Sea urchin maternal and embryonic U1 RNAs are spatially segregated in early embryos.

We have used in situ hybridization and cell fractionation methods to follow the distribution of U1 RNA and immunofluorescence microscopy to follow the distribution of snRNP proteins in oocytes, eggs, and embryos of several sea urchin species. U1 RNA and U1-specific snRNP antigens are concentrated in germinal vesicles of oocytes. Both appear to relocate after oocyte maturation because they are found primarily, if not exclusively, in the cytoplasm of mature unfertilized eggs. This cytoplasmic residence is maintained during early cleavage and U1 RNA is first detectable in nuclei of micromeres at the 16-cell stage. Between morula and gastrula stages the steady-state concentrations of both RNA and antigens gradually increase in nuclei and decrease in cytoplasm. Surprisingly, analysis of the distribution of newly synthesized U1 RNA shows that it does not equilibrate with the maternal pool. Instead new transcripts are confined to nuclei, while cytoplasmic U1 RNAs are of maternal origin. This lack of equilibration and the conversion of maternal U1 RNAs from nuclear species in oocytes to cytoplasmic in embryos suggests that these RNPs (or RNAs) are structurally altered when released to the cytoplasm at oocyte maturation.

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.

Molecular indices of cell lineage specification in sea urchin embryos.

The origins of several of the differentiated cell lineages of the advanced sea urchin embryo are well defined. Cytological application of molecular probes to three lineages, those responsible for the formation of the skeleton, the gut, and the aboral ectodermal wall of the late embryo, has demonstrated expression of lineage-specific genes long before overt morphological differentiation. These observations lead to useful generalizations regarding the processes of gene regulation that underlie the molecular biology of cell lineage specification in the embryo.

Identification of a new sea urchin ets protein, SpEts4, by yeast one-hybrid screening with the hatching enzyme promoter.

We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE. This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity. To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element. This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4. This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments. The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output. As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula.

Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis.

We have determined spatial patterns of expression of individual actin genes in embryos of the sea urchin Strongylocentrotus purpuratus. Radioactively labeled probes specific for each of five cytoplasmic-type (Cy) and the single muscle-type (M) mRNAs were hybridized in situ to sections of fixed embryos. M actin mRNA appears only late in development and is confined to a few cells associated with the coelomic rudiments. The five Cy mRNAs fall into three sets, whose times and sites of expression during development are highly distinctive. Different cell lineages express messages of one or more of these sets, but never all three. Although all Cy actin mRNAs exhibit monophasic accumulation in the RNA of whole embryos during the course of development, such accumulation in many cases results from the summation of both increases and decreases in abundance within individual sets of cells. Within the genomic linkage group CyI-CyIIa-CyIIb, expression of CyI and CyIIb appears to be co-ordinate, and quite distinct from that of CyIIa. CyI and CyIIb are expressed in all lineages at some point in embryogenesis, but confined mainly to oral ectoderm and portions of the gut of the pluteus larva. CyIIa mRNAs are restricted to mesenchyme lineages throughout late gastrula stage, and subsequently accumulate in parts of the gut. The CyIIIa and CyIIIb genes, which form a separate linkage group, are expressed only in aboral ectoderm and its precursors. Furthermore, CyIII messages are the only detectable actin mRNAs in this cell lineage after late blastula stage.

Activation of sea urchin actin genes during embryogenesis. Measurement of transcript accumulation from five different genes in Strongylocentrotus purpuratus.

The number of molecules of mRNA transcribed from each of five different actin genes are reported for developing embryos of the sea urchin Strongylocentrotus purpuratus. Transcripts of the cytoskeletal actin genes CyI, CyIIa, CyIIb and CyIIIa, and of the muscle actin gene M, were measured in unfertilized egg and embryo RNAs of cleavage, blastula, gastrula and pluteus stages. The measurements were obtained by probe excess titrations of these RNAs, using a set of single-stranded RNA probes each identifying the mRNA transcripts of a specific actin gene. These mRNAs can be identified by their distinct 3' non-translated trailer sequences. We confirm prior observations that the prevalence of actin mRNA in the unfertilized egg is low. Cytoskeletal actin genes CyI and CyIIIa each contribute 1 X 10(3) to 2 X 10(3) maternal mRNA molecules, and CyIIb contributes less than 2 X 10(2) mRNA molecules, while no detectable maternal mRNAs derive from cytoskeletal actin gene CyIIa or the muscle actin gene M. During certain periods of development, transcripts derived from the individual cytoskeletal actin genes accumulate rapidly, with kinetics specific to each mRNA. Transcripts of the muscle actin gene are absent until after gastrulation, when the initial muscle progenitor cells are formed. At late stages of development, each of the five genes studied is represented by 10(4) to 10(5) mRNA molecules per embryo. The present measurements permit calculation of the levels of each actin mRNA species in the particular cell types in which each gene functions in the fully differentiated embryo.

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.

The SpHE gene is downregulated in sea urchin late blastulae despite persistence of multiple positive factors sufficient to activate its promoter.

Previous studies of the regulatory region of the SpHE (hatching enzyme) gene of the sea urchin Strongylocentrotus purpuratus (Wei, Z., Angerer, L.M., Gagnon, M.L. and Angerer, R.C. (1995) Characterization of the SpHE promoter that are spatially regulated along the animal-vegetal axis of the sea urchin embryo. Dev. Biol. 171, 195-211) have shown that approximately 330 bp is necessary and sufficient to promote high level expression in embryos of transgenes that reproduce the spatially asymmetric pattern of endogenous gene activity along the maternally determined animal-vegetal embryonic axis. Furthermore, SpHE regulatory elements appear to be redundant since several different combinations are sufficient to elicit strong promoter activity and many subsets function like the endogenous gene only in non-vegetal cells of the blastula (Wei, Z., Angerer, L.M. and Angerer, R.C. (1997) Multiple positive cis-elements regulate the asymmetric expression of the SpHE gene along the sea urchin embryo animal-vegetal axis. Dev. Biol., 187, 71-88). Here we demonstrate by in vivo footprinting that many cis elements on the endogenous promoter are occupied when the gene is active in early blastulae, but the binding of corresponding trans factors is significantly reduced when the gene becomes inactive in late blastulae. In addition, downregulation of the promoter is accompanied by a transition from a non-nucleosomal to a nucleosome-like chromatin structure. Surprisingly, in vitro DNase I footprints of the 300 bp promoter using nuclear protein extracts from early and late blastulae are not detectably different and neither this sequence, nor a longer one extending to -1255, reproduces the loss of endogenous SpHE transcriptional activity after very early blastula stage. These observations imply that temporal repression of SpHE transcription involves a decrease in accessibility of the promoter to activators that are nevertheless present in nuclei and capable of activating transgene promoters. Temporal, but not spatial, downregulation is therefore likely to be regulated by negative activities functioning outside the -1255 promoter region which may serve as direct repressors or mediate an inactive chromatin structure.

SpSoxB1 serves an essential architectural function in the promoter SpAN, a tolloid/BMP1-related gene.

Transcription of SpAN, which encodes a secreted protease related to tolloid and BMP 1, is differentially regulated along the animal-vegetal axis of the sea urchin embryo by a maternally initiated mechanism. Regulatory sites that bind SpSoxB1 and CBF (CCAAT binding factor) are essential for strong transcriptional activity because mutations of these elements reduce promoter activity in vivo 20- and 10-fold, respectively. Here we show that multimerized SpSoxB1 elements cannot activate transcription from the SpAN basal promoter in vivo. However, like other factors containing HMG-class DNA binding domains, SpSoxB1 does induce strong bending of DNA. The CBF binding site lies abnormally far from the transcriptional start site at -200 bp. We show that the SpSoxB1 site is not required if the CCAAT element is moved 100 bp closer to the transcriptional start site, replacing the SpSoxB1 site. This supports a model in which the bending of SpAN promoter DNA by SpSoxB1 facilitates interactions between factors binding to upstream and downstream regulatory elements.

Animal-vegetal axis patterning mechanisms in the early sea urchin embryo.

We discuss recent progress in understanding how cell fates are specified along the animal-vegetal axis of the sea urchin embryo. This process is initiated by cell-autonomous, maternally directed, mechanisms that establish three unique gene-regulatory domains. These domains are defined by distinct sets of vegetalizing (beta-catenin) and animalizing transcription factor (ATF) activities and their region of overlap in the macromeres, which specifies these cells as early mesendoderm. Subsequent signaling among cleavage-stage blastomeres further subdivides fates of macromere progeny to yield major embryonic tissues. Zygotically produced Wnt8 reinforces maternally regulated levels of nuclear beta-catenin in vegetal derivatives to down regulate ATF activity and further promote mesendoderm fates. Signaling through the Notch receptor from the vegetal micromere lineages diverts adjacent mesendoderm to secondary mesenchyme fates. Continued Wnt signaling expands the vegetal domain of beta-catenin's transcriptional regulatory activity and competes with animal signaling factors, including BMP2/4, to specify the endoderm-ectoderm border within veg(1) progeny. This model places new emphasis on the importance of the ratio of maternally regulated vegetal and animal transcription factor activities in initial specification events along the animal-vegetal axis.

Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization.

We present an improved procedure for detecting poly A tracts in situ by hybridization of 3H poly U. Glutaraldehyde fixation achieves significantly higher retention of RNA and better morphologic preservation than does Carnoy's. A dramatic increase in signal to noise is obtained by prehybridization treatment of glutaraldehyde-fixed sections with proteinase K and acetic anhydride. Measurement of the increase in poly A concentration after fertilization by solution titration and by in situ hybridization are in excellent agreement indicating that in situ measurements yield accurate relative estimates of local RNA concentrations in sections. Examination of the grain density distribution in section of sea urchin eggs and cleaving embryos reveals no major cytoplasmic localization of poly A+ RNA, although nuclei show much less labelling and micromeres of 16-cell embryos have a small, but significant, reduction in poly A concentration.

Regulative development of the sea urchin embryo: signalling cascades and morphogen gradients.

Differentiation of sea urchin embryo ectoderm, endoderm and mesenchyme cells, whose anlagen are arrayed along the animal-vegetal axis, relies on both maternally regulated localized transcription factor activities and cell-cell signalling. Classic models proposed that fates are determined by opposing animal and vegetal morphogenetic gradients, whereas current models emphasize unidirectional and sequential vegetal-to-animal signalling cascades between adjacent blastomeres. Recent data support aspects of both models: the vegetal micromeres send one or more signals, which depend on a nuclear beta-catenin-dependent pathway, that both activate Notch signalling required for secondary mesenchyme fate and promote endoderm differentiation and gastrulation. This is opposed by an animalizing domain of BMP4 signals that regulates ectodermal cell fates and establishes the ectoderm-endoderm border.

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.

Regulation of protein synthesis in Tetrahymena. Quantitative estimates of the parameters determining the rates of protein synthesis in growing, starved, and starved-deciliated cells.

The calculated rate of protein synthesis for growing Tetrahymena is 360 pg/h, whereas starved cells synthesize only about 3 pg of protein/h. Within 50 min after deciliation of starved cells, the rate of protein synthesis increases to about 60 pg/h. The major mechanism to accomplish these large and rapid changes in the rate of bulk protein synthesis involves regulation of the number of messages loaded on polysomes. Logarithmically growing cells contain about 3.2 X 10(7) mRNA molecules/cell, of which approximately 60% is loaded on polysomes. Starved cells contain about 0.8 X 10(7) messages and the percentage of messages loaded is reduced to 4%. Thus, the number of loaded messages is approximately 60-fold lower in starved cells than in growing cells, although the total message content of cells in these two physiological states differs by only a factor of 4. After deciliation of starved cells, message loading increases about 10-fold. It seems likely that much of the message loaded after deciliation is derived from the large pool of nonpolysomal message in starved cells. Although large differences in message loading exist for growing, starved, and starved-deciliated cells, measurements of the rate of polypeptide elongation and the rate of message initiation indicate the translational efficiency of loaded messages (pg of protein synthesized per pg of message/unit time) is very similar under all conditions.

Single copy DNA and structural gene sequence relationships among four sea urchin species.

Measurements of the divergence of single copy DNA sequences among four sea urichin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60 degrees C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5 degrees C; S. franciscanus 51% and 3.5 degrees C; Lytechinus pictus 12% and 7.5 degrees C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06-0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.

DNA sequence organization in the mollusc Aplysia californica.

The sequence organization of the DNA of the mollusc Aplysia californica has been examined by a combination of techniques. Close-spaced interspersion of repetitive and single copy sequences occurs throughout the majority of the genome. Detailed examination of the DNA of this protostome reveals great similarities to the pattern observed in the two deuterostome organisms previously examined in detail in this laboratory, Xenopus laevis and Strongylocentrotus purpuratus. Labeled and unlabeled Aplysia DNA were prepared from developing embryos and sheared to a fragment length of 400 nucleotides. The kinetics of reassociation were studied by means of hydroxyapatite chromatography, single-strand-specific S1 nuclease, and optical methods of assay. Aplysia DNA of this fragment length contains at least five resolvable kinetic fractions. One classification of these fractions, listed with their reassociation rate constants (l M-1 sec-1) is: single copy (0.00057), slow (0.047), fast (2.58), very fast (4000), and foldback (greater than 10(5)). Sequence arrangement was deduced from: the kinetics of reassociation of DNA fragments of length 400 or 2000 nucleotides; the hyperchromicity of reassociated fragments containing duplex regions; the size of duplex regions resistant to S1 nuclease; and the reassociation of labeled fragments of various lengths with short driver fragments. More than 80% of the single copy DNA sequences are interspersed with repetitive sequences. The maximum spacing of the repeats is about 2000 nucleotides, and the average less than 1000. The very fast fraction does not show interspersion with single copy sequences or with other kinetic fractions. The foldback fraction sequences are fairly widely interspersed. The slow fraction sequences are interspersed with the fast fraction, and possibly also with the single copy DNA. The fast fraction is the dominant interspersed repetitive fraction. Its sequences are adjacent to the great majority of the single copy sequences and have an average length of about 300 nucleotides.

Multiple positive cis elements regulate the asymmetric expression of the SpHE gene along the sea urchin embryo animal-vegetal axis.

The mechanism that establishes the maternally determined animal-vegetal axis of sea urchin embryos is unknown. We have analyzed the cis-regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a beta-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195-211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic beta-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis-acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957-1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early embryo.

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.