Heart development before beating

During heart development at the pregastrula stage, prospective heart cells reside in the posterior lateral region of the epiblast layer. Interaction of tissues between the posterior epiblast and hypoblast is necessary to generate the future heart mesoderm. Signaling regulating the interaction involves fibroblast growth factor (FGF)-8, Nodal, bone morphogenetic protein (BMP)-antagonist, and canonical Wnt and acts on the posterior epiblast to induce the expression of genes specific for the anterior lateral mesoderm. At the early gastrula stage, prospective heart cells accumulate at the posterior midline and migrate to the anterior region of the primitive streak. During gastrulation, future heart cells leave the primitive streak and migrate anterolaterally to form the left and right anterior lateral plate mesoderm including the precardiac mesoderm. At this stage, prospective heart cells receive endoderm-derived signals, including BMP, FGF, and Wnt-antagonist, and thereby become committed to the heart lineage. At the neurula stage, the left and right precardiac mesoderm move to the ventral midline and fuse, resulting in the formation of a single primitive heart tube. Therefore, a two-step signaling cascade, which includes tissue interaction between epiblast and hypoblast at the blastula stage and endoderm-derived signals during gastrulation, is required to generate a beating heart.

Wnt antagonism initiates cardiogenesis in Xenopus laevis

Heart induction in Xenopus occurs in paired regions of the dorsoanterior mesoderm in response to signals from the Spemann organizer and underlying dorsoanterior endoderm. These tissues together are sufficient to induce heart formation in noncardiogenic ventral marginal zone mesoderm. Similarly, in avians the underlying definitive endoderm induces cardiogenesis in precardiac mesoderm. Heart-inducing factors in amphibians are not known, and although certain BMPs and FGFs can mimic aspects of cardiogenesis in avians, neither can induce the full range of activities elicited by the inducing tissues. Here we report that the Wnt antagonists Dkk-1 and Crescent can induce heart formation in explants of ventral marginal zone mesoderm. Other Wnt antagonists, including the frizzled domain-containing proteins Frzb and Szl, lacked this activity. Unlike Wnt antagonism, inhibition of BMP signaling did not promote cardiogenesis. Ectopic expression of GSK3beta, which inhibits beta-catenin-mediated Wnt signaling, also induced cardiogenesis in ventral mesoderm. Analysis of Wnt proteins expressed during gastrulation revealed that Wnt3A and Wnt8, but not Wnt5A or Wnt11, inhibited endogenous heart induction. These results indicate that diffusion of Dkk-1 and Crescent from the organizer initiate cardiogenesis in adjacent mesoderm by establishing a zone of low Wnt3A and Wnt8 activity.     

Early temporal-specific responses and differential sensitivity to lithium and Wnt-3A exposure during heart development.

Members of both Wnt and bone morphogenetic protein (BMP) families of signaling molecules are important in heart development. We previously demonstrated that beta-catenin, a key downstream intermediary of the canonical Wnt signaling pathway, delineates the dorsal boundary of the cardiac compartments in an anteroposterior progression. We hypothesized the progression involves canonical Wnt signaling and reflects development of the primary body axis of the embryo. A similar anteroposterior signaling wave leading to cardiac cell specification involves inductive signaling by BMP-2 synthesized by the underlying endoderm in anterior bilateral regions. Any molecule that disrupts the normal balance of Wnt and BMP concentrations within the heart field may be expected to affect early heart development. The canonical Wnt signaling step mimicked by lithium involves inactivation of glycogen synthase kinase-3beta (GSK-3beta; Klein and Melton [1996] Proc. Natl. Acad. Sci. U. S. A. 93:8455-8459). We show that lithium, Wnt-3A, and an inhibitor of GSK-3beta, SB415286, affect early heart development at the cardiac specification stages. We demonstrate that normal expression patterns of key signaling molecules as Notch-1 and Dkk-1 are altered in the anterior mesoderm within the heart fields by a one-time exposure to lithium, or by noggin inhibition of BMP, at Hamburger and Hamilton (HH) stage 3 during chick embryonic development. The severity of developmental defects is greatest with exposure to lithium or Wnt-3A at HH stage 3 and decreases at HH stage 4. Taken together, our results demonstrate that there are temporal-specific responses and differential sensitivities to lithium/Wnt-3A exposure during early heart development.     

Anterior expression of the caudal homologue cCdx-B activates a posterior genetic program in avian embryos.

Several families of regulatory genes have been implicated in anteroposterior patterning of gastrulation-stage vertebrate embryos. Members of the Drosophila caudal family of homeobox genes (Cdx) are among the earliest regulators of posterior cell fates. The regulatory cascade initiated by the caudal homologue, cCdx-B, was examined in avian embryos. During gastrulation, cCdx-B is expressed with other posterior patterning genes. In the posterior primitive streak, cCdx-B expression coincides with posteriorly expressed Hox cluster genes and Wnt family members such as Wnt-8c. The hierarchical relationship between these patterning genes was examined after anterior ectopic expression of cCdx-B. cCdx-B expression in anterior cardiogenic cells by means of adenoviral infection leads to the induction of Wnt-8c and the posterior Hox genes, Hoxa-7, Hoxc-6, and Hoxc-8. Cardiogenesis is not inhibited in cCdx-B expressing anterior lateral mesoderm, indicating that anterior cell fates are not respecified with the activation of posterior patterning genes after gastrulation. These results support an important role for cCdx-B in initiating a posterior program of gene expression that includes Wnt signaling molecules and the Hox cluster genes.     

The establishment of a somitomeric pattern in the mesoderm of the gastrulating mouse embryo

Mesoderm formation in the mouse embryo begins at 6.5–6.75 days p.c. (postcoitum) when a primitive streak is formed along the posterior side of the egg cylinder. Epiblast cells in a localized region separate from one another and spread laterally between the primitive endoderm and the rest of the epiblast. The newly formed mesoderm contributes to both embryonic and extraembryonic regions. When the endoderm is removed, a definitive somitomeric pattern is first observed in the lateral wings of mesoderm of the mid-primitive-streak-stage embryo. The sequential appearance and the placement of somitomeres in the gastrulating mouse embryo are closely related to the general changes in physical dimensions and to the pattern of tissue growth which occur during the maturation of the egg cylinder. By the late-primitive-streak stage, about four somitomeres are present in the paraxial mesoderm on either side of the embryonic axis. These somitomeres will undergo morphogenesis and give rise to the cranial segments and head mesenchyme of neurulating embryos (Meier and Tam, 1982). The midline or axial mesoderm, consisting of prechordal plate and notochord, is derived from the head process mesoderm originating from the anterior end of the primitive streak. Cells of the head process are compact and adherent to the endoderm. The early presence of a somitomeric pattern which persists and is added to throughout subsequent phases of mesoderm formation suggests that spreading mesodermal cells have relatively stable neighbor relationships. This morphological evidence supports the idea that the expansion of the mesoderm during gastrulation results from tissue growth and progressive deposition of cells from the primitive streak. Cell migration may be limited principally to nonsomitomeric mesodermal cells found in the leading edge of the spreading lateral wings.     

Cell fate decisions within the mouse organizer are governed by graded Nodal signals

It is well known that cell fate decisions in the mouse organizer region during gastrulation ultimately govern gut formation and patterning, left-right axis determination, and development of the central nervous system. Previous studies suggest that signaling pathways activated by Nodal, bone morphogenetic protein (BMP), and Wnt ligands coordinately regulate patterning of the streak and the formation of midline organizing tissues, but the specific contributions of these molecules within discrete cell lineages are poorly defined. Here we removed Smad2 activity in the epiblast, using a conditional inactivation strategy. Abrogation of Smad2 does not compromise primitive streak (PS) formation or gastrulation movements, but rather results in a failure to correctly specify the anterior definitive endoderm (ADE) and prechordal plate (PCP) progenitors. To selectively lower Nodal activity in the posterior epiblast, we generated a novel allele lacking the proximal epiblast enhancer (PEE) governing Nodal expression in the PS. As for conditional inactivation of Smad2, germ-line deletion of the PEE selectively disrupts development of the anterior streak. In striking contrast, the node and its midline derivatives, the notochord and floor plate, develop normally in both categories of mutant embryos. Finally, we show that removal of one copy of Smad3 in the context of a Smad2-deficient epiblast results in a failure to specify all axial midline tissues. These findings conclusively demonstrate that graded Nodal/Smad2 signals govern allocation of the axial mesendoderm precursors that selectively give rise to the ADE and PCP mesoderm.     

The rostro-caudal position of cardiac myocytes affect their fate.

During chick embryogenesis, cells destined to form cardiac myocytes are located within the primitive streak at stage 3 in the same relative anterior-posterior distribution as in the prelooped heart. The most rostral cells contribute to the extreme anterior pole of the heart, the bulbus cordis, and the most caudal to the extreme posterior end, the sinoatrial region. After gastrulation, these cells commit to the myocyte lineage and, retaining their relative positions, migrate to the anterior lateral plate. From stages 5 to 10 they diversify into atrial and ventricular myocytes, with the former located posteriorly and the latter, anteriorly. To determine the effect of a change in the rostro-caudal position of these cells on their diversification, anterior lateral plate mesoderm and the underlying endoderm were cut and rotated 180 degrees along the longitudinal axis, at stages 4-8. The subsequent diversification of these precursor cells into atrial and ventricular myocytes was examined using lineage-specific markers. Our results showed that altering location along the longitudinal axis through stage 6 changed the normal fate of a precursor cell. The orientation of the overlying ectoderm did not alter normal morphogenesis or determination of fate.     

Chicken dapper genes are versatile markers for mesodermal tissues,embryonic muscle stem cells,neural crest cells,and neurogenic placodes

Dapper (Dpr) proteins are context‐dependent regulators of Wnt and Tgfβ signaling. However, although inroads into their molecular properties have been made, their expression and biological function are not understood. Searching for avian Dpr genes, we found that the chicken harbors a Dpr1 and a Dpr2 paralogue only. The genes are expressed in distinct patterns at gastrulation, neurulation, and organogenesis stages of development with key expression domains being the posterior primitive streak, anterior node and notochord, presomitic mesoderm (segmental plate), lateral and cardiac mesoderm, limb mesenchyme, and neurogenic placodes for Dpr1, and anterior primitive streak, node, epithelial somites, embryonic muscle stem cells, oral ectoderm and endoderm, neural crest cells, limb ectoderm, and lung buds for Dpr2. Expression overlaps in a few tissues; however, in several tissues, expression is complementary. Developmental Dynamics 238:1166–1178, 2009. © 2009 Wiley‐Liss, Inc.     

Wnt signals from the neural tube block ectopic cardiogenesis

It has long been observed that repressive signals from the neural tube block cardiogenesis in vertebrates. Here we show that a signal from the neural tube that blocks cardiogenesis in the adjacent anterior paraxial mesoderm of stage 8-9 chick embryos can be mimicked by ectopic expression of either Wnt-3a or Wnt-1, both of which are expressed in the dorsal neural tube. Repression of cardiogenesis by the neural tube can be overcome by ectopic expression of a secreted Wnt antagonist. On the basis of both in vitro and in vivo results, we propose that Wnt signals from the neural tube normally act to block cardiogenesis in the adjacent anterior paraxial mesendoderm.     

A direct role for Wnt8 in ventrolateral mesoderm patterning

Vertebrate dorsoventral patterning requires both Wnt8 and BMP signaling. Because of their multiple interactions, discerning roles attributable specifically to Wnt8 independent of BMP has been a challenge. For example, Wnt8 represses the dorsal organizer that negatively regulates ventral BMP signals, thus Wnt8 loss‐of‐function phenotypes may reflect the combined effects of reduced Wnt8 and BMP signaling. We have taken a loss‐of‐function approach in the zebrafish to generate embryos lacking expression of both Wnt8 and the BMP antagonist Chordin. wnt8;chordin loss‐of‐function embryos show rescued BMP signaling, thereby allowing us to identify Wnt8‐specific requirements. Our analysis shows that Wnt8 is uniquely required to repress prechordal plate specification but not notochord, and that Wnt8 signaling is not essential for specification of tailbud progenitors but is required for normal expansion of posterior mesoderm cell populations. Thus, Wnt8 and BMP signaling have independent roles during vertebrate ventrolateral mesoderm development that can be identified through loss‐of‐function analysis. Developmental Dynamics 239:2828–2836, 2010. © 2010 Wiley‐Liss, Inc.     

Early endocardial formation originates from precardiac mesoderm as revealed by QH-1 antibody staining

The formation of endocardial endothelium in quail embryos was investigated using in vivo and in vitro systems. At stage 7+ (2 somite), the initial emergence of endothelial cells within the bilateral heart forming region (HFR) was detected in quail embryos by immunohistochemistry with QH-1 (an anti-quail endothelial cell marker) and confocal microscopy. We consistently observed more QH-1 positive cells in the right HFR than the left. At stage 8 (4 somite), the HFR, including QH-1 positive cells, were located in the splanchnic mesoderm after formation of the coelom. During stage 8, the HFR migrated along the margin of anterior intestinal portal in association with the endoderm. By stage 8+ (5 somite), the two HFR had fused at the midline and formed a plexus of QH-1 positive endothelial precursor cells. The definitive endocardium developed as a single, hollow, tube within this plexus. Posteriorly, QH-1 positive cells of the HFR established vascular-like connections with QH-1 positive cells that had formed outside (peripheral to) the HFR. During migration and subsequent determination, the precardiac mesoderm is continuously associated with the basement membrane of the anterior endoderm. To determine the role of endoderm on endocardial endothelial cell formation and development, precardiac mesoderm from stage 5 embryos, which does not express QH-1 antigen, was explanted onto the surface of collagen gels. When co-cultured with endoderm, the outgrowth of free cells from the mesoderm was much more extensive, many of which invaded the gel and expressed the QH-1 antigen; mesoderm cultured without endoderm did not seed nor express QH-1 antigen. These findings suggest that the segregation of endothelial and myocardial lineages may occur by an endoderm-mediated, mesenchymal formation.     

Sheep heart RNA stimulates myofibril formation and beating in cardiac mutant axolotl hearts in organ culture

In the Mexican axolotl, Ambystoma mexicanum, recessive mutant gene c, when homozygous, results in a failure of the heart to form sarcomeric myofibrils and contract normally. Previous studies have shown that purified RNA from normal anterior endoderm or from medium conditioned with anterior endoderm/pre-cardiac mesoderm has the capacity to rescue mutant hearts in organ culture. In the present study, RNA extracted from adult sheep heart was tested for its capacity to promote differentiation in the mutant axolotl hearts. Mutant hearts cultured in the presence of the sheep heart RNA in Steinberg's solution for 48 h displayed rhythmic contractions. Ultrastructural studies showed that the rescued mutant axolotl ventricular myocardial cells contained myofibrils of normal morphology. Mutant hearts cultured in Steinberg's solution alone did not beat throughout their lengths and myofibrils were not observable in the ventricles. Confocal microscopy confirmed the increase of Tropomyosin expression and formation of myofibrils in mutant hearts treated by sheep heart RNA. Thus, sheep heart RNA promotes myofibrillogenesis and the development of contractile function in embryonic cardiac mutant axolotl hearts.     

Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development

In vertebrate embryos, formation of anterior neural structures requires suppression of Wnt signals emanating from the paraxial mesoderm and midbrain territory. In Six3(-/-) mice, the prosencephalon was severely truncated, and the expression of Wnt1 was rostrally expanded, a finding that indicates that the mutant head was posteriorized. Ectopic expression of Six3 in chick and fish embryos, together with the use of in vivo and in vitro DNA-binding assays, allowed us to determine that Six3 is a direct negative regulator of Wnt1 expression. These results, together with those of phenotypic rescue of headless/tcf3 zebrafish mutants by mouse Six3, demonstrate that regionalization of the vertebrate forebrain involves repression of Wnt1 expression by Six3 within the anterior neuroectoderm. Furthermore, these results support the hypothesis that a Wnt signal gradient specifies posterior fates in the anterior neural plate.     

Antagonists of Wnt and BMP signaling promote the formation of vertebrate head muscle

Recent studies have postulated that distinct regulatory cascades control myogenic differentiation in the head and the trunk. However, although the tissues and signaling molecules that induce skeletal myogenesis in the trunk have been identified, the source of the signals that trigger skeletal muscle formation in the head remain obscure. Here we show that although myogenesis in the trunk paraxial mesoderm is induced by Wnt signals from the dorsal neural tube, myogenesis in the cranial paraxial mesoderm is blocked by these same signals. In addition, BMP family members that are expressed in both the dorsal neural tube and surface ectoderm are also potent inhibitors of myogenesis in the cranial paraxial mesoderm. We provide evidence suggesting that skeletal myogenesis in the head is induced by the BMP inhibitors, Noggin and Gremlin, and the Wnt inhibitor, Frzb. These molecules are secreted by both cranial neural crest cells and by other tissues surrounding the cranial muscle anlagen. Our findings demonstrate that head muscle formation is locally repressed by Wnt and BMP signals and induced by antagonists of these signaling pathways secreted by adjacent tissues.     

Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation

During early mouse development, the anterior visceral endoderm (AVE) secretes inhibitor and activator signals that are essential for establishing the anterior–posterior (AP) axis of the embryo and for restricting mesoderm formation to the posterior epiblast in the primitive streak (PS) region. Here we show that AVE cells have an additional morphogenetic function. These cells express the transmembrane protein FLRT3. Genetic ablation of FLRT3 did not affect the signaling functions of the AVE according to the normal expression pattern of Nodal and Wnt and the establishment of a proper AP patterning in the epiblast. However, FLRT3^−/− embryos showed a highly disorganized basement membrane (BM) in the AVE region. Subsequently, adjacent anterior epiblast cells displayed an epithelial-to-mesenchymal transition (EMT)-like process characterized by the loss of cell polarity, cell ingression, and the up-regulation of the EMT and the mesodermal marker genes Eomes, Brachyury/T, and FGF8. These results suggest that the AVE acts as a morphogenetic boundary to prevent EMT and mesoderm induction in the anterior epiblast by maintaining the integrity of the BM. We propose that this novel function cooperates with the signaling activities of the AVE to restrict EMT and mesoderm induction to the posterior epiblast.