Simulated microgravity and hypergravity attenuate heart tissue development in explant culture.

Exposure to altered gravity may disturb the cytoskeleton-cell surface-extracellular matrix (ECM) interface of embryonic cells. Development of organs such as the heart depends on dynamic interactions across cell surfaces. Fibronectin (FN), for example, a glycoprotein that links the ECM to the cytoskeleton through integrin surface receptors, is required for normal heart development. Thus, altered gravity may perturb organogenesis. We cultured precardiac explants from chick embryos in a rotating bioreactor vessel to simulate microgravity (microG), or in a tissue culture centrifuge, for 18 h during heart development. Bioreactor microG did not alter external morphology of explants, but did significantly reduce the proportion that developed contractions. Immunostaining for FN of explant sections showed that it also significantly reduced the linear extent of staining present in basement membrane regions. Analysis of ultrastructure revealed a significant reduction in the number of desmosomes per unit area and other differences. Hypergravity dramatically abolished development of contractions and altered morphogenesis. The results indicate a probable sensitivity of cardiomyogenic development involving FN to altered gravity.

Isotretinoin alters morphology, polarity, and motility of neural crest cells in culture.

Migrating neural crest cells (NCCs) contribute to a diverse array of vertebrate head and neck structures. Retinoids are proven human and animal teratogens. To elucidate isotretinoin's effects, cranial and trunk neural folds were microdissected from chick embryos and cultured. Image analysis and immunostaining were used to quantitate cell behavior. We found that a higher proportion of Stage 8, 9, and 10 treated NCCs were rounded and clustered. Medians and means for cell area, perimeter, and elongation index were lower for treated cells from Stage 9 and 10 embryos, but not from Stage 8. Cumulative medians and means for changes in area and perimeter, and cell migration were similarly lower. Thus interference with the transitory basal activity of the cytoskeleton that adjusts and determines cell-substratum adhesion, spreading, elongation, and migration may be the mechanism by which isotretinoin acts on NCCs in slightly older embryos.

An alternative model for cell sheet migration on fibronectin during heart formation.

The emergence of animal form and function depends on cell migrations in the embryo. Some migrations are accomplished by cells individually, and the mechanism of movement is predictable by contemporary models of cell adhesion and cytoskeletal function. However, other migrations occur that involve layers or sheets of cells connected by junctions, and the mechanism of migration is obscure. An example is the precardiac mesoderm, an epithelium that migrates anteriorly and ventrally in the early amniote embryo to the position of heart formation. It moves upon and is influenced by the adjacent endoderm, which has produced an extracellular matrix. The matrix contains the cell adhesion and cytoskeleton-activating glycoprotein fibronectin. Some immunolocalization studies have reported that fibronectin is arrayed in an anterior-to-posterior gradient, and it has been suggested that directional migration results from a haptotactic response of each cell to the gradient, a model derived from and supported by experiments with individual cells in culture. However, we have produced evidence from immunostaining that suggests fibronectin is arrayed as a localized anterior patch rather than a gradient. We propose an alternative model for precardiac epithelial migration in which only the anterior cells attach effectively to fibronectin. Thus adhered, their cytoskeletal contractile activity generates force which propagates throughout the layer of connected cells, and efficiently pulls them in the proper direction, following the bending and extending movements of the foregut, notochord and other structures of the head. Theoretical implications of the two models are discussed.

Early heart development in the chick embryo: effects of isotretinoin on cell proliferation, alpha-actin synthesis, and development of contractions.

Isotretinoin is a potent retinoic acid used in the treatment of skin disorders. Though very effective, it is teratogenic if administered during pregnancy, and its teratogenic effect may be related to the normal activity of retinoids as signalling molecules in the embryo. Although its exact mechanism of action is unknown, it has been suggested that it causes its characteristic pattern of defects that includes heart defects, by inhibiting the migration of neural crest cells. However, other effects on cells are known. We studied early cardiac cell proliferation using incorporation of bromodeoxyuridine (BrdU) and detection with a monoclonal anti-BrdU. Proliferation in heart tissue of whole embryo cultures was inhibited in medium with 10(-6) M isotretinoin to 62% of the control level in myocardium. We studied its effects in culture on precardiac explant development in the absence of the neural crests. Culture of precardiac mesodermal-endodermal explants revealed that development of heart vesicles from the mesoderm was little affected, but the development of heartbeat was inhibited depending on dose in the 10(-5) to 10(-7) M range. The effect on development of contractions was augmented in the presence of serum; it could be duplicated by all-trans-retinoic acid, and it was reversible. Synthesis of the alpha-actin isotype, analyzed by isoelectric focusing, was found to be inhibited or delayed. The results suggest multiple effects of retinoids on growth, morphogenesis, and differentiation of early cardiac tissue, and are discussed in relation to the potential role of retinoids in early embryogenesis.

Casein, actin, and tubulin expression during early involution in bovine and murine mammary tissue.

Hybridization methods and in vitro translation were used to examine the expression and functional condition of messenger RNA encoding caseins and cytoskeletal proteins in the mammary gland during early involution. In the mouse, steady state mRNA levels for alpha-, beta-, and gamma-caseins coordinately decreased to 20% of initial levels between 12 and 72 h after pup removal. In vitro translatability of mouse casein mRNA, as determined by immunoprecipitation, electrophoresis, and gel slice counting, revealed a pattern that closely paralleled mRNA expression. In contrast, bovine casein mRNA levels were only slightly reduced by 72 h postmilking, whereas in vitro translatability decreased by about one-half. Northern blot analysis of total mouse mammary RNA that were hybridized with probes to cytoskeletal proteins showed a gradual decrease of alpha-tubulin mRNA, but an increase in beta-actin mRNA during early involution. Two-dimensional gel analysis of in vitro translated products indicated a concordant increase in beta-gamma-actin. In the cow, beta-actin mRNA at 72 h of involution was equal to or greater than that during lactation. These results demonstrate the generally slower involution response in the cow and suggest that differing regulations are involved. Early events of cellular involution may be related to a reorganization of the cytoskeleton.