The vestigial gene has been shown to control skeletal muscle formation in Drosophila and the related Vestigial-like 2 (Vgl-2) protein plays a similar role in mice. Vgl-family proteins are thought to regulate tissue-specific gene expression by binding to members of the broadly expressed Scalloped/Tef/TEAD transcription factor family. Zebrafish have at least four Vgl genes, including two Vgl-2s, and at least three TEAD genes, including two Tead3s. We describe the cloning and expression of one member from each family in the zebrafish. A novel gene, vgl-2b, with closest homology to mouse and human vgl-2, is expressed transiently in nascent notochord and in muscle fibres as they undergo terminal differentiation during somitogenesis. Muscle cells also express a TEAD-3 homologue, a possible partner of Vgl-2b, during myoblast differentiation and early fibre assembly. Tead3a is also expressed in rhombomeres, eye and epiphysis regions.

3MC syndrome has been proposed as a unifying term to integrate the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders of unknown cause comprise a spectrum of developmental features including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability, and genital, limb and vesicorenal anomalies. In a cohort of eleven 3MC families, we identified two mutated genes COLEC11 and MASP1 both of which encode proteins within the lectin complement pathway (CL-K1 and MASP-1 & −3 respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney, and vertebral bodies. Zebrafish morphants develop pigment defects and severe craniofacial abnormalities.

Here, we show that CL-K1 serves as a key guidance cue for neural crest cell migration thus demonstrating for the first time, a role for complement pathway factors in fundamental developmental processes and the origin of 3MC syndrome.

Differentiation often requires conversion of analogue signals to a stable binary output through positive feedback. Hedgehog (Hh) signalling promotes myogenesis in the vertebrate somite, in part by raising the activity of muscle regulatory factors (MRFs) of the Myod family above a threshold. Hh is known to enhance MRF expression. Here we show that Hh is also essential at a second step that increases Myod protein activity, permitting it to promote Myogenin expression. Hh acts by inducing expression of cdkn1c (p57Kip2) in slow muscle precursor cells, but neither Hh nor Cdkn1c is required for their cell cycle exit. Cdkn1c co-operates with Myod to drive differentiation of several early zebrafish muscle fibre types. Myod in turn up-regulates cdkn1c, thereby providing a positive feedback loop that switches myogenic cells to terminal differentiation.

Poc1 shores up basal bodies to support cilia formation in Tetrahymena thermophila, zebrafish, and humans; Poc1 depletion causes phenotypes commonly seen in ciliopathies.

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis.

Summary

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.

Mrf4 (Myf6) is a basic helix-loop-helix (bHLH) myogenic regulatory transcription factor (MRF) family which also contains Myod, Myf5 and myogenin. Mrf4 is implicated in commitment of amniote cells to skeletal myogenesis and is also abundantly expressed in many adult muscle fibres. The specific role of Mrf4 is unclear both because mrf4 null mice are viable, suggesting redundancy with other MRFs, and because of genetic interactions at the complex mrf4/myf5 locus. We report the cloning and expression of an mrf4 gene from zebrafish, Danio rerio, which shows conservation of linkage to myf5. Mrf4 mRNA accumulates in a subset of terminally differentiated muscle fibres in parallel with myosin protein in the trunk and fin. Although most, possibly all, trunk muscle expresses mrf4, the level of mRNA is dynamically regulated. No expression is detected in muscle precursor cell populations prior to myosin accumulation. Moreover, mrf4 expression is not detected in head muscles, at least at early stages. As fish mature, mrf4 expression is pronounced in slow muscle fibres.