David L. Bolender

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.

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.

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.

Secretion of inhibin βA by endoderm cultured from early embryonic chicken

Abstract 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 βA were very reactive. Western blot analysis verified the presence of fibronectin and, most remarkably, inhibin βA, activin dimers of which have recently been implicated in Xenopus mesoderm induction ( Smith, Price, Van Nimmen, and Huylebrock (1990). Nature 345, 729 ).

Ultrastructural alterations associated with the initiation of follicular atresia.

SummaryTo 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.

Curriculum integration = course disintegration: What does this mean for anatomy?

Many basic scientists including anatomists are currently involved in decisions related to revisions of the undergraduate medical curriculum. Integration is a common theme in many of these decisions. As described by Harden, integration can occur along a multistep continuum from independent, discipline‐based courses to a completely interdisciplinary curriculum. For anatomy, each derivative of curricular integration can be shown to involve progressive disruptions of the temporal and topographical relationship between organ systems in a body region, of the temporal relationship with other courses in a harmonized curriculum, and of the relationships between components of organ systems when integration is implemented in thematic curricula. Drawing from our experience teaching in various types of integrated medical curricula, we encourage readers to proceed cautiously with their curricular decisions because each one can have gains and losses that may impact learning in the new format. Anat Sci Educ.

A Culture Model for Cardiac Morphogenesisa

Studies in amphibian and avian embryos have indicated that an inductive interaction between precardiac mesoderm and endoderm is required to establish a fully differentiated, functional heart.’ The nature of the inductive stimulus is unknown. Although cell-cell interactions may be involved, it is likely that macromolecular factors secreted by the interacting tissues signal inductive changes in this and other developing systems.’ To identify endodermal secretions that modulate cardiogenesis, an in vitro bioassay system is needed that ideally recapitulates the inductive processes necessary for three-dimensional heart morphogenesis. We report here that splanchnic mesoderm explanted onto hydrated collagen gel cultures with anterior endoderm from early embryos may provide such a model system.

Computerized Grading of Anatomy Laboratory Practical Examinations

At the Medical College of Wisconsin, a procedure was developed to allow computerized grading and grade reporting of laboratory practical examinations in the Clinical Human Anatomy course. At the start of the course, first year medical students were given four Lists of Structures. On these lists, numbered items were arranged alphabetically; the items were anatomical structures that could be tagged on a given lab practical examination. Each lab exam featured an anatomy laboratory component and a computer laboratory component. For the anatomy lab component, students moved from one question station to another at timed intervals and identified tagged anatomical structures. As students identified a tagged structure, they referred to a copy of the list (provided with their answer sheet) and wrote the number corresponding to the structure on their answer sheet. Immediately after the anatomy lab component, students were escorted to a computer instruction laboratory where they typed their answer numbers into a secured testing component of a learning management system that recorded their answers for automatic grading. After a brief review of examination scores and item analysis by faculty, exam scores were reported to students electronically. Adding this brief computer component to each lab exam greatly reduced faculty grading time, reduced grading errors and provided faster performance feedback for students without changing overall student performance. Anat Sci Ed 1:220–223, 2008.

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