Positional cloning and functional analysis of the SF3B1 gene in zebrafish

Zebrafish toast b460 (tst) is a recessive embryonic lethal mutation, characterized by visible defects in both neural crest and blood development. tst is involved in the neural plate border development in zebrafish embryos, resulting in the complete lack neural crest derivatives and a decrease in the number of Rohon-Beard sensory neurons. In addition, lateral plate mesoderm development is also affected in tst mutants, as development of most of its derivatives are disrupted during hematopoiesis, vasculogenesis, and cardiogenesis. Other ectodermal and mesodermal derivatives develop normally in tst mutants. The tst locus has been positionally cloned and encodes splicing factor 3b subunit 1 (sf3b1). Sequence analysis between wild-type and homozygous mutant genomic DNA identified the nucleotide mutation at 5' splicing site in the 4th intron of sf3b1 genomic DNA where T is changed to G in the tst mutant gene. The nucleotide change causes abnormal splicing in tst homozygotes with variant transcripts. The truncations are predicted to occur in the extreme N-terminal region of the protein, thus eliminating essential functional regions and are therefore predicted to be non-functional. The presence of normal transcripts and the more severe phenotype of sf3b1 morphants compared to tst mutants indicate that the tst mutation is hypomorphic. Zebrafish genome analysis shows that sf3b1 is a single-copy gene. It is expressed ubiquitously during early embryonic development in zebrafish. Studies of interactions between sf3b1 and key transcriptional regulators of neural crest development and hematopoiesis reveal that deficiency of sf3b1 function not only causes disruption in the expression levels of genes required for sublineage fate specification, but also results in abnormal pre-mRNA splicing of some of these genes. As a result, the survival, migration, and differentiation of cells derived from both neural plate border and lateral plate mesoderm are severely disrupted. These results demonstrate that the ubiquitous and essential pre-mRNA processing gene sf3b1 is required to different degrees by specific embryonic cell populations during development. Thus, while some genes are required by all cell types, the timing and degree of the requirement for such "housekeeping" genes differs for specific embryonic cell population during embryogenesis.