Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny

Quail-chick intracoelomic grafts of organ rudiments were used to study the origin of endothelia and haemopoietic cells during avian organogenesis in conjunction with the monoclonal antibody QH1 which recognizes the quail haemangioblastic lineage. Results differed according to the germ-layer constitution of the grafted rudiments. In the case of the limb buds, endothelial cells from the host invaded the graft through an angiogenic process. Haemopoietic progenitors from the host also colonized the grafted bone marrow. In contrast, rudiments of internal organs provided their own contingent of endothelial precursors, a process termed vasculogenesis. Nevertheless, haemopoietic cells in these organs were all derived from the host. In the lung, this extrinsic cell population appeared regularly scattered around the parabronchi and had a macrophage-like phenotype. In the pancreas, the granulocytes which differentiate as dense aggregates located in the wall of the largest vessels were extrinsic. Similarly in the spleen, a mesodermal primordium that develops in close association with the pancreatic endoderm, endothelial cells were intrinsic and haemopoietic cells host-derived. This study demonstrates that, in ontogeny, vascularization obeys different rules depending on which germ layer the mesoderm is associated with: in mesodermal/ectodermal rudiments angiogenesis is the rule; in mesodermal/endodermal rudiments, vasculogenesis occurs. However, in these internal organs undergoing vasculogenesis, endothelial and haemopoietic cells have separate origins. We put forward the hypothesis that the endoderm induces the emergence of endothelial cells in the associated mesoderm. Formation of blood stem cells may also involve interactions between endoderm and mesoderm, but in this case the responding capacity of the mesoderm appears restricted to the paraaortic region.     

Sequence of the twist gene and nuclear localization of its protein in endomesodermal cells of early Drosophila embryos.

The twist gene is involved in the establishment of germ layers in Drosophila embryos: twist homozygous mutant embryos fail to form the ventral furrow at gastrulation and lack mesoderm and all internal organs. We have determined the sequence of the twist gene, that contains 'CAX' repeats in its 5' moiety, and codes for a protein of 490 amino acids. We have raised anti-twist antibodies that were used to study the distribution of the twist protein in whole mounts and tissue sections of wild-type embryos. Twist protein appears to be a nuclear protein at all developmental stages. It is present over both poles and in the midventral region (endoderm and mesoderm anlagen) at cellular blastoderm stage; later in development, it is detected within the mesodermal layer until its differentiation into somatopleura and splanchnopleura in which some cells are still labelled by anti-twist antibodies.     

Transformation of sensory organs by mutations of the cut locus of D. melanogaster

The identities of two types of sensory organs in the body wall of Drosophila, namely the external sensory organs and the chordotonal organs, are under genetic control. Embryonic lethal mutations in the cut gene complex transform the external sensory organs into chordotonal organs. The neurons, as well as the support cells forming the external sensory structures, change their morphological and antigenic characteristics to those of chordotonal organs, providing genetic evidence that these two types of sensory organs are homologous. Similar transformations of external sensory organs are observed in adult mosaic flies. Analysis of mosaic larvae and flies suggests that the cut gene function is required either in or near external sensory organs in order for them to acquire their correct identity.     

Further studies of the engrailed phenotype in Drosophila.

Although most mutations at the engrailed locus of Drosophila cause embryonic death when homozygous, they are viable in clones of cells. We describe the phenotype of such clones in the eye-antenna, proboscis, humerus, wing, legs, and terminalia. When in anterior compartments the clones are normal, but in most posterior compartments they are abnormal and fail to respect the anteroposterior compartment boundary. We find that the yield of engrailed-lethal clones in posterior compartments is often significantly lower than expected, indicating that these clones are lost during development. Mutant clones are abnormal in the analia and rare in the humerus, suggesting that both structures are of posterior provenance. These results support the hypothesis that the engrailed+ gene is required exclusively in cells of posterior compartments to specify their characteristic cell affinities and pattern.     

Fate mapping study of the splanchnopleural mesoderm of the 1.5-day-old chick embryo

Various portions of the splanchnopleural mesoderm lateral to the somites of 1.5-day chick embryos were marked in ovo by local injection of Dil, and the distribution of the labelled cells in the digestive-tract mesoderm formed after 3 days' reincubation was analysed. The presumptive area of the digestive organs was confined to bands of splanchnic mesoderm lying lateral to the somites, on both sides, with a width two or three times that between the midline of the embryo and the lateral edge of the somite. Each band generally contributed cells to its own side of the digestive-tract mesoderm, except for the region around the bile duct. The anterior and posterior portion of the pre-gut area contributed cells to the anterior and posterior region of the digestive tract, respectively, but label originating from the portion furthest from the somite took the more ventral and posterior position. Thus, the presumptive areas of the respective digestive organs were located anteroposteriorly in the same order as in the digestive tract with their boundaries lying oblique to the embryonic axis.     

Elucidation of the role of activin in organogenesis using a multiple organ induction system with amphibian and mouse undifferentiated cells in vitro

Studies performed over the last century have clarified the mechanisms of organ and tissue formation. Mesoderm formation is one of the most important events in early body pattern determination during embryogenesis. In 1988, we found that activin A has mesoderm-inducing activity. As activin A could induce dorsal mesoderm formation, unlike fibroblast growth factor and bone morphogenetic protein, this factor was thought to be the molecular entity of the Spemann-Mangold organizer. Subsequently, the mechanisms of early embryogenesis have been clarified using molecular biological techniques, resulting in the identification of many genes that are involved in organ and tissue development. This finding that activin A could induce dorsal mesoderm formation spurred research into the application of agents that induce organs and tissues in vitro . In this regard, we have shown that many organ types can be induced by activin A in vitro . Moreover, we have found that other types of organs can be induced by changing the conditions of treatment. To date, more than 20 different types of tissues and organs have been successfully induced from Xenopus undifferentiated cells in vitro . In recent years, we have applied these protocols to mouse embryonic stem cells, and we have successfully induced several tissues, such as the pancreas and cardiomyocytes. We are also investigating how the pluripotency of undifferentiated stem cells is regulated. In this review, we summarize the current knowledge regarding activin as a mesoderm-inducing factor and its application for the induction of tissues and organs from undifferentiated cells. Moreover, we provide some examples of in vitro tissue differentiation from mouse embryonic stem cells, which may prove useful in regenerative medicine.     

The role of mesodermal signals during liver organogenesis in zebrafish

Three germ cell layers, the ectoderm, mesoderm and endoderm, are established during the gastrulation stage. All cell types in different organs and tissues are derived from these 3 germ cell layers at later stages. For example, skin epithelial cells and neuronal cells are derived from the ectoderm, while endothelial cells and muscle cells from the mesoderm and lung, and intestine epithelial cells from the endoderm. While in a normal situation different germ cells are destined to specific cell fates in differ...     

Development of the subsoephageal body cells and the pericardiac cells during embryogenesis with diapause in Locusta migratoria (Linnaeus 1758) (Orthoptera: Acrididae)

During Locusta migratoria embryogenesis, the yolk is progressively degraded and the resulting metabolites are released in the haemolymph. We researched the organs possibly involved in the uptake of haemolymphatic proteins. Among organs originated from mesoderm, the SOB (suboesophageal bodies) situated in the embryonic head are remarkable by a very early acquisition of differentiated cytological characters, while most other cells of the embryo are undifferentiated. The SOB quite disappear before hatching. Just before katatrepsis stage, the other organs derived from mesoderm begin to differentiate, including the PC (pericardial cells) which take over from the SOB. These cells, situated in thorax and abdomen, are developed during the dorsal close of embryo. The development and the ultrastructural changes of the SOB cells and of the PC were studied during an embryogenesis with diapause. The morphology of embryos which enter diapause is comparable with that of a continuous development at the beginning of katatrepsis. However, the cells of SOB and PC cells suffer from remarkable changes not only physiologically but cytologically. At the beginning of diapause, the proteosynthetic activity practically disappears in the SOB cells and the lysis areas appear. Nevertheless, the exchanges between these cells and the haemolymph still remain important. For the period of cold, which is necessary to the resumption of development, the aspect of the SOB cells changes and in particular the areas of lysis become less wide. When the embryo reopens its development, the SOB cells show a proteosynthetic activity and the areas of lysis disappear. The changes of the SOB cells and of the PC cells are regularized during the resumption of the development: the SOB cells which had again taken a normal activity start to regress from the stage VII on, while the PC cells take over.     

Roundabout 2 regulates migration of sensory neurons by signaling in trans

BACKGROUND: Roundabout (Robo) receptors and their ligand Slit are important regulators of axon guidance and cell migration. The development of Drosophila embryonic sense organs provides a neuronal migration paradigm where the in vivo roles of Slit and Robo can be assayed using genetics. RESULTS: Here we show that Slit-Robo signaling controls migration of Drosophila larval sensory neurons that are part of the Chordotonal (Cho) stretch receptor organs. We used live imaging to show that abdominal Cho organs normally migrate ventrally during development, whereas thoracic Cho organs do not. Robo2 overexpression in cis (in the sensory neurons) or in trans (on neighboring visceral mesoderm) transforms abdominal organs to a thoracic morphology and position by blocking migration, while loss of Slit-Robo signaling produces a reverse transformation in which thoracic organs migrate ectopically. Rescue and tissue-specific knockout experiments indicate that trans signaling by Robo2 contributes to the normal positioning of the thoracic Cho organs. The differential positioning of Cho organs between the thorax and abdomen is known to be regulated by Hox genes, and we show that the essential Hox cofactor Homothorax, represses Robo2 expression in the abdominal visceral mesoderm. CONCLUSIONS: Our results suggest that segment-specific neuronal migration patterns are directed through a novel signaling complex (the "Slit sandwich") in which Robo2 on the thoracic visceral mesoderm binds to Slit and presents it to Robo receptors on Cho neurons. The differential positioning of Cho organs between thorax and abdomen may be determined by Hox gene-mediated repression of robo2.     

Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning

A conserved molecular cascade involving Nodal signaling that patterns the laterality of the lateral mesoderm in vertebrates has been extensively studied, but processes involved in the initial break of left-right (LR) symmetry are just beginning to be explored. Here we report that Na,K-ATPase alpha2 and Ncx4a function upstream of Nodal signaling to regulate LR patterning in zebrafish. Knocking down Na,K-ATPase alpha2 and Ncx4a activity in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle (KV), is sufficient to disrupt asymmetric gene expression in the lateral plate mesoderm and randomize the placement of internal organs, indicating that the activity of Na,K-ATPase alpha2 and Ncx4a in DFCs/KV is crucial for LR patterning. High-speed videomicroscopy and bead implantation experiments show that KV cilia are immobile and the directional fluid flow in KV is abolished in Na,K-ATPase alpha2 and Ncx4a morphants, suggesting their essential role in KV ciliary function. Furthermore, we found that intracellular Ca(2+) levels are elevated in Na,K-ATPase alpha2 and Ncx4a morphants and that the defects in ciliary motility, KV fluid flow and placement of internal organs induced by their knockdown could be suppressed by inhibiting the activity of Ca(2+)/calmodulin-dependent protein kinase II. Together, our data demonstrate that Na,K-ATPase alpha2 and Ncx4a regulate LR patterning by modulating intracellular calcium levels in KV and by influencing cilia function, revealing a previously unrecognized role for calcium signaling in LR patterning.     

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.     

Mypt1-mediated spatial positioning of Bmp2-producing cells is essential for liver organogenesis

Mesodermal tissues produce various inductive signals essential for morphogenesis of endodermal organs. However, little is known about how the spatial relationship between the mesodermal signal-producing cells and their target endodermal organs is established during morphogenesis. Here, we report that a mutation in the zebrafish myosin phosphatase targeting subunit 1 (mypt1) gene causes abnormal bundling of actin filaments and disorganization of lateral plate mesoderm (LPM) and endoderm cells. As a result, the coordination between mesoderm and endoderm cell movements is disrupted. Consequently, the two stripes of Bmp2a-expressing cells in the LPM fail to align in a V-shaped pocket sandwiching the liver primordium. Mispositioning Bmp2a-producing cells with respect to the liver primordium leads to a reduction in hepatoblast proliferation and final abortion of hepatoblasts by apoptosis, causing the liverless phenotype. Our results demonstrate that Mypt1 mediates coordination between mesoderm and endoderm cell movements in order to carefully position the liver primordium such that it receives a Bmp signal that is essential for liver formation in zebrafish.     

Development of chick axial mesoderm: specification of prechordal mesoderm by anterior endoderm-derived TGFbeta family signalling

Two populations of axial mesoderm cells can be recognised in the chick embryo, posterior notochord and anterior prechordal mesoderm. We have examined the cellular and molecular events that govern the specification of prechordal mesoderm. We report that notochord and prechordal mesoderm cells are intermingled and share expression of many markers as they initially extend out of Hensen's node. In vitro culture studies, together with in vivo grafting experiments, reveal that early extending axial mesoderm cells are labile and that their character may be defined subsequently through signals that derive from anterior endodermal tissues. Anterior endoderm elicits aspects of prechordal mesoderm identity in extending axial mesoderm by repressing notochord characteristics, briefly maintaining gsc expression and inducing BMP7 expression. Together these experiments suggest that, in vivo, signalling by anterior endoderm may determine the extent of prechordal mesoderm. The transforming growth factor (beta) (TGFbeta) superfamily members BMP2, BMP4, BMP7 and activin, all of which are transiently expressed in anterior endoderm mimic distinct aspects of its patterning actions. Together our results suggest that anterior endoderm-derived TGFbetas may specify prechordal mesoderm character in chick axial mesoderm.     

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.     

Insights into the establishment of left-right asymmetries in vertebrates

The body-plan of vertebrates, while exteriorly essentially symmetric along its medio-lateral plane, displays numerous left-right differences in the disposition and placement of internal organs. Such left-right asymmetries, established during embryogenesis, are controlled by complex epigenetic and genetic cascades that impart laterality information to the different embryo structures and organ primordia. A key and evolutionarily conserved feature of these information cascades among vertebrate embryos is the left-sided transfer of information from the node to the lateral plate mesoderm during early somitogenesis stages. We review here recent evidence concerning the mechanisms that regulate the laterality of such transfer. Furthermore, we propose a model of left-right axis specification that underscores the role of the node as an integrator of laterality information and the evolutionary conservation of the mechanisms that convey such information to and from the node.     

Fate and plasticity of the endoderm in the early chick embryo

In vertebrates, the endoderm is established during gastrulation and gradually becomes regionalized into domains destined for different organs. Here, we present precise fate maps of the gastrulation stage chick endoderm, using a method designed to label cells specifically in the lower layer. We show that the first population of endodermal cells to enter the lower layer contributes only to the midgut and hindgut; the next cells to ingress contribute to the dorsal foregut and followed finally by the presumptive ventral foregut endoderm. Grafting experiments show that some migrating endodermal cells, including the presumptive ventral foregut, ingress from Hensen's node, not directly into the lower layer but rather after migrating some distance within the middle layer. Cell transplantation reveals that cells in the middle layer are already committed to mesoderm or endoderm, whereas cells in the primitive streak are plastic. Based on these results, we present a revised fate map of the locations and movements of prospective definitive endoderm cells during gastrulation.     

Drosophila atonal fully rescues the phenotype of Math1 null mice: new functions evolve in new cellular contexts

Many genes share sequence similarity between species, but their properties often change significantly during evolution. For example, the Drosophila genes engrailed and orthodenticle and the onychophoran gene Ultrabithorax only partially substitute for their mouse or Drosophila homologs. We have been analyzing the relationship between atonal (ato) in the fruit fly and its mouse homolog, Math1. In flies, ato acts as a proneural gene that governs the development of chordotonal organs (CHOs), which serve as stretch receptors in the body wall and joints and as auditory organs in the antennae. In the fly CNS, ato is important not for specification but for axonal arborization. Math1, in contrast, is required for the specification of cells in both the CNS and the PNS. Furthermore, Math1 serves a role in the development of secretory lineage cells in the gut, a function that does not parallel any known to be served by ato. We wondered whether ato and Math1 might be more functionally homologous than they appear, so we expressed Math1 in ato mutant flies and ato in Math1 null mice. To our surprise, the two proteins are functionally interchangeable.