Mesenchyme Blastula Stage Research Articles

Sphedgehog is expressed by pigment cell precursors during early gastrulation in Strongylocentrotus purpuratus.

We have sequenced the Sphedgehog (Sphh) gene from the sea urchin Strongylocentrotus purpuratus. Sphh transcripts are detected first at the mesenchyme blastula stage, and they accumulate throughout early embryogenesis. The Sphh protein is produced by precursor pigment cells during early and midgastrulation. NiCl2 inhibits pigment cell differentiation in sea urchins. Here, we show that, in S. purpuratus, nickel affects a process(es) between 17 and 24 hr of development, corresponding to the time period when Sphh mRNA is first detected. However, nickel treatment does not alter the early expression of Sphh.

LvTbx2/3: a T-box family transcription factor involved in formation of the oral/aboral axis of the sea urchin embryo.

T-box family transcription factors have been identified in many organisms and are frequently associated with patterning events during embryonic development. With an interest in the molecular basis of patterning in the sea urchin embryo, we identified several members of the T-box family in Lytechinus variegatus. Here, we report the cloning and characterization of an ortholog of the Tbx2/3 subfamily, LvTbx2/3. To characterize the spatial distribution of LvTbx2/3 protein throughout sea urchin embryogenesis, a polyclonal antiserum was generated. Nuclear localization of LvTbx2/3 initiated at the mesenchyme blastula stage and protein was present into the pluteus stage. Localization was asymmetric throughout this period and costaining with marker genes indicated that asymmetry was about the oral/aboral (O/A) axis. Asymmetric distribution of LvTbx2/3 was observed in the aboral territories of all three germ layers. In the skeletogenic mesoderm lineage, LvTbx2/3 expression was dynamic because expression appeared initially in all skeletogenic mesenchyme cells (PMCs) but, subsequently, became refined solely to the aboral ones during skeletogenesis. To determine if the aboral expression of LvTbx2/3 is linked between germ layers, and to place LvTbx2/3 in the sequence of events that specifies the O/A axis, the effects of a series of perturbations to O/A polarity on LvTbx2/3 expression in each germ layer were examined. Preventing the nuclear localization of beta-catenin, pharmacological disruption of the O/A axis with NiCl(2), overexpression of BMP2/4 and disruption of the extracellular matrix all blocked LvTbx2/3 expression in all germ layers. This indicates that expression of LvTbx2/3 in the aboral territories of each germ layer is a common aspect of O/A specification, downstream of the molecular events that specify the axis. Furthermore, blocking the nuclear localization of beta-catenin, overexpression of BMP2/4 and disruption of the extracellular matrix also prevented the oral (stomodael) expression of LvBrachyury (LvBrac) protein, indicating that the O/A axis is established by a complex series of events. Last, the function of LvTbx2/3 in the formation of the O/A axis was characterized by examining the phenotypic consequences of ectopic expression of LvTbx2/3 mRNA on embryonic development and the expression of marker genes that identify specific germ layers and tissues. Ectopic expression of LvTbx2/3 produced profound morphogenetic defects in derivatives of each germ layer with no apparent loss in specification events in those tissues. This indicates that LvTbx2/3 functions as a regulator of morphogenetic movements in the aboral compartments of the ectoderm, endoderm and mesoderm.

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 ∼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.

T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo.

Signals from micromere descendants play a crucial role in sea urchin development. In this study, we demonstrate that these micromere descendants express HpTb, a T-brain homolog of Hemicentrotus pulcherrimus. HpTb is expressed transiently from the hatched blastula stage through the mesenchyme blastula stage to the gastrula stage. By a combination of embryo microsurgery and antisense morpholino experiments, we show that HpTb is involved in the production of archenteron induction signals. However, HpTb is not involved in the production of signals responsible for the specification of secondary mesenchyme cells, the initial specification of primary mesenchyme cells, or the specification of endoderm. HpTb expression is controlled by nuclear localization of beta-catenin, suggesting that HpTb is in a downstream component of the Wnt signaling cascade. We also propose the possibility that HpTb is involved in the cascade responsible for the production of signals required for the spicule formation as well as signals from the vegetal hemisphere required for the differentiation of aboral ectoderm.

The expression of SpRunt during sea urchin embryogenesis

The runt box (runx) is a highly conserved DNA binding and protein–protein interaction domain that defines a family of heterodimeric transcription factors that regulate development in metazoans. The three mammalian runx genes are oncogenes with essential functions in normal development: Runx1 is required for hematopoiesis and is frequently mutated in human and murine leukemias; Runx2 is required for bone development and is associated with human cleidocranial dysplasia and murine leukemias; and Runx3 (the evolutionarily basal member of the mammalian family) regulates growth of the gut and functions as a tumor suppressor in the gastric epithelium ( Westendorf and Hiebert, 1999; Li et al., 2002). The sea urchin Strongylocentrotus purpuratus contains a single runx gene, SpRunt. We present here the initial structural characterization of SpRunt, and its pattern of expression during embryogenesis. SpRunt contains two introns, the locations of which are identical to those of the second and third introns from promoter P2 of the mammalian runx genes. A ∼6 kb transcript begins to accumulate during cleavage. At mesenchyme blastula stage, SpRunt transcripts are found throughout the embryo, but specifically enriched in the vegetal plate, skeletogenic mesenchyme, and part of the ectoderm. By late gastrula stage expression is localized to the endomesoderm and oral ectoderm. In the pluteus larva SpRunt transcripts remain confined to the endomesoderm and oral ectoderm, with highest levels of accumulation in the foregut and in the ciliary band. These data suggest that SpRunt expression is enhanced in proliferating cells.

New Early Zygotic Regulators Expressed in Endomesoderm of Sea Urchin Embryos Discovered by Differential Array Hybridization

Genes that are upregulated by LiCl treatment of sea urchin embryos and/or downregulated by injection into the egg of mRNA encoding an internal fragment of cadherin (Cad) were detected in a differential macroarray screen. The method was that recently described by J. P. Rast et al. (2000, Dev. Biol. 228, 270–296). Almost 105 clones from a 12-h cDNA library were screened. Measurements on internal standards showed that the screening procedure was sufficiently sensitive to afford detection of differentially expressed mRNAs of the most rare class, those present in only a few copies per average cell. The injection of Cad mRNA, which specifically blocks nuclearization of β-catenin, resulted in many-fold decreases in the levels of transcripts of a suite of marker genes expressed zygotically during endomesoderm specification. These measurements substantiated the use of Cad mRNA as the basis for a differential screen for discovery of new endomesodermal genes. By use of the newly developed BioArray software for analysis of macroarray screens, 1106 clones representing differentially expressed genes and yielding useful sequence were recovered. The 367 clones that gave significant BLASTX matches to known cellular proteins fell into 264 nonredundant sequence classes. Those of particular interest for this work were clones encoding DNA-binding transcription factors, signal transduction pathway components, proteases, kinases, and phosphatases. Quantitative PCR analysis of 66 such selected clones revealed that the large majority of these clones had been selected because they are upregulated by LiCl treatment, which affects the expression of a much greater diversity and number of genes than are involved in endomesoderm specification. Seven transcript species were identified that responded sharply to injection of Cad mRNA, and that are not represented in maternal mRNA. Six of those encode transcription factors. We focused on three transcription factor genes of this set that were previously unknown in sea urchin embryos. By whole-mount in situ hybridization, these genes are expressed in specific domains of the endomesodermal territory. They are: (1) Speve, an evenskipped orthologue expressed very early in all vegetal blastomeres and then gradually shifting to veg1 derivatives by the mesenchyme blastula stage; (2) Spgcm, an orthologue of the fruit fly gene glial cells missing, which is first expressed specifically and exclusively in part of the prospective secondary mesenchyme (mesodermal) domain at late-cleavage blastula stage; and (3) Spfoxc, which is first expressed in the early blastula only in the four small micromeres, and later only expressed in that coelomic pouch which gives rise to the mesoderm of the ventral surface of the adult rudiment.

LvDelta is a mesoderm-inducing signal in the sea urchin embryo and can endow blastomeres with organizer-like properties.

Signals from micromere descendants play a critical role in patterning the early sea urchin embryo. Previous work demonstrated a link between the induction of mesoderm by micromere descendants and the Notch signaling pathway. In this study, we demonstrate that these micromere descendants express LvDelta, a ligand for the Notch receptor. LvDelta is expressed by micromere descendants during the blastula stage, a time when signaling has been shown to occur. By a combination of embryo microsurgery, mRNA injection and antisense morpholino experiments, we show that expression of LvDelta by micromere descendants is both necessary and sufficient for the development of two mesodermal cell types, pigment cells and blastocoelar cells. We also demonstrate that LvDelta is expressed by macromere descendants during mesenchyme blastula and early gastrula stages. Macromere-derived LvDelta is necessary for blastocoelar cell and muscle cell development. Finally, we find that expression of LvDelta is sufficient to endow blastomeres with the ability to function as a vegetal organizing center and to coordinate the development of a complete pluteus larva.

The Role of Brachyury (T) during Gastrulation Movements in the Sea Urchin Lytechinus variegatus

The studies described here sought to identify and characterize genes involved in the gastrulation and morphogenetic movements that occur during sea urchin embryogenesis. An orthologue of the T-box family transcription factor, Brachyury, was cloned through a candidate gene approach. Brachyury (T) is the founding member of this T-box transcription factor family and has been implicated in gastrulation movements in Xenopus, zebrafish, and mouse embryogenesis. Polyclonal serum was generated to LvBrac in order to characterize protein expression. LvBrac initially appears at mesenchyme blastula stage in two distinct regions with embryonic expression perduring until pluteus stage. Vegetally, LvBrac expression is in endoderm and lies circumferentially around the blastopore. This torus-shaped area of LvBrac expression remains constant in size as endoderm cells express LvBrac upon moving into that circumference and cease LvBrac expression as they leave the circumference. Vegetal expression remains around the anus through pluteus stage. The second domain of LvBrac expression first appears broadly in the oral ectoderm at mesenchyme blastula stage and at later embryonic stages is refined to just the stomodael opening. Vegetal LvBrac expression depends on autonomous β-catenin signaling in macromeres and does not require micromere or veg2-inductive signals. It was then determined that LvBrac is necessary for the morphogenetic movements occurring in both expression regions. A dominant-interfering construct was generated by fusing the DNA binding domain of LvBrac to the transcriptional repression module of the Drosophila Engrailed gene in order to perturb gene function. Microinjection of mRNA encoding this LvBrac-EN construct resulted in a block in gastrulation movements but not expression of endoderm and mesoderm marker genes. Furthermore, injection of LvBrac-EN into one of two blastomeres resulted in normal gastrulation movements of tissues derived from the injected blastomere, indicating that LvBrac downstream function may be nonautonomous during sea urchin gastrulation.

Wnt gene expression in sea urchin development: heterochronies associated with the evolution of developmental mode.

The Wnt genes encode a large family of conserved secreted proteins that are widely involved in animal development. The variety and ubiquity of this ancient family suggest that Wnt genes may have been important in the evolution of animal development, including early development. To test this hypothesis, we have characterized the expression of several Wnt genes in closely related sea urchins that exhibit radically different modes of early development. Wnt-1, -4, and -5 genes exhibit several conserved molecular and developmental characteristics, both within sea urchins and with Wnt genes examined in other animals (Ferkowicz et al. 1998). Here, we demonstrate that sea urchin Wnt-5 transcripts are specifically detected by in situ hybridization in discrete embryonic, larval, and developing adult tissues and processes: (1) in a band of vegetal ectoderm in mesenchyme blastula stage embryos, (2) in the larval ciliary bands, (3) in tissues that form the early adult rudiment (left coelomic pouch and overlying vestibular ectoderm), and (4) in the developing adult radial nervous system. We find that the sites of Wnt-5 transcript accumulation are conserved in species exhibiting either indirect- or direct-developmental modes, suggesting that Wnt-5 function(s) have been conserved in sea urchin development. However, dramatic heterochronic changes in Wnt-5 gene expression have occurred in the direct-developing species that parallel the accelerated morphological changes that occur during direct development. These results suggest that heterochronic changes in the expression of conserved developmental regulatory genes, such as the Wnt family members, are agents of evolutionary change in animal development.

Cloning and Characterization of αP Integrin in Embryos of the Sea Urchin Strongylocentrotus purpuratus

Differentially expressed integrins have been shown to be involved in the intricate cell movements that occur during early development. Because the migration and movement of cells have been well characterized in sea urchin embryos, we searched for α-integrin subunits in this organism. An α integrin subunit, αP, was cloned from Strongylocentrotus purpuratus mesenchyme blastula stage mRNA by RT-PCR and RACE and found to exhibit 74–77% sequence similarity to mammalian α5, α8, αIIb, and αv integrin. The 8-kb transcript was most abundant at the prism stage, although low levels could be detected at all stages by Northern blot analysis and RT-PCR. A polyclonal antibody to this novel integrin was generated against a 100-amino-acid αP fragment fused to glutathione S-transferase and shown to recognize a 180-kDa α-integrin in the egg and in all stages of embryogenesis studied.

Origin and behaviour of pigment cells in sea urchin embryos

Sea urchin pluteus larvae contain dozens of pigment cells in their ectoderm. These pigment cells are the descendants of the veg2 blastomeres of the 60-cell stage embryo. According to the fate map made by Ruffins and Ettensohn, the prospective pigment cells occupy the central region of the vegetal plate. Most of these prospective pigment cells exclusively give rise to pigment cells. Therefore, specification of the pigment cell lineage should occur at some point between the 60-cell and mesenchyme blastula stage. However, the detailed process of the specification of the pigment lineage is unknown.When are pigment cells specified? Are cell interactions necessary for the specification? Do founder cells exist? To answer these questions, I treated embryos with Ca2+-free seawater during the cleavage stage and examined the number of pigment cells observed in pluteus larvae. Treatment at 5.5–8.5 h and especially 7.5–10.5 h postfertilisation markedly reduced the number of pigment cells. The decrease was statistically significant. On the other hand, the treatment at 3.5–6.5 h or 9.5–12.5 h never reduced the number of pigment cells. By examining the frequency of the appearance of embryos whose numbers of pigment cells were less than 20, it was also found that the numbers of pigment cells were frequently in multiples of 4. Embryos having 4, 8, 12, 16 and 20 pigment cells were more frequently observed. Statistics indicated that the frequency of appearance was not random. These results indicated that cell contacts are necessary for the specification of pigment cells and that the specification occurs from 7 to 10 h postfertilisation. The results also suggest that the founder cells, if they exist, divide twice before they differentiate into pigment cells.

Gene expression in the endoderm during sea urchin development

Understanding how the embryonic germ layers become competent to form their characteristic tissue types is a problem of fundamental importance to developmental biology. Knowledge of how the endodermal layer is first determined and then differentiates has only recently begun to accumulate. In sea urchins, several different signals have been implicated in endoderm formation, beginning as early as the fourth cleavage division and continuing until just prior to invagination of the endoderm. Recent experiments in sea urchin embryos have shown that the activity of glycogen synthase kinase 3-β and entry of β-catenin into the nucleus during cleavage stages is required for mesoderm and endoderm formation (Emily-Fenouilet al., 1998; Loganet al., 1999), implicating the Wnt signalling pathway in this process. Overexpression of β-catenin leads to an exaggeration of endoderm and mesoderm in the embryo at the expense of ectoderm (Wikramanayakeet al., 1998). Since this signal is required for both mesoderm and endoderm, some other signal must be present to differentiate between these two germ layers. Micromeres formed by the fourth cleavage division have the ability to induce endoderm (Ransick & Davidson, 1995). This induction can occur independently of the entry of β-catenin into the nucleus of the cells induced to form endoderm (Loganet al., 1999), indicating micromere induction acts through a different signalling pathway. Final determination of endoderm also requires cell interactions through the late mesenchyme blastula stage, since cells from embryos dissociated prior to that stage fail to develop into endoderm autonomously (Chen & Wessel, 1996). A sea urchin member of the hedgehog family of signalling molecules has been reported to be expressed in the vegetal plate, indicating it also may play a role in endoderm formation.

Lim1 related homeobox gene (HpLim1) expressed in sea urchin embryos.

A cDNA clone for the LIM class homeobox gene (HpLim1) of the sea urchin, Hemicentrotus pulcherrimus, was isolated. HpLim1 contains two LIM domains and a LIM-class homeodomain, and amino acid sequences of these three domains are highly homologous to corresponding domains of Lim1 of other animals. Accumulation of HpLim1 transcripts begins at hatching, and declines after the mesenchyme blastula stage. HpLim1 mRNA was localized in the vegetal plates of hatched blastulae, but it was not detectable in primary mesenchyme cells (PMC) ingressed into the blastocele. HpLim1 mRNA-injected embryos became spherical with markedly reduced gut formation, failed to express marker proteins for aboral ectoderm and mesoderm, and mainly expressed an oral ectoderm marker. These results imply that while short-term expression of HpLim1 in the vegetal plate is needed for differentiation of aboral ectoderm, endoderm and PMC, ectopic expression of HpLim1 suppresses normal differentiation directing all embryonic cells to differentiate into oral ectoderm.

Sea Urchin TgBMP2/4 Gene Encoding a Bone Morphogenetic Protein Closely Related to Vertebrate BMP2 and BMP4 with Maximal Expression at the Later Stages of Embryonic Development

We have cloned a gene fragment (namedTgBMP2/4) that encodes a protein homologous to vertebrate bone morphogenetic protein (BMP) 2 and BMP4 in the sea urchinTripneustes gratilla.This peptide sequence contains 204 amino acids with 7 conserved cysteine residues at the C-terminus of the coding region and a cluster of basic amino acids that may serve as a signal for proteolytic cleavage. Sequence comparison and phylogenetic analyses reveal that TgBMP2/4 is closely related to vertebrate BMP2 and BMP4 as well as to amphioxus BMP2/4, with similarity levels ranging from 90% to 94% at the mature C-terminal domain. Northern blot analyses show that a 6.3-kbTgBMP2/4mRNA appears first at the mesenchyme blastula stage and increases to a maximal level at the gastrula and pluteus stages. This expression pattern is different from that of a BMP2/4-related gene previously found in sea urchin.

Measurements of Mechanical Properties of the Blastula Wall Reveal Which Hypothesized Mechanisms of Primary Invagination Are Physically Plausible in the Sea UrchinStrongylocentrotus purpuratus

Computer simulations showed that the elastic modulus of the cell layer relative to the elastic modulus of the extracellular layers predicted the effectiveness of different force-generating mechanisms for sea urchin primary invagination [L. A. Davidson, M. A. R. Koehl, R. Keller, and G. F. Oster (1995)Development121, 2005–2018]. Here, we measured the composite elastic modulus of the cellular and extracellular matrix layers in the blastula wall ofStrongylocentrotus purpuratusembryos at the mesenchyme blastula stage. Combined, these two layers exhibit a viscoelastic response with an initial stiffness ranging from 600 to 2300 Pa. To identify the cellular structures responsible for this stiffness we disrupted these structures and correlated the resulting lesions to changes in the elastic modulus. We treated embryos with cytochalasin D to disrupt the actin-based cytoskeleton, nocodazole to disrupt the microtubule-based cytoskeleton, and a gentle glycine extraction to disrupt the apical extracellular matrix (ECM). Embryos treated less than 60 min in cytochalasin D showed no change in their time-dependent elastic modulus even though F-actin was severely disrupted. Similarly, nocodazole had no effect on the elastic modulus even as the microtubules were severely disrupted. However, glycine extraction resulted in a 40 to 50% decrease in the elastic modulus along with a dramatic reduction in the hyalin protein at the apical ECM, thus implicating the apical ECM as a major mechanical component of the blastula wall. This finding bears on the mechanical plausibility of several models for primary invagination.

Expression pattern of Brachyury and Not in the sea urchin: comparative implications for the origins of mesoderm in the basal deuterostomes.

This work concerns the expression of two transcription factors during the development of the sea urchin Strongylocentrotus purpuratus: SpNot, the orthologue of the vertebrate Not gene, and SpBra, the orthologue of the vertebrate Brachyury gene. SpNot transcripts are detected by in situ hybridization in the vegetal plate at the mesenchyme-blastula stage. Later the gene is expressed in the secondary mesenchyme, but expression is no longer detectable after gastrulation. SpNot is upregulated during larval development, in the invaginating vestibule of the adult rudiment. Transcripts are also found in several larva-specific tissues, including the epaulets, blastocoelar cells, and pigment cells. SpBra also displays a discontinuous pattern of expression. Much like SpNot, this gene is expressed during embryogenesis in the embryonic vegetal plate and secondary mesenchyme founder cells, and expression is then extinguished. The gene is upregulated over a week later in the feeding larva, in the vestibule of the adult rudiment. In contrast to SpNot, SpBra is also expressed in the mesoderm of both left and right hydrocoels, and it is not expressed in any larva-specific tissues. We compare the spatial expression profile determined in this study with that of the orthologous Brachyury gene in an indirectly developing enteropneust hemichordate, a representative of the sister group to the echinoderms within the deuterostomes. These observations illuminate the genetic basis underlying the process of maximal indirect development in basal deuterostomes. Finally, Brachyury appears to be an excellent marker for the progeny of the set-aside cells of the sea urchin embryo.

A Molecular Analysis of Hyalin—A Substrate for Cell Adhesion in the Hyaline Layer of the Sea Urchin Embryo

The hyaline layer of echinoderm embryos is an extraembryonic matrix that functions as a substrate for cell adhesion through early development. The major constituent of the hyaline layer is the protein hyalin, a fibrillar glycoprotein of approximately 330 kDa that multimerizes in the presence of calcium. Here we provide a molecular characterization of hyalin and identify a region of the protein that is important for its function in cell adhesion. Partial hyalin cDNAs were identified from two sea urchin species,Strongylocentrotus purpuratusandLytechinus variegatus,by screening expression libraries with monoclonal antibodies to hyalin. The cDNAs each encode a tandemly arranged series of conserved repeats averaging 84 amino acids. These hyalin repeats are as similar between the two species as they are to repeats within each species, suggesting a strong functional conservation. Analysis of this repeat shows that it is a unique sequence within the GenBank database with only weak similarity to mucoid protein sequences. The hyalin mRNA is approximately 12 kb in length and is present in developing oocytes coincident with the appearance of cortical granules, the vesicle in which the hyalin protein is specifically packaged. The mRNA is present throughout oogenesis but is rapidly lost at oocyte maturation so that eggs and early embryos have no detectable hyalin mRNA. The hyalin protein, however, remains at relatively constant levels throughout development. Thus, all the hyalin protein present during early development, when no RNA is detectable, is maternally derived and exocytosed from cortical granules at fertilization. Hyalin mRNA reaccumulates in embryos beginning at the mesenchyme blastula stage; a RNA gel blot andin situhybridization analysis of gastrulae and larvae shows a progressive confinement of hyalin mRNA to the aboral ectoderm. Recombinant hyalin containing the tandem repeat region of the protein was expressed in bacteria and is shown to serve as an adhesive substrate, almost equal to that of native hyalin, in cell adhesion assays. This adhesive activity is partially blocked by dilute hyalin monoclonal antibody Tg-HYL to the same extent as that for native hyalin. Thus, this hyalin repeat region appears to contain the ligand for the hyalin cell surface receptor. These data help explain some of the classic functions ascribed to the hyalin protein in early development and now enable investigators to focus on the mechanisms of cell interactions with the hyaline layer.

Two Otx Proteins Generated from Multiple Transcripts of a Single Gene inStrongylocentrotus purpuratus

Orthodenticle-related (Otx) proteins are a highly conserved class of homeobox-containing transcription factors found in a wide range of organisms. They function in numerous developmental events, most prominently, anterior head patterning in insects and vertebrates. In the sea urchin,Strongylocentrotus purpuratus,an orthodenticle-related protein called SpOtx is believed to direct the activation of the aboral ectoderm-specificSpec2agene and more generally the differentiation of aboral ectoderm cells. To learn more about the structure, expression, and function of SpOtx and compare its properties with those of orthologs from other species, we isolated cDNA and genomic clones containingSpOtxsequences. Here, we report that SpOtx exists in two forms (α and β) that are generated by alternative RNA splicing from a singleSpOtxgene. SpOtx(α) and SpOtx(β) had identical C-termini and homeoboxes but were entirely different in their N-terminal domains.SpOtx(α)mRNAs were transcribed from a single start site and accumulated in all cells during cleavage, but were gradually concentrated in oral ectoderm and vegetal plate territories during gastrulation. In contrast, three distinctSpOtx(β)mRNAs resulted from two separate transcriptional initiation events, and these transcripts began to accumulate at mesenchyme blastula stage primarily in ectoderm and then later were largely restricted to oral ectoderm and vegetal plate territories. DNA-binding activity for SpOtx(β) appeared later in development than SpOtx(α). Overexpression of SpOtx(α) and SpOtx(β) induced in sea urchin embryos by mRNA injection demonstrated that SpOtx(α) was able to repress the accumulation ofSpOtx(β)transcripts, whereas SpOtx(β) had no effect on the accumulation ofSpOtx(α)transcripts. These results demonstrate that novel forms of Otx are produced in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to diverse forms of Otx in vertebrates.

Comparative Analysis of Fibrillar and Basement Membrane Collagen Expression in Embryos of the Sea Urchin, Strongylocentrotus purpuratus

The time of appearance and location of three distinct collagen gene transcripts termed 1 alpha, 2 alpha, and 3 alpha, were monitored in the developing S. purpuratus embryo by in situ hybridization. The 1 alpha and 2 alpha transcripts of fibrillar collagens were detected simultaneously in the primary (PMC) and secondary (SMC) mesenchyme cells of the late gastrula stage and subsequently expressed in the spicules and gut associated cells of the pluteus stage. The 3 alpha transcripts of the basement membrane collagen appeared earlier than 1 alpha and 2 alpha, and were first detected in the presumptive PMC at the vegetal plate of the late blastula stage. The PMC exhibited high expression of 3 alpha at the mesenchyme blastula stage, but during gastrulation the level of expression was reduced differentially among the PMC. In the late gastrula and pluteus stages, both PMC and SMC expressed 3 alpha mRNA, and thus at these stages all three collagen genes displayed an identical expression pattern by coincidence. This study thus provides the first survey of onset and localization of multiple collagen transcripts in a single sea urchin species.

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos.

During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC.