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Novel Genes Critical for Hypoxic Preconditioning in Zebrafish Are Regulators of Insulin and Glucose Metabolism.

Manchenkov T, Pasillas MP, Haddad GG, Imam FB - G3 (Bethesda) (2015)

Bottom Line: Severe hypoxia is a common cause of major brain, heart, and kidney injury in adults, children, and newborns.However, mild hypoxia can be protective against later, more severe hypoxia exposure via "hypoxic preconditioning," a phenomenon that is not yet fully understood.Using a functional genomic approach, we used this zebrafish model to identify and validate five novel hypoxia-protective genes, including irs2, crtc3, and camk2g2, which have been previously implicated in metabolic regulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Neonatology, University of California San Diego School of Medicine, La Jolla, California 92093.

No MeSH data available.


Related in: MedlinePlus

Knockdown of hypoxia-induced stress candidate genes identifies a stress-sensitive phenotypic class. (A) Outline of functional acute hypoxia screen performed on morphants of the 10 candidates validated from the initial differential expression screen as described. Embryo microinjection was performed for each MO, after which control-injected morphant (ctl-MO; clear) and hypoxia target morphant (h-MO; blue) larvae were then divided into control and hypoxia-exposure (red box) groups, resulting in four different groups in total—ctl-MO injection only, ctl-MO + hypoxia, h-MO injection only, and h-MO + hypoxia. (B) Different phenotypic classes from a moderate hypoxic stress protocol sH(1.5d:30h) for the 10 genes tested by MO knockdown are shown: “no/mild” with no defects or minor defects only in the fin or air bladder (five genes, camk2g2 shown); “dev” have severe defects such as curved body axis or pericardial edema irrespective of hypoxia treatment (four genes, opsin5 shown); and “hypoxia-sensitive,” where morphant phenotype is greatly exacerbated under hypoxia (one gene, irs2). dnd1, whose expression and function are restricted to the developing gonad, is used as a control MO. See Materials and Methods for detailed phenotypic scoring criteria. Scale bar = 0.5 mm.
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fig3: Knockdown of hypoxia-induced stress candidate genes identifies a stress-sensitive phenotypic class. (A) Outline of functional acute hypoxia screen performed on morphants of the 10 candidates validated from the initial differential expression screen as described. Embryo microinjection was performed for each MO, after which control-injected morphant (ctl-MO; clear) and hypoxia target morphant (h-MO; blue) larvae were then divided into control and hypoxia-exposure (red box) groups, resulting in four different groups in total—ctl-MO injection only, ctl-MO + hypoxia, h-MO injection only, and h-MO + hypoxia. (B) Different phenotypic classes from a moderate hypoxic stress protocol sH(1.5d:30h) for the 10 genes tested by MO knockdown are shown: “no/mild” with no defects or minor defects only in the fin or air bladder (five genes, camk2g2 shown); “dev” have severe defects such as curved body axis or pericardial edema irrespective of hypoxia treatment (four genes, opsin5 shown); and “hypoxia-sensitive,” where morphant phenotype is greatly exacerbated under hypoxia (one gene, irs2). dnd1, whose expression and function are restricted to the developing gonad, is used as a control MO. See Materials and Methods for detailed phenotypic scoring criteria. Scale bar = 0.5 mm.

Mentions: Hypoxia-activated genes identified by microarray at gastrula and segmentation stages were further validated by qPCR and/or in situ hybridization (Figure 3). Genes demonstrating hypoxia-inducibility at 1 dpf, and therefore conservation of response across several phases of development, were preferentially chosen for MO knockdown in a secondary functional acute hypoxia sensitivity screen (Table 1, Figure 3). The rationale for selection of hypoxia-upregulated genes was that MO knockdown could uncover a necessary and protective role, therefore resulting in decreased resistance against sH. In contrast, the prediction and interpretation of results from knockdown of hypoxia-repressed genes were anticipated to be less straightforward and are not a focus of this study.


Novel Genes Critical for Hypoxic Preconditioning in Zebrafish Are Regulators of Insulin and Glucose Metabolism.

Manchenkov T, Pasillas MP, Haddad GG, Imam FB - G3 (Bethesda) (2015)

Knockdown of hypoxia-induced stress candidate genes identifies a stress-sensitive phenotypic class. (A) Outline of functional acute hypoxia screen performed on morphants of the 10 candidates validated from the initial differential expression screen as described. Embryo microinjection was performed for each MO, after which control-injected morphant (ctl-MO; clear) and hypoxia target morphant (h-MO; blue) larvae were then divided into control and hypoxia-exposure (red box) groups, resulting in four different groups in total—ctl-MO injection only, ctl-MO + hypoxia, h-MO injection only, and h-MO + hypoxia. (B) Different phenotypic classes from a moderate hypoxic stress protocol sH(1.5d:30h) for the 10 genes tested by MO knockdown are shown: “no/mild” with no defects or minor defects only in the fin or air bladder (five genes, camk2g2 shown); “dev” have severe defects such as curved body axis or pericardial edema irrespective of hypoxia treatment (four genes, opsin5 shown); and “hypoxia-sensitive,” where morphant phenotype is greatly exacerbated under hypoxia (one gene, irs2). dnd1, whose expression and function are restricted to the developing gonad, is used as a control MO. See Materials and Methods for detailed phenotypic scoring criteria. Scale bar = 0.5 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4478541&req=5

fig3: Knockdown of hypoxia-induced stress candidate genes identifies a stress-sensitive phenotypic class. (A) Outline of functional acute hypoxia screen performed on morphants of the 10 candidates validated from the initial differential expression screen as described. Embryo microinjection was performed for each MO, after which control-injected morphant (ctl-MO; clear) and hypoxia target morphant (h-MO; blue) larvae were then divided into control and hypoxia-exposure (red box) groups, resulting in four different groups in total—ctl-MO injection only, ctl-MO + hypoxia, h-MO injection only, and h-MO + hypoxia. (B) Different phenotypic classes from a moderate hypoxic stress protocol sH(1.5d:30h) for the 10 genes tested by MO knockdown are shown: “no/mild” with no defects or minor defects only in the fin or air bladder (five genes, camk2g2 shown); “dev” have severe defects such as curved body axis or pericardial edema irrespective of hypoxia treatment (four genes, opsin5 shown); and “hypoxia-sensitive,” where morphant phenotype is greatly exacerbated under hypoxia (one gene, irs2). dnd1, whose expression and function are restricted to the developing gonad, is used as a control MO. See Materials and Methods for detailed phenotypic scoring criteria. Scale bar = 0.5 mm.
Mentions: Hypoxia-activated genes identified by microarray at gastrula and segmentation stages were further validated by qPCR and/or in situ hybridization (Figure 3). Genes demonstrating hypoxia-inducibility at 1 dpf, and therefore conservation of response across several phases of development, were preferentially chosen for MO knockdown in a secondary functional acute hypoxia sensitivity screen (Table 1, Figure 3). The rationale for selection of hypoxia-upregulated genes was that MO knockdown could uncover a necessary and protective role, therefore resulting in decreased resistance against sH. In contrast, the prediction and interpretation of results from knockdown of hypoxia-repressed genes were anticipated to be less straightforward and are not a focus of this study.

Bottom Line: Severe hypoxia is a common cause of major brain, heart, and kidney injury in adults, children, and newborns.However, mild hypoxia can be protective against later, more severe hypoxia exposure via "hypoxic preconditioning," a phenomenon that is not yet fully understood.Using a functional genomic approach, we used this zebrafish model to identify and validate five novel hypoxia-protective genes, including irs2, crtc3, and camk2g2, which have been previously implicated in metabolic regulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Neonatology, University of California San Diego School of Medicine, La Jolla, California 92093.

No MeSH data available.


Related in: MedlinePlus