PubMed Health⌕ Search

Biomedical subjects

F H Wilt

Publications and source records attributed to F H Wilt.

At least 19 recordsLinked to original sources
PubMed2000

Expression of spicule matrix proteins in the sea urchin embryo during normal and experimentally altered spiculogenesis.

During its embryonic development, the sea urchin embryo forms an endoskeletal calcitic spicule. This instance of biomineralization is experimentally accessible and also offers the advantage of occurring within a developmental context. Here we investigate the time course of appearance and localization of two proteins among the four dozen that constitute the protein matrix of the skeletal spicule. SM50 and SM30 have been studied in some detail, and polyclonal antisera have been prepared against them (C. E. Killian and F. H. Wilt, 1996, J. Biol. Chem. 271, 9150-9159). Using these antibodies we describe here the localization and time course of accumulation of these two proteins in Strongylocentrotus purpuratus, both in the intact embryo and in micromere cultures. We also investigate the disposition of the matrix proteins, SM50, SM30, and PM27, in the three-dimensional spicule by studying changes in protein localization during experimental manipulation of isolated skeletal spicules. We conclude that SM50, PM27, and SM30 probably play different roles in biomineralization, based on their localization and patterns of expression. It is unlikely that these proteins are solely structural elements within the mineral. SM50 and PM27 may play a role in defining the extracellular space in which spicule deposition occurs, while SM30 may play a role in secretion of spicule components. Finally, we report on the effects of serum on expression of some primary mesenchyme-specific proteins in micromere cultures; withholding serum severely depresses accumulation of SM30 but has only modest effects on the accumulation of other proteins.

Animals↗
PubMed1999

Matrix and mineral in the sea urchin larval skeleton.

The endoskeletal spicules of sea urchin larvae are composed of calcite, a surrounding extracellular matrix, and small amounts of occluded matrix proteins. The spicules are formed by primary mesenchyme cells (PMCs) in the blastocoel of the embryo, where they adopt stereotypical locations, thereby specifying where spicules will form. PMCs also fuse to form cytoplasmic cords connecting the cell bodies, and it is within the cords that spicules arise. The mineral phase contains 5% Mg as well as Ca, and about 0.1% of the mass is protein. The matrix and mineral form concentric plies, and the composite has different physical properties than those of pure calcite. The calcite diffracts as a single crystal and is composed of well-ordered, but not perfectly ordered, microdomains. There is evidence for adsorption of matrix proteins to specific crystal faces at domain boundaries, which may help regulate crystal growth and texture. Immature spicules contain considerable precipitated amorphous CaCO3, and PMCs also have vesicles that contain amorphous CaCO3. This suggests the hypothesis that the cellular precursor to the spicules is actually amorphous CaCO3 stabilized in the cell by protein. The spicule s enveloped by the PMC cord, but is topologically exterior to the cell. The PMC plasmalemma is tightly applied to the developing spicules, except perhaps at the elongating tip. The characteristics, localization, and possible function of the four identified matrix proteins are discussed. SM50, SM37, and PM27 all primarily enclose the mineral, though small amounts are occluded. SM30 is found in cellular vesicles and is probably the principal occluded protein of the spicule.

Animals↗
PubMed1999

Functional organization of DNA elements regulating SM30alpha, a spicule matrix gene of sea urchin embryos.

The SM30a gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30alpha gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from -142 to -105bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in ear y blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to -122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule.

5' Untranslated Regions↗
PubMed1998

Matrix metalloproteinase inhibitors disrupt spicule formation by primary mesenchyme cells in the sea urchin embryo.

The primary mesenchyme cells of the sea urchin embryo construct an elaborate calcareous endoskeletal spicule beginning at gastrulation. This process begins by ingression of prospective primary mesenchyme cells into the blastocoel, after which they migrate and then fuse to form a syncytium. Skeleton deposition occurs in spaces enclosed by the cytoplasmic cables between the cell bodies. Experiments are described which probe the role of proteases in these early events of spicule formation and their role in the continued elaboration of the spicule during later stages of embryogenesis. We find that several inhibitors of metalloproteinases inhibit the continuation of spiculogenesis, an effect first reported by Roe et al. (Exp. Cell Res. 181, 542-550, 1989). A detailed study of one of these inhibitors, BB-94, shows that fusion of primary mesenchyme cells still occurs in the presence of the inhibitor and the formation of the first calcite granule is not impeded. Continued elaboration of the spicule, however, is completely stopped; addition of the inhibitor during the active elongation of the spicule stops further elongation immediately. Removal of the inhibitor allows resumption of spicule growth. The inhibition is accompanied by almost complete cessation of massive Ca ion transport via the primary mesenchyme cells to the spicule. The inhibitor does not prevent the continued synthesis of several spicule matrix proteins. Electron microscopic examination of inhibited primary mesenchyme cells shows an accumulation of characteristic vesicles in the cytoplasm. Gel zymography demonstrates that although most proteases in homogenates of primary mesenchyme cells are not sensitive to the inhibitor in vitro, a protease of low abundance detectable in the medium of cultured primary mesenchyme cells is inhibited by BB-94. We propose that the inhibitor is interfering with the delivery of precipitated calcium carbonate and matrix proteins to the site(s) of spicule growth.

Animals↗
PubMed1997

Looking into the sea urchin embryo you can see local cell interactions regulate morphogenesis.

The transparent sea urchin embryo provides a laboratory for study of morphogenesis. The calcareous endoskeleton is formed by a syncytium of mesenchyme cells in the blastocoel. The locations of mesenchyme in the blastocoel, the size of the skeleton, and even the branching pattern of the skeletal rods, are governed by interactions with the blastula wall. Now Guss and Ettensohn show that the rate of deposition of CaCO3 in the skeleton is locally controlled in the mesenchymal syncytium, as is the pattern of expression of three genes involved in skeleton formation. They propose that short range signals emanating from the blastula wall regulate many aspects of the biomineralization process.

Animals↗
PubMed1996

Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules.

In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

Animals↗
PubMed1996

Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus.

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.

Animals↗
PubMed1994

Genomic organization of a gene encoding the spicule matrix protein SM30 in the sea urchin Strongylocentrotus purpuratus.

We report the characterization of a genomic clone containing portions of two tandemly arranged genes that encode a spicule matrix protein, SM30, of the sea urchin Strongylocentrotus purpuratus. The isolated 18.4-kilo-base genomic clone contains the complete genomic sequence of one SM30 gene, designated SM30-alpha, and a portion of another SM30 gene, designated SM30-beta. Southern blot analysis shows that SM30 protein is encoded by a small gene family of two to four members. RNase protection assays indicate that the SM30-alpha gene is expressed at the time of spicule formation in the sea urchin embryo. In addition, mapping of SM30-alpha shows that a large single intron interrupts the coding sequence. Comparison of the nucleic acid and amino acid sequences of the SM30-alpha genomic sequence and the previously isolated SM30 cDNA reveals them to be very similar, but not identical. We also demonstrate that 2.6 kilobases of upstream sequence of SM30-alpha are sufficient to direct primary mesenchyme cell-specific expression of a reporter gene construct.

Amino Acid Sequence↗
PubMed1993

Regulation of maternal messenger RNA translation during oogenesis and embryogenesis in Urechis caupo.

We have tested the hypothesis that the selective translation of mRNAs in oocytes and embryos is controlled by when during oogenesis individual maternal mRNAs are synthesized. In an earlier paper we described the isolation of cDNA probes to 21 maternal mRNAs which accumulate with different patterns during oogenesis in Urechis caupo (E. T. Rosenthal and F. H. Wilt, 1986, Dev. Biol. 117, 55-62). Many of these probes have now been used to analyze the translation of the maternal mRNAs in growing oocytes, full-grown oocytes, and embryos. The translation of all of the mRNAs studied changes dramatically when full-grown Urechis oocytes are fertilized. Our data do not support the idea that the translation of the different maternal mRNAs depends on when they were synthesized during oogenesis. This strongly suggests that, at some level, the ability of the cell's translational machinery to distinguish between different maternal mRNAs must reside directly, or indirectly, in the mRNA sequences. We have also found that in Urechis, as in Xenopus, the selective translation of maternal mRNAs is correlated with their selective adenylation. Consequently, we have sequenced the 3' ends of 16 translationally controlled maternal mRNAs in an effort to detect consensus sequences that might regulate adenylation. No such consensus sequences have been found, suggesting that the mRNA characteristics involved in the regulation of polyadenylation may reside in a wide variety of sequences or in more subtle features, such as secondary structure.

Animals↗
PubMed1992

Phorbol esters alter cell fate during development of sea urchin embryos.

Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.

Animals↗
PubMed1991

Characterization and expression of a gene encoding a 30.6-kDa Strongylocentrotus purpuratus spicule matrix protein.

We describe here the isolation and characterization of several cDNA clones that encode a single 30.6-kDa Strongylocentrotus purpuratus spicule matrix protein designated SM30. The clones were isolated by screening a lambda gt11 cDNA library with a rabbit polyclonal antiserum raised against S. purpuratus total spicule matrix proteins. DNA sequencing reveals that the SM30 protein is acidic. RNA blot analysis shows that the cDNAs hybridize to a single 1.8-kb transcript and that there is a sharp increase in the SM30 transcript levels at middle to late mesenchyme blastula stage. SM30 transcript levels remain high through the 3-day pluteus stage. In situ hybridization analysis indicates that, within the embryo, SM30 transcript accumulation is restricted to the primary mesenchyme cells. Quantitations of SM30 transcript levels show that by the prism stage there are about 29,000 SM30 transcripts present per embryo, which averages to approximately 480 transcripts per primary mesenchyme cell. Additionally, RNA blot analysis of total RNA isolated from adult tissues shows that SM30 mRNA accumulates exclusively in mineralized tissues. These findings taken together strongly suggest that the gene corresponding to the SM30 cDNAs does in fact encode a spicule matrix protein.

Aging↗
PubMed1990

Determination of cell fate in sea urchin embryos.

Classical embryological studies have provided a great deal of information on the autonomy and stability of cell fate determination in early sea urchin embryos. However, these studies were limited by the tools available at the time, and the interpretation of the results of these experiments was limited by the lack of information available at the molecular level. Recent studies which have re-examined classical experiments at the molecular level have provided important new insights into the mechanism of determination in sea urchins, and require us to re-evaluate some long standing theories on the process of differentiation.

Animals↗
PubMed1990

Range and stability of cell fate determination in isolated sea urchin blastomeres.

We have examined the developmental potential of blastomeres isolated from either the animal (mesomeres) or vegetal (macromeres-micromeres) half of 16-cell embryos of the sea urchin Lytechinus pictus. We have also examined the effects of two known vegetalizing agents on the development of isolated mesomeres; LiCl treatment and combination with micromeres, the small blastomeres found at the vegetal pole of the 16-cell embryo. The markers for differentiation used were both morphological (invaginations, spicules and pigment cells) and molecular (gut-specific alkaline phosphatase activity, and monoclonal antibodies against antigens specific for gut and oral ectoderm). Embryoids derived from isolated mesomeres expressed markers characteristic of vegetal differentiation only at very low levels. They did express an antigen characteristic of animal development, the oral ectoderm antigen, but with an altered pattern. Isolated macromere-micromere pairs expressed all markers characteristic of vegetal development, but did not express the marker characteristic of animal development. Increasing concentrations of LiCl caused isolated mesomeres to give rise to embryoids with an increasing tendency to express vegetal markers of differentiation, and it was found that expression of different vegetal markers begin to appear at different concentrations of LiCl. LiCl also caused the marker for oral ectoderm to be expressed in a more normal pattern. Combining micromeres with mesomeres also induced mesomere derivatives to differentiate in a vegetal manner. Micromeres were not completely effective in inducing a more normal pattern of expression of the marker for oral ectoderm. The treatment of isolated mesomeres with both LiCl and micromeres produces a synergistic effect resulting in embryoids expressing markers not induced by either treatment alone.

Animals↗
PubMed1989

The accumulation and translation of a spicule matrix protein mRNA during sea urchin embryo development.

In this report we further characterize the expression of the gene that encodes the 50-kDa spicule matrix protein (SM50) during development of the sea urchin Strongylocentrotus purpuratus. Quantitative measurements of SM50 mRNA levels using the single-stranded probe excess titration technique indicate that SM50 transcript levels attain a maximum level of 8000 to 10,000 transcripts per embryo by the gastrula stage, representing 120 to 200 SM50 mRNAs per primary mesenchyme cell. Experiments analyzing run-on transcription in nuclei isolated at different stages of development indicate that the sharp increase in SM50 mRNA levels occurring at the time of primary mesenchyme ingression is concomitant with an increase in transcription of the SM50 gene. We have also analyzed the RNA sequences present on polyribosomes at different stages of development. These studies indicate that SM50 mRNA is present on polyribosomes as soon as it begins to accumulate (which is well in advance of overt spicule formation) and SM50 mRNA remains on polyribosomes through subsequent development. From estimates of the rate of SM50 protein synthesis based on these data, we calculated that the maximum amount of SM50 accumulated during development through the 4-day pluteus stage is approximately 7.4 pg/embryo. This approximation is concordant with the amount of SM50 actually found in the sea urchin embryo.

Animals↗
PubMed1989

Lithium evokes expression of vegetal-specific molecules in the animal blastomeres of sea urchin embryos.

The mechanism of determination of early embryonic cells has been investigated using sea urchin embryos. An efficacious method of isolating blastomere pairs from the animal or vegetal half of sea urchin embryos was developed. The overt differentiation of separated animal and vegetal blastomere pairs resembles that of separated animal and vegetal hemispheres isolated by manual dissection. Treatment of animal blastomeres with LiCl caused them to display a morphology resembling that of isolated vegetal blastomeres. The effects of separation of animal and vegetal blastomeres and of treatment of animal blastomeres with LiCl were examined at the molecular level using gut alkaline phosphatase and a spicule matrix protein RNA as markers of differentiation. Histochemical staining and in situ hybridization studies showed that these markers are normally only expressed in vegetal blastomeres but that their expression can be evoked in animal blastomeres by treatment with LiCl.

Alkaline Phosphatase↗
PubMed1987

Changes in the synthesis and intracellular localization of nuclear proteins during embryogenesis in the sea urchin Strongylocentrotus purpuratus.

A rapid, gentle technique is described for the isolation of nuclei from sea urchin embryos. Using this technique, we have analyzed the synthesis and accumulation of nonhistone nuclear proteins during sea urchin development by two-dimensional gel electrophoresis. Most nuclear proteins fall into one of three patterns of synthesis, which are distinguished by maximal rates of accumulation at early (prior to hatching blastula), middle (hatching blastula/gastrula), or late (prism/pluteus) stages of development. Over 60% of observed nuclear proteins undergo apparent qualitative changes in synthesis and accumulation between the 64-cell and pluteus stages. Most of these changes represent appearances of new proteins. A large number of qualitative changes occur very early in development; the period of greatest change is between the 64-cell and 200-cell stages. Over half of the proteins which first appear in the nucleus subsequent to the 64-cell stage are synthesized at stages prior to the time of their initial appearance in nuclei, but are excluded from nuclei for some time.

Animals↗
PubMed1987

Determination and morphogenesis in the sea urchin embryo.

The study of the sea urchin embryo has contributed importantly to our ideas about embryogenesis. This essay re-examines some issues where the concerns of classical experimental embryology and cell and molecular biology converge. The sea urchin egg has an inherent animal-vegetal polarity. An egg fragment that contains both animal and vegetal material will produce a fairly normal larva. However, it is not clear to what extent the oral-aboral axis is specified in embryos developing from meridional fragments. Newly available markers of the oral-aboral axis allow this issue to be settled. When equatorial halves, in which animal and vegetal hemispheres are separated, are allowed to develop, the animal half forms a ciliated hollow ball. The vegetal half, however, often forms a complete embryo. This result is not in accord with the double gradient model of animal and vegetal characteristics that has been used to interpret almost all defect, isolation and transplantation experiments using sea urchin embryos. The effects of agents used to animalize and vegetalize embryos are also due for re-examination. The classical animalizing agent, Zn2+, causes developmental arrest, not expression of animal characters. On the other hand, Li+, a vegetalizing agent, probably changes the determination of animal cells. The stability of these early determinative steps may be examined in dissociation-reaggregation experiments, but this technique has not been exploited extensively. The morphogenetic movements of primary mesenchyme are complex and involve a number of interactions. It is curious that primary mesenchyme is dispensable in skeleton formation since in embryos devoid of primary mesenchyme, the secondary mesenchyme cells will form skeletal elements. It is likely that during its differentiation the primary mesenchyme provides some of its own extracellular microenvironment in the form of collagen and proteoglycans. The detailed form of spicules made by primary mesenchyme is determined by cooperation between the epithelial body wall, the extracellular material and the inherent properties of primary mesenchyme cells. Gastrulation in sea urchins is a two-step process. The first invagination is a buckling, the mechanism of which is not understood. The secondary phase in which the archenteron elongates across the blastocoel is probably driven primarily by active cell repacking. The extracellular matrix is important for this repacking to occur, but the basis of the cellular-environmental interaction is not understood.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗