Experimental hypoxia and embryonic angiogenesis.

We examined the role of hypoxia and HIF factors in embryonic angiogenesis and correlated the degree of hypoxia with the level of HIF and VEGF expression and blood vessel formation. Quail eggs were incubated in normoxic and hypoxic (16% O(2)) conditions. Tissue hypoxia marker, pimonidazol hydrochloride, was applied in vivo for 1 hr and detected in sections with Hypoxyprobe-1 Ab. VEGF and HIF expression was detected by in situ hybridization. HIF-1alpha protein was detected in sections and by Western blot. Endothelial cells were visualized with QH-1 antibody. Hypoxic regions were detected even in normoxic control embryos, mainly in brain, neural tube, branchial arches, limb primordia, and mesonephros. The expression patterns of HIF-1alpha and HIF-1beta factors followed, in general, the Hypoxyprobe-1 marked regions. HIF-2alpha was predominantly expressed in endothelial cells. Diffuse VEGF expression was detected in hypoxic areas of neural tube, myocardium, digestive tube, and most prominently in mesonephros. Growing capillaries were directed to areas of VEGF positivity. Hypoxic regions in hypoxic embryos were larger and stained more intensely. VEGF and HIF-1 factors were proportionately elevated in Hypoxyprobe-1 marked regions without being expressed at new sites and were followed by increased angiogenesis. Our results demonstrate that normal embryonic vascular development involves the HIF-VEGF regulatory cascade. Experimentally increasing the level of hypoxia to a moderate level resulted in over-expression of HIF-1 factors and VEGF followed by an increase in the density of developing vessels. These data indicate that embryonic angiogenesis is responsive to environmental oxygen tension and, therefore, is not entirely genetically controlled.

Lectin histochemistry of microvascular endothelium in chick and quail musculature.

The lectin binding pattern of muscular microvessels in chick, quail and chick/quail chimeras was analysed. Paraffin wax sections of muscles from embryonic and adult animals were used. The biotin-labelled lectins were detected by avidin-alkaline phosphatase complex. The following lectins bound to muscular microvessels including arterioles, capillaries and venules of both species: SNA-I (Sambucus nigra agglutinin), MAA (Maackia amurensis agglutinin), AIA (Artocarpus integrifolia agglutinin), VAA-I, VAA-II and VAA-III (Viscum album agglutinin I-III), WGA (wheat germ agglutinin), LEA (Lycopersicon esculentum agglutinin). Endomysium and basement membranes of muscle fibres were also stained to a variable extent and intensity. Only SNA-I stained almost exclusively the endothelium of blood vessels. WFA (Wisteria floribunda agglutinin) bound to the quail endothelium only. MPA (Maclura pomifera agglutinin) marked vessels in adult muscles of chick and quail, but embryonic vessels were stained in quail only. Our results show that lectin histochemistry is a useful tool for visualisation of microvasculature in avian species. In particular, WFA and MPA can be used to determine the origin of endothelia in chick/quail chimeras.

Development of mechanoreceptor numbers in embryonic chick-quail chimeras.

Our experiments addressed the problem of the regulation of the number of mechanoreceptors by sensory axons and/or their peripheral target tissues. According to a previous study (Zelená et al. 1997) white leghorn chickens have more muscle spindles in the plantaris muscle (45.4+/-7.8; mean+/-SD) than the Japanese quail (35.3+/-4.8) and significantly more Herbst corpuscles in the crural region (380.0+/-85.0) than the quail (124.9+/-32.8). Embryonic chick-quail chimeras were therefore used as a model with distinct recombinations of the nerve supply and peripheral tissue for studying the developmental control of these mechanoreceptors. The chick host leg bud was replaced with a quail leg bud of equal age and vice versa on embryonic day 3, prior to the onset of innervation of the periphery. Shortly before hatching the chimeras were sacrificed and muscle spindles and Herbst corpuscles counted. Recombinations of chicken nerves with quail limb buds have shown that the richer nerve supply by chick Ia axons induced a significant increase in the number of muscle spindles in the plantaris muscles (55.5+/-13.4) of the grafted quail limb. In some instances, a similar increase in spindle numbers was also found in control legs grafted onto hosts of the same species. In the reverse type of chimera where chick embryo legs were grafted onto quail hosts, spindles developed in lower numbers (27.3+/-3.2). In that case the lower number of Ia axons in quail nerves induced a lower number of spindles in the chicken muscle. The numbers of Herbst corpuscles were, however, low in both types of chimera. Quail legs grafted onto host chick embryos contained 126.8+/-26.4 corpuscles, presumably due to a restrictive influence of the smaller crural area in the quail. Chick legs grafted onto quail hosts had only 99.6+/-34.1 crural corpuscles; the target area in chick embryo legs failed to attract more quail axons and/or to induce axonal sprouting. The developmental regulation of the number of the two types of mechanoreceptors examined in our study thus differ. While sensory axons appear to play the dominant role in the development of muscle spindles, their role seems to be restricted by hitherto unknown peripheral factors during the development of Herbst corpuscles.