Biomedical subjects

F Wilt

Publications and source records attributed to F Wilt.

At least 19 recordsLinked to original sources

PubMed2006

A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus.

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.

Amino Acid Sequence↗

PubMed2001

Proteins and saccharides of the sea urchin organic matrix of mineralization: characterization and localization in the spine skeleton.

Properties of the echinoderm skeleton are under biological control, which is exerted in part by the organic matrix embedded in the mineralized part of the skeleton. This organic matrix consists of proteins and glycoproteins whose carbohydrate component is specifically involved in the control mechanisms. The saccharide moiety of the organic matrix of the spines of the echinoid Paracentrotus lividus was characterized using enzyme-linked lectin assays (ELLAs). O-glycoproteins, different types of complex N-glycoproteins, and terminal sialic acids were detected. Sialic acids are known to interact with Ca ions and could play an important role in the mineralization process. Some of the carbohydrate components detected by ELLAs as well as two organic matrix proteins (SM30 and SM50) were localized within different subregions of the spine skeleton using field-emission scanning electron microscopy. The mappings show that some of these components are not homogeneously distributed in the different skeletal subregions. For example, some N-glycoproteins were preferentially located in the putative amorphous subregion of the skeleton, whereas some O-glycoproteins were localized in the subregion where skeletal growth is inhibited. These results suggest that the biological control exerted on the skeletal properties can be partly modulated by local differences in the organic matrix composition.

Animals↗

PubMed2000

Roles of two homotetrameric kinesins in sea urchin embryonic cell division.

To improve our understanding of the roles of microtubule cross-linking motors in mitosis, we analyzed two sea urchin embryonic kinesin-related proteins. It is striking to note that both of these proteins behave as homotetramers, but one behaves as a more compact molecule than the other. These observations suggest that these two presumptive motors could cross-link microtubules into bundles with different spacing. Both motors localize to mitotic spindles, and antibody microinjection experiments suggest that they have mitotic functions. Thus, one of these kinesin-related proteins may cross-link spindle microtubules into loose bundles that are "tightened" by the other.

Amino Acid Sequence↗

PubMed1999

Ultrastructural localization of proteins involved in sea urchin biomineralization.

Three skeletal tissues of the adult echinoid Paracentrotus lividus (the pedicellaria primordium, the test, and the tooth) were immunolabeled with three sera raised against the total mineralization organic matrix and two specific matrix proteins (SM30 and SM50) from the embryo of the echinoid Strongylocentrotus purpuratus. Two conventional chemical fixation protocols and two high-pressure freezing/freeze-substitution protocols were tested. One conventional protocol is recommended for its good preservation of the ultrastructure, and one high-pressure freezing/freeze-substitution protocol is recommended for its good retention of antigenicity. Immunolabeling was obtained in the three adult tissues. It was confined to the active skeleton-forming cells and to the structured organic matrix. The results indicate that the matrix proteins follow the classical routes of secretory protein assembly and export and suggest that SM30 and SM50 are a part of the tridimensional network formed by the organic matrix before the onset of mineralization. They show that the genetic program of part of skeletogenesis is conserved among different calcification models and developmental stages.

Animals↗

PubMed1997

Oral-aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore.

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3 h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue-specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral-aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral-aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oral-aboral axis during sea urchin development.

Animals↗

PubMed1995

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter.

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

Animals↗

PubMed1993

Cell interactions in invertebrate development.

By utilizing both genetics and physical manipulations, studies on invertebrate embryos have provided substantial information on how cell interactions influence cell fate during development. The picture emerging from these studies suggest that while there may be varied and intricate patterns of cell interactions during the development of different embryos, the signaling molecules that convey positional information across cell membranes have been highly conserved.

Animals↗

PubMed1993

A role for regulated secretion of apical extracellular matrix during epithelial invagination in the sea urchin.

Epithelial invagination, a basic morphogenetic process reiterated throughout embryonic development, generates tubular structures such as the neural tube, or pit-like structures such as the optic cup. The 'purse-string' hypothesis, which proposes that circumferential bands of actin microfilaments at the apical end of epithelial cells constrict to yield a curved epithelial sheet, has been widely invoked to explain invaginations during embryogenesis. We have reevaluated this hypothesis in two species of sea urchin by examining both natural invagination of the vegetal plate at the beginning of gastrulation and invagination induced precociously by Ca2+ ionophore. Neither type of invagination is prevented by cytochalasin D. In one species, treatment with A23187 three hours before the initiation of invagination resulted in the deposition of apical extracellular matrix at the vegetal plate, rather than invagination. This apical matrix contains chondroitin sulfate, as does the lumen of the archenteron in normal gastrulae. When the expansion of this secreted matrix was resisted by an agarose gel, the vegetal plate buckled inward, creating an archenteron that appeared 3-4 hours prematurely. Pretreatment with monensin, which blocks secretion, inhibits both Ca2+ ionophore-stimulated folding and natural invagination, demonstrating that secretion is probably required for this morphogenetic event. These results indicate that alternatives to the purse-string hypothesis must be considered, and that the directed deposition of extracellular matrix may be a key Ca(2+)-regulated event in some embryonic invaginations. A bending bilayer model for matrix-driven epithelial invagination is proposed in which the deposition of hygroscopic material into a complex, stratified extra-cellular matrix results in the folding of an epithelial sheet in a manner analagous to thermal bending in a bimetallic strip.

Animals↗

PubMed1991

Role of the extracellular matrix in tissue-specific gene expression in the sea urchin embryo.

The role of extracellular matrix (ECM) in the differentiation of tissue types was examined in embryos of Strongylocentrotus purpuratus. We have examined the expression of various tissue-specific molecular markers after disrupting the ECM by culturing embryos in the presence of beta-aminoproprionitrile fumarate (BAPN), which disrupts collagen deposition, and beta-D-xyloside, which disrupts proteoglycan metabolism. The markers examined included accumulation of primary mesenchyme-specific mRNA (SM 50); an aboral ectoderm-specific mRNA (Spec 1); and a gut-specific enzyme, alkaline phosphatase. Treatment with BAPN or beta-D-xyloside results in developmental arrest at the mesenchyme blastula stage. Although spicule formation is inhibited, the accumulation of SM 50 transcripts and the synthesis of most of the prominent spicule matrix proteins is similar to that of control embryos. Spec 1 mRNA, in contrast, while accumulating to a significant extent when collagen and proteoglycan metabolism is disrupted, does accumulate to a level somewhat lower than that seen in control embryos. Additionally, the postgastrula rise in gut-specific alkaline phosphatase is reversibly inhibited by BAPN and xyloside treatment. These results demonstrate a differential effect of the ECM on expression of tissue-specific molecular markers.

Alkaline Phosphatase↗

PubMed1991

Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus.

In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus pictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide, a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to proteins that form a unique beta-spiral supersecondary structure.

Amino Acid Sequence↗

PubMed1991

Interactions of different vegetal cells with mesomeres during early stages of sea urchin development.

It has been known from results obtained in the classical experiments on sea urchin embryos that cell isolation and transplantation showed extensive interactions between the early blastomeres and/or their descendants. In the experiments reported here a systematic reexamination of recombination of mesomeres and their progeny (which come from the animal hemisphere) with various vegetal cells derived from blastomeres of the 32- and 64-cell stage was carried out. Cells were marked with lineage tracers to follow which cell gave rise to what structures, and newly available molecular markers have been used to analyze different structures characteristic of regional differentiation. Large micromeres form spicules and induce gut and pigment cells in mesomeres, conforming to previous results. Small micromeres, a cell type not heretofore examined, gave rise to no recognizable structure and had very limited ability to evoke poorly differentiated gut tissue in mesomeres. Macromeres and their descendants, Veg 1 and Veg 2, form primarily what their normal fate dictated, though both did have some capacity to form spicules, presumably by formation from secondary mesenchyme. Macromeres and their descendants were not potent inducers of vegetal structures in animal cells, but they suppress the latent ability of mesomeres to form vegetal structures. The results lead us to propose that the significant interactions during normal development may be principally suppressive effects of mesomeres on one another and of adjacent vegetal cells on mesomeres.

Animals↗

PubMed1990

The synthesis and secretion of collagen by cultured sea urchin micromeres.

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.

Animals↗

PubMed1990

The influence of cell interactions and tissue mass on differentiation of sea urchin mesomeres.

The developmental potential of different blastomeres of the sea urchin embryo was re-examined. We have employed a new method to isolate substantial numbers of different kinds of blastomeres from 16-cell-stage embryos, and we have used newly available molecular markers to analyze possible vegetal differentiation. We have found that, while isolated mesomere pairs behave according to the classical expectations and develop into ectodermal vesicles, there is a clear effect of reaggregating two or more mesomere pairs. They survive better in long-term culture and, after prolonged periods, they display an astonishing ability to express vegetal differentiation. We also combined mesomeres with stained micromeres or macromeres from the vegetal hemisphere. Although induction of guts and spicules was observed, there was little if any effect of varying the ratio of different blastomeres on the kinds of differentiation obtained.

Animals↗

PubMed1989

Expression of an embryonic spicule matrix gene in calcified tissues of adult sea urchins.

The sea urchin spicule is composed of CaCO3 associated with an organic matrix containing at least 10 proteins. We have previously shown that one of these proteins, a 50-kDa glycoprotein (SM 50), is encoded by a gene which is only transcribed in cells of the micromere-mesenchyme lineage. In this report we examined RNA from five adult tissues: ovary, coelomocytes, intestine, tube feet, and spine for the presence of SM 50 transcripts and protein. Only RNA from cells of spine and tube feet hybridized to a SM 50-specific probe on Northern blots. These same two tissues undergo biomineralization and contain a protein which is immunologically related to the SM 50 protein. The restricted accumulation of SM 50 transcripts and protein suggests that the differentiation of biomineralizing mesenchyme cells in embryonic and adult tissue may utilize the same genetic program.

Animals↗

PubMed1989

Autonomous expression of tissue-specific genes in dissociated sea urchin embryos.

The effects of disrupting cell interactions in early development were investigated by examining the accumulation of a primary mesenchyme specific transcript (SM50) and an aboral ectoderm-specific transcript (Spec 1) in cultures of sea urchin embryos that were dissociated at early stages and then cultured in CFSW. The expression of both SM50 and Spec 1 is temporally correct and remains restricted to the appropriate cell types, even if the embryo is dissociated as early as the 2-cell stage and maintained as a suspension of single cells. This result is consistent with the idea that the specificity of expression of these two genes, each characteristic of different lineages, is strongly regulated by information in the egg. Average SM50 expression is half that of intact embryos, but Spec 1 expression is very low, only 10-20% of intact controls, suggesting some differences in the response of the two genes to lack of close cell interactions.

Animals↗

PubMed1987

A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. I. Authentication of the cloned gene and its developmental expression.

The developing sea urchin embryo forms endoskeletal CaCO3 containing spicules which are elaborated by the primary mesenchyme cells, descendants of the micromeres, beginning at gastrulation. In this and the accompanying paper [H. M. Sucov, S. Benson, J. J. Robinson, R. J. Britten, F. Wilt, and E. H. Davidson (1987) Dev. Biol. 120, 507-519] the isolation and characterization of a gene that encodes a 50-kDa spicule matrix glycoprotein that we call SM50 are described. A cloned cDNA isolated from a lambda gt11 library was used in hybrid-selected translation and hybrid arrest of translation experiments to verify that the cDNA encodes a spicule matrix protein. The cognate RNA transcript encodes a 50-kDa protein which is precipitated by polyclonal antisera against spicule matrix proteins and is present only in polyadenylated RNA at stages known to be making a spicule. The cloned cDNA sequence described in the accompanying paper was used to follow the time of expression of the cognate gene by RNA blotting analysis. The 2.2-kb mRNA is first detected at late cleavage stages and rapidly accumulates as the primary mesenchyme forms, reaching an apparent maximum concentration in the late gastrula and pluteus stages. The cDNA was also used to identify the cells that contain the transcripts by hybridization in situ. Hybridization to cellular transcripts is first detected in primary mesenchyme cells as they enter the blastocoel, and transcripts are confined to these cells during spicule formation and subsequent development.

Animals↗

PubMed1987

A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. II. Structure of the gene and derived sequence of the protein.

A lambda gt11 cDNA clone isolated by use of a polyclonal antispicule matrix protein antiserum is shown in the accompanying paper [S. C. Benson, H. M. Sucov, L. Stephens, E. H. Davidson, and F. Wilt (1987) Dev. Biol. 120, 499-506] to encode a prominent 50-kDa spicule matrix protein (SM50). This clone was used to select homologous genomic recombinants, and the structure of the gene was determined. The SM50 gene occurs once per haploid genome. It contains a single intron located within the 35th codon. A unique transcription initiation site 110 nucleotide pairs prior to the translation start signal was mapped by primer extension. The mRNA is 1895 nucleotides in length, excluding the 3' poly(A) sequence, and contains a single open reading frame 450 codons in length. Though rare in whole embryo RNA the prevalence of the SM50 mRNA is calculated to be about 1% of the total mRNA in skeletogenic mesenchyme cells. The derived peptide sequence indicates a typical N-terminal signal peptide, and an N-linked glycosylation site near the C terminus. About 45% of the length of the protein is included in a domain composed of consecutive approximate repetitions of a 13-amino-acid element, the consensus sequence of which is Trp-Val-Gly-Asp-Asn-Gln-Ala-LeuTrp-Val-IleAsp-Asn-GlnPro+ ++-ValGlu. The protein also contains an internal domain unusually rich in proline residues and a very basic C-terminal region.

Amino Acid Sequence↗

PubMed1986

The effects of aphidicolin on morphogenesis and differentiation in the sea urchin embryo.

Aphidicolin, an inhibitor of DNA polymerase alpha, blocks DNA synthesis and cell division in sea urchin embryos. The effects of this inhibition appear to be stage dependent. Blastulae treated with aphidicolin before the thickening of the vegetal plate undergo developmental arrest prior to gastrulation. The extent of inhibition of DNA synthesis varies from 60 to 93% in these embryos. However, when aphidicolin is added after the vegetal plate has thickened, development continues normally through pluteus formation, even though DNA synthesis is inhibited by greater than or equal to 90% and cell division has ceased. These observations indicate that, from the vegetal plate stage onward, morphogenesis and overt differentiation are independent of DNA synthesis and cell division.

Animals↗