Limits...
Transcriptome analysis of severe hypoxic stress during development in zebrafish.

Woods IG, Imam FB - Genom Data (2015)

Bottom Line: Although hypoxic preconditioning has been demonstrated in several model organisms and tissues including the heart and brain, its molecular mechanisms remain poorly understood.Accordingly, we used embryonic and larval zebrafish to develop a novel vertebrate model for hypoxic preconditioning, and used this model to identify conserved hypoxia-regulated transcripts for further functional study as published in Manchenkov et al. (2015) in G3: Genes / Genomes / Genetics.In this Brief article, we provide extensive annotation for the most strongly hypoxia-regulated genes in zebrafish, including their human orthologs, and describe in detail the methods used to identify, filter, and annotate hypoxia-regulated transcripts for downstream functional and bioinformatic assays using the source data provided in Gene Expression Omnibus Accession GSE68473.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Ithaca College, Ithaca, NY, USA.

ABSTRACT
Hypoxia causes critical cellular injury both in early human development and in adulthood, leading to cerebral palsy, stroke, and myocardial infarction. Interestingly, a remarkable phenomenon known as hypoxic preconditioning arises when a brief hypoxia exposure protects target organs against subsequent, severe hypoxia. Although hypoxic preconditioning has been demonstrated in several model organisms and tissues including the heart and brain, its molecular mechanisms remain poorly understood. Accordingly, we used embryonic and larval zebrafish to develop a novel vertebrate model for hypoxic preconditioning, and used this model to identify conserved hypoxia-regulated transcripts for further functional study as published in Manchenkov et al. (2015) in G3: Genes / Genomes / Genetics. In this Brief article, we provide extensive annotation for the most strongly hypoxia-regulated genes in zebrafish, including their human orthologs, and describe in detail the methods used to identify, filter, and annotate hypoxia-regulated transcripts for downstream functional and bioinformatic assays using the source data provided in Gene Expression Omnibus Accession GSE68473.

No MeSH data available.


Related in: MedlinePlus

In vivo hypoxic stress protection assay. Candidate genes were assessed for a functional role in protection from hypoxic stress through two assays, both of which were performed with targeted morphant (knockdown; red needle and red outline) and embryos injected with control morpholino oligonucleotides. For acute hypoxia, larvae were subjected to hypoxia at 1.5 dpf and evaluated for exacerbation of lethality at 5 dpf (blue bolt). For the hypoxic preconditioning assay, larvae were preconditioned through a milder hypoxia exposure at 1 dpf (yellow bolt) and re-exposed to prolonged severe hypoxia at 1.5 dpf (blue bolt).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664676&req=5

f0020: In vivo hypoxic stress protection assay. Candidate genes were assessed for a functional role in protection from hypoxic stress through two assays, both of which were performed with targeted morphant (knockdown; red needle and red outline) and embryos injected with control morpholino oligonucleotides. For acute hypoxia, larvae were subjected to hypoxia at 1.5 dpf and evaluated for exacerbation of lethality at 5 dpf (blue bolt). For the hypoxic preconditioning assay, larvae were preconditioned through a milder hypoxia exposure at 1 dpf (yellow bolt) and re-exposed to prolonged severe hypoxia at 1.5 dpf (blue bolt).

Mentions: To identify novel hypoxia protective factors, we developed a novel in vivo hypoxic stress assay in zebrafish. First, we optimized hypoxic stress parameters at two different timepoints during zebrafish development and collected control and hypoxia-exposed embryos at gastrula (shield, 6 h postfertilization or hpf) and segmentation (8 somite, 12 hpf) stages to identify shared hypoxia responses at two timepoints (Fig. 1A). We reasoned that hypoxia response genes activated at distinct developmental timepoints were likely to be enriched for evolutionarily conserved hypoxia-protective components relevant for human biology and disease. We performed RNA extraction, labeling, and hybridization to NimbleGen zebrafish microarrays and show both the raw and normalized intensity plots and histograms for these data (Fig. 1B, C). We combined data from normoxic control samples at both gastrula and segmentation in comparison with matched hypoxia-exposed samples and identified 3768 of the 37,157 transcripts measured to be differentially expressed greater than 2-fold under hypoxia (Fig. 2). To further increase the utility of this dataset for the scientific community, we provide additional annotations in this article that includes human homologs and functional properties/domains for all transcripts measured, including the 300 most hypoxia-induced and hypoxia-repressed genes passing statistical significance criteria (Supplementary Table 1, Supplementary Table 2, Supplementary Table 3). From these lists of genes, we validated the expression of a subset of individual hypoxia-induced genes using qPCR and/or in situ hybridization, with irs2 shown as a representative hypoxia-induced gene that exhibited increased and ectopic expression at both gastrula and segmentation (Fig. 3). We further tested individual expression-validated genes for hypoxia-protective function in both acute hypoxic stress buffering and hypoxic preconditioning assays in zebrafish (Fig. 4) and identified several novel hypoxia-induced genes that function to protect against acute hypoxic stress and/or provide hypoxic preconditioning protection [1].


Transcriptome analysis of severe hypoxic stress during development in zebrafish.

Woods IG, Imam FB - Genom Data (2015)

In vivo hypoxic stress protection assay. Candidate genes were assessed for a functional role in protection from hypoxic stress through two assays, both of which were performed with targeted morphant (knockdown; red needle and red outline) and embryos injected with control morpholino oligonucleotides. For acute hypoxia, larvae were subjected to hypoxia at 1.5 dpf and evaluated for exacerbation of lethality at 5 dpf (blue bolt). For the hypoxic preconditioning assay, larvae were preconditioned through a milder hypoxia exposure at 1 dpf (yellow bolt) and re-exposed to prolonged severe hypoxia at 1.5 dpf (blue bolt).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664676&req=5

f0020: In vivo hypoxic stress protection assay. Candidate genes were assessed for a functional role in protection from hypoxic stress through two assays, both of which were performed with targeted morphant (knockdown; red needle and red outline) and embryos injected with control morpholino oligonucleotides. For acute hypoxia, larvae were subjected to hypoxia at 1.5 dpf and evaluated for exacerbation of lethality at 5 dpf (blue bolt). For the hypoxic preconditioning assay, larvae were preconditioned through a milder hypoxia exposure at 1 dpf (yellow bolt) and re-exposed to prolonged severe hypoxia at 1.5 dpf (blue bolt).
Mentions: To identify novel hypoxia protective factors, we developed a novel in vivo hypoxic stress assay in zebrafish. First, we optimized hypoxic stress parameters at two different timepoints during zebrafish development and collected control and hypoxia-exposed embryos at gastrula (shield, 6 h postfertilization or hpf) and segmentation (8 somite, 12 hpf) stages to identify shared hypoxia responses at two timepoints (Fig. 1A). We reasoned that hypoxia response genes activated at distinct developmental timepoints were likely to be enriched for evolutionarily conserved hypoxia-protective components relevant for human biology and disease. We performed RNA extraction, labeling, and hybridization to NimbleGen zebrafish microarrays and show both the raw and normalized intensity plots and histograms for these data (Fig. 1B, C). We combined data from normoxic control samples at both gastrula and segmentation in comparison with matched hypoxia-exposed samples and identified 3768 of the 37,157 transcripts measured to be differentially expressed greater than 2-fold under hypoxia (Fig. 2). To further increase the utility of this dataset for the scientific community, we provide additional annotations in this article that includes human homologs and functional properties/domains for all transcripts measured, including the 300 most hypoxia-induced and hypoxia-repressed genes passing statistical significance criteria (Supplementary Table 1, Supplementary Table 2, Supplementary Table 3). From these lists of genes, we validated the expression of a subset of individual hypoxia-induced genes using qPCR and/or in situ hybridization, with irs2 shown as a representative hypoxia-induced gene that exhibited increased and ectopic expression at both gastrula and segmentation (Fig. 3). We further tested individual expression-validated genes for hypoxia-protective function in both acute hypoxic stress buffering and hypoxic preconditioning assays in zebrafish (Fig. 4) and identified several novel hypoxia-induced genes that function to protect against acute hypoxic stress and/or provide hypoxic preconditioning protection [1].

Bottom Line: Although hypoxic preconditioning has been demonstrated in several model organisms and tissues including the heart and brain, its molecular mechanisms remain poorly understood.Accordingly, we used embryonic and larval zebrafish to develop a novel vertebrate model for hypoxic preconditioning, and used this model to identify conserved hypoxia-regulated transcripts for further functional study as published in Manchenkov et al. (2015) in G3: Genes / Genomes / Genetics.In this Brief article, we provide extensive annotation for the most strongly hypoxia-regulated genes in zebrafish, including their human orthologs, and describe in detail the methods used to identify, filter, and annotate hypoxia-regulated transcripts for downstream functional and bioinformatic assays using the source data provided in Gene Expression Omnibus Accession GSE68473.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Ithaca College, Ithaca, NY, USA.

ABSTRACT
Hypoxia causes critical cellular injury both in early human development and in adulthood, leading to cerebral palsy, stroke, and myocardial infarction. Interestingly, a remarkable phenomenon known as hypoxic preconditioning arises when a brief hypoxia exposure protects target organs against subsequent, severe hypoxia. Although hypoxic preconditioning has been demonstrated in several model organisms and tissues including the heart and brain, its molecular mechanisms remain poorly understood. Accordingly, we used embryonic and larval zebrafish to develop a novel vertebrate model for hypoxic preconditioning, and used this model to identify conserved hypoxia-regulated transcripts for further functional study as published in Manchenkov et al. (2015) in G3: Genes / Genomes / Genetics. In this Brief article, we provide extensive annotation for the most strongly hypoxia-regulated genes in zebrafish, including their human orthologs, and describe in detail the methods used to identify, filter, and annotate hypoxia-regulated transcripts for downstream functional and bioinformatic assays using the source data provided in Gene Expression Omnibus Accession GSE68473.

No MeSH data available.


Related in: MedlinePlus