Fish exposed to hypoxia develop decreased heart rate, or bradycardia, the physiological significance of which remains unknown. The general muscarinic receptor antagonist atropine abolishes the development of this hypoxic bradycardia, suggesting the involvement of muscarinic receptors. In this study, we tested the hypothesis that the hypoxic bradycardia is mediated specifically by stimulation of the M(2) muscarinic receptor, the most abundant subtype in the vertebrate heart. Zebrafish (Danio rerio) were reared at two levels of hypoxia (30 and 40 Torr PO(2)) from the point of fertilization. In hypoxic fish, the heart rate was significantly lower than in normoxic controls from 2 to 10 days postfertilization (dpf). At the more severe level of hypoxia (30 Torr PO(2)), there were significant increases in the relative mRNA expression of M(2) and the cardiac type beta-adrenergic receptors (beta1AR, beta2aAR, and beta2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at 40 Torr PO(2)) or significantly attenuated (at 30 Torr PO(2)) in larvae experiencing M(2) receptor knockdown (using morpholino antisense oligonucleotides). Sham-injected larvae exhibited typical hypoxic bradycardia in both hypoxic regimens. The expression of beta1AR, beta2aAR, beta2bAR, and M(2) mRNA was altered at various stages between 1 and 4 dpf in larvae experiencing M(2) receptor knockdown. Interestingly, M(2) receptor knockdown revealed a cardioinhibitory role for the beta(2)-adrenergic receptor. This is the first study to demonstrate a specific role of the M(2) muscarinic receptor in the initiation of hypoxic bradycardia in fish.

BackgroundAs a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear.Methodology/Principal FindingsIn the current study, the effect of hypoxia on primordial germ cell (PGC) migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF) signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1), an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration.Conclusions/SignificanceThis study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1.