Significance of bone morphogenetic protein-4 function in the initial myofibrillogenesis of chick cardiogenesis

The heart is the first organ to form and function during vertebrate embryogenesis. Using a secreted protein, noggin, which specifically antagonizes bone morphogenetic protein (BMP)-2 and -4, we examined the role played by BMP during the initial myofibrillogenesis in chick cultured precardiac mesoendoderm (mesoderm + endoderm; ME). Conditioned medium from COS7 cells transfected with Xenopus noggin cDNA inhibited the expression of sarcomeric proteins (such as sarcomeric alpha-actinin, Z-line titin, and sarcomeric myosin), and so myofibrillogenesis was perturbed in cultured stage 4 precardiac ME; however, it did not inhibit the expression of smooth muscle alpha-actin (the first isoform of alpha-actin expressed during cardiogenesis). In cultured stage 5 precardiac ME, noggin did not inhibit either the formation of I-Z-I components or the expression of sarcomeric myosin, but it did inhibit the formation of A-bands. Although BMP4 was required to induce expressions of sarcomeric alpha-actinin, titin, and sarcomeric myosin in cultured stage 6 posterolateral mesoderm (noncardiogenic mesoderm), smooth muscle alpha-actin was expressed without the addition of BMP4. Interestingly, in cultured stage 6 posterolateral mesoderm, BMP2 induced the expressions of sarcomeric alpha-actinin and titin, but not of sarcomeric myosin. These results suggest that (1) BMP4 function lies upstream of the initial formation of I-Z-I components and A-bands separately in a stage-dependent manner, and (2) at least two signaling pathways are involved in the initial cardiac myofibrillogenesis: one is an unknown pathway responsible for the expression of smooth muscle alpha-actin; the other is BMP signaling, which is involved in the expression of sarcomeric alpha-actinin, titin, and sarcomeric myosin.     

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.     

Immunofluorescent studies on titin and myosin in developing hearts of normal and cardiac mutant axolotls

Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34–35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26–35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35–42. At tailbud stage-26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32–33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages. © 1994 Wiley-Liss, Inc.     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.     

Cardiomyocytes re-enter the cell cycle and contribute to heart development after differentiation from cardiac progenitors expressing Isl1 in chick embryo

Cardiomyocytes are generated from the precardiac mesoderm and the size of the heart increases dramatically during embryogenesis. However, it is unclear how differentiation and proliferation correlate in the cardiac cell line during development. Here, we show that cardiomyocytes re-entered into a proliferative state after differentiation with a concomitant cell cycle arrest in chick embryo. The cells in the course of differentiation from Isl1-positive cardiac precursors to cardiomyocytes did not proliferate, but differentiated cardiomyocytes proliferated even after the acquisition of contractile function. After differentiation, cardiomyocytes developed a proliferative potential to contribute to the increase in cell numbers during heart development. Almost all differentiated cardiomyocytes (82.8%) incorporated bromodeoxyuridine (BrdU) in vitro, indicating the ability of DNA replication. Furthermore, mitotic chromosomes were observed in the cardiomyocytes in which a sarcomeric structure was sustained in the cytoplasm. We conclude that the sequential events of the differentiation to contractile myocytes and the re-entry into the cell cycle are strictly regulated during cardiac cell maturation. These results provide an insight into the maturation mechanism of the cardiac cell line.     

The outflow tract of the heart is recruited from a novel heart-forming field.

As classically described, the precardiac mesoderm of the paired heart-forming fields migrate and fuse anteriomedially in the ventral midline to form the first segment of the straight heart tube. This segment ultimately forms the right trabeculated ventricle. Additional segments are added to the caudal end of the first in a sequential fashion from the posteriolateral heart-forming field mesoderm. In this study we report that the final major heart segment, which forms the cardiac outflow tract, does not follow this pattern of embryonic development. The cardiac outlet, consisting of the conus and truncus, does not derive from the paired heart-forming fields, but originates separately from a previously unrecognized source of mesoderm located anterior to the initial primitive heart tube segment. Fate-mapping results show that cells labeled in the mesoderm surrounding the aortic sac and anterior to the primitive right ventricle are incorporated into both the conus and the truncus. Conversely, if cells are labeled in the existing right ventricle no incorporation into the cardiac outlet is observed. Tissue explants microdissected from this anterior mesoderm region are capable of forming beating cardiac muscle in vitro when cocultured with explants of the primitive right ventricle. These findings establish the presence of another heart-forming field. This anterior heart-forming field (AHF) consists of mesoderm surrounding the aortic sac immediately anterior to the existing heart tube. This new concept of the heart outlet's embryonic origin provides a new basis for explaining a variety of gene-expression patterns and cardiac defects described in both transgenic animals and human congenital heart disease.     

Isoproterenol directs hair follicle-associated pluripotent (HAP) stem cells to differentiate in vitro to cardiac muscle cells which can be induced to form beating heart-muscle tissue sheets

Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of the follicle. Previous studies have shown that HAP stem cells can differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. HAP stem cells effected nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. The differentiation potential to cardiac muscle cells was greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol. In the present study, we observed that isoproterenol directs HAP stem cells to differentiate to cardiac muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. These results demonstrate that HAP stem cells have great potential to form beating cardiac muscle cells in tissue sheets.     

Early heart development in the chick embryo: effects of isotretinoin on cell proliferation, α-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.     

DNA synthesis, mitosis, and differentiation in cardiac myogenesis

Cardiac muscle cells obtained by trypsinizing 5-day chick embryonic heart were cultured as single cells in separate culture dishes. Using this technique, problems of heterotypic cell interactions, “overgrowth” of one cell type, etc., are eliminated. Experiments performed on these single cell cultures show that the muscle cells in the embryonic chick hearts differ in morphology, including content of cross-striated myofibrils; in ability to synthesize DNA and undergo mitosis; and in frequency of contraction. Contracting cells containing cross-striated myofibrils undergo mitosis in vitro, giving rise to spontaneously beating daughter cells. These daughter cells contain cytoplasmic fibrils, which bind fluorescein-labeled antimyosin immediately after cytokinesis. Some cardiac muscle cells from 5-day heart do not divide in culture; the rest undergo 1–5 doublings. This preliminary investigation suggests that the new muscle cells formed during cardiac growth are derived from mitotically active “overtly” differentiated cardiac muscle cells.     

Making more heart muscle

Postnatally, heart muscle cells almost completely lose their ability to divide, which makes their loss after trauma irreversible. Potential repair by cell grafting or mobilizing endogenous cells is of particular interest for possible treatments for heart disease, where the poor capacity for cardiomyocyte proliferation probably contributes to the irreversibility of heart failure. Knowledge of the molecular mechanisms that underly formation of heart muscle cells might provide opportunities to repair the diseased heart by induction of (trans) differentiation of endogenous or exogenous cells into heart muscle cells. We briefly review the molecular mechanisms involved in early development of the linear heart tube by differentiation of mesodermal cells into heart muscle cells. Because the initial heart tube does not comprise all the cardiac compartments present in the adult heart, heart muscle cells are added to the distal borders of the tube and within the tube. At both distal borders, mesodermal cell are recruited into the cardiac lineage and, within the heart tube, muscular septa are formed. In this review, the relative late additions of heart muscle cells to the linear heart tube are described and the potential underlying molecular mechanisms are discussed.     

Myofibril organisation and mitosis in cultured cardiac muscle cells.

Isolated cardiac muscle cells grown in vitro have been studied with respect to their ability to contract spontaneously and maintain myofibrillar organisation during division. These cells do not round up to undergo mitosis; division is achieved by the cell pinching itself in two in a selected area. This adaptation minimises disturbance to cell attachment sites and to myofibrils running between them. We correlated this with the persistence of beating during division and the maintenance of myofibrils with intact Z bands, even in close proximity to the nucleus, through division in many cells. Cessation of beating and disorganisation of myofibrils are therefore not prerequisites for division of cardiac muscle cells, as reported previously.     

Immunogold-labeled S-phase neoblasts, total neoblast number, their distribution, and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora)

Neoblasts in Platyhelminthes are the only cells to proliferate and differentiate into all cell types. In Macrostomum lignano, the incorporation of 5'-bromo-2'-deoxyuridine (BrdU) in neoblasts confirmed the distribution of S-phase cells in two lateral bands. BrdU labeling for light and for transmission electron microscopy (TEM) identified three populations of proliferating cells: somatic neoblasts located between the epidermis and gastrodermis (mesodermal neoblasts), neoblasts located within the gastrodermis (gastrodermal neoblasts), and gonadal S-phase cells. In adults, three stages of mesodermal neoblasts (2, 2-3, and 3) defined by their ultrastructure were found. Stage 1 neoblasts where only seen in hatchlings. These stages either were phases within the S-phase of one neoblast pool or were subsequent stages of differentiating neoblasts, each with its own cell cycle. Regular TEM and immunogold labeling provided the basis for calculating the total number of neoblasts and the ratio of labeled to non-labeled neoblasts. Somatic neoblasts represented 6.5% of the total number of cells. Of these, 27% were labeled in S-phase. Of this fraction, 33% were in stage 2, 46% in stage 2-3, and 21% in stage 3. Immunogold labeling substantiated results concerning the differentiation of neoblasts into somatic cells. Non-labeled stage 2 neoblasts were present, even after a 2-week BrdU exposure. Double labeling of mitoses and FMRF-amide revealed a close spatial relationship of mesodermal neoblasts with the nervous system. Immunogold-labeled sections showed that nearly 70% of S-phase cells were in direct contact or within 5 microm from nerve cords.     

N-cadherin localization in early heart development and polar expression of Na+,K(+)-ATPase, and integrin during pericardial coelom formation and epithelialization of the differentiating myocardium.

N-cadherin, a Ca(2+)-dependent cell adhesion molecule, has been localized previously to the mesoderm during chick gastrulation and to adherens junctions in beating avian hearts. However, a systematic study of the dynamic nature of N-cadherin localization in the critical early stages of heart development is lacking. The presented work defines the changes in the spatial and temporal expression of N-cadherin during early stages of chick heart development, principally between Hamburger and Hamilton stages 5-8, 18-29 hr of development. During gastrulation N-cadherin appears evenly distributed in the heart forming region. As development proceeds to form the pericardial coelom (stages 6, 7, and 8, i.e., between 22 and 26 hr of development) N-cadherin localization becomes restricted to the more central areas of the mesoderm. The localization also shows a periodicity that correlates closely with the distance between foci of cavities that eventually coalesce to form the coelom. This distribution suggests that N-cadherin may have a function in the sorting out of somatic and splanchnic mesoderm cells to form the coelom. This separation of the mesoderm in the embryo for the first time physically delineates the precardiac mesoderm population. Concomitant with cell sorting during coelom formation, the precardiac cells change shape and show a distinct polarity as conveyed by (1) the apical expression of N-cadherin on precardiac cell surfaces lining the pericardial coelom, (2) the primarily lateral expression of Na+,K(+)-ATPase, and (3) an enrichment of integrin (beta 1 subunit) on basal cell surfaces. The somatic mesoderm cells apparently down-regulate N-cadherin expression. N-cadherin is also absent from the precardiac cells close to the endoderm. The latter cells eventually form the endocardium, i.e., the endothelial lining of the heart. By contrast, in the tubular, beating heart N-cadherin is found throughout the myocardium. In summary, immunolocalization patterns of N-cadherin during early cardiogenesis suggest that this cell adhesion molecule has a major role in the dynamics of pericardial coelom formation. Subsequently, its continued expression during cell differentiation of the cardiomyocyte to form the myocardium, but not endocardium, suggests N-cadherin is an essential morphoregulatory molecule in heart organogenesis.