High-throughput whole mount in situ hybridization of zebrafish embryos for analysis of tissue-specific gene expression changes after environmental perturbation.

Whole mount in situ hybridization is a process that allows the visualization of gene expression (mRNA) within the cells of an intact organism. By comparing gene expression domains between organisms that have been subjected to different environmental conditions, an understanding of the cellular and tissue-specific effects of these environmental exposures can be identified. This technique is complementary to gene expression profiling techniques such as DNA microarrays which can usually provide information only on the differential levels of gene expression within an organism or tissue. In the case of whole mount in situ hybridization there is the added ability to detect differences in the distribution of cells, within a whole organism, expressing a particular gene. Subtle changes in the distribution of cells expressing a gene may not be reflected in the overall level of gene expression when RNA samples are retrieved from a whole organism and assayed. Exploitation of automation technology has made whole mount in situ hybridization a procedure that is amiable to high-throughput genomic studies. Combining automation with computer-aided image analysis makes this an efficient strategy for quantifying subtle changes in tissues and genes expression that can result from sublethal exposures to environmental toxins, for example.

Differential expression of the methyl-cytosine binding protein 2 gene in embryonic and adult brain of zebrafish.

Epigenetic gene repression occurs as the result of the interactions between DNA and a number of proteins, including methyl-cytosine binding protein 2 (MeCP2). We have isolated a 1680 bps MeCP2 cDNA from zebrafish that shows deduced amino acid identity with Xenopus and mammalian MeCP2alpha protein sequences. The zebrafish MeCP2 gene was mapped to linkage group 8 using the LN54 radiation hybrid cell panel. The genomic organizations of the zebrafish MeCP2 and mammalian MeCP2alpha are highly similar. Relatively high levels of expression of MeCP2 mRNA were found in embryos at 1 to 4 h postfertilization (hpf), after 24 hpf, and in adult brain and eyes. Whole mount in situ hybridization was performed on embryos and revealed ubiquitous MeCP2 expression during early zebrafish development. At 24 and 48 hpf, the highest levels of expression are found in the epiphysis, midbrain, ventricular zone, and the otic vesicles. In adult zebrafish, MeCP2-expressing cells were found throughout the brain. Regions that are enriched in MeCP2 mRNA include the pallium layer of the telencephalon, the preoptic area, the periventricular grey zone, lobus caudalis, and the vagus lobes. In the cerebellum, high expression is found in the Purkinje and Golgi type 2 cells.

Histone deacetylase 1 (HDAC-1) required for the normal formation of craniofacial cartilage and pectoral fins of the zebrafish.

Histone deacetylases interact with nucleosomes to facilitate the formation of transcriptionally repressed chromatin. In the present study, we show that histone deacetylase 1 (hdac-1) is expressed throughout embryonic development of the zebrafish. The expression of hdac-1 is ubiquitous in early embryos (2-16 hr postfertilization), but at later stages (36 and 48 hr postfertilization), it is primarily restricted to the branchial arches, fin bud mesenchyme, and hindbrain. We report the phenotypes of hdac-1 homozygous mutant embryos and embryos injected with an hdac-1 antisense morpholino. These embryos possess a complex phenotype affecting several embryonic structures. We observed developmental abnormalities in the heart and neural epithelial structures, including the retina and the loss of craniofacial cartilage and pectoral fins.