LvNotch signaling plays a dual role in regulating the position of the ectoderm-endoderm boundary in the sea urchin embryo

The molecular mechanisms guiding the positioning of the ectoderm-endoderm boundary along the animal-vegetal axis of the sea urchin embryo remain largely unknown. We report here a role for the sea urchin homolog of the Notch receptor, LvNotch, in mediating the position of this boundary. Overexpression of an activated form of LvNotch throughout the embryo shifts the ectoderm-endoderm boundary more animally along the animal-vegetal axis, whereas expression of a dominant negative form shifts the border vegetally. Mosaic experiments that target activated and dominant negative forms of LvNotch into individual blastomeres of the early embryo, combined with lineage analyses, further reveal that LvNotch signaling mediates the position of this boundary by distinct mechanisms within the animal versus vegetal portions of the embryo. In the animal region of the embryo, LvNotch signaling acts cell autonomously to promote endoderm formation more animally, while in the vegetal portion, LvNotch signaling also promotes the ectoderm-endoderm boundary more animally, but through a cell non-autonomous mechanism. We further demonstrate that vegetal LvNotch signaling controls the localization of nuclear beta-catenin at the ectoderm-endoderm boundary. Based on these results, we propose that LvNotch signaling promotes the position of the ectoderm-endoderm boundary more animally via two mechanisms: (1) a cell-autonomous function within the animal region of the embryo, and (2) a cell non-autonomous role in the vegetal region that regulates a signal(s) mediating ectoderm-endoderm position, possibly through the control of nuclear beta-catenin at the boundary.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

Prospective fate of early blastomeres in Hydractinia echinata (Cnidaria,Hydrozoa)

Summary Blastomeres of two-cell, four-cell, and eight-cell embryos of Hydractinia echinata were injected with horseradish-peroxidase (HRP) or fluorescein isothiocyanate (FITC)-dextran. The fate of the descendants of the injected blastomeres was followed until the planula larva had developed. The results obtained after HRP or FITC-dextran injection were essentially the same. Blastomeres are equivalent up to the four-cell stage, i.e. half-blastomeres produce half of the ectoderm of the planula larva and quarter-blastomeres give rise to one quarter of the larval ectoderm. During normal embryogenesis, the larval anterior-posterior axis corresponds to the animal-vegetal axis of the zygote. Thus, the labelled areas of larvae consisting of the progeny of injected half or quarter blastomeres normally stretch along the larval anterior-posterior axis. Normally, material giving rise to anterior or posterior larval parts, respectively, is separated at the third cleavage. Irrespective of the type of experiment, the progeny of injected blastomeres always contributed to endoderm formation, i.e. in larvae resulting from injected embryos the endoderm was more or less uniformly labelled. Application of vital stains locally to the exterior of zygotes and following these markers through first and second cleavage, produced evidence that in the vast majority of cases, the second cleavage is meridional. Offprint requests to: A. Schlawny     

A micromere induction signal is activated by beta-catenin and acts through notch to initiate specification of secondary mesenchyme cells in the sea urchin embryo

At fourth cleavage of sea urchin embryos four micromeres at the vegetal pole separate from four macromeres just above them in an unequal cleavage. The micromeres have the capacity to induce a second axis if transplanted to the animal pole and the absence of micromeres at the vegetal pole results in the failure of macromere progeny to specify secondary mesenchyme cells (SMCs). This suggests that micromeres have the capacity to induce SMCs. We demonstrate that micromeres require nuclear beta-catenin to exhibit SMC induction activity. Transplantation studies show that much of the vegetal hemisphere is competent to receive the induction signal. The micromeres induce SMCs, most likely through direct contact with macromere progeny, or at most a cell diameter away. The induction is quantitative in that more SMCs are induced by four micromeres than by one. Temporal studies show that the induction signal is passed from the micromeres to macromere progeny between the eighth and tenth cleavage. If micromeres are removed from hosts at the fourth cleavage, SMC induction in hosts is rescued if they later receive transplanted micromeres between the eighth and tenth cleavage. After the tenth cleavage addition of induction-competent micromeres to micromereless embryos fails to specify SMCs. For macromere progeny to be competent to receive the micromere induction signal, beta-catenin must enter macromere nuclei. The macromere progeny receive the micromere induction signal through the Notch receptor. Signaling-competent micromeres fail to induce SMCs if macromeres express dominant-negative Notch. Expression of an activated Notch construct in macromeres rescues SMC specification in the absence of induction-competent micromeres. These data are consistent with a model whereby beta-catenin enters the nuclei of micromeres and, as a consequence, the micromeres produce an inductive ligand. Between the eighth and tenth cleavage micromeres induce SMCs through Notch. In order to be receptive to the micromere inductive signal the macromeres first must transport beta-catenin to their nuclei, and as one consequence the Notch pathway becomes competent to receive the micromere induction signal, and to transduce that signal. As Notch is maternally expressed in macromeres, additional components must be downstream of nuclear beta-catenin in macromeres for these cells to receive and transduce the micromere induction signal.     

Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma

The sea urchin Heliocidaris erythrogramma undergoes direct development, bypassing the usual echinoid pluteus larva. We present an analysis of cell lineage in H. erythrogramma as part of a definition of the mechanistic basis for this evolutionary change in developmental mode. Microinjection of fluoresceinated tracer dye and surface marking with vital dye are used to follow larval fates of 2-cell, 8-cell, and 16-cell blastomeres, and to examine axial specification. The animal-vegetal axis and adult dorsoventral axis are basically unmodified in H. erythrogramma. Animal cell fates are very similar to those of typically developing species; however, vegetal cell fates in H. erythrogramma are substantially altered. Radial differences exist among vegetal blastomere fates in the 8-cell embryo: dorsal vegetal blastomeres contribute proportionately more descendants to ectodermal and fewer to mesodermal fates, while ventral vegetal blastomeres have a complementary bias in fates. In addition, vegetal cell fates are more variable than in typical developers. There are no cells in H. erythrogramma with fates comparable to those of the micromeres and macromeres of typically developing echinoids. Instead, all vegetal cells in the 16-cell embryo can contribute progeny to ectoderm and gut. Alterations have thus arisen in cleavage patterns and timing of cell lineage partitioning during the evolution of direct development in H. erythrogramma.     

The role of animal-vegetal interaction with respect to the determination of dorsoventral polarity in the equal-cleaving spiralian,Lymnaea palustris

Summary Spirally cleaving embryos in which the first two cleavages generate four equal-sized blastomeres remain radially symmetrical along their animal-vegetal axis until the interval between third and fourth quartet formation. At this time animal micromeres and vegetal macromeres contact each other as they elongate and occlude the central, fluid-filled cleavage cavity. The overlying micromeres focus their contacts onto one of the four macromeres, the presumptive 3D macromere, as it elongates to a central position within the embryo. We tested the hypothesis that this animal-vegetal interaction was causally involved in the determination of the symmetry properties in both the animal and vegetal hemispheres by reversibly inhibiting animal-vegetal contact at the 24 cell stage with cytochalasin-B. Embryos remained hollow throughout the treatment period and animal-vegetal interaction did not occur. After treatment, blastomere elongation occurred but no D quadrant macromere appeared and the vegetal hemisphere remained radialized. On the basis of cleavage and ciliation patterns of first quartet derivatives, treated embryos remained fully or partially radialized, showing a strong tendancy to develop as ventral quadrants. These results show that the quadrants of this equal-cleaving spiralian are not definitively determined until after the 24 cell stage and that animal-vegetal interaction is required for D quadrant determination. The mechanisms of symmetrization in the animal and vegetal hemispheres of equal-cleaving spiralians is also discussed.     

Differential stability of beta-catenin along the animal-vegetal axis of the sea urchin embryo mediated by dishevelled

beta-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. Here, we use in-vivo measurements of the half-life of fluorescently tagged beta-catenin in specific blastomeres to demonstrate a gradient in beta-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3beta-mediated phosphorylation of beta-catenin. Calculations show that the difference in beta-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. We show that dishevelled (Dsh), a key signaling protein, is required for the stabilization of beta-catenin in vegetal cells and provide evidence that Dsh undergoes a local activation in the vegetal region of the embryo. Finally, we report that GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus.     

Primary mesenchyme cell patterning during the early stages following ingression

Sea urchin primary mesenchyme cells (PMCs) ingress into the blastocoel during an epithelial-to-mesenchymal transition (EMT), migrate along the blastocoelar wall for a period of time, and then settle into a subequatorial ring to form the larval skeleton. Fluorescent-marked blastomeres alone, or in combination with blastomere recombination, were used to track the position of PMCs during the early phases of this movement. Micromeres expressing Golgi-tethered GFP (galtase-GFP) were transplanted onto TRITC-stained hosts (in place of the endogenous micromere) to observe the progeny of a single micromere. Galtase-GFP as a Golgi marker is not transferred between PMCs when the syncytium forms. Thus, the position of cells can be followed relative to beginning position for longer periods than previously reported. The PMC progeny of a single micromere do not disperse upon ingression, but instead remain in a closely associated cluster. Generally, progeny of a single micromere remain in the quadrant of origin. In total, greater than approximately 94% of labeled PMCs remain within the local region of ingression. By contrast, when a transplanted micromere is placed at the vegetal plate after removing all 4 host micromeres, the resultant PMCs ingress and migrate into all 4 quadrants. Similarly, if 1 blastomere is injected at the 2-cell stage, and later the 2 unlabeled micromeres are removed at the 16-cell stage, the remaining PMCs ingress into all 4 quadrants of the vegetal plate. We conclude that the normal restriction of PMCs to a quadrant is due to mechanical constraint from other micromere-PMCs. If a labeled micromere is placed ectopically at the macromere/mesomere boundary, the PMC progeny ingress ectopically and migrate longitudinally along the animal-vegetal axis only. Injection of galtase-GFP into one blastomere at the 4-cell stage shows a 2-step pattern of localization. At late mesenchyme blastula and early gastrula stages, greater than 90% of GFP-expressing PMCs remain in the injected quadrant, while at mid- to late-gastrula stage and beyond, more PMCs are found outside the injected quadrant. The migration that sets up the asymmetry of the larval skeleton first occurs around mid- to late-gastrula stages, when some PMCs from an aboral quadrant migrate to the adjacent oral quadrant. In all, these data combined with previous data suggest that freshly ingressed PMCs migrate along a longitudinal path toward the animal pole and back toward the vegetal pole. Beginning at mid- to late-gastrula stage, PMCs utilize oral-aboral cues from the ectoderm for the first time. At this time, some aboral PMCs migrate into the adjacent oral quadrant to assist in the formation of the ventrolateral cluster.     

Involvement of Tcf/Lef in establishing cell types along the animal-vegetal axis of sea urchins

Members of the Tcf/Lef family interact with β-catenin to activate programs of gene expression during development. Recently β-catenin was shown to be essential for establishing cell fate along the animal-vegetal axis of the sea urchin embryo. To examine the role of Tcf/Lef in sea urchins we cloned a Strongylocentrotus purpuratus Tcf/Lef homolog. Expression of SpTcf/Lef was maximal when β-catenin became localized to nuclei of vegetal blastomeres, consistent with its acting in combination with β-catenin to specify vegetal cell fates. Expression of a dominant-negative SpTcf/Lef inhibited primary and secondary mesenchyma, endoderm, and aboral ectoderm formation in a manner similar to that observed when nuclear accumulation of β-catenin was prevented. Our results suggest that SpTcf/Lef functions by interacting with β-catenin to specify cell fates along the sea urchin animal-vegetal axis. Received: 6 July 1999 / Accepted: 27 August 1999     

Transient appearance of Strongylocentrotus purpuratus Otx in micromere nuclei: Cytoplasmic retention of SpOtx possibly mediated through an α-actinin interaction

At the 16-cell stage, the sea urchin embryo is partitioned along the animal-vegetal axis into eight mesomeres, four macromeres, and four micromeres. The micromeres, unlike the other blastomeres, are autonomously specified to produce skeletogenic mesenchymal cells and are also required to induce the vegetal-plate territory. A long-held belief is that micromeres inherit localized maternal determinants that endow them with their cell autonomous behavior and inducing capabilities. Here, we present evidence that an orthodenticle-related protein, SpOtx appears transiently in nuclei of micromeres but not in nuclei of mesomeres and macromeres. At later stages of development, SpOtx was translocated into nuclei of all cells. To address the possibility that SpOtx was retained In the cytoplasm at early developmental stages we searched for cytoplasmic proteins that interact with SpOtx. A proline-rich region of SpOtx resembling an SH3-binding domain was used to screen an embryo cDNA expression library, and a cDNA clone was isolated and shown to be α-actinin. A yeast two-hybrid analysis showed a specific interaction between the proline-rich region of SpOtx and a putative SH3 domain of the sea urchin α-actinin. Because micromeres lack an actin-based cytoskeleton, the results suggested that, at the vegetal pole of the 16-cell stage embryo, SpOtx was translocated into micromere nuclei, whereas in other blastomeres SpOtx was actively retained in the cytoplasm by binding to α-actinin. The transient appearance of SpOtx in micromere nuclei may be associated with the specification of micromere cell fate. © 1996 Wiley-Liss, Inc.     

Mesoderm and ectoderm lineages in the crustacean Parhyale hawaiensis display intra-germ layer compensation

In Parhyale hawaiensis, the first three divisions are holoblastic and asymmetric, resulting in an embryo comprised of eight cells—four macromeres and four micromeres. Lineage studies performed at this stage demonstrate that the progeny of each cell contribute to specific portions of different germ layers. However, it is not known if this lineage pattern means a given blastomere is committed to its specific fate, indicative of mosaic development, or if regulation can occur between blastomere progeny so that the loss of a blastomere could be compensated for during development. Furthermore, if compensation occurs, what would be the source of such replacement? To investigate these possibilities, we performed ablation experiments at the eight-cell stage. We find that loss of blastomeres results in compensation. To determine the compensation pattern, we combined ablation and cell lineage tracing to reveal that progeny of mesoderm and ectoderm producing blastomeres display intra-germ layer compensation. Furthermore, by ablating lineages later in development, we identify a key interval between gastrulation and germband elongation after which compensation no longer occurs. Our results suggest that Parhyale possesses a mechanism to assess the status of mesoderm and ectoderm formation and alter development to replace the missing portions of these lineages.     

Isolation and characterization of beta-catenin downstream genes in early embryos of the ascidian Ciona savignyi

Nuclear localization of beta-catenin is most likely the first step of embryonic axis formation or embryonic cell specification in a wide variety of animal groups. Therefore, the elucidation of beta-catenin target genes is a key research subject in understanding the molecular mechanisms of the early embryogenesis of animals. In Ciona savignyi embryos, nuclear accumulation of beta-catenin is the first step of endodermal cell specification. Previous subtractive hybridization screens of mRNAs between beta-catenin-overexpressed embryos and nuclear beta-catenin-depleted embryos have resulted in the identification of beta-catenin downstream genes in Ciona embryos. In the present study, I characterize seven additional beta-catenin downstream genes, Cs-cadherinII, Cs-protocadherin, Cs-Eph, Cs-betaCD1, Cs-netrin, Cs-frizzled3/6, and Cs-lefty/antivin. All of these genes were expressed in vegetal blastomeres between the 16-cell and 110-cell stages, although their spatial and temporal expression patterns were different from one another. In situ hybridizations and real-time PCR revealed that the expression of all of these genes was up-regulated in beta-catenin-overexpressed embryos, and down-regulated in beta-catenin-suppressed embryos. Therefore, the accumulation of beta-catenin in the nuclei of vegetal blastomeres activates various vegetally expressed genes with potentially important functions in the specification of these cells.     

Localized activity of Ca2+-stimulated ATPase and transcellular ionic currents during mesoderm induction in embryos ofLymnaea stagnalis (Mollusca)

Summary InLymnaea stagnalis, mesoderm induction occurs at the 24-cell stage, when the apical tip of the macromere 3D establishes a close contact with a number of micromeres. Via its tip, the macromere 3D is supposed to receive an inductive signal from the micromeres, resulting in the determination of the mesodermal stem cell 4d at the next division. In view of the possibility that transcellular ionic currents might somehow be involved, either in the processes that bring about this particular configuration of blastomeres or in the induction process itself, we mapped the electric field around the embryo during the 24-cell stage, using a vibrating probe. We detected a reversal of the current direction as compared to the uncleaved egg, whilst the polarity of the field along the animal-vegetal axis was maintained. We also mapped the localization of Ca²⁺-stimulated AT-Pase, an enzyme that drives the Ca²⁺-efflux from the cell. We found that this enzyme is localized exclusively along the cytoplasmic face of the apical plasma membrane of macromere 3D, and that its presence is restricted to the period from 110 to 135 min after the fifth cleavage, when there is close contact between macormere 3D and the micromeres. Since the localization of the Ca²⁺-stimulated ATPase coincides both in time and space with the induction of the mesoderm-mother cell, we suggest that localized calcium fluxes may play a role in this induction process.     

Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four-armed plutei. After feeding, the larvae developed into eight-armed plutei. The left-right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal-vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five-fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.     

Deuterostome evolution: early development in the enteropneust hemichordate, Ptychodera flava

SUMMARY Molecular and morphological comparisons indicate that the Echinodermata and Hemichordata represent closely related sister‐phyla within the Deuterostomia. Much less is known about the development of the hemichordates compared to other deuterostomes. For the first time, cell lineage analyses have been carried out for an indirect‐developing representative of the enteropneust hemichordates, Pty‐ chodera flava. Single blastomeres were iontophoretically labeled with DiI at the 2‐ through 16‐cell stages, and their fates followed through development to the tornaria larval stage. The early cleavage pattern of P. flava is similar to that of the direct‐developing hemichordate, Saccoglossus kowalevskii, as well as that displayed by indirect‐developing echinoids. The 16‐celled embryo contains eight animal “mesomeres,” four slightly larger “macromeres,” and four somewhat smaller vegetal “micromeres.” The first cleavage plane was not found to bear one specific relationship relative to the larval dorsoventral axis. Although individual blastomeres generate discrete clones of cells, the appearance and exact locations of these clones are variable with respect to the embryonic dorsoventral and bilateral axes. The eight animal mesomeres generate anterior (animal) ectoderm of the larva, which includes the apical organ; however, contributions to the apical organ were found to be variable as only a subset of the animal blastomeres end up contributing to its formation and this varies from embryo to embryo. The macromeres generate posterior larval ectoderm, and the vegetal micromeres form all the internal, endomesodermal tissues. These blastomere contributions are similar to those found during development of the only other hemichordate studied, the direct‐developing enteropneust, S. kowalevskii. Finally, isolated blastomeres prepared at either the two‐ or the four‐cell stage are capable of forming normal‐appearing, miniature tornaria larvae. These findings indicate that the fates of these cells and embryonic dorsoventral axial properties are not committed at these early stages of development. Comparisons with the developmental programs of other deuterostome phyla allow one to speculate on the conservation of some key developmental events/mechanisms and propose basal character states shared by the ancestor of echinoderms and hemichordates.