All four twist genes of zebrafish have partially redundant, but essential, roles in patterning the craniofacial skeleton

Twist proteins are highly conserved transcription factors expressed in the cephalic neural crest of all vertebrates and may therefore constitute part of the molecular machinery that controls head morphogenesis. The zebrafish genome carries four twist genes - twist1a, twist1b, twist2 and twist3. To explore whether all four have roles in development of the craniofacial skeleton, we attenuated their expression individually and in groups using antisense morpholino oligonucleotides. All morphant types exhibited variously abnormal or absent head cartilages, demonstrating that all zebrafish twist paralogs are essential for chondrogenesis during formation of the head skeleton. We found a degree of functional conservation between the teleost and mammalian twist genes, for example in patterning of the jaw and of periorbital crest-derived structures. It suggests that understanding evolution of twist genes may be informative for understanding the evolution of the vertebrate head. Further morphant analysis showed that while all four genes promote chondrogenesis, twist1a has a dual function and can also serve as a chondrogenic inhibitor. Comparing the morphant and RNA in situ data showed that in some structures, despite their high homology and indistinguishable spacio-temporal expression, the twist paralogs had disparate or opposing roles. In all morphants, we observed cartilage defects ranging from borderline normal to full agenesis. On one hand, this indicates partial redundancy among twist paralogs. On the other hand, such phenotypic spectrum suggests that twist genes are active at multiple stages of progression from an osteochondroprogenitor along the chondrogenic lineage.