C. Cristofre Martin

Specific craniofacial cartilage dysmorphogenesis coincides with a loss of dlx gene expression in retinoic acid-treated zebrafish embryos

Treatments of zebrafish embryos with retinoic acid (RA), a substance known to cause abnormal craniofacial cartilage development in other vertebrates, result in dose- and stage-dependent losses of dlx homeobox gene expression in several regions of the embryo. Dlx expression in neural crest cells migrating from the hindbrain and in the visceral arch primordia is particularly sensitive to RA treatment. The strongest effects are observed when RA is administered prior to or during crest cell migration but effects can also be observed if RA is applied when the cells have entered the primordia of the arches. Losses of dlx expression correlate either with the loss of cartilage elements originating from hindbrain neural crest cells or with abnormal morphology of these elements. Cartilage elements that originate from midbrain neural crest cells, which do not express dlx genes, are less affected. Taken together with the observation that the normal patterns of visceral arch dlx expression just prior to cartilage condensation resemble the morphology of the cartilage elements that are about to differentiate, our results suggest that dlx genes are an important part of a multi-step process in the development of a subset of craniofacial cartilage elements.

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. Developmental Dynamics 231:647–654, 2004.

Genotype-specific modifiers of transgene methylation and expression in the zebrafish, Danio rerio

Previous reports involving mammalian systems, particularly mice, have demonstrated the existence of cis- and trans-acting modifiers of transgene methylation. These modifiers are thought to be important in dominance modification, genome imprinting and cellular expression mosaicism. Their potential role in the penetrance and severity of many complex human diseases could be of even greater significance. In the present investigation we demonstrate that modifiers that act in a similar fashion to those identified in mice also exist in a non-mammalian vertebrate, the zebrafish Danio rerio. We also provide evidence that the transgene methylation pattern may be influenced by the sex of the individual and environmental modulators such as temperature and sodium butyrate. These data support the theory that this type of dominance modification is mechanistically similar to position effect variegation in Drosophila. Furthermore, these data suggest evolutionary conservation of the modifiers, at least within vertebrates, and imply that they and their actions are important in normal vertebrate development.

Differentiation trees, a junk DNA molecular clock, and the evolution of neoteny in salamanders

Obligate neotenic salamanders die if forced to metamorphose. We suggest that this can be explained by assuming: 1) their “excess” DNA is “junk” DNA; 2) the “adult” specifying portion of the DNA becomes junk DNA and is available for repeated duplication. This suggests a “new” junk DNA molecular clock. We obtain remarkable agreement in “predicting” the amount of DNA per nucleus in present day non‐obligate neotene salamanders from this molecular clock. These observatons are consistent with the idea that the development of these animals is describable in terms of differentiation trees whose branches (gene cascades) corresponding to adult somatic tissues accumulate deleterious mutations over evolutionary time. We show that the amount of DNA per nucleus increases linearly with the phylogenetic age of salamander families. The lack of constraints by natural selection, on unused adult branches, may account for the large amount of so‐called “junk DNA” in obligate neotenic salamanders. The effects of this excess DNA, via increased cell size, suggest a positive feedback, ecophysiological explanation for such junk DNA: adaptation to cool water environments is enhanced by the lower metabolism associated with more DNA, larger cells and slower developmental time.

Expression of the chaperonin 10 gene during zebrafish development

Abstract We have isolated a cDNA encoding chaperonin 10 (cpn10) from the zebrafish. Using northern, western, and in situ hybridization analysis, we observed that the cpn10 gene is expressed uniformly and ubiquitously throughout embryonic development of the zebrafish. Upregulation of cpn10 expression was observed following exposure of zebrafish embryos to a heat shock of 1 hour at 37°C compared to control embryos raised at 27°C. The extracellular form of Cpn10 called early pregnancy factor (EPF), found in the serum of pregnant mammals, was not detected in the serum of either male or female zebrafish. These expression studies suggest that Cpn10 plays a general role in zebrafish development as well as being consistent with the hypothesis that EPF is involved in the embryo implantation process in mammals.

Not Just a Fishing Trip - Environmental Genomics Using Zebrafish

Genetic diversity is the raw material needed by a species allowing adaptation to changing environmental conditions and thus ensuring long-term sustainability. The development of technologies for environmental genomics provides us with the opportunity to link information, at the whole genome level, with the response of an organism to its natural environment. Over the past 15 years a small tropical fish native to the rivers of India and south Asia, the zebrafish (Danio rerio), has become one of the most popular vertebrate model systems. Zebrafish are abundant and many populations exist that are reproductively isolated. They evolved under distinct environments, and this have lead to genetic diversity and, as a consequence, has created genotypic and phenotypic differences between the populations. For this fish species, a large number of molecular and genomic tools have been developed. As a result, the zebrafish has emerged as a popular model for the study of embryonic development and genetics as well as the study human disease counterparts. The advantages that zebrafish possess, in addition to newly developed large scale screening assays, such as automated in situ hybridization and transgenics for example, has lead to researchers using zebrafish to study toxicogenomics and environmental genomics. Researchers have identified molecular and biochemical pathways, which may not have been observable using standard methods, that are disrupted by some toxin exposures and environmental stressors. These studies will allow us to potentially formulate specific predictions on how vertebrate organisms and populations may be affected by both man- made and natural changes in the environment.

A role for modifier genes in genome imprinting.

It is, by now, an old observation that the phenotype elicited by a mutant allele at a particular locus is not the sole result of the DNA sequence of that allele. The truth of this statement may be easily recognized by comparing different individuals within the same family, each of whom has the same genetic disease. Siblings who have neurofibromatosis, for example, all share the same disease allele at the NF1 locus but the manifestations of disease (number and size of cafe au lait spots, number and size of neurofibromas, presence of neurofibrosarcoma, etc.) among the siblings may vary dramatically (Easton et al. 1993). These variations in disease phenotype between individuals who carry the same mutant allele have been subsumed under the mechanistically vague concept that many diseases exhibit variable expressivity (Thompson et al. 1991). Such differences in disease phenotype may result from many factors, including stochastic processes and environmental influences, but at least some of these differences are thought to result from the collective action of additional genetic factors that differ between individuals. These additional genetic factors are said to modify the phenotype and are, therefore, called modifier genes.