Axis Of Sea Urchin Embryos Research Articles

Canonical and non-canonical Wnt signaling pathways define the expression domains of Frizzled 5/8 and Frizzled 1/2/7 along the early anterior-posterior axis in sea urchin embryos

The spatiotemporal expression of Frizzled receptors is critical for patterning along the early anterior-posterior axis during embryonic development in many animal species. However, the molecular mechanisms that regulate the expression of Frizzled receptors are incompletely understood in any species. In this study, I examine how the expression of two Frizzled receptors, Fzl1/2/7 and Fzl5/8, is controlled by the Wnt signaling network which directs specification and positioning of early regulatory states along the anterior-posterior (AP) axis of sea urchin embryos. I used a combination of morpholino- and dominant negative-mediated interference to knock down each Wnt signaling pathway involved in the AP Wnt signaling network. I found that the expression of zygotic fzl5/8 as well as that of the anterior neuroectoderm gene regulatory network (ANE GRN) is activated by an unknown broadly expressed regulatory state and that posterior Wnt/β-catenin signaling is necessary to down regulate fzl5/8's expression in posterior blastomeres. I show that zygotic expression of fzl1/2/7 in the equatorial ectodermal belt is dependent on an uncharacterized regulatory mechanism that works in the same cells receiving the TGF-β signals patterning this territory along the dorsal-ventral axis. In addition, my data indicate that Fzl1/2/7 signaling represses its own expression in a negative feedback mechanism. Finally, we discovered that a balance between the activities of posterior Wnt8 and anterior Dkk1 is necessary to establish the correct spatial expression of zygotic fzl12/7 expression in the equatorial ectodermal domain during blastula and gastrula stages. Together, these studies lead to a better understanding of the complex interactions among the three Wnt signaling pathway governing AP axis specification and patterning in sea urchin embryos.

Differential regulation of disheveled in a novel vegetal cortical domain in sea urchin eggs and embryos: implications for the localized activation of canonical Wnt signaling.

Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved cytoarchitectural domain that specifies the AV axis in metazoan ova.

Structure, regulation, and function of micro1 in the sea urchin Hemicentrotus pulcherrimus.

The animal-vegetal axis of sea urchin embryos is morphologically apparent at the 16-cell stage, when the mesomeres, macromeres, and micromeres align along it. At this stage, the micromere is the only autonomously specified blastomere that functions as a signaling center. We used a subtraction PCR survey to identify the homeobox gene micro1 as a micromere-specific gene. The micro1 gene is a representative of a novel family of paired-like class homeobox genes, along with PlHbox12 from Paracentrotus lividus and pmar1 from Strongylocentrotus purpuratus. In the present study, we showed that micro1 is a multicopy gene with six or more polymorphic loci, at least three of which are clustered in a 30-kb region of the genome. The micro1 gene is transiently expressed during early cleavage stages in the micromere. Recently, nuclear beta-catenin was shown to be essential for the specification of vegetal cell fates, including micromeres, and the temporal and spatial coincidence of micro1 expression with the nuclear entry of beta-catenin is highly suggestive. We demonstrated that micro1 is a direct target of beta-catenin. In addition, we showed that micro1 is necessary and sufficient for micromere specification. These observations on the structure, regulation, and function of micro1 lead to the conclusion that micro1 and pmar1 (and potentially PlHbox12) are orthologous.

Specification of secondary mesenchyme-derived cells in relation to the dorso-ventral axis in sea urchin blastulae.

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg2-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg2 descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.

Transient activation of the micro1 homeobox gene family in the sea urchin ( Hemicentrotus pulcherrimus) micromere.

The animal-vegetal (A-V) axis of sea urchin embryos is morphologically evident at the 16-cell stage of development. Mesomeres, macromeres, and micromeres are arrayed along the A-V axis. The vegetal micromere differentiates into the skeletogenic mesenchyme and functions as a signaling center. To date, no zygotic or maternally specified gene with restricted expression in the micromere at the 16-cell stage has been reported. We performed subtraction PCR and dot blot hybridization using poly(A)+ RNA extracted from the micromere (tester) and the mesomere (driver) in order to identify micromere-specific genes. Using a cDNA fragment identified in this screen, we isolated four similar but distinct cDNA clones from a library, which corresponded to a group of genes that we refer to as the micro1 family. The micro1 family encoded putative transcription factors with a homeodomain which had 87-95% identity between family members. The most highly conserved protein was encoded by PlHbox12 from Paracentrotus lividus (71-76% identity among family members). Northern blot hybridization and in situ hybridization demonstrated that micro1 was transiently activated during the early cleavage stages and that the transcript was restricted to the micromere. Thus, the expression domain was complementary to that of PlHbox12 along the A-V axis. The micro1 gene family has at least six loci, including polymorphic alleles, which are probably clustered in the genome. PlHbox12 and micro1 constitute a novel family of paired-like class homeobox genes. Phylogenetic analyses suggest that PlHbox12/micro1 evolved exceptionally rapidly.

Micromere descendants at the blastula stage are involved in normal archenteron formation in sea urchin embryos.

Several lines of evidence suggest that micromere signaling plays a key role in endo-mesoderm differentiation along the animal-vegetal (A-V) axis in sea urchin embryos. A recent study has suggested that the activity of micromeres of inducing endoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula stage in the echinoids Scaphechinus mirabiris and Hemicentrotus pulcherrimus. In the present study, to confirm the inductive capacity of the micromere descendants in normal development, the timing of initiation of gastrulation and the elongation rate of the archenteron were examined in both micromereless embryos and in micromereless embryos cultured until the hatching blastula stage and then recombined with micromere descendants of the same age. The micromereless embryos consistently exhibited a delay in the initiation of gastrulation and a decrease in elongation rate of the archenteron, as compared with those in controls. In contrast, when the micromereless embryos cultured until the hatching blastula stage were recombined with micromere descendants of the same age, the recombinant embryos exhibited rescue of both the delay in initiation of gastrulation and a decrease in elongation rate of the archenteron. The delayed expression of alkaline phosphatase activity, an endoderm-specific marker, in the micromereless embryos was also rescued in the recombinant embryos. The recombined micromere descendants formed the larval spicules in the same schedule as that observed in the controls. These results indicate that at the hatching blastula stage, micromere descendants emanate a signal(s) required for normal gastrulation of the presumptive endo-mesodermal region.

Competence of the animal cap to react with the inductive signal from micromere descendants in the hatching blastula stage of echinoid embryos

Micromere signalling is a key to understanding the developmental mechanisms underlying endomesoderm differentiation along the A-V axis in sea urchin embryos. A recent study has shown that micromere activity in inducing endo-mesoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula (HB) stage in the echinoids Scaphechinus mirabilis and Hemicentrotus pulcherrimus. This study focused mainly on the timing of emission of the inductive signal by the micromere descendants. The timing of animal cap competent to react to the inductive signal from micromeres was not specifically investigated.In the present study, we examined the competence of mesomere descendants at the HB stage to react to the inductive signal of micromere descendants by producing recombinant embryos of the descendants of mesomeres and micromeres.Adults of the sand dollar Scaphechinus mirabilis and the sea urchin Hemicentrotus pulcherrimus were collected along the shore of Shiraishi Island, Okayama Prefecture and in the vicinity of the Misaki Marine Biological Station, Kanagawa Prefecture, respectively. The fertilised eggs were separated into two groups immediately after removal of the fertilisation membranes. One group was cultured in normal artificial seawater (ASW), and the other in ASW containing 50 mg/ml of rhodamine B isothiocyanate (RITC). At the early 16-cell stage unlabelled embryos were transferred to calcium-free seawater (CFSW) and dissected in the equatorial plane with a glass needle to isolate animal caps consisting of eight mesomeres. Embryos labeled with RITC were also transferred to CFSW at the same stage and dissected to isolate four micromeres. An isolated animal cap and a quartet of micromeres were cultured separately in ASW and recombined at the stage corresponding to the HB of control (undisturbed) embryos.

Identification of a new sea urchin ets protein, SpEts4, by yeast one-hybrid screening with the hatching enzyme promoter.

We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE. This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity. To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element. This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4. This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments. The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output. As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula.

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.

Multiple PositivecisElements Regulate the Asymmetric Expression of theSpHEGene along the Sea Urchin Embryo Animal–Vegetal Axis

The mechanism that establishes the maternally determined animal–vegetal axis of sea urchin embryos is unknown. We have analyzed thecis-regulatory elements of theSpHEgene ofStrongylocentrotus purpuratus,which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C.,Dev. Biol.171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negativeciselement is responsible for the lack of vegetalSpHEtranscription. Surprisingly, even short segments of the regulatory region containing only several identifiedciselements also direct nonvegetal expression. Furthermore, theSpHEbasal promoter functions effectively in vegetal cells in combination withcis-acting elements derived from the PMC-specific gene,SM50.We conclude that the spatial pattern ofSpHEtranscription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression ofSM50also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H.,Development121, 1957–1970, 1995). A chimeric promoter containing bothSpHEandSM50regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which theSpHEandSM50genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early embryo.

Early mRNAs, spatially restricted along the animal-vegetal axis of sea urchin embryos, include one encoding a protein related to tolloid and BMP-1.

The cloning and characterization of cDNAs representing four genes or small gene families that are coordinately expressed in a spatially restricted pattern during the very early blastula (VEB) stage of sea urchin development are presented. The VEB genes encode multiple transcripts that are expressed transiently in embryos of Strongylocentrotus purpuratus between 16-cell stage and hatching, with peak abundance 12 to 15 hours post-fertilization (approximately 150-250 cells). The VEB transcripts share the same spatial pattern in the early blastula embryo: they are asymmetrically distributed along the animal-vegetal axis but their distribution around this axis is uniform. Thus, the VEB transcripts are the earliest messages to reveal asymmetry along the primary axis in the sea urchin embryo. The temporal and spatial patterns of VEB transcript accumulation are not consistent with involvement of these gene products in cell division or in tissue-specific functions. Furthermore, VEB messages cannot be detected in either ovary or adult tissues, suggesting that these genes function exclusively during embryogenesis. We suggest that the VEB genes function in constructing the early blastula. Two VEB genes encode metalloendoproteases: one (SpHE) is hatching enzyme and the other (SpAN) is similar to bone morphogenetic protein-1 (BMP-1; Wozney et al., Science 242: 1528-1534, 1988) and the Tolloid gene product (tld) (Shimell et al., Cell 67: 459-482, 1991). Several lines of evidence suggest that the VEB genes are regulated directly by factors or regulatory activities localized along the maternally specificed animal-vegetal axis.

Determination of dorso-ventral axis in early embryos of the sea urchin, Hemicentrotus pulcherrimus

To elucidate a relationship between early cleavage planes and dorso-ventral (DV)-axis of sea urchin embryos, a fluorescent dye, Lucifer Yellow CH, was iontophoretically introduced into one blastomere at the 2-cell stage, and the location of the progeny cells was determined in the half-labeled prism larvae by examining the embryos from the animal pole. The boundary plane which divides the embryonic tissue into the labeled and nonlabeled parts was (1) coincident with, (2) perpendicular to, or (3) obliquely crossing the larval plane of bilateral symmetry. The oblique boundaries took only two angles mutually symmetrical with regard to the DV-axis of embryos. Combining these labeling patterns, the tissue of prism larvae could be divided into 8 sectors around the animal-vegetal axis. When the 2-cell stage embryos with different diameters of sister blastomeres were labeled with the dye, one end of the boundary plane was again found at one of the 8 boundary points noticed in equally cleaved embryos, while the other was observed to fall in the middle of a sector. These results indicate that the DV-axis of the embryo is established according to the spatial arrangement of blastomeres during the 5–6th cleavage stages when blastomeres align in 8 rows in meridional direction. It was also suggested that intercellular communication takes part in the determination of the fate of individual founder blastomeres during the two subsequent cleavages, i.e., 7–8th cleavage stages.