FGF-2-induced imbalance in early embryonic heart cell proliferation: a potential cause of late cardiovascular anomalies.

BACKGROUND: This laboratory previously demonstrated that placement of fibroblast growth factor-2 (FGF-2)-soaked beads adjacent to the developing ventricle at stage 24 caused cardiovascular anomalies by embryonic day 15. We sought to characterize early cellular changes that may suggest mechanisms for the abnormalities observed at day 15. Because levels of both myocyte proliferation and immunohistochemically detectable endogenous FGF-2 begin to decline before stage 24 in untreated embryos, it was of interest to determine whether exogenous FGF-2 might maintain cardiac myocyte proliferation at or near peak levels. METHODS: Chick embryos were incubated to stage 18 (2.8 days), at which time beads soaked in phosphate-buffered saline (PBS) or 100 microg/ml FGF-2 were placed adjacent to the developing ventricle and development was allowed to continue. After 3 days (stage 29), bromodeoxyuridine (BrdU) was applied to mark dividing cells, followed by double fluorescent assessments to detect relative numbers of dividing and nondividing cells. RESULTS: Quantitative image analysis, using Metamorph software, showed that exogenous FGF-2 caused a 62% increase in the overall number of dividing cells (P < 0.01), concomitant with a 25% increase in total cell number (cell density: P < 0.05). Expressed in relative terms, these changes corresponded to a 25% increase in the proliferation labeling index: 30% of all cells were proliferating in FGF-treated hearts, in contrast with only 24% in control hearts. CONCLUSIONS: Taken together, these data suggest that an FGF-induced imbalance in myocardial cell proliferation at early developmental stages of heart development causes cardiovascular anomalies during late embryogenesis.

Postmortem blood tests for HIV, HBV, and HCV in a body donation program.

A retrospective analysis of the results of blood tests conducted on body donors received by the Anatomical Gift Registry of the Medical College of Wisconsin (MCW) was performed. Over the 5-year period from April 1992 through March 1997 a total of 785 body donors were tested for Human Immunodeficiency Virus (HIV) and Hepatitis B and C Viruses (HBV and HCV). Eighteen of the 785 donors (2.3%) tested positive for one of these infectious agents. Two donors were positive for HIV, six were positive for HBV and ten were positive for HCV. The death certificates and files of those donors who tested positive were reviewed and the results are presented here. Blood testing prior to the use of the body donors is an effective and reasonable way of identifying the presence of these infectious agents, thus reducing the risk to those who work with cadavers. The cost for the testing at MCW is about $60 per donor.

Teratogenic effects of implanting fibroblast growth factor-2-soaked beads in the cardiac region of the stage 24 chick embryo.

The identification of fibroblast growth factor-2 (FGF-2), and other family members, in a variety of embryonic tissue extracts has implicated these growth factors as participants in many embryonic events, including cardiogenesis. The present study was conducted in an attempt to characterize the effects of exogenous FGF-2 on the development of the avian heart. Heparin acrylic beads, each soaked in 100 micrograms/ml FGF-2, were applied to the Hamburger and Hamilton [(1951) J. Morphol. 88:49-92] stage 24 (day 4 of incubation) chick heart, near the primitive ventricle. The embryos were allowed to develop until stage 41 (day 15), at which time they were observed for the presence of gross external and internal anomalies. Crown rump length (CRL), wet weight (WW), and various heart measurements were obtained and compared. The survival rate of the control group was significantly higher (P < 0.05) than that for the experimental group and the anomaly rate for the control group was significantly lower (P < 0.01) than that for the experimental group. The experimental group exhibited a wide range of anomalies, most of which were cardiac in nature; however, anomalies of the ventral thoracoabdominal wall were also noted. Our data suggest that application of exogenous FGF-2 has teratogenic effects on cardiac development as well as on the development of the ventral body wall of the avian embryo.

Combined BMP-2 and FGF-4, but neither factor alone, induces cardiogenesis in non-precardiac embryonic mesoderm.

Previous work in this laboratory has shown that endoderm cells in the heart forming region (HFR endoderm) of the chicken embryo induce terminal cardiac differentiation in explanted precardiac mesoderm cells. Immunostaining patterns indicating that HFR endoderm cells express Drosophila decapentaplegic (dpp)-like antigens prompted a degenerate polymerase chain reaction (PCR) screen to identify cDNAs in the dpp subgroup of the transforming growth factor-beta (TGF-beta) family. Among 50 clones of PCR products that have been sequenced, over half have identity with bone morphogenetic protein-2 (BMP-2). No other TGF-beta cDNAs have been detected, suggesting that BMP-2 is the major dpp subgroup protein synthesized by HFR endoderm cells. However, BMP-2 protein did not promote survival of either precardiac or non-precardiac mesoderm cells in culture. Whereas FGF-4 supports cardiogenesis in precardiac mesoderm, it did not induce cardiogenesis in nonprecardiac mesoderm, although explant viability was maintained. In contrast to the isolated effects of these growth factors, treatment of non-precardiac mesoderm with combined BMP-2 and FGF-4 induced cardiogenesis in the majority of explants, as revealed by the formation of a rhythmically contractile multicellular vesicle that expresses sarcomeric alpha-actin. These findings suggest that BMP-2 and FGF-4 possess respective differentiative and proliferative activities, the combination of which specifies cells to the cardiac lineage.

Effects of ectoderm co-culture and conditioned medium on the limb mesoderm in vitro.

Undissociated mesoderm placed onto collagen gels forms three subpopulations of mesenchyme which differentiate along myogenic, chondrogenic and fibrogenic phenotypes. Co-culture with ectoderm appears to inhibit the formation of distinct cartilage elements and myotubes by interfering with the differentiation of chondrogenic and fibrogenic progenitors. Addition of CCM enriched in ES antigens enhances the effects of the ectoderm on chondrogenesis. Culture in the presence of CCM alone retards chondrogenesis and almost completely inhibits myogenesis. These results suggest that the primary effect of ectoderm or CCM in our culture system is on myogenic and chondrogenic differentiation, and ES antigens, if responsible for these effects, appear to have no role in pattern formation.

Secretion of inhibin beta A by endoderm cultured from early embryonic chicken.

Although several reports have indicated a role for endoderm in the regulation of heart development, the mechanism remains unknown. To begin characterization of endoderm-secreted proteins, explants from postgastrulation (Hamburger-Hamilton stage 5/6-8) chicken embryos were cultured in defined medium. Fluorography of SDS-PAGE gels revealed a pattern of synthesized, secreted proteins that was independent of time in culture or embryonic stage when explants were removed. Approximately 10 labeled bands were detected, the most prominent of which migrated at 17, 25, and 200 kDa. ELISA analysis revealed that while acidic and basic fibroblast growth factor-like antigens were barely detectable, fibronectin and inhibin beta A were very reactive. Western blot analysis verified the presence of fibronectin and, most remarkably, inhibin beta A, activin dimers of which have recently been implicated in Xenopus mesoderm induction (Smith, Price, Van Nimmen, and Huylebrock (1990). Nature 345, 729.)

Localization of bFGF-like proteins as punctate inclusions in the preseptation myocardium of the chicken embryo.

Immunocytochemistry has been employed to map the appearance of bFGF-like proteins in precardiac and preseptation myocardial cells between stages 6 and 15 of chicken embryogenesis. Stage 6 embryos exhibited no staining, with the exception of a subtle signal in endoderm cells. At subsequent stages, staining was observed only in cells of the developing myocardium, first appearing at the time of heart tube fusion (stage 9+) as punctate cytoplasmic aggregates. While the expression of bFGF-like antigen was temporally similar to that of myosin heavy chain, their staining patterns differed in that bFGF-like proteins were nonsarcomeric and did not extend into the inflow or outflow tracts. Western blotting of heparin agarose affinity-isolated proteins from stage 15 hearts revealed an antigen migrating at approximately 19 kDa. In contrast with the unique localization of bFGF-like proteins in myocardial cells, FGF receptor (FGFR) staining was widely distributed in the embryo; however, concentrated deposits of FGFR were detected in endothelial and myocardial cells, which diminished in the myocardium but not in the endothelium by stage 15. These results suggest that FGF-like proteins may have autocrine and/or paracrine functions during early cardiac morphogenesis.

Morphogenesis of precursor subpopulations of chicken limb mesenchyme in three dimensional collagen gel culture.

Although homogeneous in appearance, several lines of evidence suggest early (stage 17-19) limb mesenchymal cells are committed to particular cell lineages, e.g., myogenic or chondrogenic. However, subsequent expression of cell or tissue phenotype in the developing limb does not occur in a randomized process but rather in a spatially specific pattern. The potential regulatory mechanisms controlling the "patterned" expression of tissue phenotype in the limb have not been resolved. The purpose of this study was to determine if, prior to the formation of an apical ectodermal ridge, nondissociated limb mesenchyme has inherent morphogenetic potential to form nonrandomized patterns of tissue organization. The hypotheses to be tested were that, if provided a spatially permissive culture environment, 1) mesenchymal cells committed to a particular lineage would segregate into precursor (sub)populations prior to overt expression of phenotype and 2) the ultimate expression of a tissue phenotype may be regulated, in part, by histogenic interactions between the precursor cell groups. For these studies, mesoblasts (intact mesenchyme minus ectoderm) from stage 17-19 hindlimb buds were explanted intact to the surface of a 1-3 mm thick hydrated lattice of repolymerized type I collagen and incubated for 2-11 days. Examination of cultures at variable intervals revealed three distinct temporal sequences (periods) which were arbitrarily termed early morphogenesis (0-3 days), cytodifferentiation (3-5.5 days), and primitive tissue formation (5.5-11 days) based on similarities to in situ limb development. By the end of the first period, the mesenchymal cells had sorted into three distinct precursor populations: 1) an epithelial-like outgrowth of premyogenic and prefibrogenic cells at the surface of the gel lattice (termed the "surface subset") which circumscribed, 2) a centrally positioned prechondrogenic condensate ("central subset"), and overlaid 3) a dispersed, population of free cells that invaded the collagen lattice ("seeded subset"). Subsequent cytodifferentiation led to the appearance of multinucleated myotubes within the surface subset and chondrification of the central subset. Cells of the seeded subset remained dispersed within the collagen lattice. Primitive histogenic events were initiated during the final period of development including 1) at sites where surface cells established boundaries with the central subset, collectives or "bundles" of variable sized myotubes were formed which became partially ensheathed by the attenuated processes of fibroblastlike cells; and 2) a secondary site of chondrogenic activity was initiated within the gel lattice at the boundary between the central and seeded cell populations. Transformation of seeded fibroblasts into chondroblasts accompanied expansion of the secondary chondrogenic element within the gel lattice.(ABSTRACT TRUNCATED AT 400 WORDS)

Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles.

As an initial step in characterizing the function of basal lamina components during muscle cell differentiation and innervation in vivo, we have determined immunohistochemically the pattern of expression of three components--laminin, proteins related to agrin (an acetylcholine receptor (AChR)-aggregating protein), and a heparan sulfate proteoglycan--during the development of chick embryo hindlimb muscles. Monoclonal antibodies against agrin were used to purify the protein from the Torpedo ray and to characterize agrin-like proteins from embryonic and adult chicken. In early hindlimb buds (stage 19), antibodies against laminin and agrin stained the ectodermal basement membrane and bound to limb mesenchyme with a generalized, punctate distribution. However, as dorsal and ventral premuscle masses condensed (stage 22-23), mesenchymal immunoreactivity for laminin and agrin-like proteins, but not the proteoglycan, became concentrated in these myogenic regions. Significantly, the preferential accumulation of these molecules in myogenic regions of the limb preceded by 1-2 days the appearance of muscle-specific proteins, myoblast fusion, and muscle innervation. All three basal lamina components were preferentially associated with all AChR clusters from the time we first observed them on newly formed myotubes at stage 26. Localization of these antigens in three-dimensional collagen gel cultures of limb mesenchyme, explanted prior to innervation of the limb, paralleled the staining patterns seen during limb development in the embryo. These results indicate that basal lamina molecules intrinsic to limb mesenchyme are early markers for myogenic and synaptic differentiation, and suggest that these components play important roles during the initial phases of myogenesis and synaptogenesis.

Distribution of basement membrane antigens in cryopreserved early embryonic hearts.

The early embryonic heart is composed of two cylindrical epithelial layers, an inner endothelium and an outer myocardium. The cardiac jelly (CJ), an acellular accumulation of extracellular matrix (ECM), fills the space between the two epithelia. During development of the heart, a portion of the endothelial cells of the atrioventricular (AV) region differentiate into mesenchyme cells in a temporally and spacially specific manner. Although contiguous with those in the AV region, endothelial cells lining the ventricle never form mesenchyme in situ. At present, the mechanisms controlling the biphasic differentiation of the endothelium and the subsequent migration of cardiac mesenchymal cells are poorly understood. Although the CJ lies between two epithelial and is spatially equivalent to a basement membrane (BM), it has not traditionally been considered to be organized into a BM-like structure. The potential significance of this observation to developmental biology lies in the possibility that BM or their individual components (i.e., fibronectin (FN), laminin (LM), type IV collagen, and heparin sulfate proteoglycan (HSPG] may function as the regulatory site of epithelial differentiation and morphogenesis. A cryofixation technique was developed in order to determine the in situ immunohistochemical distribution of the BM components in the CJ. Results indicated that the CJ exists as the fusion between a larger myocardially derived BM having a lamina densa and an extended reticular lamina and an attenuated, endothelial-associated BM composed only of a lamina densa. Except for FN, the individual BM components were not all present during early stages, but instead appeared in a sequential manner, suggesting that all components of an adult-type BM are not required to initiate the assembly of a structural and functional BM during development. In the AV canal and outflow tract (OT), FN appeared as a progressively expanding gradient of material with the greatest density nearer the myocardium.

The distribution and spatial organization of the extracellular matrix encountered by mesencephalic neural crest cells.

Cephalic neural crest (NC) cells enter a cell-free space (CFS) that contains an abundant extracellular matrix (ECM). Numerous in vitro investigations have shown that extracellular matrices can influence cellular activities including NC cell migration. However, little is known about the actual ECM composition of the CFS in vivo, how the components are distributed, or the nature of NC cell interactions with the CFS matrix. Using ultrastructural, autoradiographic, and histochemical techniques we analyzed the composition and spatial organization of the ECM found in the CFS and its interaction with mesencephalic NC cells. We have found that a specific distribution of glycoproteins and sulfated polyanions existed within the CFS prior to the translocation of NC cells and that this ECM was modified in areas occupied by NC. The interaction between the ECM components and the NC cells was not the same for all NC cells in the population. Subpopulations of the NC cell sheet became associated with ECM of the ectoderm (basal lamina) while other NC cells became associated with the ECM of the CFS. Trailing NC cells (NC cells that emerge after the initial appearance of NC cells) encountered a modified ECM due to extensive matrix modifications by the passage of the initial NC cell population.

Structural analysis of extracellular matrix prior to the migration of cephalic neural crest cells.

Cephalic neural crest cells enter cell free areas containing abundant extracellular matrix (ECM). Previous histochemical studies have identified both sulfated and non-sulfated glycosaminoglycans within this matrix. In the present study, ultrastructural examination of the ECM demonstrated an anastomosing network of pleomorphic, cetyl pyridinium chloride-dependent strands within cell free spaces and in association with the basement membrane of the surface ectoderm. Thin section analysis revealed that the strands consisted of three components: (1) 3-5 nm filament meshwork; (2) electron dense amorphous material and (3) 30 nm granules. In contrast, the ECM associated with the basement membrane consisted principally of a continuum of electron dense, amorphous material. The molecular ordering of ECM within crest cell pathways was compared to the well-characterized, hyaluronate-rich, premigratory matrix of cardiac jelly.

Ultrastructural alterations associated with the initiation of follicular atresia.

To identify and describe ovarian follicles committed to undergo follicular degeneration (atresia), immature rats were primed with pregnant mare serum gonadotropin (PMSG). After PMSG treatment, preovulatory follicles develop but subsequently degenerate. Prior to the appearance of pyknotic nuclei (Stage I of atresia), degenerative changes were observed in focal areas of the granulosa cell layer. These changes include "blebbing" of the cytoplasm and alterations in the shape of the granulosa cells. The appearance of these degenerative changes coincides with a decrease in ovarian concentrations of estradiol and testosterone. Since estrogens and androgens maintain the follicle, the decline in estradiol and testosterone could be responsible for the further degenerative alterations that lead to complete deterioration of the preovulatory follicle. In Stage I atretic follicles, lysosome-derived autophagic vacuoles develop and macrophages invade both the thecal and granulosa cell layers. The combined actions of the autophagic vacuoles and macrophages could destroy both the granulosa-cell and thecal layers and thereby transform the preovulatory follicle into an ovarian cyst.

A histochemical analysis of polyanoinic compounds found in the extracellular matrix encountered by migrating cephalic neural crest cells.

Neural crest cells destined to form craniofacial primordia initially are "seeded" into and subsequently migrate through the extracellular matrix (ECM) of a cell free space (CFS) between the surface ectoderm and the underlying mesoderm. Utilizing histochemical procedures for polyanionic compounds, we have demonstrated that both sulfated and nonsulfated glycosaminoglycans (GAG) are present in the CFS of the cephalic region of the chick embryo and that their distribution and structural organization vary with the passage of neural crest or mesodermally derived (MD) mesenchymal cells through it. In stages 7 and 8 embryos a predominance of fine filamentous strands composed primarily on nonsulfated, carboxyl-rich GAG is seen spanning intercellular spaces between adjacent tissues and MD mesenchymal cells. In older embryos (stages 9 and 10) much of the filamentous material is replaced by coarse fibrillar strands or amorphous material which coats the surfaces of MD mesenchymal and neural crest cells as they invade the CFS. Using enzymatic digestions (Streptomyces and testicular hyaluronidase) and the critical electrolyte concentration procedure, data suggest that the fine filamentous matrix onto which the neural crest cells migrate consists mainly of hyaluronate with lesser amounts of chondroitin and some sulfated GAG present. The coarse fibrillar matrix that appears after passage of either neural crest or MD mesenchymal cells through the original CFS contains strongly sulfated polyanionic material, predominantly chondroitin sulfates A, C. Since GAG is located ubiquitously within the ECM of embryos at various stages, the role of GAG, if any, in the transfer of developmental information may be of a general nature (ie. stimulus of motility) rather than of specific morphogenetic cues (for specific differentiation into craniofacial primordia).