Biomedical subjects

Patrick H Krone

Publications and source records attributed to Patrick H Krone.

7 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.

Animals↗

PubMed2006

Accumulation and elimination of cadmium in larval stage zebrafish following acute exposure.

A number of recent studies have examined the impact of acute cadmium exposure on early zebrafish development at the morphological, cellular, and molecular levels. However, no information on the accumulation and elimination of cadmium during early life stages of zebrafish development has been available. Here we have quantified cadmium accumulation in larval zebrafish (Danio rerio) by graphite furnace atomic absorption spectroscopy following short-term acute exposure and recovery periods. Zebrafish (80 h postfertilization) were exposed to various concentrations of cadmium (0.2, 1.0, 5.0, 25, 125 microM) for 3 h. Cadmium accumulation in larvae increased with exposure concentration. After exposure at 5.0, 25, and 125 microM cadmium, the fish were allowed to recover in freshwater for 0, 12, or 24 h. Cadmium content did not show a statistically significant decrease over the recovery period when exposed to 5.0 or 25 microM cadmium, whereas significant losses over the recovery period were observed following 125 microM exposure. These results suggest that the larval zebrafish decrease total cadmium body burden only following relatively high short-term acutely toxic exposures.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

PubMed2002

Expression and genomic organization of the zebrafish chaperonin gene complex.

Chaperonin 10 and chaperonin 60 monomers exist within the multimeric mitochondrial chaperonin folding complex with a stoichiometry of 2:1. This complex is located in the mitochondrial matrix, where it aids in the folding and acquisition of the tertiary structure of proteins. We have previously isolated the cpn10 cDNA in zebrafish (Danio rerio), and demonstrated that it is ubiquitously expressed during embryonic development and transcriptionally upregulated after exposure to heat shock. In the present study, we have isolated a cDNA encoding chaperonin 60 (cpn60) from zebrafish, and have shown that it is similarly expressed uniformly and ubiquitously throughout early embryonic development of zebrafish. Upregulation of cpn60 expression was also observed after exposure of zebrafish embryos to a heat shock of 1 h at 37 degrees C compared with control embryos raised at 27 degrees C. The induction of the cpn60 heat shock response was greatest after 1 h of heat shock, whereas significant decreases of cpn60 mRNA were observed within 2 h following a return to 27 degrees C. We subsequently isolated genomic DNA sequences for both of these genes, and show that they are also arranged in a head-to-head organization and share a common bidirectional promoter that contains a single heat shock element (HSE). Our database analysis shows that this head-to-head organization is also found in human (Homo sapiens), rat (Rattus norvegicus), pufferfish (Fugu rubripes), and Caenorhabditis elegans, but not in Drosophila or yeast (Saccharomyces cerevisiae). The data suggest that the genomic organization of the cpn gene complex has been conserved across the vertebrates.

Amino Acid Sequence↗

PubMed2002

Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line.

The toxic effects of cadmium and other heavy metals have been well established, and many of these and other environmental pollutants are known to be embryotoxic or teratogenic. However, it has proven difficult to identify individual cells that respond to toxicants among the wide range of cell populations in an intact animal, particularly during early development when cells are continually changing their molecular and physiologic characteristics as they differentiate. Here we report the establishment of an in vivo system that uses hsp70 gene activation as a measure of cadmium toxicity in living early larvae of transgenic zebrafish carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. We demonstrate that eGFP expression in this strain of fish acts as an accurate and reproducible indicator of cell-specific induction of hsp70 gene expression. Furthermore, the transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early-life-stage toxicity and is sensitive enough to detect cadmium at doses below the median combined adverse effect concentration and the median lethal concentration. The stable nature of this transgenic line should allow for extremely rapid and reproducible toxicologic profiling of embryos and larvae throughout development.

Animals↗