GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo

In the sea urchin embryo, the animal-vegetal axis is defined before fertilization and different embryonic territories are established along this axis by mechanisms which are largely unknown. Significantly, the boundaries of these territories can be shifted by treatment with various reagents including zinc and lithium. We have isolated and characterized a sea urchin homolog of GSK3beta/shaggy, a lithium-sensitive kinase which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos including Xenopus. The effects of overexpressing the normal and mutant forms of GSK3beta derived either from sea urchin or Xenopus were analyzed by observation of the morphology of 48 hour embryos (pluteus stage) and by monitoring spatial expression of the hatching enzyme (HE) gene, a very early gene whose expression is restricted to an animal domain with a sharp border roughly coinciding with the future ectoderm / endoderm boundary. Inactive forms of GSK3beta predicted to have a dominant-negative activity, vegetalized the embryo and decreased the size of the HE expression domain, apparently by shifting the boundary towards the animal pole. These effects are similar to, but even stronger than, those of lithium. Conversely, overexpression of wild-type GSK3beta animalized the embryo and caused the HE domain to enlarge towards the vegetal pole. Unlike zinc treatment, GSK3beta overexpression thus appeared to provoke a true animalization, through extension of the presumptive ectoderm territory. These results indicate that in sea urchin embryos the level of GSKbeta activity controls the position of the boundary between the presumptive ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos. GSK3beta and probably other downstream components of the Wnt pathway thus mediate patterning both along the primary AV axis of the sea urchin embryo and along the dorsal-ventral axis in Xenopus, suggesting a conserved basis for axial patterning between invertebrate and vertebrate in deuterostomes.     

beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal-vegetal axis in sea urchin embryos are largely unknown. Nuclear beta-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that beta-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that beta-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal-vegetal axis. Our results also reveal similarities between the sea urchin animal-vegetal axis and the vertebrate dorsal-ventral axis, suggesting that these axes share a common evolutionary origin.     

The betaL integrin subunit is necessary for gastrulation in sea urchin embryos

Integrins are a family of cell adhesion molecules reported to mediate cellular interactions essential for normal embryonic morphogenesis. Here we describe a beta integrin subunit that is expressed during early embryogenesis in the sea urchin embryo and appears to be necessary for normal development. The deduced amino acid sequence of betaL is similar to vertebrate beta integrin subunits, but is most closely related to the sea urchin betaG subunit. Northern blots show that betaL is expressed at all stages with maximum expression beginning during gastrulation. Immunolocalization and in situ RNA hybridization show that in blastulae betaL is expressed in the blastoderm and by the ring of bottle cells in the vegetal plate during the initial phase of gastrulation. Presumptive secondary mesenchyme cells express high levels of betaL throughout elongation of the archenteron and in the pluteus betaL is expressed by blastocoelar cells, skeletal mesenchyme, and pigment cells. Antibodies and Fab fragments against betaL block spreading of dissociated embryonic cells on RGD (arginine-glycine-aspartate)-containing substrates. Treating embryos with anti-betaL antibodies blocks the initial phase of gastrulation and interferes with the organization of actin filaments. Prior to gastrulation, the antibodies cause thickening of the blastoderm and later in development defects in skeletal patterning result. Probing for antibody in treated embryos indicates that it penetrates the ectoderm to cells within the blastocoel and is actively endocytosed. We propose that betaL forms receptors that bind to RGD-containing ligands and anchors actin filaments. These receptors appear to be essential in several aspects of morphogenesis.     

FGF is a prospective competence factor for early activin-type signals in Xenopus mesoderm induction

Normal pattern formation during embryonic development requires the regulation of cellular competence to respond to inductive signals. In the Xenopus blastula, vegetal cells release mesoderm-inducing factors but themselves become endoderm, suggesting that vegetal cells may be prevented from expressing mesodermal genes in response to the signals that they secrete. We show here that addition of low levels of basic fibroblast growth factor (bFGF) induces the ectopic expression of the mesodermal markers Xbra, MyoD and muscle actin in vegetal explants, even though vegetal cells express low levels of the FGF receptor. Activin, a potent mesoderm-inducing agent in explanted ectoderm (animal explants), does not induce ectopic expression of these markers in vegetal explants. However, activin-type signaling is present in vegetal cells, since the vegetal expression of Mix.1 and goosecoid is inhibited by the truncated activin receptor. These results, together with the observation that FGF is required for mesoderm induction by activin, support our proposal that a maternal FGF acts at the equator as a competence factor, permitting equatorial cells to express mesoderm in response to an activin-type signal. The overlap of FGF and activin-type signaling is proposed to restrict mesoderm to the equatorial region.     

Anterior endoderm is a specific effector of terminal cardiac myocyte differentiation of cells from the embryonic heart forming region

The ability of anterior lateral plate mesoderm cells in the heart-forming region (HFR) of stage 6 chicken embryos to respond to cardiogenic stimuli from cells in adjacent germ layers has been investigated using explants cultured under defined conditions. Two types of explantation were evaluated: those in which two germ layers were explanted in contiguity, and those in which germ layers were isolated and co-cultured. Two parameters--contractility and expression of sarcomeric alpha-actin--were monitored to evaluate the terminal differentiation of cardiac myocytes. Contiguously explanted anterior endoderm/mesoderm became multilayered and underwent terminal differentiation within 2 days. By contrast, although contiguous anterior ectoderm/mesoderm or posterior endoderm/mesoderm co-explants also became multilayered, these explants did not differentiate, up to 5 days. To ascertain the cardiogenic potential of cells from different regions of the embryo, individual germ layers were isolated and co-cultured by placing the explants in separate areas of the culture chamber. These determinations demonstrated that anterior, but not posterior, endoderm effected differentiation of anterior mesoderm. As before, mesoderm in both types of co-culture survived and became multilayered; by contrast, mesoderm did not survive when cultured in isolation. These experiments provide evidence that anterior endoderm regulates the terminal differentiation, as opposed to growth, of presumptive cardiac myocytes in mesoderm cells from the anterior lateral plate. Finally, anterior endoderm was co-cultured with mesoderm from the posterior half of the embryo, which does not contain an HFR. The failure of these co-cultured explants to differentiate infers that pre-cardiac myoblasts in stage 6 anterior mesoderm are previously specified to respond to the terminal cardiogenic effects of endoderm.     

Alterations in colonic mucosal vessels in patients with cirrhosis and noncirrhotic portal hypertension

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus. The protein has been named Pl-200K or Hp-200K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

The origins of primitive blood in Xenopus: implications for axial patterning

The marginal zone in Xenopus laevis is proposed to be patterned with dorsal mesoderm situated near the upper blastoporal lip and ventral mesoderm near the lower blastoporal lip. We determined the origins of the ventralmost mesoderm, primitive blood, and show it arises from all vegetal blastomeres at the 32-cell stage, including blastomere C1, a progenitor of Spemann's organizer. This demonstrates that cells located at the upper blastoporal lip become ventral mesoderm, not solely dorsal mesoderm as previously believed. Reassessment of extant fate maps shows dorsal mesoderm and dorsal endoderm descend from the animal region of the marginal zone, whereas ventral mesoderm descends from the vegetal region of the marginal zone, and ventral endoderm descends from cells located vegetal of the bottle cells. Thus, the orientation of the dorsal-ventral axis of the mesoderm and endoderm is rotated 90( degrees) from its current portrayal in fate maps. This reassessment leads us to propose revisions in the nomenclature of the marginal zone and the orientation of the axes in pre-gastrula Xenopus embryos.     

Origin and development of the zebrafish endoderm

The segregation of cells into germ layers is one of the earliest events in the establishment of cell fate in the embryo. In the zebrafish, endoderm and mesoderm are derived from cells that involute into an internal layer, the hypoblast, whereas ectoderm is derived from cells that remain in the outer layer, the epiblast. In this study, we examine the origin of the zebrafish endoderm and its separation from the mesoderm. By labeling individual cells located at the margin of the blastula, we demonstrate that all structures that are endodermal in origin are derived predominantly from the more dorsal and lateral cells of the blastoderm margin. Frequently marginal cells give rise to both endodermal and mesodermal derivatives, demonstrating that these two lineages have not yet separated. Cells located farther than 4 cell diameters from the margin give rise exclusively to mesoderm, and not to endoderm. Following involution, we see a variety of cellular changes indicating the differentiation of the two germ layers. Endodermal cells gradually flatten and extend filopodial processes forming a noncontiguous inner layer of cells against the yolk. At this time, they also begin to express Forkhead-domain 2 protein. Mesodermal cells form a coherent layer of round cells separating the endoderm and ectoderm. In cyclops-mutant embryos that have reduced mesodermal anlage, we demonstrate that by late gastrulation not only mesodermal but also endodermal cells are fewer in number. This suggests that a common pathway initially specifies germ layers together before a progressive sequence of determinative events segregate endoderm and mesoderm into morphologically distinct germ layers.     

Analysis of intracellular trafficking and interactions of cytoplasmic HIV-1 Rev mutants in living cells

Single blastomeres of the sixth-cleavage veg1 and veg2 tiers of Strongylocentrotus purpuratus embryos were labeled with DiI lineage tracer, and the disposition of the progeny was followed through the blastula and gastrula stages in order to determine their respective endodermal and ectodermal contributions. In the endoderm of postgastrula embryos, veg1-derived cells constituted nearly all of the prospective hindgut and about half of the prospective midgut, while veg2-derived cells made up the prospective foregut and half the midgut. Oral veg1 clones consistently contributed more cells to endoderm than aboral veg1 clones. Oral veg1 clones extended along the archenteron up to the foregut region, while aboral veg1 clones contributed only small numbers of hindgut cells but large patches of ectoderm cells that extended out to the prospective larval vertex. The oral/aboral asymmetry in veg1 allocations was also demonstrated using chimeric embryos, the animal halves of which were labeled with a rhodamine-dextran. Lineages expressing the vegetal plate marker Endo16 were more precisely determined by combining lineage tracer injection with whole-mount in situ hybridization. Endo16 expression was found in all cells that are going to participate in gastrulation. Recruitment of new cells to the Endo16 domain occurs in advance of the actual invagination of those cells. During the blastula stages Endo16 expression expands radially until all cells in the veg2 lineages express this gene. The first phase of gastrulation, including the normal buckling of the vegetal plate and primary invagination of the archenteron, involves only the Endo16-expressing cells of the veg2 lineages. As the archenteron begins to elongate, marking the onset of the second phase of gastrulation, there is an asymmetric expansion of Endo16 into the veg1-derived cells that will contribute to the hindgut and midgut in accordance with lineage tracing observations. The results indicate a relatively late specification of veg1-derived cells, resulting in late recruitment to the periphery of the vegetal plate territory as gastrulation proceeds. Differential recruitment of veg1-derived cells on the oral side of the embryo introduces an oral bias to gastrulation by disproportionately increasing the number of cells on the oral side that are competent to participate in gastrulation.     

Analysis of 3- and 6-linked sialic acids in mixtures of gangliosides using blotting to polyvinylidene difluoride membranes, binding assays, and various mass spectrometry techniques with application to recognition by Helicobacter pylori

We report the isolation and characterization of a new inhibitory Smad in Xenopus, which we have designated as Xenopus Smad7. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-beta-like signals is mediated by receptor ALK-4 and we show that Smad7 blocks signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway.     

Differentiation of myoendocrine cardiac cells from presumptive heart mesoderm explants of Bufo arenarum

To investigate which factors are involved in the differentiation of Bufo arenarum heart myoendocrine cells, we studied the distribution of Atrial Natriuretic Peptide (ANP) immunoreactivity in hearts formed from presumptive cardiac mesoderm explanted at early embryo stages. Explants isolated from different embryonic stages throughout neurulation were cultured in vitro with or without the pharyngeal endoderm, and in other cases transplanted to the caudal region of embryos at similar stages. We utilized immunohistochemical and morphological techniques to assess myoendocrine cardiac cell differentiation. Development of heart beat and positive tropomyosin immunolabeling were considered signs of cardiac tissue differentiation. Our results confirm that explants of cardiac mesoderm cultured with endoderm showed a greater and more complete level of cardiac differentiation than those of mesoderm alone, and this coincides with the staining pattern of tropomyosin. ANP immunostaining revealed that cardiac cells containing ANP were scarce in those cultures without endoderm. On the contrary, in both cultured and grafted explants containing endoderm, ANP immunostaining was intensive and well-distributed in the cardiac tube, and in some cases restricted to one side of the formed heart. We conclude that the endoderm regulates cardiac cell differentiation, and in this way, is involved in the development of the heart myoendocrine system.     

Neurulation in amniote vertebrates: a novel view deduced from the use of quail-chick chimeras

Two apparently different mechanisms successively contribute to the formation of the neural tube in the avian embryo: bending of the neural plate during the primary neurulation in the cephalo-cervico-thoracic region and cavitation of the medullary cord during the secondary neurulation in the lumbo-sacral region. During both these processes, gastrulation continues by the caudal regression of Hensen's node--also called cordoneural hinge in the secondary neurulation. Labeling of Hensen's node or cordoneural hinge by the quail chick marker system revealed that this structure, which is the equivalent of the dorsal blastoporal lip of the Amphibian embryo, i.e., of the Spemann's organizer, gives rise to the midline cells of the three germ layers: the floor plate of the neural tube, the notocord and the dorsal cells of the intestinal endoderm. Caudally to the organizer, both in primary and secondary neurulation, the presumptive territory of the alar plates of the future neural tube overlies the precursors of the paraxial mesoderm. Regression of Hensen's node bisects the ectoderm in two bilateral neural plates leaving in its wake the floor plate, the notocord and the dorsal endoderm.     

The one-eyed pinhead gene functions in mesoderm and endoderm formation in zebrafish and interacts with no tail

The zebrafish locus one-eyed pinhead (oep) is essential for the formation of anterior axial mesoderm, endoderm and ventral neuroectoderm. At the beginning of gastrulation anterior axial mesoderm cells form the prechordal plate and express goosecoid (gsc) in wild-type embryos. In oep mutants the prechordal plate does not form and gsc expression is not maintained. Exposure to lithium, a dorsalizing agent, leads to the ectopic induction and maintenance of gsc expression in wild-type embryos. Lithium treatment of oep mutants still leads to ectopic gsc induction but not maintenance, suggesting that oep acts downstream of inducers of dorsal mesoderm. In genetic mosaics, wild-type cells are capable of forming anterior axial mesoderm in oep embryos, suggesting that oep is required in prospective anterior axial mesoderm cells before gastrulation. The oep gene is also essential for endoderm formation and the early development of ventral neuroectoderm, including the floor plate. The loss of endoderm is already manifest during gastrulation by the absence of axial-expressing cells in the hypoblast of oep mutants. These findings suggest that oep is also required in lateral and ventral regions of the gastrula margin. The sonic hedgehog (shh).gene is expressed in the notochord of oep animals. Therefore, the impaired floor plate development in oep mutants is not caused by the absence of the floor plate inducer shh. This suggests that oep is required downstream or in parallel to shh signaling. The ventral region of the forebrain is also absent in oep mutants, leading to severe cyclopia. In contrast, anterior-posterior brain patterning appears largely unaffected, suggesting that underlying prechordal plate is not required for anterior-posterior pattern formation but might be involved in dorsoventral brain patterning. To test if oep has a wider, partially redundant role, we constructed double mutants with two other zebrafish loci essential for patterning during gastrulation. Double mutants with floating head, the zebrafish Xnot homologue, display enhanced floor plate and adaxial muscle phenotypes. Double mutants with no tail (ntl), the zebrafish homologue of the mouse Brachyury locus, display severe defects in midline and mesoderm formation including absence of most of the somitic mesoderm. These results reveal a redundant function of oep and ntl in mesoderm formation. Our data suggest that both oep and ntl act in the blastoderm margin to specify mesendodermal cell fates.     

Patterning the Xenopus blastula

This review starts from the classical standpoint that there are at least two separable processes acting with respect to axis formation and tissue specification in the early Xenopus embryo: a UV-insensitive event establishing a postgastrula embryo consisting of three concentric germ layers, ectoderm, mesoderm and endoderm, all of a ventral character; and a UV-sensitive event producing tissue of a dorsal type, including somites, notochord and neural tissue, and concomitantly establishing the dorsoventral and anteroposterior axes. The experimental evidence suggesting the molecular basis of the dorsal and ventral pathways is reviewed.     

Expression of type IV collagen-degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis

Type IV collagen-degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600-fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non-collagenous proteins of the extracellular matrix. Chelators such as 1,10-phenanthroline or Na2EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen-degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus-larva stage. The specific inhibitors of collagen biosynthesis 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide and tricyclodecane-9-yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen-degrading activity to non-detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen-degrading activity.