Changing patterns of cytokeratins and vimentin in the early chick embryo

The distribution of cytokeratins and vimentin intermediate filaments in the first 48 h of chick development has been determined using immunofluorescent labelling. During formation of the germ layers, cytokeratin expression is associated with the appearance of an integral epithelium (ectoderm), whereas vimentin expression is associated with cells that detach and migrate from this epithelium to form endoderm and mesoderm. Subsequently, vimentin persists in the endoderm and mesoderm and the tissues derived therefrom, such as the somites and developing heart, throughout the period of study. The appearance of cytokeratins at later stages of development occurs in some epithelia such as the ectoderm, endoderm, lateral plate and epimyocardium but not others including the neural plate, neural tube and somites. Expression of cytokeratins in endoderm and mesenchymal tissues occurs in tandem with vimentin. In conclusion, vimentin expression is related to its distribution in the epiblast before germ layer formation. Its initial appearance may be related to the motile behaviour of cells about to ingress through the primitive streak. The appearance of cytokeratin filaments, however, does not reflect germ layer derivation but rather the need for an epithelial sheet.     

Impact of node ablation on the morphogenesis of the body axis and the lateral asymmetry of the mouse embryo during early organogenesis.

The node of the mouse gastrula is the major source of the progenitor cells of the notochord, the floor plate, and the gut endoderm. The node may also play a morphogenetic role since it can induce a partial body axis following heterotopic transplantation. The impact of losing these progenitor cells and the morphogenetic activity on the development of the body axes was studied by the ablation of the node at late gastrulation. In the ablated embryo, an apparently intact anterior-posterior body axis with morphologically normal head folds, neural tube, and primitive streak developed during early organogenesis. Cell fate analysis revealed that the loss of the node elicits de novo recruitment of neural ectoderm and somitic mesoderm from the surrounding germ-layer tissues. This leads to the restoration of the neural tube and the paraxial mesoderm. However, the body axis of the embryo was foreshortened and somite formation was retarded. Histological and gene expression studies reveal that in most of the node-ablated embryos, the notochord in the trunk was either absent or interrupted, and the floor plate was absent in the ventral region of the reconstituted neural tube. The loss of the node did not affect the differentiation of the gut endoderm or the formation of the mid- and hindgut. In the node-ablated embryo, expression of the Pitx2 gene in the lateral plate mesoderm was no longer restricted to the left side but was found on both sides of the body or was completely absent from the lateral plate mesoderm. Therefore, the loss of the node results in the failure to delineate the laterality of the body axis. The node and its derivatives therefore play a critical role in the patterning of the ventral neural tube and lateral body axis but not of the anterior-posterior axis during early organogenesis.     

Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis

We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes.     

Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate

During embryonic development, organs arise along the gut tube as a series of buds in a stereotyped anterior-posterior (A-P) pattern. Using chick-quail chimeras and in vitro tissue recombination, we studied the interactions governing the induction and maintenance of endodermal organ identify focusing on the pancreas. Though several permissive signals in pancreatic development have been previously identified, here we provide evidence that lateral plate mesoderm sends instructive signals to the endoderm, signals that induce expression of the pancreatic genes Pdx1, p48, Nkx6.1, glucagon, and insulin. Moreover, this instructive signal directs cells to form ectopic insulin-positive islet-like clusters in endoderm that would otherwise form more rostral organs. Once generated, endocrine cells no longer require interaction with mesoderm, but nonendocrine cells continue to require permissive signals from the mesoderm. Stimulation of activin, BMP, or retinoic acid signaling is sufficient to induce Pdx1 expression in endoderm anterior to the pancreas. Lateral plate mesoderm appears to pattern the endoderm in a posterior-dominant fashion as first noted in the patterning of the neural tube at the same embryonic stage. These findings argue for a central role of the mesoderm in coordinating the A-P pattern of all three primary germ layers.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).     

Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development

In amniotes, the pancreatic mesenchyme plays a crucial role in pancreatic epithelium growth, notably through the secretion of fibroblast growth factors. However, the factors involved in the formation of the pancreatic mesenchyme are still largely unknown. In this study, we characterize, in zebrafish embryos, the pancreatic lateral plate mesoderm, which is located adjacent to the ventral pancreatic bud and is essential for its specification and growth. We firstly show that the endoderm, by expressing the fgf24 gene at early stages, triggers the patterning of the pancreatic lateral plate mesoderm. Based on the expression of isl1, fgf10 and meis genes, this tissue is analogous to the murine pancreatic mesenchyme. Secondly, Fgf10 acts redundantly with Fgf24 in the pancreatic lateral plate mesoderm and they are both required to specify the ventral pancreas. Our results unveil sequential signaling between the endoderm and mesoderm that is critical for the specification and growth of the ventral pancreas, and explain why the zebrafish ventral pancreatic bud generates the whole exocrine tissue.     

Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population

During embryogenesis the central and peripheral nervous systems arise from a neural precursor population, neurectoderm, formed during gastrulation. We demonstrate the differentiation of mouse embryonic stem cells to neurectoderm in culture, in a manner which recapitulates embryogenesis, with the sequential and homogeneous formation of primitive ectoderm, neural plate and neural tube. Formation of neurectoderm occurs in the absence of extraembryonic endoderm or mesoderm and results in a stratified epithelium of cells with morphology, gene expression and differentiation potential consistent with positionally unspecified neural tube. Differentiation of this population to homogeneous populations of neural crest or glia was also achieved. Neurectoderm formation in culture allows elucidation of signals involved in neural specification and generation of implantable cell populations for therapeutic use.     

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.     

Elongation of axolotl tailbud embryos requires GPI-linked proteins and organizer-induced, active, ventral trunk endoderm cell rearrangements

Application of phosphatidylinositol-specific phospholipase C to early tailbud stage axolotl embryos reveals that a specific subset of morphogenetic movements requires glycosylphosphatidylinositol (GPI)-linked cell-surface proteins. These include pronephric duct extension, "gill bulge" formation, and embryonic elongation along the anteroposterior axis. The work of Kitchin (1949, J. Exp. Zool. 112, 393-416) led to the conclusion that extension of the notochord provided the motive force driving anteroposterior stretching in axolotl embryos, elongation of other tissues being a passive response. We therefore conjectured that axial mesoderm cells might display the GPI-linked proteins required for elongation of the embryo. However, we show here that removal of most of the neural plate and axial and paraxial mesoderm prior to neural tube closure does not prevent elongation of ventrolateral tissues. Tissue-extirpation and tissue-marking experiments indicate that elongation of the ventral trunk occurs via active, directed tissue rearrangements within the endoderm, directed by signals emanating from the blastopore region. Extension of both dorsal and ventral tissues requires GPI-linked proteins. We conclude that elongation of axolotl embryos requires active cell rearrangements within ventral as well as axial tissues. The fact that both types of elongation are prevented by removal of GPI-linked proteins implies that they share a common molecular mechanism.     

T-Box genes and developmental decisions that cells make

During gastrulation in vertebrate embryos, three definitive germ layers (ectoderm, mesoderm, and endoderm) are formed by organized and coordinated cell movements. In zebrafish, further subdivision of the mesoderm gives rise to the axial, adaxial and paraxial mesoderm. The axial mesoderm contributes to the prechordal plate and notochord whereas the adaxial and paraxial cells give rise to slow and fast muscles, respectively (Devoto et al., 1996; Blagden et al., 1997; Currie and Ingham, 1998). An inductive interaction in which the notochord plays an essential role will also provide an input in forming other specialized types of tissue contributing to the axial structures: the floor plate located dorsally to the notochord in the ventral spinal cord and the hypochord located ventrally of the notochord and deriving probably from the endoerm. It is known that despite the difference in developmental roles (Str?hle et al., 1993; Krauss et al., 1993), the floor plate and hypochord co-express a number of common molecular markers (Jan et al., 1995; our unpublished results) that may illustrate a certain similarity of their origin. Their close proximity to the notochord determines specialized features of these structures that differ substantially from the rest of the neural tube and endoderm, correspondingly. Once formed under the influence of the notochordal signaling, the floor plate will acquire an ability, similar to the notochord, to express genes of the Hedgehog family and several other groups of genes and to induce specification of ventral cell types in the neural tube during later development (for review, see Korzh, 1998). The biology of the hypochord is much less understood. It seems that the hypochord develops slightly later than the floor plate. It may be required for proper positioning of the dorsal aorta as well as induction of some other endoderm derivatives.     

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.     

Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo

Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube.     

Mechanisms of neurulation: traditional viewpoint and recent advances

In this review article, the traditional viewpoint of how neurulation occurs is evaluated in light of recent advances. This has led to the formulation of the following fundamentals: (1) neurulation, specifically neural plate shaping and bending, is a multifactorial process resulting from forces both intrinsic and extrinsic to the neural plate; (2) neurulation is driven by both changes in neuroepithelial cell shape and other form-shaping events; and (3) forces for cell shape changes are generated by both the cytoskeleton and other factors. Several cell behaviors within the neural plate have been elucidated. Future challenges include identifying cell behaviors within non-neuroepithelial tissues, determining how intrinsic and extrinsic cell behaviors are orchestrated into coordinated morphogenetic movements and elucidating the molecular mechanisms underlying such behaviors.     

Patterning of the avian intermediate mesoderm by lateral plate and axial tissues

Amniote kidney tissue is derived from the intermediate mesoderm (IM), a strip of mesoderm that lies between the somites and the lateral plate. While much has been learned concerning the later events which regulate the differentiation of IM into tubules and other types of kidney tissue, much less is known concerning the earlier events which regulate formation of the IM itself. In the current study, the chick pronephros was used as a model system to identify tissues that play a role in patterning the IM and the critical time periods during which such patterning events take place. Explant studies revealed that the prospective pronephric IM is already specified to express kidney genes by stage 6, shortly after its gastrulation through the primitive streak, and earlier than previously reported. Transplant and explant experiments revealed that the lateral plate contains an activity that can repress IM formation in tissues that are already specified to express IM genes. In contrast, Hensen's node can promote formation of IM in the lateral plate. Paraxial tissues (presomitic mesoderm plus neural plate and notochord) were found to influence the morphogenesis of the nephric duct, but did not induce IM tissue to an appreciable extent. Combining lateral plate and paraxial tissue in vivo or in vitro led to induction of IM genes in the paraxial mesoderm but not in the lateral plate mesoderm. Based on these results and those of others, we propose a two-step model for the patterning of the IM. While tissue is still in the primitive streak, the prospective IM is relatively uncommitted. By stage 6, shortly after cells leave the primitive streak, a field of cells is generate which is specified to give rise to IM (Step 1). Subsequently, competing signals from the lateral plate and axial tissues modulate the number of cells that commit to an IM fate (Step 2).     

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.     

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.