Timothy P. Fitzharris

Structural analysis of endocardial cytodifferentiation

Abstract Embryonic hearts 8–40 somites (10.0–13.5 days) where subjected to transmission and scanning ultrastructural and histological examination to monitor pathways of endocardial differentiation. Primitive endocardium (day 10.0 or 8 somites) consisted of closely packed cells with smooth luminal surfaces except for cilia at cellular interfaces. Internally these cells possessed minimal secretory potential as indicated by undilated rough-surfaced endoplasmic reticulum (RER) and undeveloped Golgi complexes. The latter had nondistended lamellae and a low population of small (100-nm) bristle-coated and uncoated vesicles. Cytodifferentiation of the primitive endocardium was biphasic. In the outflow tract and AV canal, areas of future cushion tissue (cardiac mesenchyme) formation, endocardium was transformed by day 11.5 (16–18 somites) into cells with amplified secretory potential as evidenced by dilation of RER cisternae, hypertrophy of Golgi lamellae and augmented formation of Golgi vesicles consisting primarily of larger (150–200 nm) uncoated types. The luminal surfaces in these areas became convoluted and flattened while the extracellular (cardiac jelly) surface developed blebs and was studded with globular and fibrillar strands of matrical material. Surface topography and serial sectioning of the initial sites of cushion tissue formation suggested the latter was actually a derivative of endocardium having augmented secretory potential. Conversely, endocardium approached by invaginating myocardial trabeculae (atrium and ventricle) appeared to lose secretory potential as indicated by its (1) progressive attenuation of the cytoplasm, (2) reduction or complete loss of surface projections and associated globular and fibrillar material, (3) regression of RER and Golgi complexes, and (4) acquisition of cystic foci in otherwise nondilated RER. Results therefore indicated that both endocardial surface and internal features could be related to developmental changes in microenvironment and function.

Structural analyses on the matrical organization of glycosaminoglycans in developing endocardial cushions

Abstract Extracellular glycosaminoglycans (GAG) were examined in embryonic rat valvular primordia (cushion tissue) to determine if there are specific, in situ, intermolecular associations of GAG and if the passage of migrating cushion cells alters matrical organization. Precursor incorporation studies and colloidal iron staining controlled by acidified methylation, pH, and polysaccharidase digestion indicated that both hyaluronate (HA) and chondroitin sulfate (CHS) were secreted into the premigratory matrix with the predominant GAG being HA. Premigratory matrix was revealed by scanning electron microscopy after routine fixation as a microfibrillar stroma; addition of cetylpyridinium chloride (CPCL) to the fixative resulted in the retention of an additional matrical component superimposed upon the microfibrillar stroma. TEM analysis of the CPCL-dependent matrix revealed that it was composed of intertwined 3-nm filaments, electron-dense, amorphous material, and 30-nm granules. Collagen-like microfibrils were associated primarily with the filamentous component of the CPCL-dependent matrix. Ultracytochemical results obtained with dialyzed iron binding regulated by pH and polysaccharidase and protease digestion suggests that the 3-nm filaments contain HA and the granules contain both CHS and protein. Commensurate with cushion cell formation and migration, X-ray dispersive analysis and polyanionic histochemical criteria indicated increased deposition of CHS in the postmigratory matrix (i.e., matrix transversed by cells). Ultrastructurally, the CPCL-dependent components of the postmigratory matrix became progressively restructured within the wedge of migrating cells. In contrast to premigratory matrix, fewer 3-nm filaments were evident, while 30-nm granules heavily studded the collagen-like microfibrils. Physical fixation controls confirmed the variations between pre- and postmigratory matrices. These results suggest that modification in the matrix organization of embryonic heart GAG may be correlated with the migration of cushion tissue mesenchyme.

Structural analysis of cell: Matrix association during the morphogenesis of atrioventricular cushion tissue☆

Abstract Translocation of an endocardially seeded cushion cell progeny across a broad acellular expanse of extracellular matrix (ECM) constitutes a fundamental morphogenetic event in the development of atrioventricular (AV) cushion pads, the primordia of membraneous septa and cardiac valves. Transmission, scanning, and high-voltage electron microscopy together with light microscopic examination of living or fixed tissues were utilized to determine if (1) one component of the ECM more than any other interacted with the motility-like appendages of cushion cells in such a manner as to suggest a physical substratum; (2) any ECM components were organized into polarized “tracks” which could serve to guide cells centrifugally; and (3) cell:ECM associations varied among the cells comprising the migratory wave. Results indicated that two morphologically identifiable matrix components, microfibrils and a continuum of solid pleomorphic strands of heterogeneous composition called cetylpyridinium chloride (CPCL)-dependent matrix, comprised the bulk of the premigratory ECM. Contact of the premigratory matrix by cushion cells at the leading edge (pioneer cells) of the migratory wave coincided with modification in composition of the CPCL matrix and alignment of microfibrils into polarized tracks (an event seemingly dependent on motility appendage formation, since cells lacking processes after cytochalasin B treatment had altered track associations). Trailing cushion cells uniformly populated the ECM, never piled up against the myocardium, had no track associations, formed numerous cell to cell associations, and were coated with a granular remnant of disrupted CPCL-dependent matrix. The foregoing data suggest that active in vivo translocation and subsequent stabilization of cushion cells involve alignment and compositional changes in the premigratory ECM, events linked temporally with the passage of pioneer cells.

Differentiation of the chorionic epithelium of chick embryos maintained in shell-less culture

In order to determine whether the chorionic epithelium (CE) of the chorioallantoic membrane (CAM) of chick embryos maintained in shell-less culture might be suitable for studies of calcium translocation and/or tissue grafting, membranes from 10- to 18-day embryos were studied histologically using Nomarski differential interference optics on living, unfixed tissue and ultrastructurally using scanning and transmission electron microscopy. Differentiation of the CAM of cultured embryos appeared similar to thatin ovo. At 10 days, an electron-lucent squamous cell layer covered much of the CE. Intraepithelial capillaries were separated from the chorionic surface by a relatively thick (>5-μm) cytoplasmic layer. By 12 days, dense, vacuolated, apparently degenerating cells covered much of the CE. At later stages (14 to 16 days), degenerating cells were seen infrequently and most capillaries were covered by thin (

Active calcium transport in the chick chorioallantoic membrane requires interaction with the shell membranes and/or shell calcium.

Abstract Direct interaction of the chorioallantoic membrane (CAM) with the overlying Ca 2+ -rich shell membranes is necessary for maximal stimulation of Ca 2+ transport by the CAM. First, CAM developing in the absence of the shell and shell membranes (in shell-less culture or in artificial air space preparations), which demonstrates normal cytodifferentiation, develops significantly reduced Ca 2+ transport compared to either equatorial or air space CAM in ovo . Second, exposure of shell-less cultured CAM to isolated sections of Ca 2+ -rich shell membranes for 5–7 days, but not for 30 min or 16 hr, resulted in a significant stimulation of Ca 2+ transport in the immediately subjacent CAM but was without effect on transport function in peripheral sections of cultured CAM exposed to shell membranes. Neither elevated CO 2 levels nor a permissive humoral environment was sufficient to maximally stimulate transport. Additionally, we have demonstrated significant regional differences in Ca 2+ transport between equatorial and air space CAM in ovo . Our data directly demonstrate that stimulation of Ca 2+ transport is a time dependent, local phenomenon, requiring the presence of the shell membranes and/or shell Ca 2+ on the ectodermal side of the CAM during development.

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.

Cellular migration through the cardiac jelly matrix: A stereoanalysis by high-voltage electron microscopy

Abstract Formation and migration of cushion tissue in the developing chick heart was analyzed by scanning and high-voltage electron microscopic stereoanalysis. Two methods of fixation which enhance the preservation of water-soluble components of the extracellular matrix (cardiac jelly) were employed: 1% tannic acid in 3% glutaraldehyde (TAG) and 1% cetylpyridinium chloride (CPC) in 3% glutaraldehyde. Our results indicated that the preservation of the cell: matrix interaction exhibited by endocardial cells and migrating cushion tissue is dependent upon the method of fixation. In TAG-fixed embryos, filopodial extensions from the endocardium as well as filopodia of pioneering cells are most often associated with microfibrillar components of the matrix, whereas in CPC-fixed material these same cellular extensions are found in association with pleomorphic anastomosing strands rich in hyaluronate. Following these initial cell:matrix interactions by both the endocardium and pioneering cells, trailing cells invade the extracellular matrical region and clearly encounter in both types of fixation a different microenvironment in which to engage in cell:matrical associations. These observations support the hypothesis that filopodial probing by endocardial cells and pioneering cells results in macromolecular reorderings of the matrix and thus suggest an additional function for filopodia beyond translocation of the cells.

Control of cell migration in atrioventricular pads during chick early heart development: Analysis of cushion tissue migration in vitro☆

Abstract The formation of the valvular and septal primordia of the embryonic heart depends upon the migration of endocardial cushion tissue mesenchyme (CT) to populate the cardiac jelly (CJ) in specific heart regions (e.g., atrioventricular (AV) pads). It has been proposed that the migration of CT may be directed by macromolecules of the CJ. In this study, [ 3 H]thymidine-labeled endocardial (EC) and CT cells were transplanted onto intact pre- and postmigratory AV pads in vitro to test whether the compositional or structural changes known to occur in the cardiac jelly during development influence the migration of cushion tissue cells. After transplantation of labeled donor cells, host AV pads were fixed, embedded, and sectioned, and autoradiography was performed to determine the distribution of labeled donor cells within the host CJ. The experiments indicate that transplanted mural EC cells remain primarily at the AV pad surface, while grafted CT cells of all developmental ages rapidly invade both developmentally young and older AV pads. Furthermore, CT cells readily migrate in a direction opposite to that of cells in vivo when transplanted to inverted AV pads from which the myocardium has been removed. It is concluded that the CJ matrix, which is clearly a suitable framework for CT cell migration, provides no direct cues to determining the polarity or extent of migration.

Effects of beta-aminopropionitrile fumurate (BAPN) on early heart development.

Abstract Collagen is a major constituent of the extracellular matrix (ECM) of the early developing heart. The role of mature cross-linked collagen has not been demonstrated in cardiac morphogenesis, although investigators using other developmental systems have suggested that collagen can act as an inducer. The injection of 500 μg of BAPN (beta-aminopropionitrile fumurate), a known inhibitor of cross-linking, into the pericardial region of chick embryos prior to valvular and septal morphogenesis (Stage 12 to 14) was without effect on the appearance or migration of cushion tissue mesenchyme in the ECM. However, by Stage 22 to 23 BAPN-treated embryos exhibited truncal aneurysms which resulted in death. Ultrastructural examination of the hearts of BAPN-treated embryos showed an alteration in the cell surface of both the endocardium and myocardium as evidenced by the deposition of very electron dense material. Biochemical analysis confirmed that BAPN-treated organisms were indeed lathyritic. These studies suggest that the continued synthesis and deposition of mature cross-linked collagen is not required for initiation and migration of cushion tissue mesenchyme nor for the appearance and maintenance of specific endocardial shape changes referred to as “flutes”. It is suggested that collagen is primarily a structural macromolecule in the early heart, rather than an inducer. These studies further suggest that, in addition to its effect on collagen cross-linking, BAPN may cause cardiac anomalies by modifying collagen: proteoglycan interaction within the ECM.

The histochemistry and structure of tentacle cartilage tissues in the marine polychaete, Sabella melanostigma

SummaryThe cartilages (or “chondroid” tissue) in tentacles of the polychaete annelid, Sabella melanostigma, have been examined by electron microscopy and a series of histochemical techniques for the demonstration of mucopolysaccharides and protein end-groups. The ultrastructural studies indicated that the cartilages possess an investing layer of dense connective tissue which differs significantly from the matrix material secreted between the chondrocytes. The cartilage matrix was positive for acidic mucins with levels of sulfation above those of mammalian chondroitins A and C. This matrix as well as the investing connective tissue were intensely PAS-positive. Sabella cartilage was also stained intensely by methods for demonstrating tryptophan, tyrosine, side-chain carboxyl groups, disulfide groups, and amino groups. It was not stained by the procedure for sulfhydryl groups. Some evolutionary aspects of cartilage and chondroid tissues were discussed.