Biomedical subjects

Tyler G Evans

Publications and source records attributed to Tyler G Evans.

4 recordsLinked to original sources

PubMed2006

Heat shock factor 1 is required for constitutive Hsp70 expression and normal lens development in embryonic zebrafish.

Heat shock factors (HSFs) are the major transcription factors responsible for heat-induced upregulation of heat shock protein (Hsp) genes. All three mammalian HSFs (HSF1, HSF2, HSF4) have also been shown to be required for normal mammalian development. It is currently unknown if HSFs play similarly important roles during normal development of non-mammalian vertebrates. In the present study, a morpholino modified antisense oligonucleotide (MO) approach targeted against hsf1 mRNA (hsf1-MO) was used to examine the requirement of HSF1 in zebrafish development. Embryos depleted of HSF1 displayed a reproducible small eye phenotype characterized by an immature lens and a disorganized retinal structure. These defects were strikingly similar to those observed when constitutive, lens specific Hsp70 expression was reduced through the microinjection of MO targeting hsp70. The data suggest that HSF1 is involved in regulating constitutive lens specific expression of hsp70 in the embryonic zebrafish. This conclusion is supported by a marked reduction in Hsp70 protein in hsf1-MO injected embryos. Microinjection of MO targeted to hsf2 mRNA (hsf2-MO) did not result in a small eye phenotype in a significant number of embryos. These data also suggest that HSF1 and HSF2 play distinct roles in non-mammalian vertebrates, similarly to what has been demonstrated previously in mouse.

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PubMed2005

Zebrafish Hsp70 is required for embryonic lens formation.

Heat shock proteins (Hsps) were originally identified as proteins expressed after exposure of cells to environmental stress. Several Hsps were subsequently shown to play roles as molecular chaperones in normal intracellular protein folding and targeting events and to be expressed during discrete periods in the development of several embryonic tissues. However, only recently have studies begun to address the specific developmental consequences of inhibiting Hsp expression to determine whether these molecular chaperones are required for specific developmental events. We have previously shown that the heat-inducible zebrafish hsp70 gene is expressed during a distinct temporal window of embryonic lens formation at normal growth temperatures. In addition, a 1.5-kb fragment of the zebrafish hsp70 gene promoter is sufficient to direct expression of a gfp reporter gene to the lens, suggesting that the hsp70 gene is expressed as part of the normal lens development program. Here, we used microinjection of morpholino-modified antisense oligonucleotides (MOs) to reduce Hsp70 levels during zebrafish development and to show that Hsp70 is required for normal lens formation. Hsp70-MO-injected embryos exhibited a small-eye phenotype relative to wild-type and control-injected animals, with the phenotype discernable during the second day of development. Histological and immunological analysis revealed a small, underdeveloped lens. Numerous terminal deoxynucleotidyl transferase-mediated dUTP-fluoroscein nick-end labeling (TUNEL)-positive nuclei appeared in the lens of small-eye embryos after 48 hours postfertilization (hpf), whereas they were no longer apparent in untreated embryos by this age. Lenses transplanted from hsp70-MO-injected embryos into wild-type hosts failed to recover and retained the immature morphology characteristic of the small-eye phenotype, indicating that the lens phenotype is lens autonomous. Our data suggest that the lens defect in hsp70-MO-injected embryos is predominantly at the level of postmitotic lens fiber differentiation, a result supported by the appearance of mature lens organization in these embryos by 5 days postfertilization, once morpholino degradation or dilution has occurred.

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PubMed2003

Heat shock gene expression and function during zebrafish embryogenesis.

Recent work in the zebrafish, Danio rerio, indicates that heat shock genes are expressed in unique spatial patterns under non-stress conditions. In particular, hsp90alpha is expressed during the normal differentiation of striated muscle fibres, and hsp70-4 is expressed during normal lens development in the eye. Furthermore, disruption of the activity of either of these genes or their protein products gives rise to unique embryonic phenotypes that result from failures in proper somitic muscle development and lens development, respectively. Embryonic hsp70-4 expression is also activated in a cell-specific manner following heavy metal exposure. This has allowed for the development of a hsp70-4/eGFP reporter gene system in stable transgenic zebrafish that serves as a reliable yet extremely quick indicator of cell-specific toxicity in the context of the multicellular, living embryo.

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PubMed2002

The heat-inducible zebrafish hsp70 gene is expressed during normal lens development under non-stress conditions.

In the present study, we show that the stress-inducible hsp70 gene in zebrafish is strongly and specifically expressed during normal lens formation from 28 to 38 hours post-fertilization, and is subsequently downregulated by 2 days of age. Only weak constitutive hsp70 mRNA signal was sporadically observed in other embryonic tissues. Similarly, transgenic fish carrying a 1.5 kb fragment of the hsp70 promoter linked to eGFP exhibited fluorescence only in the lens. In contrast, both the endogenous hsp70 gene and the transgene were strongly expressed throughout the embryo following heat shock at the same developmental stages.

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