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ABSTRACT: The specification of an appropriate number of cardiomyocytes from the lateral plate mesoderm requires a careful balance of both positive and negative regulatory signals. To identify new regulators of cardiac specification, we performed a phenotype-driven ENU mutagenesis forward genetic screen in zebrafish. In our genetic screen we identified a zebrafish ctr9 mutant with a dramatic reduction in myocardial cell number as well as later defects in primitive heart tube elongation and atrioventricular boundary patterning. Ctr9, together with Paf1, Cdc73, Rtf1 and Leo1, constitute the RNA polymerase II associated protein complex, PAF1. We demonstrate that the PAF1 complex (PAF1C) is structurally conserved among zebrafish and other metazoans and that loss of any one of the components of the PAF1C results in abnormal development of the atrioventricular boundary of the heart. However, Ctr9, Cdc73, Paf1 and Rtf1, but not Leo1, are required for the specification of an appropriate number of cardiomyocytes and elongation of the heart tube. Interestingly, loss of Rtf1 function produced the most severe defects, resulting in a nearly complete absence of cardiac precursors. Based on gene expression analyses and transplantation studies, we found that the PAF1C regulates the developmental potential of the lateral plate mesoderm and is required cell autonomously for the specification of cardiac precursors. Our findings demonstrate critical but differential requirements for PAF1C components in zebrafish cardiac specification and heart morphogenesis.
Developmental Biology 02/2011; 353(1):19-28. · 4.07 Impact Factor
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ABSTRACT: Leo1 is a component of the Polymerase-Associated Factor 1 (PAF1) complex, an evolutionarily conserved protein complex involved in gene transcription regulation and chromatin remodeling. The role of leo1 in vertebrate embryogenesis has not previously been examined. Here, we report that zebrafish leo1 encodes a nuclear protein that has a similar molecular structure to Leo1 proteins from other species. From a genetic screen, we identified a zebrafish mutant defective in the leo1 gene. The truncated Leo1(LA1186) protein lacks a nuclear localization signal and is distributed mostly in the cytoplasm. Phenotypic analysis showed that while the initial patterning of the primitive heart tube is not affected in leo1(LA1186) mutant embryos, the differentiation of cardiomyocytes at the atrioventricular boundary is aberrant, suggesting a requirement for Leo1 in cardiac differentiation. In addition, the expression levels of markers for neural crest-derived cells such as crestin, gch2, dct and mitfa are greatly reduced in leo1(LA1186) mutants, indicating a requirement for Leo1 in maintaining the neural crest population. Consistent with this finding, melanocyte and xanthophore populations are severely reduced, craniofacial cartilage is barely detectable, and mbp-positive glial cells are absent in leo1(LA1186) mutants after three days of development. Taken together, these results provide the first genetic evidence of the requirement for Leo1 in the development of the heart and neural crest cell populations.
Developmental Biology 02/2010; 341(1):167-75. · 4.07 Impact Factor
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ABSTRACT: Hedgehog (Hh) signalling is essential for several aspects of embryogenesis. In Drosophila, Hh transduction is mediated by a cytoplasmic signalling complex that includes the putative serine-threonine kinase Fused (Fu) and the kinesin Costal 2 (Cos2, also known as Cos), yet Fu does not have a conserved role in Hh signalling in mammals. Mouse Fu (also known as Stk36) mutants are viable and seem to respond normally to Hh signalling. Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 orthologue, and linked Fu to known structural components of the central pair apparatus, providing evidence for the first regulatory component involved in central pair construction. We also demonstrated that zebrafish Fu is required both for Hh signalling and cilia biogenesis in Kupffer's vesicle. Mouse Fu rescued both Hh-dependent and -independent defects in zebrafish. Our results delineate a new pathway for central pair apparatus assembly, identify common regulators of Hh signalling and motile ciliogenesis, and provide insights into the evolution of the Hh cascade.
Nature 04/2009; 459(7243):98-102. · 36.28 Impact Factor
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Nature 03/2009; 459(7243):98-102. · 36.28 Impact Factor
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Drug Discovery Today Disease Models 01/2008; 5(3):135-140.
