Cavefish cope with environmental hypoxia by developing more erythrocytes and overexpression of hypoxia inducible genes

Dark caves lacking primary productivity can expose subterranean animals to hypoxia. We used the surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs of Astyanax mexicanus as a model for understanding the mechanisms of hypoxia tolerance in the cave environment. Primitive hematopoiesis, which is restricted to the posterior lateral mesoderm in other teleosts, also occurs in the anterior lateral mesoderm in Astyanax, potentially pre-adapting surface fish for hypoxic cave colonization. Cavefish have enlarged both hematopoietic domains and develop more erythrocytes than surface fish, which are required for normal development in both morphs. Laboratory induced hypoxia suppresses growth in surface fish but not in cavefish. Both morphs respond to hypoxia by overexpressing hypoxia-inducible factor 1 (hif1) pathway genes, and some hif1 genes are constitutively upregulated in normoxic cavefish to similar levels as in hypoxic surface fish. We conclude that cavefish cope with hypoxia by increasing erythrocyte development and constitutive hif1 gene overexpression.

Cavefish Increase Red Blood Cell Development and Reprogram Metabolism as Adaptations to Environmental Hypoxia

The teleost Astyanax mexicanus is a single species with surface dwelling (surface fish) and cave dwelling (cavefish) morphs. Constructive and regressive traits have evolved in cavefish as adaptations for survival in perpetual darkness. In addition to darkness, cavefish must cope with de-oxygenated aquatic environments. Blood cell quantification and expression of hematopoietic marker genes indicated that cavefish have more erythrocytes than surface fish, and that this increase has a developmental basis. In contrast to zebrafish and other teleost embryos, in which erythrocyte formation is restricted to the posterior lateral mesoderm, the anterior as well as the posterior lateral mesoderm is involved in red blood cell formation in Astyanax embryos, and both of these hematopoietic domains are expanded in cavefish embryos. Erythroid development in the anterior mesoderm may be a pre-adaptation for surface fish to successfully colonize hypoxic cave environments. We also show that cavefish are less sensitive to phenylhydrazine-induced erythrocyte ablation than surface fish, suggesting a functional advantage of increased red blood cells. By mimicking a hypoxic cave environment in the laboratory, we further demonstrate that cavefish respond to hypoxia differently than surface fish. Surface fish with fewer red blood cells use overall metabolic depression to counteract hypoxia, whereas cavefish with larger numbers of erythrocytes respond to hypoxia by switching to anaerobic metabolism. These results suggest that cavefish may have adapted to hypoxic environments by enhancing the capacity to form erythrocytes and reprogramming metabolism.SummaryAstyanax mexicanus cavefish adapt to life in hypoxic cave environments by evolving the capacity to increase red blood cell numbers during early development and reprogramming metabolism to favor anaerobic processes.

Conditional deletion of WT1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice

Congenital diaphragmatic hernia (CDH) is a severe birth defect. Wt1-null mouse embryos develop CDH but the mechanisms regulated by WT1 are unknown. We have generated a murine model with conditional deletion of WT1 in the lateral plate mesoderm, using the G2 enhancer of the Gata4 gene as a driver. 80% of G2-Gata4Cre;Wt1fl/fl embryos developed typical Bochdalek-type CDH. We show that the posthepatic mesenchymal plate coelomic epithelium gives rise to a mesenchyme that populates the pleuroperitoneal folds isolating the pleural cavities before the migration of the somitic myoblasts. This process fails when Wt1 is deleted from this area. Mutant embryos show Raldh2 downregulation in the lateral mesoderm, but not in the intermediate mesoderm. The mutant phenotype was partially rescued by retinoic acid treatment of the pregnant females. Replacement of intermediate by lateral mesoderm recapitulates the evolutionary origin of the diaphragm in mammals. CDH might thus be viewed as an evolutionary atavism.

Genetic Analyses in Zebrafish Reveal That Angiopoietin-Like Proteins 1 and 2 Are Required for HSC Development during Embryogenesis

Recent studies have shown that members of the angiopoietin-like protein (angptl) family can help stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptl for HSC development has yet to be determined. Here, the effects of morpholino (MO) knockdown of angptl1, angptl2 or the combination of both, were assessed during zebrafish development. Single MO knockdown of either angptl1 or angptl2 resulted in a decrease in c-myb positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf). Embryos injected with combined angptl1 and angptl2 MOs (double morphants) had an even more severe phenotype, in which both c-myb and runx1 positive HSCs were almost completely absent. In an effort to discern the onset of the phenotype, early HSC markers were evaluated. The double morphants had decreased expression of scl and lmo2 at 8–10 somite stages (ss) in the bilateral stripes of the lateral mesoderm region from which hematopoietic and vascular progenitors are thought to arise. To determine if angptl are sufficient to expand HSCs, angptl1 and angptl2 mRNA were injected into one-cell stage embryos and HSC markers were evaluated. Angptl1 and angptl2 mRNA significantly increased c-myb and runx1 positive HSCs in the AGM. Interestingly, addition of thrombopoietin, a potent stem cell growth factor, with angptl1 and angptl2 further increased HSCs in the AGM, implying a synergistic effect of these growth factors during HSC development. Angptl regulation of HSC development may occur by stimulating the production of the hemogenic endothelium. MO knockdown of either angptl1 or angptl2 alone did not alter vascular development significantly, while MO knockdown of both angptl1 and angptl2 resulted in a disruption of intersegmental blood vessel sprouting at 28 hpf. The arterial and venous specification in these double morphants was also disrupted at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4. These results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium. Furthermore, since Vegf signaling is indispensable for proper endothelial specification and definitive HSC formation, we found that in the double morphants, somite expression of vegf165 mRNA was decreased early in development. Although angptl1 mRNA expression was undetectable at this stage, angptl2 mRNA expression was localized in the yolk sac extension and posterior spinal cord, appearing in close proximity to the vegf positive somites. This suggests that in these locations, angptl may stimulate Vegf production in the embryo to participate in the induction of hemogenic endothelium and subsequently lead to HSC formation. Taken together, our data demonstrates that the angptl are required for HSC development by coordinating the production of a functional hemogenic endothelium that can lead to the formation of HSCs.