Analysis of the endocardium and cardiac jelly in truncal development in the cardiac lethal mutant axolotl Ambystoma mexicanum.

Recessive mutant gene c in axolotls results in a failure of the heart to function because of abnormal embryonic induction processes. The myocardium in this mutant lacks organized sarcomeric myofibrils. The present study was undertaken to determine if developmental abnormalities were evident in other areas of the heart besides the myocardium. A detailed comparative survey of the structure of developing normal and mutant hearts, including the endocardium, its cellular derivatives, and the extracellular matrix, known as cardiac jelly, showed that in the mutant there are fewer than the normal number of endocardial cells lining the heart lumen, the number of mesenchyme cells is reduced, and the cardiac jelly area is greatly enlarged in the posterior part of the truncus adjacent to the ventricle.

Nonrandom association of cellular antigens with HTLV-III virions.

Retroviruses are known to incorporate cellular antigens as they bud from infected cells. To identify the cellular antigens that associate with the AIDS-retrovirus, we evaluated a preparation of HTLV-III antigens with a panel of monoclonal antibodies reactive with a variety of antigens expressed on the H9 T-cell line used to produce the virus. Only monoclonal antibodies that identified HLA class-II antigens, beta-2 microglobulin, and a single anti-HLA class-I antibody were reactive in an ELISA of solubilized HTLV-III virus. No reactivity was seen with 11 monoclonal antibodies to T-cell antigens or with five antibodies to determinants on HLA class-I A or B molecules. These data suggest that on H9 cells the association of budding HTLV-III virions with cellular antigens may be a nonrandom process in which some HLA antigens, particularly class-II antigens, are selectively incorporated into the viral envelope. It is possible that a selective association of HLA class II antigens with budding HTLV-III virions may also occur for T cells infected in vivo, and could have relevance for the pathogenesis of this virus.

Endocytosis in the embryonic chick chorionic epithelium.

The chorionic epithelium of the developing chick embryo consists of two major cell types, villus cavity (VC) and sinus-covering (SC) cells. Ultrastructurally, VC cells contain apical microvilli with numerous underlying vesicles, whereas SC cells possess thin cytoplasmic processes (containing occasional vesicles) overlying adjacent blood sinuses. This study was performed to determine whether vesicles observed in VC and SC cells are endocytic in nature and whether endocytosis is limited to either VC or SC cells. Exposure of air space chorionic epithelium in ovo to microperoxidase (MRP-11) resulted in endocytic uptake of marker by the thin cytoplasmic processes of 70% of SC cells. Vesicles containing MRP-11 reaction product were also observed in approximately 20% of VC cells. Exposure of the chorionic epithelium of shell-less cultured embryos to horseradish peroxidase (HRP) resulted in endocytic uptake of HRP in approximately 80% of SC cells and in 33% of VC cells. Thus, VC and SC cells demonstrate endocytic activity both in ovo and in shell-less culture. In ovo, endocytosis may be involved in the recovery of water and/or ions from the eggshell or shell membranes.

Morphogenesis of human cardiac outflow.

The developmental anatomy of human cardiac outflow was studied in a series of 16 normal embryos (gestational days 29-39, crown-rump length 6-20 mm, stages 14-19). Structural features and kinetics during truncal septation (TS) were described from external photographs, serial histological sections, and computer graphic reconstructions of selected tissues. Early in the period studied, the tubular myocardium ensheathed the single cardiac lumen and spiralling conotruncal ridges, which were filled with mesenchymal cells during days 31-33. As TS began (late stage 16), the aorticopulmonary (AP) septum appeared across the dorsal wall of the aortic sac between arches IV and VI. Mesenchymal condensations formed within the AP septum, crossing the lumen bifurcation to extend along the truncal ridges to the myocardium. During days 35-37, the cephalic margin of the myocardium grew or folded in toward these mesenchymal condensations between the developing valves and within the nearby conal ridges, which appeared to fuse to separate the subvalvular outflow channels by day 39. These observations are consistent with studies in chicks and rats which suggest that mesenchymal condensations or cell death foci interact with the distal myocardial rim during TS to form a structural septation complex dividing the two arterial streams.

Myocyte transplantation for treatment of complete heart block.

We hypothesize that it may be possible to treat complete heart block by transplanting right atrial myocytes directly into the ventricular wall where they may set the rate of ventricular contraction at their own intrinsic rate. The biologic background for this conjecture has already been determined to a large extent. However, a few critical unanswered biologic questions must be addressed before the development of this suggested new therapy becomes a mere technologic exercise.

Selected views of early heart development by scanning electron microscopy.

This tutorial on cardiac development is designed to acquaint the novice student of embryology with the key events that occur during cardiogenesis. Each of the following events is depicted through a series of scanning electron micrographs which convey the spatial relationships between minute, yet essential structures: fusion of paired heart tubes; looping; partitioning of the common atrium, the atrioventricular canal, the primitive ventricle and the outflow tract. The cellular and biochemical mechanisms responsible for these events cannot be adequately determined nor illustrated by SEM and therefore will not be considered in detail herein.

Computer reconstruction of serial sections.

A computer graphics system is described which reconstructs three-dimensional images from serial sectional data. Microscopic sectional tracings are first digitized and coded with a microcomputer (APPLE II-Plus). The data are transferred to a main frame facility for reconstruction and the final result is displayed on a high-resolution color monitor (Hewlett-Packard 9845C). Depth cueing of the image is enhanced by edge ribboning and area filling. The system is very simple to operate and yet flexible enough to allow selective portions of the tissue sample to be reconstructed and displayed in various orientations.

Effects of varying chamber construction and embryo pre-incubation age on survival and growth of chick embryos in shell-less culture.

Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of the eggshell and shell membranes. The technique allows direct access to and continuous observation of cultured embryos almost to the time of hatching. The plastic wrap/culture tripod technique described in this paper allows normal embryonic growth and differentiation from 48 hours (in ovo pre-incubation age) through at least 10 days of total incubation. As the duration of in ovo pre-incubation is decreased below 36 hours, there is a concomitant increase in the percentage of grossly abnormal embryos associated with decreased survival and retarded growth and differentiation. Survival of embryos pre-incubated for 72 hours through 13 and 18 days of total incubation is greater than 80% and 40%, respectively. The wrap/tripod technique allows substantially better survival and more normal development of cultured embryos than does the petri dish technique of Auerbach et al. (1974). Embryonic growth in 7.8-cm-diameter chambers is significantly greater than in either larger (10.6 cm) or smaller (5.2 cm) diameter chambers. Some gas exchange through the culture chamber walls appears necessary for optimal embryonic survival and growth. Suspending egg contents in either Safeway or Handi-Wrap plastic wrap (both of intermediate permeability) in 7.8-cm tripods resulted in superior growth and/or survival compared to suspension in Silastic sheeting (high permeability), Saran Wrap (low permeability), glass dishes (nonpermeable), or glass dishes lined with an inner layer of Safeway wrap(nonpermeable).

Origin and migration of cushion tissue in the developing heart.

The origin of cushion tissue mesenchyme in the developing chick heart was investigated by three basic methods: use of an inert metabolic marker; time lapse recording of organ-cultured heart regions; and scanning stereo-microscopy. All three approaches support the hypothesis that the endocardium is the progenitor of cushion tissue mesenchyme. Additional observations show a cell:matrix interaction by the endocardium prior to the formation of mesenchyme. It is postulated that this activity in some manner alters the underlying matrix and helps to initiate and maintain migratory activity of the mesenchyme.

Cultivation of microvascular endothelial cells from human preputial skin.

A procedure is described for the isolation and cultivation of microvascular endothelium from human skin. Neonatal foreskins are pooled, washed, minced, and dissociated by a mixture of collagenase and dispase. Microvascular endothelium, liberated in the form of intact capillary fragments, is incompletely separated from fibroblasts and epidermal cells by sieving through nylon mesh, followed by velocity sedimentation on 5% bovine serum albumin. The endothelium-enriched fraction has been maintained in primary culture for up to 3 weeks. The resulting epithelioid colonies have been characterized morphologically by both light and transmission electron microscopy and manifest all of the structural features that distinguish other, large-vessel endothelia in culture. In addition, immunohistochemical studies using an indirect fluorescent antibody technique demonstrate that these cells contain the endothelium-specific product, Factor VIII antigen.

Origin of cushion tissue in the developing chick heart: cinematographic recordings of in situ formation.

Differential interference microscopy and time-lapse cinematography were used to determine unequivocally the origin of cushion tissue cells migrating in situ in the atrioventricular region of the embryonic chick heart. These studies have verified the presumed endocardial origin of cushion tissue mesenchyme.

Morphogenesis of the truncus arteriosus of the chick embryo heart: tissue reorganization during septation.

Septation of the truncus arteriosus of the normal chick embryo heart was surveyed systematically with the light microscope. Tissue from replicate samples at successive periods of development was sectioned within an arbitrary coordinate system based on positional reference points along the external surface of the heart. Correlation of several aspects of tissue morphology within this spatial and temporal reference-frame yielded a new description of tissue associations and kinetics during septation. A stable complex of tissue structures appeared in the downstream, distal truncus at Stage 25 and persisted throughout the septation process. This complex consisted of (1) the cephalic margin of the myocardial sheath, and (2) the adjacent bifurcation of the vascular lumen, linked together by (3) the newly condensed Y-shaped strap of cells forming the aorticopulmonary septum. The apparent motion of this septation-complex toward the ventricle(s), the appearance within the thoracic cavity of the adjacent segments of the aortic arches, and measures of tissue length and width suggested that septation was accompanied, and perhaps initiated, by increased tension along the truncus. The truncal ridges remained upstream from the complex, with mesenchymal condensations beneath the endocardium differentiating into the definitive semilunar valves. Downstream from the bifurcation, mesenchyme in the aortic arch region condensed around the separate lumens to form the smooth muscular tunica media of the great arteries. The epicardium developed in a caudocephalic direction along the heart tube. Vagal innervation approached the heart cephalocaudally. Capillaries formed along the dividing truncus in both directions. Autoradiography following 3H-thymidine labelling demonstrated reduced DNA synthetic activity in the cephalic margin of the myocardium and aorticopulmonary septum, compared with the associated loose mesenchyme.

Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.

The appearance and migration of mesenchymal cushion tissue within the truncus arteriosus of the normal 2.5 to 6-day chick embryo heart was surveyed systemically with the light microscope. Series of cross-sections taken from replicate hearts at successive developmental stages allowed comparison of the following qualitative and quantitative aspects of early truncal morphogenesis. Mesenchyme within the truncus was derived from two distinct sources. The first mesenchyme appeared to migrate caudally into the cardiac jelly of the distal truncus from the nearby aortic arch region, coincident with slowing of the anterior elongation of the heart tube (Hamburger-Hamilton Stage 17-18). A second, separate mesenchymal population, derived from endocardium, began to fill the conus and proximal truncus in a radial direction, coicident with expansion of the bulbs cordis (Stage 12-19). The measured kinetics of relative cell numbers, distributions, and mitotic indices suggest substantial contributions from both sources. By Stage 26, the conotruncal region was filled with mesenchyme, which then condensed to form the anlagen of three future structures: the semilunar valves, the aorticopulmonary septum, and the tunica media of the great arteries.