Inhibition of Wnt activity induces heart formation from posterior mesoderm

In the chick, heart mesoderm is induced by signals from the anterior endoderm. Although BMP-2 is expressed in the anterior endoderm, BMP activity is necessary but not sufficient for heart formation. Previous work from our lab has suggested that one or more additional factors from anterior endoderm are required. Crescent is a Frizzled-related protein that inhibits Wnt-8c and is expressed in anterior endoderm during gastrulation. At the same stages, expression of Wnt-3a and Wnt-8c is restricted to the primitive streak and posterior lateral plate, and is absent from the anterior region where crescent is expressed. Posterior lateral plate mesoderm normally forms blood, but coculture of this tissue with anterior endoderm or infection with RCAS-crescent induces formation of beating heart muscle and represses formation of blood. Dkk-1, a Wnt inhibitor of a different protein family, similarly induces heart-specific gene expression in posterior lateral plate mesoderm. Furthermore, we have found that ectopic Wnt signals can repress heart formation from anterior mesoderm in vitro and in vivo and that forced expression of either Wnt-3a or Wnt-8c can promote development of primitive erythrocytes from the precardiac region. We conclude that inhibition of Wnt signaling promotes heart formation in the anterior lateral mesoderm, whereas active Wnt signaling in the posterior lateral mesoderm promotes blood development. We propose a model in which two orthogonal gradients, one of Wnt activity along the anterior-posterior axis and the other of BMP signals along the dorsal-ventral axis, intersect in the heart-forming region to induce cardiogenesis in a region of high BMP and low Wnt activity.     

Testin interacts with vangl2 genetically to regulate inner ear sensory cell orientation and the normal development of the female reproductive tract in mice

Results: We identified Testin as a Vangl2‐interacting protein through a 2‐hybrid screen with a cochlea cDNA library. Testin is enriched to cell–cell boundaries in the presence of Vangl2 in cultured cells. Genetic inactivation of Testin leads to abnormal hair cell orientation in the vestibule and cellular patterning defects in the cochlea. In addition, Testin genetically interacts with Vangl2 to regulate hair cell orientation in the cochlea and the opening of the vaginal tract. 相似文献   

Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo

Fgf8 and Fgf4 encode FGF family members that are coexpressed in the primitive streak of the gastrulating mouse embryo. We have analyzed the phenotype of Fgf8(-/-) embryos and discovered that they fail to express Fgf4 in the streak. In the absence of both FGF8 and FGF4, epiblast cells move into the streak and undergo an epithelial-to-mesenchymal transition, but most cells then fail to move away from the streak. As a consequence, no embryonic mesoderm- or endoderm-derived tissues develop, although extraembryonic tissues form. Patterning of the prospective neuroectoderm is greatly perturbed in the mutant embryos. Anterior neuroectoderm markers are widely expressed, at least in part because the anterior visceral endoderm, which provides signals that regulate their expression, is not displaced proximally in the absence of definitive endoderm. Posterior neuroectoderm markers are not expressed, presumably because there is neither mesendoderm underlying the prospective neuroectoderm nor a morphologically normal node to provide the inductive signals necessary for their expression. This study identifies Fgf8 as a gene essential for gastrulation and shows that signaling via FGF8 and/or FGF4 is required for cell migration away from the primitive streak.     

Scanning electron microscopy of the development of the mesoderm layer in chick embryos

Summary The distribution of mesoderm, the structure of mesoderm cells and relationship between mesoderm and ectoderm were examined by SEM in embryos at stages 3 to 5. The mesoderm was displayed by removal of the endoderm and by fracturing the embryos through mesoderm containing regions. Within the mesoderm layer four zones could be distinguished by their cell shape and arrangement — the primitive streak, a multilayered compact area around the margins of the area pellucida, multilayered and loosely arranged cells near the primitive streak and a flattened monolayer of cells around the advancing lateral and anterior edges of the mesoderm sheet. Secretion of basement membrane by the ectoderm was seen to precede the arrival of mesoderm cells. This suggests that ectoderm alone can synthesize basement membrane without mesodermal contribution.     

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 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.     

Beta-catenin signaling marks the prospective site of primitive streak formation in the mouse embryo.

Beta-catenin signaling has been shown to be involved in triggering axis formation in several organisms, including Xenopus and zebrafish. Genetic analysis has demonstrated that the Wnt/beta-catenin signaling pathway is also involved in axis formation in the mouse, since a targeted deletion of beta-catenin results in embryos that have a block in anterior-posterior axis formation, fail to initiate gastrulation, and do not form mesoderm. However, because beta-catenin is ubiquitously expressed, the precise time and cell types in which this signaling pathway is active during early embryonic development remain unknown. Thus, to better understand the role of the Wnt/beta-catenin signaling pathway in axis formation and mesoderm specification, we have examined both the distribution and signaling activity of beta-catenin during early embryonic development in the mouse. We show that the N-terminally nonphosphorylated form of beta-catenin as well as beta-catenin signaling is first detectable in the extraembryonic visceral endoderm in day 5.5 embryos. Before the initiation of gastrulation at day 6.0, beta-catenin signaling is asymmetrically distributed within the epiblast and is localized to a small group of cells adjacent to the embryonic--extraembryonic junction. At day 6.5 and onward, beta-catenin signaling was detected in the primitive streak and mature node. Thus, beta-catenin signaling precedes primitive streak formation and is present in epiblast cells that will go on to form the primitive streak. These results support a critical role for the Wnt/beta-catenin pathway in specifying cells to form the primitive streak and node in the mammalian embryo as well as identify a novel domain of Wnt/beta-catenin signaling activity during early embryogenesis.     

Fate diversity of primitive streak cells during heart field formation in ovo.

In amniote embryos, cells from a rostral portion of the primitive streak migrate anterolaterally and establish the heart field mesoderm, from which two cardiac cell lineages, cardiomyocytes and endocardial endothelial cells, differentiate. The endoderm underlying the heart field has been postulated as the source of several paracrine factors that may serve to induce each of these cell types. However, it has been unclear how these signal molecules, which are expressed broadly in the endoderm, instruct individual cells of the heart field mesoderm to enter either the cardiomyocyte lineage or the endocardial cell lineage. To clarify lineage relationships of these two cardiac cell types, the fate of chicken primitive streak cells was traced for the first time in ovo. By using replication-defective retroviral-mediated gene transfer, we demonstrate that cells in the rostral half of Hamburger and Hamilton (HH) stage 3 primitive streak generate a daughter population that proliferates and migrates into the heart field, differentiating into either endocardial or myocardial cells, but not both cell types. The results suggest that the rostral portion of the primitive streak at HH stage 3 consists of at least two distinct subpopulations, entering either the cardiomyocyte lineage or the endocardial cell lineage. Thus, in ovo these two cell lineages of the heart are already segregated within the primitive streak, significantly before their migration to the heart field. When the precardiomyocytes and pre-endocardial cells arrive at the heart field, each mesodermal cell subpopulation may be permissive to paracrine signal(s) from underlying endoderm to initiate their terminal differentiation into either muscle or endothelial cells.     

Development of the chick embryo endoderm studied by S.E.M

Summary The endoderm of a series of chick embryos from the unincubated egg to Hamburger and Hamilton stage 5 was examined by scanning electron microscopy (SEM). During this period the endoderm develops from a few scattered cells to a complete epithelial layer. Prior to the formation of the primitive streak endoderm cells can be observed delaminating from the ectoderm. These cells are round and have few processes except where they contact each other. At stage 2 cells appear in the endoderm over the primitive streak which have broad flat processes. This suggests that the cells originate directly from the streak. Away from the streak the endoderm cells are either smooth or have short microvilli. In later streak stages a mixture of smooth and some microvillous cells form a hexagonal pattern. This pattern is occasionally modified and holes are found in the endoderm with cell processes protruding from below the endoderm level. Sometimes whole cells, smaller and rounder than the majority of the endoderm cells are associated with this disturbance of the pattern. These cells are connected to the mesoderm by a long cytoplasmic process and it is suggested that they could be cells entering the endoderm from the middle layer, having accompanied the mesoderm cells through the primitive streak.     

Differential expression of the seven-pass transmembrane cadherin genes Celsr1-3 and distribution of the Celsr2 protein during mouse development.

Drosophila Flamingo (Fmi) is an evolutionally conserved seven-pass transmembrane receptor of the cadherin superfamily. Fmi plays multiple roles in patterning neuronal processes and epithelial planar cell polarity. To explore the in vivo roles of Fmi homologs in mammals, we previously cloned one of the mouse homologs, mouse flamingo1/Celsr2. Here, we report the results of our study of its embryonic and postnatal expression patterns together with those of two other paralogs, Celsr1 and Celsr3. Celsr1-3 expression was initiated broadly in the nervous system at early developmental stages, and each paralog showed characteristic expression patterns in the developing CNS. These genes were also expressed in several other organs, including the cochlea, where hair cells develop planar polarity, the kidney, and the whisker. The Celsr2 protein was distributed at intercellular boundaries in the whisker and on processes of neuronal cells such as hippocampal pyramidal cells, Purkinje cells, and olfactory neurons. Celsr2 is mapped to a distal region of the mouse chromosome 3. We discussed possible functions of seven-pass transmembrane cadherins in mouse development.     

The formation of the embryonic mesoderm in the early post-implantation mouse embryo

Summary The formation of the mesoderm in early post-implantation mouse embryos is described and analysed. The outgrowth of the mesoderm was found to depend on the changes in the shape of the embryonic ectoderm, which lead to a relative displacement of the primitive streak in the caudal direction. The primitive streak deposits its cells laterally in the case of the lateral mesoderm, and medially in the case of the headprocess. In doing so, the primitive streak leaves a trail of mesoderm cells. This means that mesoderm cells do not migrate actively from the caudally located primitive streak towards more frontal positions in the embryo. This is confirmed by the results of scanning electron microscopy, which revealed that mesoderm cells show no polarity at all in the caudofrontal direction. In may therefore be concluded that these cells probably do not migrate.Studies on the cell-cycle parameters of the embryonic ectoderm, showed that mesoderm cells-to-be are probably recruited not only from the proliferation zone, but also from the lateral ectoderm. It is oostulated that the lateral ectoderm gives rise, via the largest part of the primitive streak, to most of the mesoderm cells, whereas the proliferation zone gives rise to the head-process mesoderm, via the anterior part of the primitive streak.     

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.     

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.     

The primitive streak

Summary The emphasis of this review is on the primitive streak of the chick embryo, collated with such information as is available on the mouse embryo. Little modern work has been published on any reptile primitive streak.The following topics are considered: — evolutionary significance; formation of the primitive streak; ingression and de-epithelialisation; the basal lamina; migration from the primitive streak of the endoderm and mesoderm; the role of the extracellular matrix; changes in cell adhesiveness; regression of the primitive streak and its role in body patterning; the primitive streak and induction.     

Differentiation of the embryonic disc,amnion, and yolk sac in the rhesus monkey

The inner cell mass of the blastocyst has differentiated into epiblast and hypoblast (primitive endoderm) prior to implantation. Since endoderm cells extend beyond the epiblast, it can be considered that both parietal and visceral endoderm are present. At implantation, epiblast cells begin to show marked evidence of polarity. They form a spherical aggregate with their basal ends toward the basal lamina and apical ends toward the interior. The potential for an internal space is formed by this change in polarity of the cells. No cytological evidence of separation of those cells that will form amniotic epithelium from the rest of the epiblast is seen until a cavity begins to form. The amniotic epithelium is originally contiguous with overlying cytotrophoblast, and a diverticulum remains in this position during early development. Epiblast forms a pseudostratified columnar epithelium, but dividing cells are situated toward the amniotic cavity rather than basally. The first evidence of a trilaminar disc occurs when a strand of cells contiguous with epiblast is found extending toward visceral endoderm. These presumptive mesoderm cells are undifferentiated, whereas extraembryonic mesoderm cells are already a distinct population forming extracellular materials. After implantation, visceral endoderm cells proliferate forming an irregular layer one to three cells thick. Visceral endoderm cells have smooth apical surfaces, but very irregular basal surfaces, and no basal lamina. At the margins of the disc, visceral endoderm is continuous with parietal endoderm and reflects back over the apices of the marginal visceral endoderm cells. This sacculation by visceral endoderm cells precedes pinching off of the secondary yolk sac from the remaining primary yolk sac.     

Signaling hierarchy downstream of retinoic acid that independently regulates vascular remodeling and endothelial cell proliferation

We previously demonstrated that during vascular morphogenesis, retinoic acid (RA) is required for the control of endothelial cell proliferation and capillary plexus remodeling. Herein, we investigate the mechanisms by which RA regulates these processes in the yolk sac. We found that although the enzyme required for RA production during early embryogenesis, retinaldehyde dehydrogenase-2 (Raldh2), was expressed in the visceral endoderm, RA receptors alpha1 and alpha2 were expressed in endothelial cells in the mesoderm, indicating that they are direct targets of RA. In Raldh2(-/-) embryos, there was down-regulation of TGF-beta1, fibronectin (Fn) and integrin alpha5, which was associated with decreased visceral endoderm survival and production of VEGF-A, Indian hedgehog (IHH), and bFGF. Exogenous provision of RA or Fn to Raldh2(-/-) explants in whole mouse embryo culture restored vascular remodeling, visceral endoderm survival, as well as integrin alpha5 expression and its downstream signaling that controls endothelial growth. Exogenous provision of visceral endoderm-derived factors (VEGF-A, IHH, and bFGF) failed to rescue endothelial cell proliferative control but collectively promoted vascular remodeling, suggesting that these processes are independently regulated via a signaling hierarchy downstream of RA.