Fred H. Wilt

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.

Mechanism of calcite co-orientation in the sea urchin tooth.

Sea urchin teeth are remarkable and complex calcite structures, continuously growing at the forming end and self-sharpening at the mature grinding tip. The calcite (CaCO(3)) crystals of tooth components, plates, fibers, and a high-Mg polycrystalline matrix, have highly co-oriented crystallographic axes. This ability to co-orient calcite in a mineralized structure is shared by all echinoderms. However, the physico-chemical mechanism by which calcite crystals become co-oriented in echinoderms remains enigmatic. Here, we show differences in calcite c-axis orientations in the tooth of the purple sea urchin ( Strongylocentrotus purpuratus ), using high-resolution X-ray photoelectron emission spectromicroscopy (X-PEEM) and microbeam X-ray diffraction (muXRD). All plates share one crystal orientation, propagated through pillar bridges, while fibers and polycrystalline matrix share another orientation. Furthermore, in the forming end of the tooth, we observe that CaCO(3) is present as amorphous calcium carbonate (ACC). We demonstrate that co-orientation of the nanoparticles in the polycrystalline matrix occurs via solid-state secondary nucleation, propagating out from the previously formed fibers and plates, into the amorphous precursor nanoparticles. Because amorphous precursors were observed in diverse biominerals, solid-state secondary nucleation is likely to be a general mechanism for the co-orientation of biomineral components in organisms from different phyla.

Phase transitions in biogenic amorphous calcium carbonate

Crystalline biominerals do not resemble faceted crystals. Current explanations for this property involve formation via amorphous phases. Using X-ray absorption near-edge structure (XANES) spectroscopy and photoelectron emission microscopy (PEEM), here we examine forming spicules in embryos of Strongylocentrotus purpuratus sea urchins, and observe a sequence of three mineral phases: hydrated amorphous calcium carbonate (ACC·H2O) → dehydrated amorphous calcium carbonate (ACC) → calcite. Unexpectedly, we find ACC·H2O-rich nanoparticles that persist after the surrounding mineral has dehydrated and crystallized. Protein matrix components occluded within the mineral must inhibit ACC·H2O dehydration. We devised an in vitro, also using XANES-PEEM, assay to identify spicule proteins that may play a role in stabilizing various mineral phases, and found that the most abundant occluded matrix protein in the sea urchin spicules, SM50, stabilizes ACC·H2O in vitro.

Erythropoiesis in the Chick Embryo: The Role of Endoderm

The endoderm can be separated from the ectomesoderm of the area vasculosa of the early chick embryo. The development of the separated germ layers in organ cultures shows that the endoderm is necessary for the normal formation of blood islands by mesodermal cells. In the absence of endoderm the mesodermal cells undergo erythropoiesis in dispersed groups of cells but do not form endothelium.

REGULATION OF THE INITIATION OF CHICK EMBRYO HEMOGLOBIN SYNTHESIS.

The chick embryo begins rapid synthesis of hemoglobin in the blood islands of the extraembryonic mesoderm at the seven-somite stage (34 hours incubation at 38°C). Explants of potential hemoglobin synthesizing tissues were placed in organ cultures for 2 to 72 hours. The capacity of the explants from the head-fold and later stages of the embryo to form hemoglobin was not eliminated by high concentrations of 5-bromodeoxyuridine, 5-fluorouracil or actinomycin D. However, the hemoglobin-forming system becomes somewhat resistant to 8-azaguanine after attaining the seven-somite stage. Fractionation of labeled RNA by sucrose density-gradient centrifugation showed that virtually all RNA synthesis except some turnover of 4 to 6 s RNA was eliminated within two hours after exposure to actinomycin D. This was confirmed autoradiographically. 8- Azaguanine reduced incorporation of uridine into ribosomal and messenger RNA populations by about 70%, and [8- 14 C]azaguanine was incorporated exclusively into 4 s RNA. It is concluded that the final transcription necessary for elaboration of a complete erythrocyte containing hemoglobin may occur prior to the head-fold stage. The initiation of hemoglobin synthesis at the seven-somite stage is probably regulated at the translational level.

Biomineralization of the Spicules of Sea Urchin Embryos

Abstract The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium cal-cite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.

Tissue interaction and the formation of the first erythroblasts of the chick embryo.

Abstract 1. 1. The ectomesoderm of the area vasculosa of the chick embryo of the definitive primitive streak stage was cultured with or without endoderm present on the opposite side of a Millipore filter. 2. 2. The development of blood islands was much more prominent in the ectomesoderm combined with endoderm across the filter than in control experiments without endoderm. 3. 3. No exchange of cells, however, between the interacting tissues was observed. A stainable material penetrating about one-fourth of the thickness of the filter was observed. 4. 4. The ectomesoderm taken from the posterior half of the area opaca cultured in direct contact with the endoderm from a blood island free area anterior to the head fold showed a prominent development of blood islands. 5. 5. The ectomesoderm cultivated in contact with the endoderm lethally treated with heat did not show distinct blood islands. The data are discussed in relation to the nature of tissue interactions.

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 [S]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.

The dynamics of maternal poly(A)-containing mRNA in fertilized sea urchin eggs

Cytoplasmic polyadenylated RNA with the characteristics of sequestered mRNA exists in the unfertilized sea urchin egg. Following egg activation, the amount of poly(A) doubles, but total RNA content stays constant. Chromatography of the RNA on poly(U)-Sepharose shows that the amount of RNA that bears a poly(A) tract increases slightly (approximately 20-30%) during the 2 hr after fertilization. When a cDNA transcript of the poly(A)+ mRNA from 2 hr zygotes is reacted against poly(A)+ RNA from either eggs or zygotes, the kinetics of reassociation of the two preparations seem identical; hence the RNA sequences bearing poly(A) are the same in eggs and zygotes. Measurement of the length of the poly(A) tract in eggs and zygotes shows an increase in number average length from about 45 bases to 60 bases. Measurement of tract length of poly(A) in two cell zygotes by adenosine/AMP ratios of radioactive RNA shows that the poly(A) tract of the zygote is solely accounted for radioactive RNA, indicating extensive turnover of the poly(A). It is concluded that the poly(A) tract in these cells is subject to both lengthening and shortening, with the former predominating in this instance. the increase in poly(A) does not involve polyadenylation of different sequences, but is due to an increase in the number of polyadenylated sequences and the length of the poly(A) tracts that they bear.

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.