Early development of the cardio-respiratory system in the model animals zebrafish (danio rerio) and xenopus laevis analysed with non-invasive computer assisted image analysis

Independent of species, the cardiovascular system is the first functioning component of the developing vertebrate embryos. In the studies presented here the cardiovascular and respiratory responses to environmental, genetic and epigenetic perturbations have been examined in detail to understand the relationships between cardiac and respiratory performance, haematopoiesis for embryonic or larval stages in the popular model animals zebrafish and Xenopus. In contrast to mammalian embryos, which largely depend on convective oxygen transport, zebrafish and Xenopus larvae because of their small body mass obtain sufficient amounts of oxygen via bulk diffusion during the first part of their development. This permits to study genetic mutants even with extreme defective phenotypes of the cardio-respiratory system. Due to the tiny size of the animals new non invasive techniques had to be used and developed in order to perform all the measurements necessary for the characterization of the functional development of the cardio-respiratory system. Most of these techniques are based on the use of microscopic video imaging methods combined with a digital data analysis with custom designed software individually developed for the different experiments. The experiments clearly revealed that almost all of the components of the circulatory system start working very early during development. While sympatho-vagal control of heart rate could only be demonstrated at 12 dpf in zebrafish larvae, ventilation as well as heart rate became sensitive to hypoxic stimulation as early as 4 dpf. Accordingly, in the early stages, in which sympatho-vagal control of the circulatory system was not yet established, peripheral resistance and cardiac activity was under hormonal control, and responsiveness to adrenaline or acetylcholine was established already at 4 day post fertilization (dpf). Nitric oxide induced vasoactivity was observed on 7 dpf. With respect to ventilatory control the combination of classical blocking experiments and immunohistochemistry gave strong evidence for a late onset of NMDA receptor-mediated ventilatory control in zebrafish larvae. The chemoreflex became MK801 sensitive at 8 dpf, but did not completely rely on a glutamatergic transmission until 13 dpf. Hypoxia induced stimulation of angiogenesis was not observed until 15 dpf, while chronic swim training increased the vascularization indexes at this stage of development. On the other hand, red cell concentration was elevated in hypoxic animals at 15 dpf, but not in trained animals. Swim training also increased mitochondrial density of muscle cells. Experiments with different parental feeding regimes revealed that cardiovascular parameters are very sensitive to be affected by maternal influences. As a consequence breeding and feeding protocol has to be carefully standardized for animals which serve for physiological measurements. The microtechniques developed for the studies presented here have the capability to be upscaled for high throughput screening of cardiovascular parameters i. e. for industrial drug screening.