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

Members of the Tcf/Lef family interact with beta-catenin to activate programs of gene expression during development. Recently beta-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 beta-catenin became localized to nuclei of vegetal blastomeres, consistent with its acting in combination with beta-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 beta-catenin was prevented. Our results suggest that SpTcf/Lef functions by interacting with beta-catenin to specify cell fates along the sea urchin animal-vegetal axis.

Requirement of SpOtx in cell fate decisions in the sea urchin embryo and possible role as a mediator of beta-catenin signaling.

We show here that the homeodomain transcription factor SpOtx is required for endoderm and aboral ectoderm formation during sea urchin embryogenesis. SpOtx target genes were repressed by fusing the SpOtx homeodomain to an active repression domain of Drosophila Engrailed. The Engrailed-SpOtx fusion protein reduced the expression of endoderm- and aboral ectoderm-specific genes and inhibited the formation of endoderm and aboral ectoderm cell types. Coexpressing activated beta-catenin with Engrailed-SpOtx did not overcome the inhibition of endoderm and aboral ectoderm formation, suggesting that SpOtx functioned either downstream of or parallel to nuclear beta-catenin. Embryos expressing C-cadherin, which blocks nuclear translocation of beta-catenin, have defects in endoderm and aboral ectoderm formation. Coexpressing SpOtx with C-cadherin restored aboral ectoderm-specific gene expression and aboral ectoderm morphology, but with C-cadherin present, SpOtx was not sufficient for endoderm formation. Our results show that SpOtx plays a key role in the activation of aboral ectoderm- and endoderm-specific gene expression and, in addition, suggest that SpOtx mediates some of beta-catenin's functions in endoderm and aboral ectoderm formation.

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.

Disruption of gastrulation and oral-aboral ectoderm differentiation in the Lytechinus pictus embryo by a dominant/negative PDGF receptor.

Little is known about the cell signaling involved in forming the body plan of the sea urchin embryo. Previous work suggested that PDGF-like and EGF-like receptor-mediated signaling pathways are involved in gastrulation and spiculogenesis in the Lytechinus pictus embryo. Here we show that expression of the human PDGF receptor-beta lacking the cytoplasmic domain disrupted development in a manner consistent with a dominant/negative mechanism. The truncated PDGF receptor-beta inhibited gut and spicule formation and differentiation along the oral-aboral axis. The most severely affected embryos arrested at a developmental stage resembling mesenchyme blastula. Coinjection into eggs of RNA encoding the entire human PDGF receptor-beta rescued development. The truncated PDGF receptor-beta caused the aboral ectoderm-specific genes LpS1 and LpC2 to be repressed while an oral ectoderm-specific gene, Ecto-V, was expressed in all ectoderm cells. The results support the hypothesis that a PDGF-like signaling pathway plays a key role in the intercellular communication required for gastrulation and spiculogenesis, and in cell commitment and differentiation along the oral-aboral axis.

Multiple signaling events specify ectoderm and pattern the oral-aboral axis in the sea urchin embryo.

In the sea urchin embryo, the animal-vegetal axis is established during oogenesis and the oral-aboral axis is specified sometime after fertilization. The mechanisms by which either of these axes are specified and patterned during embryogenesis are poorly understood. Here, we investigated the role of cellular interactions in the specification of the ectoderm territories and polarization of the ectoderm along the oral-aboral axis. Isolated animal halves (mesomeres), which are fated to give rise to oral and aboral ectoderm, developed into polarized embryoids that expressed an oral ectoderm-specific marker uniformly. These embryoids also produced neuron-like cells and serotonergic neurons, suggesting that mesomeres are autonomously specified as oral ectoderm. Mesomere-derived embryoids did not express any aboral ectoderm-specific markers, although we previously showed that aboral ectoderm-specific genes can be induced by 25 mM lithium chloride, which also induced endoderm formation (Wikramanayake, A. H., Brandhorst, B. P. and Klein, W. H.(1995). Development 121, 1497-1505). To ascertain if endoderm formation is a prerequisite for induction of aboral ectoderm by lithium and for normal ectoderm patterning in animal halves, we modulated the lithium treatment to ensure that no endoderm formed. Remarkably, treating animal halves with 10 mM LiCl at approximately 7 hours postfertilization resulted in embryoids that displayed oral-aboral axis patterning in the absence of endoderm. Application of 25 mM LiCl to animal halves at approximately 16 hours postfertilization, which also did not induce endoderm, resulted in polarized expression of the aboral ectoderm-specific LpS1 protein, but global expression of the Ecto V antigen and no induction of the stomodeum or ciliary band. These results suggest that at least two signals, a positive inductive signal to specify the aboral ectoderm and a negative suppressive signal to inactivate oral ectoderm-specific genes in the prospective aboral ectoderm territory, are needed for correct spatial expression of oral and aboral ectoderm-specific genes. Transmission of both these signals may be prerequisite for induction of secondary ectodermal structures such as the ciliary band and stomodeum. Thus, differentiation of ectoderm and polarization of the oral-aboral axis in Lytechinus pictus depends on cellular interactions with vegetal blastomeres as well as interactions along the oral-aboral axis.

Very early and transient vegetal-plate expression of SpKrox1, a Krüppel/Krox gene from Stronglyocentrotus purpuratus.

All endodermal and mesenchymal cells of the sea urchin embryo descend from the vegetal plate, a thickened epithelium of approximately 50 cells arising at the early blastula stage. Cell types that derive from the vegetal plate are specified conditionally by inductive interactions with underlying micromeres, but the molecular details of vegetal-plate specification remain unresolved. In a search for regulatory proteins that have roles in vegetal-plate specification, a screen was performed to clone Krüppel/Krox-related genes from a Strongylocentrotus purpuratus embryo cDNA library. One newly identified clone, named SpKrox1, contained four zinc fingers and a leucine zipper domain. SpKrox1 expression was low in unfertilized eggs, increased severalfold to the early blastula stage and decreased between the early gastrula and pluteus stages. SpKrox1 mRNA was first seen in macromeres of 16-cell stage embryos and was restricted to cells of the developing vegetal plate thereafter. Vegetal-plate expression corresponded to a ring of cells around the blastopore and overlapped the expression patterns of other genes with potential roles in vegetal plate-specification. As the vegetal-plate cells invaginated into the blastopore, SpKrox1 expression was lost, suggesting that its role was not in endoderm differentiation per se but rather in the initial establishment of the vegetal plate.

Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein.

While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified. SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene. Here, we present evidence that SpOtx has the potential to alter cell fates. SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation. Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate. At least three distinct domains on SpOtx, the homeobox and regions in the N-terminal and C-terminal halves of the protein, were required for the morphological alterations. These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor. Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate.

Transient appearance of Strongylocentrotus purpuratus Otx in micromere nuclei: cytoplasmic retention of SpOtx possibly mediated through an alpha-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 alpha-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 alpha-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 alpha-actinin. The transient appearance of SpOtx in micromere nuclei may be associated with the specification of micromere cell fate.

Autonomous and non-autonomous differentiation of ectoderm in different sea urchin species.

During early embryogenesis, the highly regulative sea urchin embryo relies extensively on cell-cell interactions for cellular specification. Here, the role of cellular interactions in the temporal and spatial expression of markers for oral and aboral ectoderm in Strongylocentrotus purpuratus and Lytechinus pictus was investigated. When pairs of mesomeres or animal caps, which are fated to give rise to ectoderm, were isolated and cultured they developed into ciliated embryoids that were morphologically polarized. In animal explants from S. purpuratus, the aboral ectoderm-specific Spec1 gene was activated at the same time as in control embryos and at relatively high levels. The Spec1 protein was restricted to the squamous epithelial cells in the embryoids suggesting that an oral-aboral axis formed and aboral ectoderm differentiation occurred correctly. However, the Ecto V protein, a marker for oral ectoderm differentiation, was detected throughout the embryoid and no stomodeum or ciliary band formed. These results indicated that animal explants from S. purpuratus were autonomous in their ability to form an oral-aboral axis and to differentiate aboral ectoderm, but other aspects of ectoderm differentiation require interaction with vegetal blastomeres. In contrast to S. purpuratus, aboral ectoderm-specific genes were not expressed in animal explants from L. pictus even though the resulting embryoids were morphologically very similar to those of S. purpuratus. Recombination of the explants with vegetal blastomeres or exposure to the vegetalizing agent LiCl restored activity of aboral ectoderm-specific genes, suggesting the requirement of a vegetal induction for differentiation of aboral ectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)