Abstract

1. Whole mount In Situ hybridization of the zebra fish ortholog of human AP000553.6 Whole mount in situ hybridization Human chromosome 22 Zebrafish as a model system Human Chromosome 22 Abstract Whole mount in situ hybridization of ssDNA probes for the ZF1 gene Percentage Identity Plot Conclusion Alkaline phosphatase-conjugated anti-DIG antibody Human Chromosome 22 encodes 223 predicted genes that are identical to known human cDNAs, protein sequences or know genes, 228 pseudogenes, 130 are immunoglobulin gene segments or immunoglobulin pseudogenes and 34 genes with either no known function and /or a known expression profile. This distribution of predicted genes is comparable to that observed over the entire human genomic sequence. In an effort to begin determining the function of these 34 genes with either no known function and /or a known expression profile, our laboratory has developed a combined comparative genomic sequencing and in situ whole mount zebrafish hybridization approach, initially focusing these human chromosome 22 predicted genes. Through comparative genomic sequencing we have detected and confirmed many of the previously undefined but predicted human chromosome 22 genes and a detailed cross species analysis of the genes of human chromosome 22 in the Cat Eye and DiGeorge syndrome regions, the BCR region and the NF2 region with chimpanzee, baboon, bovine, mouse and zebrafish will be presented. To elucidate the expression pattern of the unknown genes, we systematically have been identifying the zebrafish homologs of these unknown genes and generating both sense and antisense ssDNA probes by unidirectional (single primer) amplification for whole mount in situ hybridization to developing zebrafish embryos. The probes for these in situ studies, were chosen after a reciprocal blast comparison of the predicted human chromosome 22 genes to the recently available compilation of zebrafish predicted genes from the Sanger Institute’s zebrafish whole genome shotgun assembly version 2. With a blast expect score threshold to 10-3, we observed that approximately 70 of the human chromosome 22 coding genes had zebrafish homologues. When we lower the threshold to 10, we observed 85 of the human chromosome 22 coding genes had zebrafish homologues. Probes constructed for several predicted but unknown zebrafish orthologs of human chromosome 22 gene exons indicate distinct expression profiles in developing brain (either throughout the brain or its outer edge), the otic placode (the auditory (hearing) and vestibular (balance) organ of the fish, equivalent to the inner ear of Amniotes), as well as the pectoral fin region, the notochord and the myotomes. The results of these whole mount in situ hybridizations on 12, 24, 48 and 72hpf zebrafish embryos for several labeled DNA probes, as well as the multiple sequnce alignments of representative human and zebrafish genes are presented. This research was funded in part by a grant from the NIH-NHGRI. Sense probe Human chromosome 22: • Have a short, ~ 3 month to reproductive maturity. • Can be easily bred in the lab in large numbers. • Are small in size - an adult is just a few centimeters long. • Have an ~ 5 day embryonic development period from fertilized egg to a swimming fish. • The embryos are transparent making it easy to see internal organs during development. • Is well established as a resource for genetic studies. • The Sanger Institute is completing the genome sequence, which presently is ~50% complete and publicly available. • More than 90 % of the predicted human genes have a zebra fish ortholog. No probe Antisense probe BCIP* + NBT** • 70% of the human chromosome 22 coding genes had zebrafish homologues, with up to 80% sequence similarity in the coding region but low conservation in the non-coding region. • Preliminary studies on zebrafish homologues of seven unknown human genes by whole mount in situ hybridization of zebrafish embryos shows their expression in the brain and behind the eyes. • Studies on the sensitivity of a given probe of variable length at different staining period showed that probes as small as 100bp showed the same hybridization specificity when compared to probes of larger length. Generation of probe from exon specific PCR was carried out with high efficiency. • The whole mount in situ hybridization was successfully scaled to a 96-wells microtiter plate format. • The zebra fish is an ideal system in which to investigate protein expression profiles for genes that are human orthologs. • This research has been funded by a grant from the NIH-NHGRI. DIG-labeled ssDNA or RNA probe 22p With the completion of human chromosome 22: Among 935 identified genes 223 are known genes 228 are pseudogenes 130 are Ig segment ※ The second smallest chromosome. Antisense probe Sense probe No probe 12 hpf ※ p arm encodes only rRNA. P P PIP Percentage Identity plot showing conservation of Dna sequence between Human and Chimpanzee, Cow, Rat, Mouse, Fugu, Zebrafish. ※ q arm (33.4 Mb) contains approximately 850 genes. 120hpf notochord notochord 24 hpf Sequencing of human chromosome 22: 3’ UTR region where sense and anti-sense probe were generated for whole mount in situ hybridization. Digoxigeninlabel uridine Wash ※ Covering 97% of chromosome 22. Wash P 48hpf 22q ※ 11 contigs (10 gaps). otic vesicle notochord notochord mRNA ※ Less than 1 error in 50,000bp. 48 hpf Our focus is on the remaining 354 predicted but unkown-function genes. ※ Genes span 39% of sequence. Protein-coding regions span 3% of sequence. Repetitive sequences span 42% of sequence. 24hpf 1. Add digoxigenin-labeled probe complementary to RNA of interest 2. Add alkaline phosphatase-conjugated antibody that binds to digoxigenin 3. Add BCIP + NBT that turns dark purple dye when dephosphorylated by the alkaline phosphatase thereby coloring the cell notochord otic vesicle 72 hpf Comparative map human chromosome 22q11.1-q11.22, and the syntenic BAC clones on the chimp, baboon, cow, mouse, and zebrafish using Generic Genome Browser (http://www.gmod.org/). The region of homology and the color codes are indicated on the top and bottom respectively. A percentage identity plot showing conservation of coding region across the species. Human sequence was set as the reference. liver Antisense probe hybridization to the notochord, otic vesicle, and liver, with sense probe hybridization to the notochord Only antisense probe hybridization to the Otic Placode *BCIP = 5 bromo-4-chloro-indoxyl phosphate **NBT = nitro-blue-tetrazolium Summary of in situ hybridization studies: Gene Antisense probe Sense probe Dj508I15.c22.5 Brain - Phf5a-like gene Brain - KIAA0819-ZF1 Otic placode - KIAA0819-ZF2 Hind brain, Otic placode, - and pectoral fin NM_032775 Otic placode and - swim bladder DGCR8 Hind brain, Hind brain, Branchial arches,pectoral fin Heart, and pectoral fin AP000553.6 Notochord, liver Notochord Hind brain, and Otic placode Comparative analysis of Human Chromosome 22 syntenic regions in Chimpanzee, Baboon, Bovine, Mouse and Zebrafish and Expression Profiling of human homologs in Zebrafish using Whole Mount in situ hybridizaion. Axin Hua, Shelly Oommen, Christopher Lau, Jianfeng Li, Hung-Chun Yu, Trang Do, Bruce A. Roe Department of Chemistry and Biochemistry University of Oklahoma, 101 E. Constellation, Norman, 73019.OK