Distal cis-acting elements restrict expression of the CyIIIb actin gene in the aboral ectoderm of the sea urchin embryo

The distal region of the S. purpuratus actin CyIIIb gene, between −400 and −1400 nucleotides, contains at least three distinct cis-acting elements (C1R, C1L and E1) which are necessary for correct expression of fusion reporter genes in transgenic sea urchin embryos. The contribution of these elements in the temporal and spatial regulation of the gene was analyzed by single and double site-directed mutagenesis in fusion constructs which carry the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter. Following microinjection of the transgenes in sea urchin embryos, the activity of the mutants was compared to the wild type in time and space by measuring CAT activity at the blastula and pluteus embryonic stages and by in situ hybridization to the CAT mRNA at pluteus stage. Our results indicate that E1 involved in the temporal regulation of CyIIIb and that all three elements are necessary and sufficient to confer aboral (dorsal) ectoderm specificity to the proximal promoter. This is achieved by suppressing the promoter's activity in all other tissues by the cooperative interaction of the cis-acting elements. The C1R element, binding site of the nuclear receptors SpCOUP-TF and SpSHR2, is by itself sufficient to restrict expression in the ectoderm, whereas the aboral ectoderm restricted expression requires in addition the presence of both C1L adn E1. It is therefore evident, that the actin CyIIIb gene is exclusively expressed in the aboral ectoderm by a combinatorial repression in all other cell lineages of the developing embryo.     

The isolation of isohistones by preparative gel electrophoresis from embryos of the sea urchin Parechinus angulosus

Ten isohistones from the embryo of the sea urchin Parechinus angulosus have been isolated by preparative gel electrophoresis and characterised by electrophoretic mobility in two detergent systems, amino acid composition and partial sequences. This brings the total number of different histones identified which are synthesized at one or the other time during the life cycle of the sea urchin to a minimum of 24 structurally characterised polypeptides.     

Very early and transient vegetal-plate expression of SpKrox1, a Krüppel/Krox gene from Strongylocentrotus purpuratus

All endodermal and mesenchymal cells of the sea urchin embryo descend from the vegetal plate, a thickened epithelium of approximately 50 cells arising at the early blastula stage. Cell types that derive from the vegetal plate are specified conditionally by inductive interactions with underlying micromeres, but the molecular details of vegetal-plate specification remain unresolved. In a search for regulatory proteins that have roles in vegetal-plate specification, a screen was performed to clone Krüppel/Krox-related genes from a Strongylocentrotus purpuratus embryo cDNA library. One newly identified clone, named SpKrox1, contained four zinc fingers and a leucine zipper domain. SpKrox1 expression was low in unfertilized eggs, increased severalfold to the early blastula stage and decreased between the early gastrula and pluteus stages. SpKrox1 mRNA was first seen in macromeres of 16-cell stage embryos and was restricted to cells of the developing vegetal plate thereafter. Vegetal-plate expression corresponded to a ring of cells around the blastopore and overlapped the expression patterns of other genes with potential roles in vegetal plate-specification. As the vegetal-plate cells invaginated into the blastopore, SpKrox1 expression was lost, suggesting that its role was not in endoderm differentiation per se but rather in the initial establishment of the vegetal plate.     

The contractility of elongated microvilli in early sea urchin embryos

Summary Elongated microvilli attach the early sea urchin embryo to the fertilization envelope and support it in a concentric position within the perivitelline space. The contractility of the elongated microvilli was demonstrated in several ways. (1) During normal cleavage, these microvilli change their length to adapt to the change in shape and numbers of blastomeres. (2) When treated with calcium-free sea water, embryos become eccentrically located and the microvilli extend further than normal on one side; when returned to normal sea water, the embryos become centered again. (3) Several agents cause the fertilization envelope to become higher and thinner than normal and the elongated microvilli to extend correspondingly if treated within ten min after fertilization. In some cases, both elongated microvilli and fertilization envelope return to normal size when returned to normal sea water. (4) Fertilization in a papain solution causes the elongated microvilli and the fertilization envelope to contract to the surface of the embryo. (5) Refertilization after the papain-induced contraction can bring about the elongation of these microvilli and the elevation of the fertilization envelope a second time. It was also shown that elongated microvilli are extended immediately upon fertilization, at the same time as the short microvilli. The firm adherence of the tips of elongated microvilli to the fertilization envelope by means of extracellular matrix fibers is shown in a high voltage electron microscope stereoimage. This allows us to understand why it is that when the elongated microvilli extend or contract, the fertilization envelope also extends and contracts accordingly.     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signaling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.     

Direct measurement of histone peptide elongation rate in cleaving sea urchin embryos

Regulation of protein synthesis can be exercised at a number of levels. One of the more experimentally difficult levels to approach has been the measurement of peptide elongation rate. This paper presents a new application of the cyanogen bromide (CNBr) cleavage of proteins in a direct measurement of histone peptide elongation rate in cleaving sea urchin embryos (Strongylocentrotus purpuratus). The data indicate an elongation rate (at 15°C) for histones H2B and H1α of 0.69 and 0.80 codons per s, respectively. These values fall within the range of previously published values of average peptide elongation rate for total protein in these cells. This method should be generally applicable to many systems for which the measurement of peptide elongation rate may provide a key to the understanding of the regulation of protein synthesis.     

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.     

Electron microscope visualization of giant polysomes in sea urchin embryos

Chromatin spreading techniques have been applied to the electron microscopic visualization of polysomes in sea urchin (Strongylocentrotus purpuratus) eggs and embryos. Polysomes of giant size are commonly found after the 8-cell stage. The largest seen, from an early gastrula, was 13.6 m in length, carried 277 ribosomes, with a message calculated to contain 6.49×10⁴ nucleotides and potentially to encoded 2.38×10⁶ daltons of peptide. Polysomes are rare and very large ones absent from lysates of unfertilized eggs. Giant polysomes appear in 4- to 8-cell stages and are common in 16-cell stages and thereafter. They are of two forms: a compact form with no spacing between ribosomes characteristic of stages through early mesenchyme blastulae, and an extended form found only after late mesenchyme blastulae. Both have potential for massive informational content. Some of each type have ribosome-free tails at one end, as long as 733 Å in the compact forms, and 7,890 Å in the extended ones. Occasionally they have a single array of fibrous material increasing from one end of a polysome to the other, interpreted to be nascent peptide chains. Polysomes are not found after brief, mild exposure of lysates to RNase A, or from embryos treated with puromycin. Very large polysomes are present in lysates of blastulae exposed since fertilization to actinomycin D, cycloheximide, or cordycepin. They appear in parthenogenetically activated or fertilized enucleate merogones, but are absent from unactivated merogones, demonstrating that egg masked messages can generate them. A potential embryological significance of giant, potentially polycistronic polysomes is suggested.