The effects of retinoic acid on heart formation in the early chick embryo.

The vitamin A derivative retinoic acid has previously been shown to have teratogenic effects on heart development in mammalian embryos. The craniomedial migration of the precardiac mesoderm during the early stages of heart formation is thought to depend on a gradient of extracellular fibronectin associated with the underlying endoderm. Here, the effects of retinoic acid on migration of the precardiac mesoderm have been investigated in the early chick embryo. When applied to the whole embryo in culture, the retinoid inhibits the craniomedial migration of the precardiac mesoderm resulting in a heart tube that is stunted cranially, while normal or enlarged caudally. Similarly, a local application of retinoic acid to the heart-forming area disrupts the formation of the cardiogenic crescent and the subsequent development of a single mid-line heart tube. This effect is analogous to removing a segment of endoderm and mesoderm across the heart-forming area and results in various degrees of cardia bifida. At higher concentrations of retinoic acid and earlier developmental stages, two completely separate hearts are produced, while at lower concentrations and later stages there are partial bifurcations. The controls, in which the identical operation is carried out except that dimethyl sulphoxide (DMSO) is used instead of the retinoid, are almost all normal. We propose that one of the teratogenic effects of retinoic acid on the heart is to disrupt the interaction between precardiac cells and the extracellular matrix thus inhibiting their directed migration on the endodermal substratum.     

Mesodermal cell migration during Xenopus gastrulation

The adhesive glycoprotein fibronectin (FN), which is a component of the network of extracellular matrix fibrils on the inner surface of the blastocoel roof (BCR), has been proposed to play a major role in directing mesodermal cell migration during amphibian gastrulation. In the first part of this paper, the adhesion of Xenopus mesodermal cells to FN in vitro is examined. Cells from several mesoderm regions, which differ in developmental fate and morphogenetic activity, are able to bind specifically to the RGD cell-binding site of FN. Dorsal mesodermal cell adhesion to FN varies along the anterior-posterior (a-p) axis: adhesion is strongest in the anterior head mesoderm, and gradually decreases posteriorly. This a-p gradient of mesodermal adhesiveness to FN does not change during mesodermal involution, and is reflected in the morphology of mesodermal explants on FN. An a-p strip of mesoderm develops a spreading, leading anterior margin and a compact, retracting posterior end, thus moving slowly and directionally over the FN substrate at about 0.8 micron/min. Although dissociated cells from all levels of the dorsal mesodermal axis adhere to FN, only the anterior, leading prospective head mesoderm cells migrate as single cells on a FN substrate in vitro. Locomotion by means of lamelliform protrusions occurs at an average rate of about 1.5 micron/min. Cells of the more posterior axial mesoderm merely shift position at random without substantial net translocation, and preinvolution mesoderm cells are completely stationary. On the BCR, the in vivo substrate for mesodermal cell migration, dissociated prospective head mesoderm cells spread and migrate as on FN in vitro, at 2.2 microns/min. In the presence of an RGD peptide which inhibits cell-FN interaction, cells remain globular and do not spread. They are still motile, but change direction frequently, which leads to less efficient net translocation. Apparently, interaction with the RGD cell-binding site of FN and concomitant spreading of head mesoderm cells is required for the stabilization of cell locomotion. In contrast to the directional migration of the mesoderm cell population toward the animal pole in the embryo, the pathways of dissociated cells on the BCR are randomly oriented. Coherent explants of migratory mesoderm do not move at all on the BCR, although they translocate on FN in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

A role for fibronectin in the migration of avian precardiac cells. I. Dose-dependent effects of fibronectin antibody

An anterior-posterior concentration difference of fibronectin associated with the endoderm in early chick embryos has been implicated in the directional migration of precardiac mesoderm cells. We have examined the effect of increasing concentrations of an antibody to fibronectin (FN) to test the essentiality of FN to precardiac cell migration. For controls embryos were incubated in the presence of antibodies produced against several other extracellular components, such as laminin and anti-collagen types I and IV, as well as against integrin, a cell surface FN receptor. Embryos were also incubated in the presence of a high concentration of exogenous FN, as well as in the presence of an RGD-containing synthetic pentapeptide that is recognized by the FN receptor. After incubation of chick embryos in various concentrations of anti-FN (5 to 80 micrograms/ml), a dose-dependent effect of anti-fibronectin was observed, whereby heart development was arrested at high concentrations of anti-FN. Early developmental stages were more susceptible to lower antibody concentrations than later stages. Incubation in the presence of the RGD-containing synthetic peptide resulted in partial cardiabifida. None of the antibodies serving as controls affected cell migration or early heart development. These results support the hypothesis that FN is a major component in the migratory pathway and plays a role in the directional migration of precardiac cells to the embryonic midline.     

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.     

The restriction of the heart morphogenetic field in Xenopus laevis

We have examined the spatial restriction of heart-forming potency in Xenopus laevis embryos, using an assay system in which explants or explant recombinates are cultured in hanging drops and scored for the formation of a beating heart. At the end of neurulation at stage 20, the heart morphogenetic field, i.e., the area that is capable of heart formation when cultured in isolation, includes anterior ventral and ventrolateral mesoderm. This area of developmental potency does not extend into more posterior regions. Between postneurula stage 23 and the onset of heart morphogenesis at stage 28, the heart morphogenetic field becomes spatially restricted to the anterior ventral region. The restriction of the heart morphogenetic field during postneurula stages results from a loss of developmental potency in the lateral mesoderm, rather than from ventrally directed morphogenetic movements of the lateral mesoderm. This loss of potency is not due to the inhibition of heart formation by migrating neural crest cells. During postneurula stages, tissue interactions between the lateral mesoderm and the underlying anterior endoderm support the heart-forming potency in the lateral mesoderm. The lateral mesoderm loses the ability to respond to this tissue interaction by stages 27-28. We speculate that either formation of the third pharyngeal pouch during stages 23-27 or lateral inhibition by ventral mesoderm may contribute to the spatial restriction of the heart morphogenetic field.     

The restriction of the heart morphogenetic field in Xenopus laevis

We have examined the spatial restriction of heart-forming potency in Xenopus laevis embryos, using an assay system in which explants or explant recombinates are cultured in hanging drops and scored for the formation of a beating heart. At the end of neurulation at stage 20, the heart morphogenetic field, i.e., the area that is capable of heart formation when cultured in isolation, includes anterior ventral and ventrolateral mesoderm. This area of developmental potency does not extend into more posterior regions. Between postneurula stage 23 and the onset of heart morphogenesis at stage 28, the heart morphogenetic field becomes spatially restricted to the anterior ventral region. The restriction of the heart morphogenetic field during postneurula stages results from a loss of developmental potency in the lateral mesoderm, rather than from ventrally directed morphogenetic movements of the lateral mesoderm. This loss of potency is not due to the inhibition of heart formation by migrating neural crest cells. During postneurula stages, tissue interactions between the lateral mesoderm and the underlying anterior endoderm support the heart-forming potency in the lateral mesoderm. The lateral mesoderm loses the ability to respond to this tissue interaction by stages 27–28. We speculate that either formation of the third pharyngeal pouch during stages 23–27 or lateral inhibition by ventral mesoderm may contribute to the spatial restriction of the heart morphogenetic field.     

Fibronectin visualized by scanning electron microscopy immunocytochemistry on the substratum for cell migration in Xenopus laevis gastrulae

In amphibian gastrulae, scanning electron microscopy (SEM) has shown the presence of a network of extracellular fibrils on the inner aspect of the ectoderm layer, which serves as the substratum for migration by the presumptive mesoderm cells. In vitro experiments have shown that the fibril network promotes attachment and migration by mesoderm cells, and probably guides the migration by contact guidance. Filopodia of the migrating cells showed preferential attachment to the fibrils. Use of a colloidal gold probe for SEM immunocytochemistry has shown that fibrils observed by SEM contain fibronectin, probably as a major component. This provides direct evidence that the extracellular matrix containing fibronectin provides the substratum and guides cell migration in morphogenetic movement.     

Establishment of substratum polarity in the blastocoel roof of the Xenopus embryo

The fibronectin fibril matrix on the blastocoel roof of the Xenopus gastrula contains guidance cues that determine the direction of mesoderm cell migration. The underlying guidance-related polarity of the blastocoel roof is established in the late blastula under the influence of an instructive signal from the vegetal half of the embryo, in particular from the mesoderm. Formation of an oriented substratum depends on functional activin and FGF signaling pathways in the blastocoel roof. Besides being involved in tissue polarization, activin and FGF also affect fibronectin matrix assembly. Activin treatment of the blastocoel roof inhibits fibril formation, whereas FGF modulates the structure of the fibril network. The presence of intact fibronectin fibrils is permissive for directional mesoderm migration on the blastocoel roof extracellular matrix.     

Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo

This fate-mapping study reveals that the progenitors of all major parts of the embryonic gut are already present in endoderm of the early-head-fold to early-somite stage (1-9 somites) mouse embryo. The anterior endoderm contributes primarily to the anterior intestinal portal of the early-organogenesis stage (16-19 somites) embryo. Endoderm cells around and lateral to the node are allocated to the open “midgut” region of the embryonic gut. The posterior (post-nodal) endoderm contributes not only to the posterior intestinal portal but also the open “midgut”. Descendants of the posterior endoderm span a length of the gut from the level of the 3rd-5th somites to the posterior end of the embryonic gut. The formation of the anterior and posterior intestinal portals is accompanied by similar repertoires of morphogenetic tissue movement. We also discovered that cells on contralateral sides of the anterior endoderm are distributed asymmetrically to the dorsal and ventral sides of the anterior intestinal portal, heralding the acquisition of laterality by the embryonic foregut.     

Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos

The development of the embryonic vasculature is examined here using a monoclonal antibody, QH-1, capable of labelling the presumptive endothelial cells of Japanese quail embryos. Antibody labelling is first seen within the embryo proper at the 1-somite stage. Scattered labelling of single cells appears ventral to the somites and at the lateral edges of the anterior intestinal portal. The dorsal aorta soon forms a continuous cord at the ventrolateral edge of the somites and continues into the head to fuse with the ventral aorta forming the first aortic arch by the 6-somite stage. The rudiments of the endocardium fuse at the midline above the anterior intestinal portal by the 3-somite stage and the ventral aorta extends craniad. Intersomitic arteries begin to sprout off of the dorsal aorta at the 7-somite stage. The posterior cardinal vein forms from single cells which segregate from somatic mesoderm at the 7-somite stage to form a loose plexus which moves mediad and wraps around the developing Wolffian duct in later stages. These studies suggest two modes of origin of embryonic blood vessels. The dorsal aortae and cardinal veins apparently arise in situ by the local segregation of presumptive endothelial cells from the mesoderm. The intersomitic arteries, vertebral arteries and cephalic vasculature arise by sprouts from these early vessel rudiments. There also seems to be some cell migration in the morphogenesis of endocardium, ventral aorta and aortic arches. The extent of presumptive endothelial migration in these cases, however, needs to be clarified by microsurgical intervention.     

Motile behavior and protrusive activity of migratory mesoderm cells from the Xenopus gastrula.

During Xenopus gastrulation, the mesoderm migrates across a fibronectin (FN)-containing substrate, the inner surface of the blastocoel roof (BCR). A possible role for FN is to promote the extension of cytoplasmic processes which serve as locomotory organelles for mesoderm cells. To test this idea, the interaction of prospective head mesoderm (HM) cells with FN was examined in vitro. Nonattached HM cells extend filiform processes from an active region of the cell surface. This spontaneous activity is modulated by cell attachment to FN. Additional active regions appear, and cytoplasmic lamellae extend from these sites, leading to cell spreading and translocation. Thus, although FN seems not to induce processes de novo, it modulates a spontaneous protrusive activity to yield the extension of lamellae along the substrate surface. As putative locomotory organelles, HM cell protrusions were characterized functionally. They adhere rapidly and selectively to in situ substrates, preferentially to FN, and retract upon attachment. During translocation, the passive cell body is moved by the activity of the protrusions. Lamellae continuously extend, retract, or split into parts. This leads to an intermittent, nonpersistent mode of translocation. The polarity of HM cells, as expressed in the arrangement of protrusions, bears no constant relationship to the orientation of the cell body, and a cell can change its direction of movement without a corresponding rotation of the cell body. This may be relevant with respect to the mechanism by which mesoderm cells translate guidance cues of the BCR into a polarized, oriented cell structure during directional migration in situ.     

Directional mesoderm cell migration in the Xenopus gastrula.

The movement of the dorsal mesoderm across the blastocoel roof of the Xenopus gastrula is examined. We show that different parts of the mesoderm which can be distinguished by their morphogenetic behavior in the embryo are all able to migrate independently on the inner surface of the blastocoel roof. The direction of mesoderm cell migration is determined by guidance cues in the extracellular matrix of the blastocoel roof and by an intrinsic tissue polarity of the mesoderm. The mesodermal polarity shows the same orientation as the external guidance cues and is strongly expressed in the more posterior mesoderm. The guidance cues of the extracellular matrix are recognized by all parts of the dorsal mesoderm and even by nonmesodermal cells from other regions of the embryo. The extracellular matrix consists of a network of fibronectin-containing fibrils. The adhesiveness of this matrix does not vary along the axis of mesoderm movement, excluding haptotaxis as a guidance mechanism in this system. However, an intact fibronectin fibril structure is necessary for directional mesoderm cell migration. When the assembly of fibronectin into fibrils is inhibited, mesoderm explants still migrate on the amorphous extracellular matrix, but no longer directionally. It is proposed that polarized extracellular matrix fibrils may normally guide the migrating mesoderm to its target region.     

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.     

The effect of 5-bromodeoxyuridine (BrdU) on cardiac muscle differentiation

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.     

The anterior heart-forming field: voyage to the arterial pole of the heart

Studies of vertebrate heart development have identified key genes and signalling molecules involved in the formation of a myocardial tube from paired heart-forming fields in splanchnic mesoderm. The posterior region of the paired heart-forming fields subsequently contributes myocardial precursor cells to the inflow region or venous pole of the heart. Recently, a population of myocardial precursor cells in chick and mouse embryos has been identified in pharyngeal mesoderm anterior to the early heart tube. This anterior heart-forming field gives rise to myocardium of the outflow region or arterial pole of the heart. The amniote heart is therefore derived from two myocardial precursor cell populations, which appear to be regulated by distinct genetic programmes. Discovery of the anterior heart-forming field has important implications for the interpretation of cardiac defects in mouse mutants and for the study of human congenital heart disease.     

Development of cardiac beat rate in early chick embryos is regulated by regional cues

The mesoderm of each of the paired lateral heart-forming regions (HFRs) in the stage 5-7 chick embryo includes prospective conus (pre-C), ventricle (pre-V), and sinoatrial (pre-SA) cells, arranged in a rostrocaudal sequence (C-V-SA). With microsurgery we divided each HFR into three rostrocaudally arranged segments. After 24 hr of further incubation, each segment differentiated into a spontaneously beating vesicle of heart tissue to form a multiheart embryo. The cardiac vesicles in these embryos expressed left-right and rostrocaudal beat rate gradients: the left caudal pre-SA mesoderm produced tissue with the fastest beat rate of the six while the rostral vesicle formed from right pre-C was the slowest. In another operation, we prevented the HFRs from fusing in the midline by cutting through the anterior intestinal portal at stage 8, to produce cardia bifida (CB) embryos with an independently beating half-heart on each side. In these cases, the left half-heart of 87.2% of CB embryos beat faster than the right, confirming the left-right difference in intrinsic beat rate. To assess whether the future beat rate of each region is already determined in the st 5-7 HFR, we exchanged rectangular fragments of left pre-SA mesoderm and attached endoderm with right pre-C fragments to yield a left HFR with the sequence C-V-C and a right HFR with the sequence SA-V-SA. A CB operation was subsequently performed on these exchange embryos to prevent fusion of the lateral HFRs. Preconus mesoderm, transplanted to the pre-SA region, differentiated into tissue with a rapid beat rate, while pre-SA mesoderm relocated to the preconus region formed heart tissue with a slow spontaneous rate typical of the conus. In 73% of the exchange CB embryos, the left half-heart beat faster than the right, despite the origins of its mesoderm. The exchanged mesoderm developed a rate that was appropriate for its new location rather than the site of origin of the mesodermal fragment. In a third set of operations, we implanted a fragment of st 15 differentiated conus tissue into a site lateral to the left caudal HFR in st 5, 6, and 7 embryos, and subsequently performed CB operations on them. The implant caused the adjacent half-heart to develop with a slower beat rate than in unoperated or sham-operated controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immuno-electron-microscopic study of fibronectin in gastrulating amphibian embryos

Summary The ultrastructural organization of fibronectin (FN) in early amphibian embryos (Ambystoma mexicanum, Pleurodeles waltlii) was studied with the use of antibodies directed against amphibian plasmatic FN. Scanning and transmission electron microscopy combined with immunogold labeling of FN revealed that the extracellular matrix that covers the inner surface of the ectodermal layer consists of FN-containing fibrils. During gastrulation, the mesodermal cells appear to be devoid of FN. These cells extend filopodia adhering to the FN-containing fibrils and are spreading along them. These findings suggest that FN may be involved in contact formation between mesodermal cells and the extracellular matrix that serves as a substratum for migration.     

PDGF signalling controls the migration of mesoderm cells during chick gastrulation by regulating N-cadherin expression

In the early chick embryo, Pdgfa is expressed in the epiblast, outlining the migration route that mesoderm cells expressing the receptor, Pdgfralpha, follow to form somites. Both expression of a dominant-negative PDGFRalpha and depletion of endogenous PDGFRalpha ligands through injection of PDGFRalpha-Fc fragments, inhibit the migration of mesoderm cells after their ingression through the primitive streak. siRNA-mediated downregulation of Pdgfa expression in the epiblast on one side of the streak strongly blocks the migration of mesoderm cells into that side. Beads soaked in PDGFA elicit a directional attractive movement response in mesoderm cells, showing that PDGFA can provide directional information. Surprisingly, however, PDGF signalling is also required for directional movement towards other attractants, such as FGF4. PDGF signalling controls N-cadherin expression on mesoderm cells, which is required for efficient migration. PDGF signalling activates the PI3 kinase signalling pathway in vivo and activation of this pathway is required for proper N-cadherin expression.     

The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)