PubMed Health⌕ Search

Biomedical subjects

L M Angerer

Publications and source records attributed to L M Angerer.

At least 37 records · Page 2Linked to original sources
PubMed1995

An orthodenticle-related protein from Strongylocentrotus purpuratus.

Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus. The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.

Amino Acid Sequence↗
PubMed1995

Characterization of the SpHE promoter that is spatially regulated along the animal-vegetal axis of the sea urchin embryo.

To understand how the maternally determined animal-vegetal polarity of the sea urchin embryo is established, we have begun to examine the regulatory apparatus of the gene encoding the Strongylocentrotus purpuratus hatching enzyme (SpHE). Previous studies have shown that the pattern of SpHE mRNA accumulation reflects the animal-vegetal developmental axis in that transcription is strongly upregulated during early cleavage in more animal blastomeres, but not in those around the maternally specified vegetal pole of the 16-cell embryo [Reynolds et al., Development 114, 769-786 (1992)]. Tests of SpHE promoter function in vivo using chloramphenicol acetyltransferase and beta-galactosidase enzymatic reporters define a regulatory region within several hundred nucleotides of the transcription initiation site. This region is sufficient to mediate both strong expression in the early blastula and spatially correct transcription. However, neither this region nor longer upstream sequences are sufficient to reproduce the transcriptional downregulation after very early blastula stage that is observed for endogenous genes. Biochemical assays of protein-DNA interactions within the regulatory region identify at least nine sites binding at least six different factors. These cis elements include Otx (an orthodenticle homologue), CCAAT, ets-related, and three unidentified motifs. Deletions and/or replacements of these cis-elements, alone and in combination, indicate that no single factor is essential for SpHE promoter activity, but instead that various combinations of subsets of these elements are capable of eliciting levels of transcription similar to those of the unaltered regulatory region. This density of regulatory elements is consistent with the intense transcription of endogenous SpHE genes during cleavage.

Animals↗
PubMed1994

Distinct pattern of embryonic expression of the sea urchin CyI actin gene in Tripneustes gratilla.

Cloning and sequencing of Tripneustes gratilla genomic DNA and cDNA encoding a developmentally regulated, embryonic messenger RNA, referred to as Tg616, revealed an actin-encoding gene orthologous to the CyI actin gene described from Strongylocentrotus purpuratus. Tg616 and SpCyI share: (1) 150 nucleotides of highly conserved sequence 5' of the transcription start site, (2) 95% nucleotide sequence identity in the protein encoding regions, which specify identical amino acid residues in 375 of 377 positions, and (3) extensive nucleotide sequence identity in the 3' untranslated region of their messenger RNAs. Tg616 was therefore designated TgCyI. In situ hybridization shows sequential activation of TgCyI in various cells of the embryo. TgCyI mRNA becomes abundant in primary and secondary mesenchyme cells as they prepare to enter the blastocoel, in prospective aboral ectoderm cells at blastula stage, in gut cells during gut differentiation, and in oral ectoderm at pluteus stage. This pattern of embryonic gene expression is more complex than any of the major patterns of developmentally upregulated genes observed in S. purpuratus embryos and is distinct from SpCyI expression which is progressively restricted to the gut and oral ectoderm.

Actins↗
PubMed1994

The univin gene encodes a member of the transforming growth factor-beta superfamily with restricted expression in the sea urchin embryo.

We have identified a gene in the sea urchin Strongylocentrotus purpuratus that encodes a member of the transforming growth factor beta (TGF-beta) gene superfamily. We have named the gene univin, and it is the first member of this superfamily to be reported in echinoderms. The cDNA sequence predicts a 383-amino-acid residue protein with 7 cysteine residues characteristic of members of this superfamily and with a cluster of basic residues appropriately situated to signal proteolytic cleavage. Sequence comparisons place univin in the bone morphogenetic protein (BMP) group of the TGF-beta superfamily along with the vertebrate BMPs, decapentaplegic protein from Drosophila, and Vg-1 from Xenopus. Analyses of univin expression in early embryos by RNA blots and in situ hybridization revealed the highest levels of expression in the egg and prehatching blastula. During late cleavage stages, univin mRNA accumulation is progressively restricted to a circumequatorial band. Expression is further restricted during gastrulation when univin transcripts are detected primarily in the presumptive foregut and ciliated band. By pluteus stage, signals are detectable only in these cell types. The restricted temporal and spatial patterns of expression of univin during early blastula stages parallel those of SpAN, which encodes an astacin-like protease related to tolloid and BMP-1 (Reynolds et al., 1992). The fact that these proteases are thought to function in the proteolytic activation of TGF-beta-related proteins that, respectively, regulate Drosophila embryonic dorsal-ventral patterning and vertebrate bone development suggests that SpAN and univin could also have critical roles in early developmental decisions in the sea urchin embryo.

Amino Acid Sequence↗
PubMed1993

Major temporal and spatial patterns of gene expression during differentiation of the sea urchin embryo.

We have investigated the temporal and spatial patterns of accumulation of mRNAs randomly selected from the sea urchin gastrula polyadenylated RNA population. Three different assays show that the predominant temporal pattern of expression, exhibited by about three-fourths of these messages, consists of a large (mean 80-fold) increase in mRNA abundance between egg and gastrula stages. Most mRNAs are present in the maternal population and are detectable on blots as single mature-sized messages; however, a large number of high-molecular-weight, heterodisperse transcripts containing these same sequences also exist in the egg cytoplasm. The majority of gastrula messages are not embryo specific but are present in total adult urchin RNA at concentrations similar to those in embryos. Fine-scale RNA blot analysis indicates that the majority of mRNAs begin to accumulate at very early blastula stages, although there is considerable diversity in the time when these messages reach peak abundance. Most gastrula mRNAs are also spatially regulated during development. The observed distributions can be categorized into three major functional or regulatory classes: (1) Forty percent of mRNAs accumulate in cells which are cycling or preparing for growth. (2) About one-third of the messages accumulate in one or more differentiating cell types. (3) Only slightly more than one-fourth of the messages are present in all cell types throughout development. Most tissue-specific messages are relatively abundant, indicating that the differentiated functions of cells are executed through mRNAs operating at the level of hundreds of copies per cell. In contrast, most rare messages are expressed in most or all cell types, in which they function at only a few copies per cell. All messages which begin to accumulate before hatching blastula stage are initially distributed broadly, and their distribution becomes progressively restricted during embryogenesis. In contrast, all messages which begin to accumulate after the onset of gastrulation accumulate only in discrete subsets of cells. The results presented here illustrate much more extensive temporal regulation of gene expression during sea urchin embryogenesis than previously detected. This is accompanied by spatial regulation of expression of most genes which is itself temporally modulated as the cellular requirements for cell division and differentiation change during development.

Age Factors↗
PubMed1992

Centrifugal elutriation of large fragile cells: isolation of RNA from fixed embryonic blastomeres.

In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96, 94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed non-uniformly among these cells.

Animals↗
PubMed1992

Tissue-restricted accumulation of a ribosomal protein mRNA is not coordinated with rRNA transcription and precedes growth of the sea urchin pluteus larva.

We have identified an mRNA that encodes a protein, SpS24, of the small ribosomal subunit in the sea urchin, Strongylocentrotus purpuratus. RNA blot and in situ hybridization analyses show that the SpS24 gene is active during early oogenesis, downregulated in the mature egg and during cleavage, and reactivated in the early blastula. The mRNA then increases in abundance at least 100-fold. Later in development, expression of SpS24 mRNA becomes restricted primarily to cells in the oral ectoderm and endoderm of the pluteus larva, and the message is undetectable in aboral ectoderm cells and most mesenchyme cells. To determine whether transcription of the ribosomal RNA genes occurs at a higher rate in oral ectoderm and endoderm tissues, a probe for the transcribed spacer was used in RNase protection and in situ hybridization assays. High concentrations of rRNA-processing intermediates were observed in unfertilized eggs and shown to reside primarily, if not exclusively, in the cytoplasm. The spatial and temporal distributions of these sequences strongly suggest that they are associated with heavy bodies. New embryonic rRNA transcripts are first detectable at the very early blastula stage. In later embryos, the content of this transcribed spacer sequence is similar in all but a few cells, which implies that they synthesize rRNA at a similar low rate. Comparison of available estimates of rRNA transcription rate with the potential rate of SpS24 protein synthesis, calculated from SpS24 mRNA prevalence, shows that oral ectoderm and endoderm cells have the capacity to synthesize 15- to 30-fold more SpS24 protein than is required to keep pace with rRNA synthesis in these cells. Because the sea urchin embryo develops from an egg to a pluteus larva in the absence of growth, this stockpiling of SpS24 mRNA anticipates rather than accompanies the onset of growth, which does not begin until after feeding. Upregulation of this gene is therefore part of the developmental program, rather than a physiological response to nutrient availability.

Amino Acid Sequence↗
PubMed1992

Posttranscriptional regulation of ectoderm-specific gene expression in early sea urchin embryos.

During development of the sea urchin Strongylocentrotus purpuratus embryo, transcription of the Spec1 and actin CyIIIa genes is activated and the corresponding mRNAs accumulate specifically in ectoderm cells. We show that in gastrulae this tissue specificity of mRNA accumulation is regulated largely if not entirely at a posttranscriptional level. We used RNAase protection assays with intron and exon probes to measure the levels of nuclear precursors and mature message, respectively, in total RNA from embryo fractions enriched for ectoderm (Ect) or endoderm+mesenchyme (E/M) cells. These measurements demonstrate that E/M cells, which do not accumulate Spec1 and actin CyIIIa mRNAs, contain high levels of intron transcripts, indicating that cells of the E/M tissues transcribe these genes. At later stages, transcripts containing intron sequences are restricted to ectoderm cells. These results indicate that there is a transition from posttranscriptional to transcriptional regulation of tissue-specific mRNA accumulation during the gastrula stage. Measurements of transcription rate by nuclear run-on assays substantiate this conclusion for Spec1 and extend it to two other genes, SpEGFI and Spec2c, which also encode ectoderm-specific mRNAs. Posttranscriptional regulation was not observed for the SM50 gene whose mRNA accumulates only in primary mesenchyme cells, or for actin CyI which is expressed predominantly in E/M cells of gastrulae.

Animals↗
PubMed1992

Early mRNAs, spatially restricted along the animal-vegetal axis of sea urchin embryos, include one encoding a protein related to tolloid and BMP-1.

The cloning and characterization of cDNAs representing four genes or small gene families that are coordinately expressed in a spatially restricted pattern during the very early blastula (VEB) stage of sea urchin development are presented. The VEB genes encode multiple transcripts that are expressed transiently in embryos of Strongylocentrotus purpuratus between 16-cell stage and hatching, with peak abundance 12 to 15 hours post-fertilization (approximately 150-250 cells). The VEB transcripts share the same spatial pattern in the early blastula embryo: they are asymmetrically distributed along the animal-vegetal axis but their distribution around this axis is uniform. Thus, the VEB transcripts are the earliest messages to reveal asymmetry along the primary axis in the sea urchin embryo. The temporal and spatial patterns of VEB transcript accumulation are not consistent with involvement of these gene products in cell division or in tissue-specific functions. Furthermore, VEB messages cannot be detected in either ovary or adult tissues, suggesting that these genes function exclusively during embryogenesis. We suggest that the VEB genes function in constructing the early blastula. Two VEB genes encode metalloendoproteases: one (SpHE) is hatching enzyme and the other (SpAN) is similar to bone morphogenetic protein-1 (BMP-1; Wozney et al., Science 242: 1528-1534, 1988) and the Tolloid gene product (tld) (Shimell et al., Cell 67: 459-482, 1991). Several lines of evidence suggest that the VEB genes are regulated directly by factors or regulatory activities localized along the maternally specificed animal-vegetal axis.

Animals↗
PubMed1991

Expression of two mRNAs encoding EGF-related proteins identifies subregions of sea urchin embryonic ectoderm.

Many proteins containing domains related to epidermal growth factor (EGF) function in intercellular interactions that mediate specification of cell fate. We have used in situ hybridization to show that the expression of two EGF-related genes (SpEGF I and SpEGF II) is restricted to the same subset of ectodermal cells in sea urchin pluteus larvae. However, the concentration of EGF I mRNA in different epithelial cells of aboral ectoderm and postoral facial epithelium is constant while that of EGF II mRNA is highly modulated. RNase protection assays show that both genes are activated during the period when ectoderm funder cells are established, i.e., between fourth and fifth and between fifth and sixth cleavages for EGF I and EGF II, respectively. By mesenchyme blastula stage EGF I mRNA reaches maximum abundance (800-1000 copies/expressing cell) as a result of a high transcription rate, while EGF II mRNA peaks at about half that concentration by gastrula stage. EGF I expression begins at early stages of oogenesis while EGF II expression appears to be confined to embryogenesis.

Animals↗
PubMed1989

Unusual pattern of accumulation of mRNA encoding EGF-related protein in sea urchin embryos.

A sea urchin (Strongylocentrotus purpuratus) messenger RNA encoding a protein (SpEGF2) related to epidermal growth factor (EGF) was identified. The full-length complementary DNA sequence predicts a protein with an unusually simple structure, including four tandem EGF-like repeats and a hydrophobic leader, but lacking a potential transmembrane domain. Sequence similarities suggest that the peptides are homologous to two peptides from a different sea urchin species, which cause a classic developmental defect, exogastrulation, when added to the seawater outside of embryos. The SpEGF2 messenger RNA begins to accumulate at blastula stage, and in pluteus larvae it is distributed in discrete regions of ectoderm that are not congruent with known histological borders. One region corresponds to that expressing the homeodomain-containing protein, SpHbox1. The structure of the SpEGF2 protein and the pattern of accumulation of its messenger RNA suggest that it may have important functions as a secreted factor during development of sea urchin embryos.

Animals↗
PubMed1989

Structure and tissue-specific developmental expression of a sea urchin arylsulfatase gene.

Arylsulfatases are a group of enzymes that remove sulfate moieties from a diverse set of substrates including glycoproteins, steroids, and cerebrosides. We have isolated recombinant cDNA clones corresponding to an arylsulfatase (SpARS) message that encodes an abundant protein of pluteus larvae of the sea urchin Strongylocentrotus purpuratus. Although vertebrate arylsulfatases have broad tissue distributions, in situ hybridization with a probe for SpARS shows that the sea urchin message accumulates in the embryo only in the single cell type of aboral ectoderm and its precursors. The message is first detectable by RNase protection assays around hatching blastula stage and accumulates through pluteus larva stage. The open reading frame of cDNA clones is 1701 nt long and encodes a deduced protein with a predicted molecular mass of 61 kDa. Analysis of corresponding genomic DNA clones reveals that the pre-mRNA contains six exons. Consistent with the fact that arylsulfatase enzyme activity is extracellular, this polypeptide has a hydrophobic leader sequence and three potential glycosylation sites. Furthermore, hybridization in situ shows that in blastulae arylsulfatase message is preferentially concentrated around nuclei at the basal sides of cells. The S. purpuratus sequence is very similar to that recently reported for the same enzyme from Hemicentrotus pulcherrimus and 30% of the amino acid residues are also identical to those of both human arylsulfatase C (steroid sulfatase) and arylsulfatase A. Sequence relationships among these four mRNAs suggest that, assuming equal rates of evolution, the duplication separating the human genes occurred at about the time of separation of the echinoderm and vertebrate lineages.

Age Factors↗
PubMed1989

Progressively restricted expression of a homeo box gene within the aboral ectoderm of developing sea urchin embryos.

A homeo box-containing gene, Hbox1 is expressed in an unusual and highly conserved spatial pattern in embryos of two different species of sea urchin, Tripneustes gratilla and Strongylocentrotus purpuratus. Hybridization in situ shows that this mRNA accumulates initially throughout the aboral ectoderm; however, between blastula and pluteus stages, the region containing Hbox1 mRNA retracts gradually until only a small area around the vertex is labeled in pluteus larvae. Aboral ectoderm appears cytologically uniform and also accumulates uniform levels of other tissue-specific mRNAs. Therefore, the Hbox1 pattern reveals a previously unsuspected heterogeneity of aboral ectoderm cells and a polarity within this tissue. In S. purpuratus, the Hbox1 gene product probably is not involved in initial specification of cell fate, as this message does not achieve a significant fraction of its peak abundance until almost hatching blastula stage, well after the time aboral ectoderm cells have initiated a tissue-specific program of gene expression. RNA blot and RNase protection analyses revealed low levels of Hbox1 mRNA in all adult tissues examined. However, this message was not detectable in mature eggs, suggesting that the Hbox1 gene does not have a maternal function. In addition to highly conserved spatial and temporal patterns of expression, the homeo box genes of these two urchin species also are conserved highly in sequences outside the homeo domain, despite the divergence of these two species (30-45 my). Two notable features of the protein shared with several vertebrate homeo proteins are a short conserved sequence encoded by an exon upstream of that encoding the homeo domain and a large region of high serine and proline content.

Amino Acid Sequence↗
PubMed1989

Altered expression of spatially regulated embryonic genes in the progeny of separated sea urchin blastomeres.

We have examined the importance of the extracellular environment on the ability of separated cells of sea urchin embryos (Strongylocentrotus purpuratus) to carry out patterns of mRNA accumulation and decay characteristic of intact embryos. Embryos were dissociated into individual blastomeres at 16-cell stage and maintained in calcium-free sea water so that daughter cells continuously separated. Levels of eleven different mRNAs in these cells were compared to those in control embryos when the latter reached mesenchyme blastula stage, by which time cells in major regions of the intact embryo have assumed distinctive patterns of message accumulation. Abrogation of interactions among cells resulted in marked differences in accumulation and/or turnover of the individual mRNAs, which are expressed with diverse temporal and spatial patterns of prevalence in intact embryos. In general, separated cells are competent to execute initial events of mRNA accumulation and decay that occur uniformly in most or all blastomeres of the intact embryo and are likely to be regulated by maternal molecules. The ability of separated cells to accumulate mRNAs that appear slightly later in development depends upon the presumptive tissue in which a given mRNA is found in the normal embryo. Messages that normally accumulate in cells at the vegetal pole also accumulate in dissociated cells either at nearly normal levels or at increased levels. In one such case, that of actin CyIIa, which is normally restricted to mesenchyme cells, in situ hybridization demonstrates that the fraction of dissociated cells expressing this message is 4- to 5-fold higher than in the normal embryo. In contrast, separated cells accumulate significant levels of a message expressed uniformly in the early ectoderm but are unable to execute accumulation and decay of different messages that distinguish oral and aboral ectodermal regions. These data are consistent with the idea that interactions among cells in the intact embryo are important for both positive and negative control of expression of different genes that are early indicators of the specification of cell fate.

Animals↗
PubMed1988

Spec2 genes of Strongylocentrotus purpuratus. Structure and differential expression in embryonic aboral ectoderm cells.

Members of the Spec gene family are expressed during embryonic development of the sea urchin, Strongylocentrotus purpuratus. The family encodes proteins related to the calmodulin/troponin C/myosin light chain group of calcium binding proteins and one gene, Spec1, has been studied extensively in our laboratory. In this paper, we analyze other members of the family, collectively termed Spec2 genes. We make use of several hybridization probes derived from Spec1 and Spec2 cDNA clones, which recognize different members of the family. Genomic DNA gel blot and slot blot analyses show that there are approximately eight Spec genes in the S. purpuratus genome. The structures of three Spec2 genes, Spec2a, Spec2c and Spec2d, are described. A 60 kb (kb = 10(3) bases or base-pairs) region of the genome contains the linked Spec1-Spec2c genes and two separate 20 kb regions contain the Spec2a and Spec2d genes. Six members of a repetitive sequence family are dispersed at various locations among the genes. The transcriptional initiation sites of the three Spec2 genes are mapped, and 400 to 500 base-pairs of 5'-flanking DNA sequenced. All three Spec2 genes initiate transcription approximately 120 base-pairs upstream from the 3' end of the first exon. In contrast, the 5' end of the Spec1 transcript begins about 107 base-pairs farther upstream, so it contains 5' untranslated sequences that correspond to non-transcribed 5'-flanking sequences of the Spec2 genes. There is little similarity among the sequences upstream from the CAP site of the Spec2 genes except the TATA consensus sequence and a repeating trinucleotide, AAC. Measurements of Spec mRNA levels during embryogenesis show that Spec1 mRNA begins to accumulate at the early blastula stage and is the most abundant; Spec2a/Spec2c mRNAs begin accumulating several hours later at the late blastula-early gastrula stage and reach about 40 to 60% the levels of Spec1; and Spec2d mRNAs accumulate mostly during the gastrula and pluteus stages with levels reaching only 2% those of Spec1. In situ hybridization with probes that recognize either all Spec2 mRNAs or only Spec2d mRNAs show that, like Spec1, these mRNAs are restricted to aboral ectoderm cells and their precursors. The Spec gene family represents a group of related genes whose mRNAs all accumulate in the same cell type but at different times and to different levels during embryogenesis.

Animals↗
PubMed1988

A histone H1 protein in sea urchins is encoded by a poly(A)+ mRNA.

Typical histone genes lack intervening sequences and encode small mRNAs (400-800 nucleotides) with short leader and trailer regions. Most histone mRNAs are not polyadenylylated but rather terminate in a highly conserved stem and loop structure. The early, late, and testis-specific histone genes of sea urchins, described to date, have this typical histone gene structure. We have identified an unusual H1 gene, H1-delta, in sea urchins that encodes a poly(A)+ mRNA. This mRNA is one of a group of polyadenylylated transcripts homologous with H1 gene probes. The sequence of H1-delta had been determined. H1-delta encodes a different H1 protein. Although the temporal expression of H1-delta mRNA is similar to that of other late H1 (beta and gamma) mRNAs, its spatial distribution at the time of maximal accumulation is distinct and confirms that H1-delta is regulated differently than other H1 genes.

Amino Acid Sequence↗
PubMed1988

Expression of a collagen gene in mesenchyme lineages of the Strongylocentrotus purpuratus embryo.

We have previously described cloning of an exon of a sea urchin collagen gene and shown that its expression is temporally regulated during embryogenesis, beginning during blastula formation. We have now localized the protein encoded by the gene and the sites of its mRNA synthesis in the developing embryo. Antibody to a synthetic peptide reacts with a 208,000 Mr protein that is digestible by collagenase. Fractionation of pluteus stage embryos demonstrates that the protein is localized primarily with cells that form the syncytium of primary mesenchyme that elaborates the larval endoskeleton; furthermore, immunofluorescence localizes the epitope to the periphery of the endoskeleton in situ. Transcripts of the gene accumulate only in mesenchyme cells, especially those of the primary mesenchyme lineage. Measurements of absolute transcript abundance show that collagen mRNA is present in blastula primary mesenchyme cells at 600-700 copies per cell and at about fourfold lower amounts in other mesenchyme cells.

Animals↗