The zebrafish Danio rerio has become an important model organism for the study of vertebrate biology, being well suited for developmental and genetic studies. Large-scale genetic screens have identified hundreds of mutant phenotypes, many of which may serve as models of human disease (Warren and Fishman, 1998; Barut and Zon, 2000). However, studies focussing on the physiology of the developing zebrafish embryo or larvae are scarce, and our understanding of the basic physiology of D. rerio lags far behind our knowledge of the genetics (Burggren and Keller, 1997). Blood circulation in all vertebrates starts early in development, and the first heart beat is typically observed before the heart is completely differentiated (Pelster and Bemis, 1991). Despite this early onset of cardiac activity and blood circulation, the physiological function of blood convection has been questioned (Pelster and Burggren, 1996; Pelster, 1999). In small larvae such as zebrafish, diffusion of oxygen through the body surface alone appears to be sufficient to meet the metabolic needs of the animal (Territo and Burggren, 1998; Territo and Altimiras, 1998; Pelster, 1999; Gielen and Kranenbarg, 2002). This demonstrates that coupling between metabolism and convective oxygen transport is not yet established in the early larval stages, and Rombough (2002) suggests that ion- and osmoregulatory functions may require blood flow much earlier in development than metabolism. Hypoxic conditions are observed in the flowing and stagnant waters that are the natural environment of the tropical zebrafish. The coupling of convective oxygen transport and metabolic activity ensures sufficient oxygen supply to the cells and prevents oxygen shortages at the organ level. Accordingly, in adult animals hypoxia itself acts a stimulus and induces profound changes in cardiac activity and peripheral resistance, and even stimulates erythropoiesis. If this coupling is not yet established in early developmental stages, it could mean that hypoxia does not act as a stimulus in early developmental stages. In a recent study in zebrafish we were able to demonstrate that long before coupling between metabolic requirements and blood flow is established, environmental hypoxia can be sensed and induces stimulation of cardiac activity (Jacob et al., 2002). A reduction in the oxygen- carrying capacity of the blood, however, had no effect on cardiac activity. Thus, hypoxia does exert a signaling effect, 1299 The Journal of Experimental Biology 206, 1299-1307 © 2003 The Company of Biologists Ltd doi:10.1242/jeb.00249 This is the first study to use a combination of digital imaging techniques and vital video microscopy to study hypoxia-induced changes in blood cell concentration, angiogenesis and blood redistribution in entire animals. Zebrafish Danio rerio, which are known to be independent of convective oxygen transport until about 2 weeks post-fertilization, were raised under chronic hypoxia (P O ∑=8.7·kPa) starting at 1 day after fertilization (d.p.f.) until 15 d.p.f. In control animals, the concentration of red cells (i.e. the number of red cells per nl blood) remained constant until 7 d.p.f., and than decreased by approximately 70% until 15 d.p.f. In hypoxic animals, however, the concentration of red cells remained significantly elevated compared to control animals at 12 and 15 d.p.f. Assuming that the hemoglobin content of the red cells is similar, hypoxic animals have a higher oxygen carrying capacity in their blood. Red cell distribution within the various parts of the circulatory system, taken as an indicator for blood distribution, revealed a significant modification in the number of blood cells perfusing the organs in hypoxic animals. At 12 d.p.f., gut perfusion was reduced by almost 50% in hypoxic animals, while perfusion of the segmental muscle tissue was increased to 350% of control values. No significant changes in brain perfusion were observed under these conditions. At 15 d.p.f., the reduction in gut perfusion was abolished, although muscle perfusion was still significantly elevated. At this time, growth of hypoxic animals was less compared to control animals, revealing that hypoxia had become deleterious for further development. The vascular bed of various organs was not obviously different in hypoxic animals compared to normoxic animals. Key words: ontogeny, erythropoiesis, angiogenesis, hypoxia, digital video imaging, zebrafish, Danio rerio. Summary Introduction Non-invasive imaging of blood cell concentration and blood distribution in zebrafish Danio rerio incubated in hypoxic conditions in vivo Thorsten Schwerte, Dietmar Überbacher and Bernd Pelster Institute for Zoology and Limnology, University of Innsbruck, Austria Author for correspondence (e-mail: thorsten.schwerte@uibk.ac.at) Accepted 16 January 2003