Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart

Endogenous patterns of retinoic acid (RA) signaling in avian cardiac morphogenesis were characterized by localized expression of a key RA-synthetic enzyme, RALDH2, which displayed a biphasic pattern during heart development. RALDH2 immunoreactivity was initially apparent posterior to Hensen's node of stage 5-6 embryos and subsequently in somites and unsegmented paraxial and lateral plate mesoderm overlapping atrial precursors in the cardiogenic plate of stage 9- embryos. Initial RALDH2 synthesis in the posterior myocardium coincided with activation of the AMHC1 gene, a RA-responsive marker of inflow heart segments. A wave of RALDH2 synthesis then swept the myocardium in a posterior-to-anterior direction, reaching the outflow tract by stage 13, then fading from the myocardial layer. The second phase of RALDH2 expression, initiated at stage 18 in the proepicardial organ, persisted in migratory epicardial cells that completely enveloped the heart by stage 24. Early restriction of RALDH2 expression to the posterior cardiogenic plate, overlapping RA-inducible gene activation, provides evidence for commitment of posterior avian heart segments by localized production of RA, whereas subsequent RALDH2 expression exclusively in the migratory epicardium suggests a role for the morphogen in ventricular expansion and morphogenesis of underlying myocardial tissues.     

Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation

The developmental fate of cells in the epiblast of early-primitive-streak-stage mouse embryos was assessed by studying the pattern of tissue colonisation displayed by lac Z-expressing cells grafted orthotopically to nontransgenic embryos. Results of these fate-mapping experiments revealed that the lateral and posterior epiblast contain cells that will give rise predominantly to mesodermal derivatives. The various mesodermal populations are distributed in overlapping domains in the lateral and posterior epiblast, with the embryonic mesoderm such as heart, lateral, and paraxial mesoderm occupying a more distal position than the extraembryonic mesoderm. Heterotopic grafting of presumptive mesodermal cells results in the grafted cells adopting the fate appropriate to the new site, reflecting a plasticity of cell fate determination before ingression. The first wave of epiblast cells that ingress through the primitive streak are those giving rise to extraembryonic mesoderm. Cells that will form the mesoderm of the yolk sac and the amnion make up a major part of the mesodermal layer of the midprimitive-streak-stage embryo. Cells that are destined for embryonic mesoderm are still found within the epiblast, but some have been recruited to the distal portion of the mesoderm. By the late-primitive-streak-stage, the mesodermal layer contains only the precursors of embryonic mesoderm. This suggests that there has been a progressive displacement of the midstreak mesoderm to extraembryonic sites, which is reminiscent of that occurring in the overlying endodermal tissue. The regionalisation of cell fate in the late-primitive-streak mesoderm bears the same spatial relationship as their ancestors in the epiblast prior to cell ingression. This implies that both the position of the cells in the proximal-distal axis and their proximity to the primitive streak are major determinants for the patterning of the embryonic mesoderm. © 1995 Wiley-Liss, Inc.     

Retinoic acid signalling is required for specification of pronephric cell fate

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.     

Retinoid signaling and cardiac anteroposterior segmentation

Summary: Establishment of anterior–posterior polarity is one of the earliest decisions in cardiogenesis. Specification of anterior (outflow) and posterior (inflow) structures ensures proper connections between venous system and inflow tract and between arterial tree and outflow tract. The last few years have witnessed remarkable progress in our understanding of cardiac anteroposterior patterning. Molecular cloning and subsequent studies on RALDH2, the key embryonic retinaldehyde dehydrogenase in retinoic acid (RA) synthesis, provided the missing link between teratogenic studies on RA deficiency and excess and normal chamber morphogenesis. We discuss work establishing the foundations of our current understanding of the mechanisms of cardiac anteroposterior segmentation, the reasons why early evidence pointing to the role of RA in anteroposterior segmentation was overlooked, and the key experiments unraveling the role of RA in cardiac anteroposterior segmentation. We have also integrated recent experiments in a model of cardiac anteroposterior patterning in which RALDH2 expression determines anteroposterior boundaries in the heart field. genesis 31:97–104, 2001. © 2001 Wiley‐Liss, Inc.     

FGF8/17/18 functions together with FGF9/16/20 during formation of the notochord in Ciona embryos

Fibroblast growth factor (FGF) signalling has been implicated in the generation of mesoderm and neural fates in chordate embryos including ascidians and vertebrates. In Ciona, FGF9/16/20 has been implicated in both of these processes. However, in FGF9/16/20 knockdown embryos, notochord fate recovers during later development. It is thus not clear if FGF signalling is an essential requirement for notochord specification in Ciona embryos. We show that FGF-MEK-ERK signals act during two distinct phases to establish notochord fate. During the first phase, FGF signalling is required during an asymmetric cell division to promote notochord at the expense of neural identity. Consistently, ERK1/2 is specifically activated in the notochord precursors following this cell division. Sustained activation of ERK1/2 is then required to maintain notochord fate. We demonstrate that FGF9/16/20 acts solely during the initial induction step and that, subsequently, FGF8/17/18 together with FGF9/16/20 is involved in the following maintenance step. These results together with others' show that the formation of a large part of the mesoderm cell types in ascidian larvae is dependent on signalling events involving FGF ligands.     

A retinoic acid synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse

Most retinoic acid (RA) in the embryonic mouse is generated by three retinaldehyde dehydrogenases (RALDHs). RALDH1 (also called E1, AHD2 or ALDH1) is expressed in the dorsal retina, and RALDH2 (V2, ALDH11) generates most RA in the embryonic trunk. The third one, RALDH3 (V1), synthesizes the bulk of RA in the head of the early embryo. We show here that RALDH3 is a mouse homologue to ALDH6, an aldehyde dehydrogenase cloned from adult human salivary gland (Hsu, L.C., Chang, W.-C., Hiraoka, L., Hsien, C.-L., 1994. Molecular cloning, genomic organization, and chromosomal localization of an additional human aldehyde dehydrogenase gene, ALDH6. Genomics 24, 333-341), which was recently reported to act as a RALDH (Yoshida, A., Rzhetsky, A., Hsu, L.C., Chang, C., 1998. Human aldehyde dehydrogenase gene family. Eur. J. Biochem. 251, 549-557). RALDH3 expression begins in the surface ectoderm over the optic recess. In rapidly changing expression patterns it labels the appearance of several ectodermal structures: it marks the formation of the lens and the olfactory organ from ectodermal placodes, and it delineates the beginning eyelid field. Within the optic vesicle, RALDH3 is expressed in the ventral retina and the dorsal pigment epithelium. In the telencephalon, RALDH3 is expressed at high levels in the lateral part of the ganglionic eminence. From here it extends via the piriform cortex into the lower part of the septum. Of the three RALDHs, RALDH3 shows the strongest predilection for epithelia.     

Distinct modes of floor plate induction in the chick embryo

To begin to reconcile models of floor plate formation in the vertebrate neural tube, we have performed experiments aimed at understanding the development of the early floor plate in the chick embryo. Using real-time analyses of cell behaviour, we provide evidence that the principal contributor to the early neural midline, the future anterior floor plate, exists as a separate population of floor plate precursor cells in the epiblast of the gastrula stage embryo, and does not share a lineage with axial mesoderm. Analysis of the tissue interactions associated with differentiation of these cells to a floor plate fate reveals a role for the nascent prechordal mesoderm, indicating that more than one inductive event is associated with floor plate formation along the length of the neuraxis. We show that Nr1, a chick nodal homologue, is expressed in the nascent prechordal mesoderm and we provide evidence that Nodal signalling can cooperate with Shh to induce the epiblast precursors to a floor-plate fate. These results indicate that a shared lineage with axial mesoderm cells is not a pre-requisite for floor plate differentiation and suggest parallels between the development of the floor plate in amniote and anamniote embryos.     

Dynamic control of head mesoderm patterning

The embryonic head mesoderm gives rise to cranial muscle and contributes to the skull and heart. Prior to differentiation, the tissue is regionalised by the means of molecular markers. We show that this pattern is established in three discrete phases, all depending on extrinsic cues. Assaying for direct and first-wave indirect responses, we found that the process is controlled by dynamic combinatorial as well as antagonistic action of retinoic acid (RA), Bmp and Fgf signalling. In phase 1, the initial anteroposterior (a-p) subdivision of the head mesoderm is laid down in response to falling RA levels and activation of Fgf signalling. In phase 2, Bmp and Fgf signalling reinforce the a-p boundary and refine anterior marker gene expression. In phase 3, spreading Fgf signalling drives the a-p expansion of MyoR and Tbx1 expression along the pharynx, with RA limiting the expansion of MyoR. This establishes the mature head mesoderm pattern with markers distinguishing between the prospective extra-ocular and jaw skeletal muscles, the branchiomeric muscles and the cells for the outflow tract of the heart.     

Retinoic acid activates myogenesis in vivo through Fgf8 signalling

Retinoic acid (RA) has been shown to regulate muscle differentiation in vitro. Here, we have investigated the role of RA signalling during embryonic myogenesis in zebrafish. We have altered RA signalling from gastrulation stages onwards by either inhibiting endogenous RA synthesis using an inhibitor of retinaldehyde dehydrogenases (DEAB) or by addition of exogenous RA. DEAB reduces expression of the myogenic markers myoD and myogenin in somites, whereas RA induces increased expression of these genes and strongly induces premature myoD expression in the presomitic mesoderm (psm). The expression dynamics of myf5 in presomitic and somitic mesoderm suggest that RA promotes muscle differentiation, a role supported by the fact that RA activates expression of fast myosin, while DEAB represses it. We identify Fgf8 as a major relay factor in RA-mediated activation of myogenesis. We show that fgf8 expression in somites and anterior psm is regulated by RA, and find that in the absence of Fgf8 signalling in the acerebellar mutant RA fails to promote myoD expression. We propose that, in the developing embryo, localised synthesis of RA by Raldh2 in the anterior psm and in somites activates fgf8 expression which in turn induces the expression of myogenic genes and fast muscle differentiation.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

Smarcd3b and Gata5 promote a cardiac progenitor fate in the zebrafish embryo

Development of the heart requires recruitment of cardiovascular progenitor cells (CPCs) to the future heart-forming region. CPCs are the building blocks of the heart, and have the potential to form all the major cardiac lineages. However, little is known regarding what regulates CPC fate and behavior. Activity of GATA4, SMARCD3 and TBX5 - the `cardiac BAF' (cBAF) complex, can promote myocardial differentiation in embryonic mouse mesoderm. Here, we exploit the advantages of the zebrafish embryo to gain mechanistic understanding of cBAF activity. Overexpression of smarcd3b and gata5 in zebrafish results in an enlarged heart, whereas combinatorial loss of cBAF components inhibits cardiac differentiation. In transplantation experiments, cBAF acts cell autonomously to promote cardiac fate. Remarkably, cells overexpressing cBAF migrate to the developing heart and differentiate as cardiomyocytes, endocardium and smooth muscle. This is observed even in host embryos that lack endoderm or cardiac mesoderm. Our results reveal an evolutionarily conserved role for cBAF activity in cardiac differentiation. Importantly, they demonstrate that Smarcd3b and Gata5 can induce a primitive, CPC-like state.     

Retinoic acid signaling is required for a critical early step in zebrafish pancreatic development

The mechanisms that subdivide the endoderm into the discrete primordia that give rise to organs such as the pancreas and liver are not well understood. However, it is known that retinoic acid (RA) signaling is critical for regionalization of the vertebrate embryo: when RA signaling is either prevented or augmented, anteroposterior (AP) patterning of the CNS and mesoderm is altered and major developmental defects occur. We have investigated the role of RA signaling in regionalization of the zebrafish endoderm. Using a mutant that prevents RA synthesis and an antagonist of the RA receptors, we show that specification of both the pancreas and liver requires RA signaling. By contrast, RA signaling is not required for the formation of the endodermal germ layer or for differentiation of other endodermal organs. Timed antagonist and RA treatments show that the RA-dependent step in pancreatic specification occurs at the end of gastrulation, significantly earlier than the expression of known markers of pancreatic progenitors. In addition to being required for pancreatic specification, RA has the capacity to transfate anterior endoderm to a pancreatic fate.     

FGF signalling controls the timing of Pax6 activation in the neural tube

We have recently demonstrated that Pax6 activation occurs in phase with somitogenesis in the spinal cord. Here we show that the presomitic mesoderm exerts an inhibitory activity on Pax6 expression. This repressive effect is mediated by the FGF signalling pathway. The presomitic mesoderm displays a decreasing caudorostral gradient of FGF8, and grafting FGF8-soaked beads at the level of the neural tube abolishes Pax6 activation. Conversely, when FGF signalling is disrupted, Pax6 is prematurely activated in the neural plate. We propose that the progression of Pax6 activation in the neural tube is controlled by the caudal regression of the anterior limit of FGF activity. Hence, as part of its posteriorising activity, FGF8 downregulation acts as a switch from early (posterior) to a later (anterior) state of neural epithelial development.     

The differential response to Fgf signalling in cells internalized at different times influences lineage segregation in preimplantation mouse embryos

Lineage specification in the preimplantation mouse embryo is a regulative process. Thus, it has been difficult to ascertain whether segregation of the inner-cell-mass (ICM) into precursors of the pluripotent epiblast (EPI) and the differentiating primitive endoderm (PE) is random or influenced by developmental history. Here, our results lead to a unifying model for cell fate specification in which the time of internalization and the relative contribution of ICM cells generated by two waves of asymmetric divisions influence cell fate. We show that cells generated in the second wave express higher levels of Fgfr2 than those generated in the first, leading to ICM cells with varying Fgfr2 expression. To test whether such heterogeneity is enough to bias cell fate, we upregulate Fgfr2 and show it directs cells towards PE. Our results suggest that the strength of this bias is influenced by the number of cells generated in the first wave and, mostly likely, by the level of Fgf signalling in the ICM. Differences in the developmental potential of eight-cell- and 16-cell-stage outside blastomeres placed in the inside of chimaeric embryos further support this conclusion. These results unite previous findings demonstrating the importance of developmental history and Fgf signalling in determining cell fate.