ske-T, a T-box gene expressed in the skeletogenic mesenchyme lineage of the sea urchin embryo.

T-box transcription factors regulate many developmental processes. Here we report the cloning and expression analysis of ske-T, a novel sea urchin T-box gene. The distribution of the maternal ske-T transcript is uniform in the egg and early embryonic stages while zygotic expression is restricted to the skeletogenic mesenchyme lineage.

Expression pattern of Brachyury in the embryo of the sea urchin Paracentrotus lividus.

Brachyury is a key transcription factor whose homologs have been identified in many animal species. Different Brachyury expression patterns have been observed amongst echinoderms. We have isolated PlBra, the Brachyury ortholog from the sea urchin Paracentrotus lividus and analyzed its expression during development. PlBra is first expressed at the end of cleavage in a ring of cells at the border between the presumptive endoderm and mesoderm territories. At later stages, PlBra is expressed around the blastopore and in the stomodaeum area as in most basal deuterostomes.

Selective expansion of intraepithelial lymphocytes expressing the HLA-E-specific natural killer receptor CD94 in celiac disease.

BACKGROUND & AIMS: Celiac disease is a gluten-induced enteropathy characterized by the presence of gliadin-specific CD4(+) T cells in the lamina propria and by a prominent intraepithelial T-cell infiltration of unknown mechanism. The aim of this study was to characterize the subset(s) of intraepithelial lymphocytes (IELs) expanding during active celiac disease to provide insights into the mechanisms involved in their expansion. METHODS: Flow-cytometric analysis of isolated IELs and/or immunohistochemical staining of frozen sections were performed in 51 celiac patients and 50 controls with a panel of monoclonal antibodies against T-cell and natural killer (NK) receptors. In addition, in vitro studies were performed to identify candidate stimuli for NK receptor expression. RESULTS: In normal intestine, different proportions of IELs, which were mainly T cells, expressed the NK receptors CD94/NKG2, NKR-P1A, KIR2D/3D, NKp46, Pen5, or CD56. During the active phase of celiac disease, the frequency of CD94(+) IELs, which were mostly alphabeta T cells, was conspicuously increased over controls. In contrast, the expression of other NK markers was not modified. Furthermore, expression of CD94 could be selectively induced in vitro by T-cell receptor activation and/or interleukin 15, a cytokine produced by intestinal epithelial cells. CONCLUSIONS: The gut epithelium favors the development of T cells that express NK receptors. In active celiac disease, there is a specific and selective increase of IELs expressing CD94, the HLA-E-specific NK receptor that may be related to T-cell receptor activation and/or interleukin 15 secretion.

Differential regulation of normal and tumoral breast epithelial cell growth by fibroblasts and 1,25-dihydroxyvitamin D3.

Mesenchymal-epithelial interactions are of paramount importance during normal and tumoral breast developments. We have investigated the paracrine growth regulation of normal and tumoral breast epithelial cells by fibroblasts derived from normal or pathological breast tissues. In some cases, breast cancer MCF-7 cells or normal epithelial cells in primary culture were cocultured with fibroblasts in a Transwell system allowing diffusible factor exchanges. Alternatively, conditioned medium produced by fibroblast cultures was added to epithelial cell cultures. Fibroblasts were shown to stimulate the proliferation of normal and carcinoma cells through paracrine mechanisms. However, the paracrine exchanges appeared to be different in normal versus tumoral breast epithelial cell growth regulation. Moreover, vitamin D-related compounds that have been proposed as anti-tumoral drugs were studied for their ability to affect normal and tumoral mammary epithelial cell proliferation and to interfere with the growth-regulatory activity of fibroblasts. Whereas vitamin D compounds inhibited MCF-7 cell growth, they led to a marked stimulation of the proliferation of normal mammary epithelial cells. Moreover, it was shown that the vitamin D analog EB 1089 can block the mitogenic effect of fibroblast-conditioned medium on tumoral but not normal breast epithelial cells. The differential effects of vitamin D compounds on cell proliferation provide further data in favor of the different behaviours of normal and tumoral mammary epithelial cells. The potential therapeutic use of vitamin D derivatives in the treatment of breast cancer is supported by these results but their growth-stimulatory properties on normal epithelial cells cannot be overlooked.

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos.

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.

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

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

Positive regulation of normal and tumoral mammary epithelial cell proliferation by fibroblasts in coculture.

In the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.

Organization of the proximal promoter of the hatching-enzyme gene, the earliest zygotic gene expressed in the sea urchin embryo.

The hatching enzyme (HE) gene is the earliest zygotic gene expressed in the sea urchin embryo. To investigate the regulation of the HE gene activity, 5' flanking DNA and the 5' untranslated leader were inserted upstream of reporter genes whose expression was monitored in vivo during development after transfer into eggs. By deletion analysis we showed that no more than 3 kb of flanking sequence are required for correct expression of transgenes. The proximal region of 0.5 kb does not precisely control spatial restriction but drives expression at a nearly maximal level. The proximal promoter was searched extensively for sites of protein-DNA interactions by DNAse protection and gel-shift methods. The 12 sites identified form 3 groups: core promoter; central region; and distal region. The central region bears three sites that contain a direct or inverted CCAAT box. Mutation and deletion analysis showed that, in addition to the core-promoter elements, the two most-distal CCAAT-containing sites are indispensable for promoter activity. These sites bind the same set of proteins, which are abundant in the nuclei of cleavage embryos.

Early gene expression along the animal-vegetal axis in sea urchin embryoids and grafted embryos.

The HE gene is the earliest strictly zygotic gene activated during sea urchin embryogenesis. It is transiently expressed in a radially symmetrical domain covering the animal-most two-thirds of the blastula. The border of this domain, which is orthogonal to the primordial animal-vegetal axis, is shifted towards the animal pole in Li+-treated embryos. Exogenous micromeres implanted at the animal pole of whole embryos, animal or vegetal halves do not modify the extent and localization of the HE expression domain. In grafted embryos or animal halves, the Li+ effect is not affected by the presence of ectopic micromeres at the animal pole. A Li+-induced shift of the border, similar to that seen in whole embryos, occurs in embryoids developing from animal halves isolated from 8-cell stage embryos or dissected from unfertilised eggs. Therefore, the spatial restriction of the HE gene is not controlled by the inductive cascade emanating from the micromeres and the patterning along the AV-axis revealed by Li+ does not require interactions between cells from the animal and vegetal halves. This suggests that maternal primary patterning in the sea urchin embryo is not limited to a small vegetal center but extends along the entire AV axis.

Structure of the gene encoding the sea urchin blastula protease 10 (BP10), a member of the astacin family of Zn2+-metalloproteases.

Blastula protease 10 (BP10), a metalloprotease of the astacin family, is secreted at the blastula stage by the sea urchin embryo. The BP10 gene shows a precise temporal and spatial regulation during embryogenesis. It has been cloned from a sea urchin lambda genomic library and the transcription unit has been entirely sequenced. It spans 6kb and contains seven exons (2.8 kb) and six introns (3.2 kb). Sequence comparison and phylogeny analysis show that BP10 belongs to a sub-family of molecular proteins which all play a role during development. In the two cases where the exon/intron organization of the gene is known (BP10 and tolloid), the modular structure of the protein is not reflected at the gene level, which indicates that this sub-family probably did not evolve by exon shuffling.

Structure of the sea urchin hatching enzyme gene.

The sea urchin embryo develops from an encased to a free-living larva by secreting at an early stage the hatching enzyme, a metalloprotease which hydrolyses a protective envelope derived from the egg extracellular matrix. Genomic clones containing the entire hatching enzyme gene were isolated from a lambda phage sea urchin library and the complete sequence of the transcription unit was determined. The hatching enzyme gene spans 6.3 kb and comprises 9 exons. The exon/intron organization of the hatching enzyme gene is similar but not identical to those of the vertebrate collagenases and stromelysins. The position and/or phase of several introns are different even in the N-terminal moiety where similarity between echinoderm and vertebrate enzymes was first detected. The active-center domain is encoded by a 1-1 class exon whose sequence, length and borders are highly conserved and might be considered as coding for a protein module. Adjacent to the active-center exon, the hatching enzyme gene has an additional 1-1 exon which codes for a threonine-rich region. This provides further evidence that the matrix-degrading metalloproteinases evolved by shuffling exons of the 1-1 class. Phylogeny analysis indicates a close relationship between the sea urchin and vertebrate enzymes.

Cell-autonomous expression and position-dependent repression by Li+ of two zygotic genes during sea urchin early development.

The expression of two zygotic genes (HE and BP10) during sea urchin embryogenesis was previously found to be early, transient, spatially restricted and controlled at the transcriptional level. Here we studied how the expression of these genes is affected when cell interactions are abolished by dissociating blastomeres and when development is perturbed by treatment with Li+. We found that in isolated blastomeres, transient transcriptional activity (HE) is unchanged and both genes apparently function in the appropriate cell type. Thus HE/BP10 expression is largely cell-autonomous and should rely on maternal factors unevenly distributed in the egg. Treatment with lithium does not affect the temporal control but decreases the transcriptional activity and the size of the domain of expression of the HE/BP10 genes. As the Li+ concentration increases, the border of the domain is progressively shifted towards the animal pole. This alteration of the spatial pattern is the earliest molecular evidence of a change in cell fate detectable only much later by morphological criteria, and reveals a gradient of sensitivity to Li+ along the animal--vegetal axis. These results suggest that the activity of the HE/BP10 genes is strongly dependent on spatially organized maternal information controlling early development.

Spatial expression of the hatching enzyme gene in the sea urchin embryo.

The sea urchin embryo at the blastula stage hatches from its protective fertilization envelope which is degraded by a secreted protease, the hatching enzyme. We have previously purified the hatching enzyme from Paracentrotus lividus (Lepage and Gache (1989). J. Biol. Chem. 264, 4787-4793), cloned its cDNA, and analyzed the temporal expression of its gene (Lepage and Gache (1990). EMBO J. 9, 3003-3012). We study here the temporal and spatial expression of the hatching enzyme gene in whole embryos by immunolabeling with an affinity-purified polyclonal antibody and by in situ hybridization using nonradioactive RNA probes. The timing of expression is consistent with our data on the activation of the gene, the mRNA accumulation in the blastula, and the role of the enzyme. Immunolabeling was observed only in blastula stage embryos; neither before the 128-cell stage nor after hatching. The distribution of the enzyme varies with time from a diffuse labeling around the nucleus to a punctate localization between the nucleus and the apical face of the blastomeres, and finally at the time of hatching, to a submembranous apical location. Not all the cells of an embryo are labeled. The presence of the hatching enzyme is restricted to a sharply delimited continuous territory spanning about two-thirds of the blastula. The orientation of this territory has been determined with respect to the animal-vegetal axis of the embryo using as a landmark the subequatorial pigmented band of the P. lividus species. The synthesis of the hatching enzyme only takes place in the animal-most two-thirds of the blastula. By in situ hybridization, the mRNA coding for the hatching enzyme is only detected in early blastulas, in a limited area having the same size and shape as the territory in which the protein is found. Thus the hatching enzyme gene is likely to be spatially controlled at the transcriptional level: its expression is restricted to a region of the blastula that corresponds roughly to the presumptive ectoderm territory. To date, the hatching enzyme gene products constitute the earliest molecular markers of the sea urchin embryo spatial organization along the primordial egg axis.

Spatial and temporal expression pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Drosophila dorsal-ventral patterning gene tolloid.

A cDNA clone coding for a sea urchin embryonic protein was isolated from a prehatching blastula lambda gt11 library. The predicted translation product is a secreted 64 x 10(3) Mr enzyme designated as BP10. The protein contains several domains: a signal peptide, a putative propeptide, a catalytic domain with an active center typical of a Zn(2+)-metalloprotease, an EGF-like domain and two internal repeats similar to repeated domains found in the C1s and C1r serine proteases of the complement cascade. The BP10 protease is constructed with the same domains as the human bone morphogenetic protein BMP-1, a protease described as a factor involved in bone formation, and as the recently characterized product of the tolloid gene which is required for correct dorsal-ventral patterning of the Drosophila embryo. The transcription of the BP10 gene is transiently activated around the 16- to 32-cell stage and the accumulation of BP10 transcripts is limited to a short period at the blastula stage. By in situ hybridization with digoxygenin-labelled RNA probes, the BP10 transcripts were only detected in a limited area of the blastula, showing that the transcription of the BP10 gene is also spatially controlled. Antibodies directed against a fusion protein were used to detect the BP10 protein in embryonic extracts. The protein is first detected in early blastula stages, its level peaks in late cleavage, declines abruptly before ingression of primary mesenchyme cells and remains constant in late development. The distribution of the BP10 protein during its synthesis and secretion was analysed by immunostaining blastula-stage embryos. The intracellular localization of the BP10 staining varies with time. The protein is first detected in a perinuclear region, then in an apical and submembranous position just before its secretion into the perivitelline space. The protein is synthesized in a sharply delimited continuous territory spanning about 70% of the blastula. Comparison of the size and orientation of the labelled territory in the late blastula with the fate map of the blastula stage embryo shows that the domain in which the BP10 gene is expressed corresponds to the presumptive ectoderm. Developing embryos treated with purified antibodies against the BP10 protein and with synthetic peptides derived from the EGF-like domain displayed perturbations in morphogenesis and were radialized to various degrees. These results are consistent with a role for BP10 in the differentiation of ectodermal lineages and subsequent patterning of the embryo. On the basis of these results, we speculate that the role of BP10 in the sea urchin embryo might be similar to that of tolloid in Drosophila. We discuss the idea that the processes of spatial regulation of gene expression along the animal-vegetal in sea urchin and dorsal-ventral axes in Drosophila might have some similarities and might use common elements.

Early expression of a collagenase-like hatching enzyme gene in the sea urchin embryo.

The hatching enzyme is a developmentally regulated protease secreted at the blastula stage by the sea urchin embryo to digest its protective envelope. A nearly full-length cDNA clone (HE6) encoding the entire sequence of the hatching enzyme was isolated from a prehatching blastula lambda gt11 cDNA library. The 1761 bp open reading frame codes for a preprohatching enzyme with an 18 amino acid signal sequence, a 148 amino acid activation peptide and a 421 amino acid mature enzyme which has homologies with the mammalian collagenases. Transcripts of the hatching enzyme gene are not detected in the unfertilized egg, they accumulate during the cleavage stages and disappear at hatching. This transient expression results from a transcriptional control. Thus the hatching enzyme mRNA is not a maternal gene product but a transcript synthesized at a very early stage from the zygotic genome.

Purification and characterization of the sea urchin embryo hatching enzyme.

The sea urchin hatching enzyme provides an interesting model for the control of gene expression during early development. In order to study its properties and developmental regulation, the hatching enzyme of the species Paracentrotus lividus has been purified. The fertilization envelopes of the embryos were digested before hatching by a crude culture supernatant previously made. The enzyme was then solubilized by 1 M NaCl and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and purified by hydrophobic chromatography on Procion-agarose. A 470-fold increase in specific activity was obtained. The kinetic parameters of the proteolytic activity using dimethylcasein as substrate are: Km = 120 micrograms x ml-1, Vm = 200 mumol x min-1 x mg-1, and kcat = 180 s-1 at 500 mM NaCl, 10 mM CaCl2, pH 8.0, at 35 degrees C. The purified enzyme is highly active on fertilization envelopes: at 20 degrees C and 500 mM NaCl, 10 mM CaCl2, pH 8.0, 100 ng of enzyme completely denudes embryos in about 20 min under standard conditions. The molecular mass of the enzyme was estimated as 57 kDa by gel filtration, 51 kDa by gel electrophoresis, and 52 kDa by amino acid analysis. The hatching enzyme was shown to be a glycoprotein which autolyzes to a 30-kDa inactive form. Antibodies raised against the 51- or 30-kDa forms reacted with both these forms. Immunoblotting experiments showed that the hatching supernatants contain important amounts of the autolyzed species.

Calcium-mediated release of glucanase activity from cortical granules of sea urchin eggs.

Confluent monolayers of sea urchin eggs were bonded to culture dishes coated with protamine sulfate. The cytoplasm was then sheared away by a jet of isosmotic buffer. About 326,000 circular fragments of individual egg cortices (430 micrograms protein) remained bound to each dish. The fragments are composed of cortical granules (CG), plasma membrane, and vitelline layer. A single dish contains 7.7 X 10(8) CG and is referred to as a CG lawn (CGL). Ca2+-EGTA buffers of estimated free-Ca2+ concentrations (0.06-25.7 microM) were applied to CGL and samples removed and assayed for the CG marker enzyme exo-beta (1 leads to 3)-glucanohydrolase (glucanase). Estimated free-Ca2+ concentrations above 2.75 microM caused the total release of the glucanase to the supernatant within 4 min. The half-maximal rate of appearance of glucanase occurred in 2.5 microM Ca2+. At all Ca2+ concentrations tested, the appearance of enzyme activity exhibited sigmoidal kinetics. The visual disappearance of CG correlated with the appearance of glucanase in the Ca2+ buffer. In response to Ca2+ the CG probably lyse, fuse with adjacent CG, or fuse with the underlying plasma membrane. The calmodulin antagonist trifluoperazine inhibited Ca2+-mediated glucanase release from CGL (I50 8 microM). The sensitivity of the CGL to Ca2+ in the 1-10 microM range is rapidly lost during incubation of CGL in the isolation buffer. ATP and low temperature retard the rate of loss of Ca2+ sensitivity. These secretory granules are a model for studying the mechanism of Ca2+-induced secretion. In addition, they contain structural proteins and enzymes which function in the fertilization process. CGL preparations should be useful in studies dealing with the processing of CG components after their release in response to micromolar concentrations of Ca2+.

Monoclonal antibodies to the sea urchin egg vitelline layer inhibit fertilization by blocking sperm adhesion.

Thirty-one mouse hybridomas were produced against the vitelline layer (VL) of the egg of the sea urchin S. purpuratus. Ascites fluids of eight of the 31 bound to the VL surface in the high ionic strength conditions of sea water. Binding was specific to the VL, since immunofluorescence showed that the antibodies elevated from the egg surface with the fertilization envelope after activation with ionophore A23187. Antibody binding was strictly species-specific, the eggs of L. pictus showing no reaction. An immunoperoxidase surface-binding assay showed a wide range in the amount of each monoclonal antibody binding to the VL surface at saturation. All eight monoclonals inhibit fertilization by inhibiting the binding of sperm to the VL. None of the eight ascites fluids reacted with egg jelly. The inhibition of fertilization correlates positively with amount of antibody binding the egg surface. In contrast to the effects of polyclonal rabbit antisera raised against whole eggs or egg cortices, these eight monoclonal antibodies to the VL do not induce the wrinkling of the egg, the cortical granule reaction, the centering of pronuclei, or any other visual indication of metabolic activation.

Transport of methionine in sea-urchin sperm by a neutral amino-acid carrier.

A carrier-mediated transport for L-methionine and other neutral amino acids exists in sperm of the sea urchin Lytechinus pictus. The initial rate of L-methionine entry is a Michaelis-Menten function of the methionine concentration in the external medium. The maximum velocity is low [V = 250 pmol h-1 (10(9) sperm)-1 at 22 degrees C] and the affinity is high (Km = 6-10 microM). The initial rate of transport under steady-state exchange conditions is also a Michaelis-Menten function of the external concentration of methionine. The Km determined by this method is about 14 microM. Neutral amino acids compete with L-methionine transport as shown by initial velocity measurements. These results indicate that L-methionine transport is a carrier-mediated process. The temperature dependence of the process is approximately 84 kJ (20 kcal) mol-1 K-1, which is not compatible with a simple diffusion mechanism, but in the range of values usually found for a mediated transport. The transport is largely Na+-independent and does not depend on Ca2+, K+ or H+ gradients. It is only partially sensitive to KCN, showing it is mainly independent of oxidative phosphorylation. The steady-state internal methionine concentration is not a linear function of the external amino acid concentration. This suggests that an exit by diffusion competes with a carrier-mediated concentrative transport in a cellular compartment. This mediated transport is compared to those of higher animal cells.