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Dev Biol
2003 Feb 01;2541:50-67. doi: 10.1016/s0012-1606(02)00029-5.
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Fluorescent labeling of endothelial cells allows in vivo, continuous characterization of the vascular development of Xenopus laevis.
Levine AJ
,
Munoz-Sanjuan I
,
Bell E
,
North AJ
,
Brivanlou AH
.
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Appropriate blood supply and vascular development are necessary in development and in cancer, heart disease, and diabetes. Here, we report the use of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) to label endothelial cells and characterize the vasculature of live Xenopus embryos. The atlas we have created provides a detailed map of normal vascular development against which perturbations of normal patterning can be compared. By following the development of the intersomitic vessels in real-time, we show that, while rostrocaudal gradient of maturing intersomitic vessels occurs, it is not absolute. In addition, the comparative study of the ontogeny of nerve bundles from the spinal cord of transgenic Xenopus embryos expressing green fluorescent protein in the nervous system and blood vessels demonstrates a strong anatomical correlation in neurovascular development. These studies provide the basis for understanding how the vascular system forms and assumes its complicated stereotypical pattern in normal development and in disease.
Fig. 1. DiI-Ac-LDL labels the vasculature of Xenopus embryos. (a) 10 magnification confocal image and (b) 40 magnification confocal image of vessels
in a stage 43 embryo. Both images represent single z-slices of 52.0 and 1.4 m, respectively. The granular staining of endothelial cells can be observed
(arrowhead). (c) a 40 wide field microscopy image of DiI-Ac-LDL labeling of the vascular vitelline network (vvn) in a 10- m frozen section of a stage
33/34 embryo. (d) Colabeling of the DiI-Ac-LDL label with a subset of cells expressing the endothelial marker, flk-1 (black arrowheads indicate cells with
both labels) on the same 10- m frozen section. Unless otherwise indicated, the fluorescent DiI-Ac-LDL signal is indicated as red.
Fig. 2. Vasculature of stage 33/34 embryos at the initiation of the heart beat. Confocal images of (a) DIC and fluorescence and (b) fluorescence alone showing the heart (ht), first aortic arches (aa-3), eye, anterior (acv), and posterior (pcv) cardinal veins which drain into the pronephric sinus (k) and Duct of Cuvier (dc), and vascular vitelline network (vvn). The width of each z-slice is 23.5 m.
Fig. 3. Vascular system of stage 35–38 embryos. Confocal images (z-width of 93.9 m) of (a) DIC and fluorescence and (b) fluorescence only of a stage 36 embryo, showing the early internal carotid artery (ica), the further development of the eyevasculature, extension of the posterior cardinal vein (pcv), early intersomitic sprouts (isv), and the formation of the fourth and fifth aortic arches (aal, 5 arrows; aa2–4 arrowheads). The splitting of the fourth aortic arch is indicated by a yellow arrowhead. (c) Lateral view of the head and trunkvasculature of stage 35/36 embryo showing early intersomitic veins (isv) sprouting off of the posterior cardinal vein (pcv) and the formation of the vena capitis medialis (vcm). This image was taken at 5 magnification on a Zeiss Axioplan. (d) The dorsal head of a stage 37/38 embryo showing the development of the anterior (acev), middle (mcv), and posterior (pcev) cerebral arteries. The metencephalic artery (mta) is also forming at this stage and runs with the middle cerebral vein. These vessels all cross the brain dorsally between the bilateral vena capitis medialis veins (vcm). This confocal image was taken at 5 magnification with a z-width of 36.6 m.
Fig. 4. Vasculature of stage 40–41 embryos. Confocal images (z-width 43.4 m) of (a) DIC and fluorescence and (b) fluorescence only of a stage 40 embryo showing the vena capitis lateralis (vcl) and the vena capitis medialis (vcm) and the further sprouting of the intersomitic vessels (isa, isv). (c) Confocal image (z-width 38.4 m) of a dorsolateral view of the head of stage 41 embryo showing the anterior cerebral vein crossing the head (acev) and the many small capillaries that cross the head between the cerebral veins, as well as the dominance of the vcl over the vcm. (d) Ventral view of a stage 41 embryogut showing the subintestinal vein (siv) draining into the left omphalomesenteric vein (lom), taken with wide-field microscopy.
Fig. 5. Vasculature of stage 43 embryos. (a) Confocal image (z-width 24.9 m) of the profile of stage 43 embryo showing the musculoabdominal vein crossing from the ventral aspect of the head to the Duct of Cuvier, the right side position of the subintestinal vein, and the further definition and capillary sprouting of the intersomitic vessels. (b) Confocal image (z-width 42.3 m) of a dorsolateral view of stage 42/43 embryohead showing ophthalmic artery (oa) entering the eye and the crossing of the external jugular vein (ejv) from the ventralhead to the Duct of Cuvier. The lymph heart (lh) may also be seen. (c) Wide-field microscopy image of a dorsal view of lateralhead of stage 43 embryo showing ophthalmic artery (oa) supplying blood to the eye and the ophthalmic vein (ov) draining the eye. The relationship of the ophthalmic artery arching over the ophthalmic vein can be seen. The ophthalmic vein drains into the internal jugular vein (ijv) whose tributaries include the anterior part of the cerebral vein that loops around the olfactory bulb. (d) Ventral view of the gut of a stage 43 embryo showing the subintestinal vein on the right side of the gut, draining the gastric vein (wide-field microscopy). (e) Lateral view of the dorsal longitudinal anastomosing vessel (dlav) draining into the merged posterior cardinal vein (pcv) by the dominant left branch (lb) and the lesser right branch (rb) (wide-field microscopy).
Fig. 6. Vasculature of stage 46 embryo. (a) Confocal image (z-width 1.0 m) of a lateral view of a stage 46 embryo showing the nasociliary artery and the expanding capillary networks of the dorsal head and between the intersomitic vessels (isv). (b, c) Ventral views of stage 46 embryos showing the extensive vascular supply to the filter apparatus, particularly the ventralmost venous drainage into the musculoabdominal veins (mab). The musculoabdominal veins drain into the Duct of Cuvier (dc) lateral to the heart. The persisting aortic arches (3–5) can also be seen (aa3– 5) and supply the external carotid artery (eca) and the pulmonary artery. (b) A confocal image (z-width 66.9 m) and (c) was taken with wide-field microscopy. (d) Dorsal view of the tail of a stage 46 embryo showing the dorsal longitudinal anastomosing vessel (dlav) with paired intersomitic vessels sprouting off of the dlav at regular intervals (wide-field microscopy). (e) A 10- m frozen section of a stage 46 tail showing the existence of a pair of intersomitic arteries (isa) and veins (isv) at the same intersomitic junction and the arrangement of the isa medial to the isv (taken with wide-field microscopy).
Fig. 7. Vasculature of the dorsal head of stage 46 embryos. (a) Dorsal view showing the proximal relationship of the internal carotid artery (ica) and the internal jugular vein (ijv). In addition, the ventral external jugular vein (ejv) can be seen crossing dorsolaterally to drain into the Duct of Cuvier and pronephric sinus (k). (b) Dorsolateral view of the head of a stage 46 embryo. (a) and (b) were obtained using a Zeiss Axioplan. (c) A dorsolateral view obtained using confocal microscopy (z-width 30.0 m).
Fig. 8. Development of the vasculature of the pronephric sinus from stage 33/34 to stage 46 showing the progressive folding of the sinus into a tangled knotof vessels. (a–d) Magnifications of confocal images with z-widths of 23.6, 37.8, 43.4, and 22.0 m, respectively. (a –d ) are tracings of (a–d).
Fig. 9. Negative image of progressive outgrowth of the intersomitic vessels in a live embryo. Embryos were injected at stage 33/34 with DiI-Ac-LDL, reanesthetized every 1.5–3 h, and imaged using a Zeiss Axioplan (widefield microscopy). A typical time series is shown with representative images from stage 33/34 (time 0), and 1.5, 3, 6, and 13 h later. The third intersomitic vessel is indicated as a reference for following single vessel outgrowth. The image is presented as a negative black/white image, where black represents the fluorescent signal.
Fig. 10. Codevelopment of somitic nerves and intersomitic vessels. Single 12.4- m z-slices from a confocal image of a stage 46 embryo expressing a tau-gfp construct and injected with DiI-Ac-LDL. (a) Both the somitic nerves (green) and the intersomitic veins (isv) are visualized and can be seen in close proximity (20 magnification). (b) and (c) are individual slices in the same z-stack (10 magnification) showing colocalization of the somitic nerves with the veins (b) and not arteries (c).
Fig. 12. Stereotypical development of the vascular system. (a) Dorsal view of the dorsal longitudinal anastomosing vessel (dlav) showing the regular pattern of paired intersomitic vessels at each somitic junction. (b) Ventral view of the posterior cardinal vein (pcv) showing the repeated pattern of intersomtic veins branching into a triple capillary system. (a) A 10 magnification taken with a Zeiss Axioplan. (b) A projection of a z-stack (z-width 27.9 m) taken with a confocal microscope.
