The functional relationship between ectodermal and mesodermal segmentation in the crustacean, Parhyale hawaiensis

In arthropods, annelids and chordates, segmentation of the body axis encompasses both ectodermal and mesodermal derivatives. In vertebrates, trunk mesoderm segments autonomously and induces segmental arrangement of the ectoderm-derived nervous system. In contrast, in the arthropod Drosophila melanogaster, the ectoderm segments autonomously and mesoderm segmentation is at least partially dependent on the ectoderm. While segmentation has been proposed to be a feature of the common ancestor of vertebrates and arthropods, considering vertebrates and Drosophila alone, it is impossible to conclude whether the ancestral primary segmented tissue was the ectoderm or the mesoderm. Furthermore, much of Drosophila segmentation occurs before gastrulation and thus may not accurately represent the mechanisms of segmentation in all arthropods. To better understand the relationship between segmented germ layers in arthropods, we asked whether segmentation is an intrinsic property of the ectoderm and/or the mesoderm in the crustacean Parhyale hawaiensis by ablating either the ectoderm or the mesoderm and then assaying for segmentation in the remaining tissue layer. We found that the ectoderm segments autonomously. However, mesoderm segmentation requires at least a permissive signal from the ectoderm. Although mesodermal stem cells undergo normal rounds of division in the absence of ectoderm, they do not migrate properly in respect to migration direction and distance. In addition, their progeny neither divide nor express the mesoderm segmentation markers Ph-twist and Ph-Even-skipped. As segmentation is ectoderm-dependent in both Parhyale and holometabola insects, we hypothesize that segmentation is primarily a property of the ectoderm in pancrustacea.     

FGF8-like1 and FGF8-like2 encode putative ligands of the FGF receptor Htl and are required for mesoderm migration in the Drosophila gastrula

BACKGROUND: Mesoderm migration in the Drosophila gastrula depends on the fibroblast growth factor (FGF) receptor Heartless (Htl). During gastrulation Htl is required for adhesive interactions of the mesoderm with the ectoderm and for the generation of protrusive activity of the mesoderm cells during migration. After gastrulation Htl is essential for the differentiation of dorsal mesodermal derivatives. It is not known how Htl is activated, because its ligand has not yet been identified. RESULTS: We performed a genome-wide genetic screen for early zygotic genes and identified seven genomic regions that are required for normal migration of the mesoderm cells during gastrulation. One of these genomic intervals produces upon its deletion a phenocopy of the htl cell migration phenotype. Here we present the genetic and molecular mapping of this genomic region. We identified two genes, FGF8-like1 and FGF8-like2, that encode novel FGF homologs and were only partially annotated in the Drosophila genome. We show that FGF8-like1 and FGF8-like2 are expressed in the neuroectoderm during gastrulation and present evidence that both act in concert to direct cell shape changes during mesodermal cell migration and are required for the activation of the Htl signaling cascade during gastrulation. CONCLUSIONS: We conclude that FGF8-like1 and FGF8-like2 encode two novel Drosophila FGF homologs, which are required for mesodermal cell migration during gastrulation. Our results suggest that FGF8-like1 and FGF8-like2 represent ligands of the Htl FGF receptor.     

Mesodermal cell migration during Xenopus gastrulation

The adhesive glycoprotein fibronectin (FN), which is a component of the network of extracellular matrix fibrils on the inner surface of the blastocoel roof (BCR), has been proposed to play a major role in directing mesodermal cell migration during amphibian gastrulation. In the first part of this paper, the adhesion of Xenopus mesodermal cells to FN in vitro is examined. Cells from several mesoderm regions, which differ in developmental fate and morphogenetic activity, are able to bind specifically to the RGD cell-binding site of FN. Dorsal mesodermal cell adhesion to FN varies along the anterior-posterior (a-p) axis: adhesion is strongest in the anterior head mesoderm, and gradually decreases posteriorly. This a-p gradient of mesodermal adhesiveness to FN does not change during mesodermal involution, and is reflected in the morphology of mesodermal explants on FN. An a-p strip of mesoderm develops a spreading, leading anterior margin and a compact, retracting posterior end, thus moving slowly and directionally over the FN substrate at about 0.8 micron/min. Although dissociated cells from all levels of the dorsal mesodermal axis adhere to FN, only the anterior, leading prospective head mesoderm cells migrate as single cells on a FN substrate in vitro. Locomotion by means of lamelliform protrusions occurs at an average rate of about 1.5 micron/min. Cells of the more posterior axial mesoderm merely shift position at random without substantial net translocation, and preinvolution mesoderm cells are completely stationary. On the BCR, the in vivo substrate for mesodermal cell migration, dissociated prospective head mesoderm cells spread and migrate as on FN in vitro, at 2.2 microns/min. In the presence of an RGD peptide which inhibits cell-FN interaction, cells remain globular and do not spread. They are still motile, but change direction frequently, which leads to less efficient net translocation. Apparently, interaction with the RGD cell-binding site of FN and concomitant spreading of head mesoderm cells is required for the stabilization of cell locomotion. In contrast to the directional migration of the mesoderm cell population toward the animal pole in the embryo, the pathways of dissociated cells on the BCR are randomly oriented. Coherent explants of migratory mesoderm do not move at all on the BCR, although they translocate on FN in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

The spatio-temporal distribution of single-stranded breaks in nuclear DNA in sections of clawed toad embryos during gastrulation and neurulation

Spatial and temporal pattern and quantities of nicks in nuclear DNA during gastrulation and neurulation was studied using nick-translation in sections of Xenopus laevis embryos. Specific changes in the number of nicks in different mesoderm and ectoderm regions were detected during embryogenesis. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and inner ectoderm layer of early and middle gastrula. The gradient was inverted during transition from gastrula to neurula. At the same time dorso-ventral (in mesoderm) and ventro-dorsal (in outer ectoderm layer) gradients of nuclear labelling were increased. The intensity of nuclear labelling in all parts of embryo as a whole was remarkably higher during neurulation as compared with gastrulation. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and ectoderm during neurulation. A connection between the nicks and differentiation status of the cells during early embryogenesis in amphibians is suggested.     

SEM analysis of amphibian mesodermal migration

Summary At the end of gastrulation, the lateral mesoderm of amphibian embryos migrates ventrally between the ectoderm and the endoderm. The present study is an examination of the morphology of the leading cells of the mesodermal sheet and of the substratum over which they move (the inner surface of the ectoderm). The cells of the leading edge of the mesoderm are generally round, with very short and narrow flattened projections in the forward direction. These projections do not have a ruffled morphology, regardless of whether fixation is carried out before or after the ectoderm and mesoderm are dissected away from the endoderm. The inner surface of the ectoderm is covered with fine (450–500A) filamentous extracellular material and the ectoderm cells sometimes extend cytoplasmic processes (approx. 0.1 wide) onto the leading surface of the mesoderm or onto adjacent ectoderm cells. These studies indicate that the morphology of cell migration in amphibians is closer to that seen inFundulus than to that characteristic of chick or mammalian cells.This paper is dedicated to the memory of Mac V. Edds, Jr., who warmly encouraged the developmental biologists of the Pioneer Valley     

Functions for Drosophila brachyenteron and forkhead in mesoderm specification and cell signalling.

The visceral musculature of the larval midgut of Drosophila has a lattice-type structure and consists of an inner stratum of circular fibers and an outer stratum of longitudinal fibers. The longitudinal fibers originate from the posterior tip of the mesoderm anlage, which has been termed the caudal visceral mesoderm (CVM). In this study, we investigate the specification of the CVM and particularly the role of the Drosophila Brachyury-homologue brachyenteron. Supported by fork head, brachyenteron mediates the early specification of the CVM along with zinc-finger homeodomain protein-1. This is the first function described for brachyenteron or fork head in the mesoderm of Drosophila. The mode of cooperation resembles the interaction of the Xenopus homologues Xbra and Pintallavis. Another function of brachyenteron is to establish the surface properties of the CVM cells, which are essential for their orderly migration along the trunk-derived visceral mesoderm. During this movement, the CVM cells, under the control of brachyenteron, induce the formation of one muscle/pericardial precursor cell in each parasegment. We propose that the functions of brachyenteron in mesodermal development of Drosophila are comparable to the roles of the vertebrate Brachyury genes during gastrulation.     

The epithelial-mesenchymal transition of the Drosophila mesoderm requires the Rho GTP exchange factor Pebble

Drosophila pebble (pbl) encodes a Rho-family GTP exchange factor (GEF) required for cytokinesis. The accumulation of high levels of PBL protein during interphase and the developmentally regulated expression of pbl in mesodermal tissues suggested that the primary cytokinetic mutant phenotype might be masking other roles. Using various muscle differentiation markers, we found that Even skipped (EVE) expression in the dorsal mesoderm is greatly reduced in pbl mutant embryos. EVE expression in the dorsalmost mesodermal cells is induced in response to DPP secreted by the dorsal epidermal cells. Further analysis revealed that this phenotype is likely to be a consequence of an earlier defect. pbl mutant mesodermal cells fail to undergo the normal epithelial-mesenchymal transition (EMT) and dorsal migration that follows ventral furrow formation. This phenotype is not a secondary consequence of failed cytokinesis, as it is rescued by a mutant form of pbl that does not rescue the cytokinetic defect. In wild-type embryos, newly invaginated cells at the lateral edges of the mesoderm extend numerous protrusions. In pbl mutant embryos, however, cells appear more tightly adhered to their neighbours and extend very few protrusions. Consistent with the dependence of the mesoderm EMT and cytokinesis on actin organisation, the GTP exchange function of the PBL RhoGEF is required for both processes. By contrast, the N-terminal BRCT domains of PBL are required only for the cytokinetic function of PBL. These studies reveal that a novel PBL-mediated intracellular signalling pathway operates in mesodermal cells during the transition from an epithelial to migratory mesenchymal morphology during gastrulation.     

Shield formation at the onset of zebrafish gastrulation

During vertebrate gastrulation, the three germ layers, ectoderm, mesoderm and endoderm are formed, and the resulting progenitor cells are brought into the positions from which they will later contribute more complex tissues and organs. A core element in this process is the internalization of mesodermal and endodermal progenitors at the onset of gastrulation. Although many of the molecules that induce mesendoderm have been identified, much less is known about the cellular mechanisms underlying mesendodermal cell internalization and germ layer formation. Here we show that at the onset of zebrafish gastrulation, mesendodermal progenitors in dorsal/axial regions of the germ ring internalize by single cell delamination. Once internalized, mesendodermal progenitors upregulate E-Cadherin (Cadherin 1) expression, become increasingly motile and eventually migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. When E-Cadherin function is compromised, mesendodermal progenitors still internalize, but, with gastrulation proceeding, fail to elongate and efficiently migrate along the epiblast, whereas epiblast cells themselves exhibit reduced radial cell intercalation movements. This indicates that cadherin-mediated cell-cell adhesion is needed within the forming shield for both epiblast cell intercalation, and mesendodermal progenitor cell elongation and migration during zebrafish gastrulation. Our data provide insight into the cellular mechanisms underlying mesendodermal progenitor cell internalization and subsequent migration during zebrafish gastrulation, and the role of cadherin-mediated cell-cell adhesion in these processes.     

Xenopus embryonic cell adhesion to fibronectin: position-specific activation of RGD/synergy site-dependent migratory behavior at gastrulation

During Xenopus laevis gastrulation, the basic body plan of the embryo is generated by movement of the marginal zone cells of the blastula into the blastocoel cavity. This morphogenetic process involves cell adhesion to the extracellular matrix protein fibronectin (FN). Regions of FN required for the attachment and migration of involuting marginal zone (IMZ) cells were analyzed in vitro using FN fusion protein substrates. IMZ cell attachment to FN is mediated by the Arg-Gly-Asp (RGD) sequence located in the type III-10 repeat and by the Pro-Pro-Arg- Arg-Ala-Arg (PPRRAR) sequence in the type III-13 repeat of the Hep II domain. IMZ cells spread and migrate persistently on fusion proteins containing both the RGD and synergy site sequence Pro-Pro-Ser-Arg-Asn (PPSRN) located in the type III-9 repeat. Cell recognition of the synergy site is positionally regulated in the early embryo. During gastrulation, IMZ cells will spread and migrate on FN whereas presumptive pre-involuting mesoderm, vegetal pole endoderm, and animal cap ectoderm will not. However, animal cap ectoderm cells acquire the ability to spread and migrate on the RGD/synergy region when treated with the mesoderm inducing factor activin-A. These data suggest that mesoderm induction activates the position-specific recognition of the synergy site of FN in vivo. Moreover, we demonstrate the functional importance of this site using a monoclonal antibody that blocks synergy region-dependent cell spreading and migration on FN. Normal IMZ movement is perturbed when this antibody is injected into the blastocoel cavity indicating that IMZ cell interaction with the synergy region is required for normal gastrulation.     

FGF signalling and the mechanism of mesoderm spreading in Drosophila embryos

FGF signalling is needed for the proper establishment of the mesodermal cell layer in Drosophila embryos. The activation of the FGF receptor Heartless triggers the di-phosphorylation of MAPK in the mesoderm, which accumulates in a graded fashion with the highest levels seen at the dorsal edge of the mesoderm. We have examined the specific requirement for FGF signalling in the spreading process. We show that only the initial step of spreading, specifically the establishment of contact between the ectoderm and the mesoderm, depends upon FGF signalling, and that unlike the role of FGF signalling in the differentiation of heart precursors this function cannot be replaced by other receptor tyrosine kinases. The initiation of mesoderm spreading requires the FGF receptor to possess a functional kinase domain, but does not depend upon the activation of MAPK. Thus, the dispersal of the mesoderm at early stages is regulated by pathways downstream of the FGF receptor that are independent of the MAPK cascade. Furthermore, we demonstrate that the activation of MAPK by Heartless needs additional cues from the ectoderm. We propose that FGF signalling is required during the initial stages of mesoderm spreading to promote the efficient interaction of the mesoderm with the ectoderm rather than having a long range chemotactic function, and we discuss this in relation to the cellular mechanism of mesoderm spreading.     

Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.     

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.     

Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak.

Two mouse genes, Evx-1 and Evx-2, each encoding a homeodomain closely related to that of the Drosophila even-skipped gene were isolated using a PCR-based strategy. The structure and sequence of these genes are described. Mapping studies localized Evx-1 to chromosome 6, near the Hox-1 gene cluster, and Evx-2 to chromosome 2, near the Hox-4 cluster. The evolutionary implications of these linkages are discussed. RNA in situ hybridization analysis of Evx expression in embryos demonstrated a striking pattern of Evx-1 expression during gastrulation, whereas Evx-2 RNA could not be detected at any stage by this technique. Evx-1 RNA is first detected shortly before the onset of gastrulation in a region of ectoderm containing cells that will soon be found in the primitive streak. This localized expression of Evx-1 provides the first molecular evidence for regional differences in the mouse embryonic ectoderm before gastrulation. Throughout gastrulation, Evx-1 expression is limited to cells near and within the streak and that expression is graded, with a posterior-to-anterior decrease in the level of RNA. Based on fate-mapping studies indicating that different types of mesoderm emerge from different regions of the primitive streak and our observation that high levels of expression are localized to the region that will give rise to extraembryonic and ventral mesoderm, we speculate that Evx-1 plays a role in the dorsoventral specification of mesodermal cell fate.     

Generation of the germ layers along the animal-vegetal axis in Xenopus laevis

After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis.     

The role of FGF signaling in guiding coordinate movement of cell groups

Cell migration influences cell-cell interactions to drive cell differentiation and organogenesis. To support proper development, cell migration must be regulated both temporally and spatially. Mesoderm cell migration in the Drosophila embryo serves as an excellent model system to study how cell migration is controlled and influences organogenesis. First, mesoderm spreading transforms the embryo into a multilayered form during gastrulation and, subsequently, cells originating from the caudal visceral mesoderm (CVM) migrate along the entire length of the gut. Here we review our studies, which have focused on the role of fibroblast growth factor (FGF) signaling, and compare and contrast these two different cell migration processes: mesoderm spreading and CVM migration. In both cases, FGF acts as a chemoattractant to guide cells’ directional movement but is likely not the only signal that serves this role. Furthermore, FGF likely modulates cell adhesion properties since FGF mutant phenotypes share similarities with those of cell adhesion molecules. Our working hypothesis is that levels of FGF signaling differentially influence cells’ response to result in either directional movement or changes in adhesive properties.     

Sdf1/Cxcr4 signaling controls the dorsal migration of endodermal cells during zebrafish gastrulation

During vertebrate gastrulation, both mesodermal and endodermal cells internalize through the blastopore beneath the ectoderm. In zebrafish, the internalized mesodermal cells move towards the dorsal side of the gastrula and, at the same time, they extend anteriorly by convergence and extension (C&E) movements. Endodermal cells showing characteristic filopodia then migrate into the inner layer within the hypoblast next to the yolk syncytial layer (YSL). However, little is known about how the movement of endodermal cells is regulated during gastrulation. Here we show that sdf1a- and sdf1b-expressing mesodermal cells control the movements of the cxcr4a-expressing endodermal cells. The directional migration of endodermal cells during gastrulation is inhibited by knockdown of either cxcr4a or sdf1a/sdf1b (sdf1). We also show that misexpressed Sdf1 acts as a chemoattractant for cxcr4a-expressing endodermal cells. We further found, using the endoderm-specific transgenic line Tg(sox17:EGFP), that Sdf1/Cxcr4 signaling regulates both the formation and orientation of filopodial processes in endodermal cells. Moreover, the accumulation of phosphoinositide 3,4,5-trisphosphate (PIP(3)), which is known to occur at the leading edge of migrating cells, is not observed at the filopodia of endodermal cells. Based on our results, we propose that sdf1-expressing mesodermal cells, which overlie the endodermal layer, guide the cxcr4a-expressing endodermal cells to the dorsal side of the embryo during gastrulation, possibly through a PIP(3)-independent pathway.     

Drosophila ventral furrow morphogenesis: a proteomic analysis

Ventral furrow formation is a key morphogenetic event during Drosophila gastrulation that leads to the internalization of mesodermal precursors. While genetic analysis has revealed the genes involved in the specification of ventral furrow cells, few of the structural proteins that act as mediators of ventral cell behavior have been identified. A comparative proteomics approach employing difference gel electrophoresis was used to identify more than fifty proteins with altered abundance levels or isoform changes in ventralized versus lateralized embryos. Curiously, the majority of protein differences between these embryos appeared well before gastrulation, only a few protein changes coincided with gastrulation, suggesting that the ventral cells are primed for cell shape change. Three proteasome subunits were found to differ between ventralized and lateralized embryos. RNAi knockdown of these proteasome subunits and time-dependent difference-proteins caused ventral furrow defects, validating the role of these proteins in ventral furrow morphogenesis.     

The role of FGF signaling in guiding coordinate movement of cell groups: Guidance cue and cell adhesion regulator?

Cell migration influences cell-cell interactions to drive cell differentiation and organogenesis. To support proper development, cell migration must be regulated both temporally and spatially. Mesoderm cell migration in the Drosophila embryo serves as an excellent model system to study how cell migration is controlled and influences organogenesis. First, mesoderm spreading transforms the embryo into a multilayered form during gastrulation and, subsequently, cells originating from the caudal visceral mesoderm (CVM) migrate along the entire length of the gut. Here we review our studies, which have focused on the role of fibroblast growth factor (FGF) signaling, and compare and contrast these two different cell migration processes: mesoderm spreading and CVM migration. In both cases, FGF acts as a chemoattractant to guide cells’ directional movement but is likely not the only signal that serves this role. Furthermore, FGF likely modulates cell adhesion properties since FGF mutant phenotypes share similarities with those of cell adhesion molecules. Our working hypothesis is that levels of FGF signaling differentially influence cells’ response to result in either directional movement or changes in adhesive properties.