Hematopoietic defects and positional cloning of cas002 mutant. (A-B) Light microscope images of zebrafish wild-type (WT) and cas002 embryos at 3 dpf. (C-F) WISH analysis of cmyb expression in WT and cas002 embryos at indicated development stages. Black arrows indicate thymus, kidney marrow and CHT. (C′-D′) enlarged CHT regions in C and D. (G) Genetic mapping of the cas002 region on chromosome 3. Bulk segregation analysis locates cas002 mutation to Chr. 3. Fine mapping using SSLPs narrows down to a region between markers 219-BX-5 and 220-CU-6, containing kri1l and four other genes as indicated. (H) The sequencing results of kri1l cDNA from mutant embryos show a 38 bp deletion (MU) compared with kri1l cDNA from WT embryos. (I, J) The sequencing result of kri1l genomic DNA shows a T-G transversion at the kri1l exon 1-intron 1 consensus splicing donor site (I), which causes a frame shift (H) and a premature stop codon leading to the production of a truncated Kri1l protein (J). (K) Synteny between zebrafish kri1l I and human KRI1 loci. (Left) Six genes, including KRI1, are located within a genomic region on human chromosome 19. (Right) Six zebrafish homologs are listed according to their map positions on chromosome 3 (Ensembl website). CHT, caudal hematopoietic tissue; KM, kidney marrow; Mb, mega base; T, thymus; het, heterozygote.
|
