname: Georgy
authors.affiliation: Bone Marrow Research Laboratories, Royal Melbourne Hospital, Parkville, VIC 3050, Australia.
authors.forename: Jacinta
authors.initials: J
authors.lastname: Caddy
authors.affiliation: Bone Marrow Research Laboratories, Royal Melbourne Hospital, Parkville, VIC 3050, Australia; Laboratory of Signal Transduction, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Wilanowski
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia; Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Lieschke
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia.
authors.forename: Karen
authors.initials: K
authors.lastname: Doggett
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia; Colon Molecular and Cell Biology Laboratory, Ludwig Institute for Cancer Research, Melbourne-Parkville Branch, Parkville, VIC 3050, Australia.
authors.forename: Joan K
authors.initials: JK
authors.lastname: Heath
authors.affiliation: Department of Medicine, Monash University Central Clinical School, Prahran, VIC 3181, Australia; Alfred Hospital, Prahran, VIC 3181, Australia.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Jane
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D019332:Endothelin-1 / Q000235:genetics* / Q000378:metabolism; D004843:Epistasis, Genetic; D005147:Facial Bones / Q000254:growth & development / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D006801:Humans; D008954:Models, Biological; D009432:Neural Crest / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015398:Signal Transduction; D012886:Skull / Q000254:growth & development / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D019332:Endothelin-1; C579350:GRHL3 protein, zebrafish; D012333:RNA, Messenger; D029961:Zebrafish Proteins
keywords: Craniofacial defects; Endoderm; Endothelin-1; Grainyhead-like; Pharynx; grhl3