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Xenopus laevis provides a number of advantages to studies on cardiovascular development. The embryos are fairly large, are easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole-mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of cardiac development in Xenopus, seeking to identify antibodies and fixatives that allow easy examination of the developing heart. We have used monoclonal antibodies (mAbs) raised against bovine cardiac troponin T and chicken tropomyosin to visualize cardiac muscle, a goat antibody recognizing bovine type VI collagen to stain the lining of vessels, and the JB3 mAb raised against chicken fibrillin, which allows the visualization of a variety of cardiovascular tissues during early development. Results from embryonic stages 24-46 are presented.
FIG. 1. Cardiac muscle development in Xenopus. Confocal images of the heart regions of six stages of embryos (27-46) are presented (ventral views), each adjacent to a diagrammatic representation of an embryo (Nieuwkoop and Faber, 1967) at the corresponding stage. Prior to microscopy, stage 27-33 embryos were immunolabeled with anti-tropomyosin (CH1) and stages 356 with anti-cardiac troponin T (CT3). The secondary antibody used for all stages was conjugated to Cy5. Each confocal image represents a digitally colored compilation of 30-53 optical sections taken (in gray scale) 5 um apart. All confocal images were taken at the same magnification and are presented with the anterior of the embryo at the top. Scale bar, 100 um
FIG.2.Nonmuscle cardiac development in Xenopus. Images representthecardiac regions of embryos (stage 27 and stage 35) immunostained with anti-fibrillin (JB3). The secondary antibody was conjugated to Alexa 568. Ventral views of the heart regions of embryos are presented with the anterior of the embryo at the top. The stage 27 (A) and stage 35 (B) images were form ed by the digital merging of 28 and 36 optical sections 4 and 5 um apart, respectively (taken in gray scale), and digitally colored. At stage 27, the endocardial tube is not completely formed. The unlabeled arrowheads point to unpartitioned secreted material at the embryos midline. By stage 35 (B), a single heart tube has formed into a S shape; the sinus venosus is paired. Scale bar, 100um.
FIG. 3. Milestones of early cardiac development in Xenopus visualized by confocal images of double-immunostained embryos. Embryos selected to represent a chronology of stages were double labeled with anti-tropomyosin (CH1) (red) and anti-fibrillin (JB3) (green) and optically sectioned. For each stage, the set of optical sections (28 to 40 sections, 4 to 6 um thick) was digitally compiled in increments of 4 to 6 sections. Resulting optical thick sections that aid in the visualization of Xenopus heart development are presented. The order of presentation for stages with m ore than one image is ventral to dorsal. Image A is a parasagittal view of a stage 24 hemisectioned embryo. The stage 26 embryo in B is shown from the leftlateral view. Images of stages 28 (C and D) and 31 (E) are ventral views of heart regions with the anterior of the em bryos placed at the top. At stage 24, the heart anlage is present but not immunohistochemically active. By stage 26, the first presence of antibody reactivity is noted near the ventral midline (ventral midline lies between arrowheads). At stage 28 (C and D), differentiation of the heart anlage is clear; the pericardial cavity can be discerned. Stage 31 (E)he heart region extends ventrally beyond the pericardial cavity and is slightly bent laterally to the embryoright; the myocardial wall does not yet close. Scale bars as denoted.
FIG. 4. Milestones of early cardiac development in Xenopus (continued). Embryos were double labeled with anti-cardiac troponin T (CT3) (red) and anti-fibrillin (JB3) (green) and optically sectioned. For each stage, the set of optical sections (35 to 53 sections 5 to 7 um thick) was digitally compiled in increments of 6 to 9 sections. Resulting ventral views of optical thick sections are presented. By stage 35 (A and B) the myocardial wall is closed and the heart tube has become twisted in a S shape; the chambers are now distinct. Stage 41 (C and D) The myocardium thickens and develops trabeculae; the atrium is posterior to the ventricle. At stage 44 (E and F) the single atrium begins to be partitioned by the forming septum and is pushed slightly anterior to the ventricle. Scale bar, 100um.
FIG. 5. Ventral-to-dorsal progression through a stage 46 Xenopus heart. Images were digitally created from a series of 48 optical sections (taken 5um apart) through a double-labeled stage 46 Xenopus heart (red, CT3; green, JB3). Image A represents the most ventral 8 images of this series, digitally merged; image B, the next 8, etc. The relative positioning of heart structures to one another, as well as an individual structure order of emergence within the series, can be established: the truncus arteriosus and ventricle are always most ventral (A). Progressing dorsally, the spiral valve (B) appears within the truncus. Next, the single atrioventricular valve (C) is seen overlying the atrial septum (D and E). Midway through the series (C and D), the first views of the atria, as well as the aortic branches, are viewed. Both atria and the sinus venosus are seen more dorsally (D and E). Finally, the back wall of the atria is seen, positioned most dorsal (F). Scale bar, 100 um.
FIG. 6. Early vasculature in Xenopus. Ventral view of a double-labelled stage 46 Xenopus embryo (red, anti-cardiac tropon in T (CT3); green, anti-type VI collagen). Image represents 45 optical sections through the embryo, each 10 um thick, digitally merged and colored. The first three branches on the left side of the paired aortic arches are easily distinguished with the antibody to type VI collagen, as are all chambers and the truncus arteriosus with the antibody to cardiac troponin T. Scale bar, 100 um
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