Constantin N. Flytzanis

Very early and transient vegetal plate expression of SpKrox1, a Kruppel/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.

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 is 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 promoters 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 and 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.

Biochemical characterization of gonadal development in the shrimp Penaeus vannamei

Abstract 1. 1. Mini-two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze polypeptides extracted from eggs and gonadal tissues from different developmental stages of male and female P. vannamei shrimp. 2. 2. Gel patterns of polypeptides from immature and mature ovaries showed notable differences. 3. 3. Several biochemical differences between ovarian and egg samples indicated that the majority of egg polypeptides may be synthesized towards the end of oogenesis. 4. 4. Five polypeptides present in the eggs, immature and mature ovaries may be synthesized early in oogenesis. 5. 5. Qualitative polypeptide differences between testes from adult and juvenile shrimp suggest differential expression of genes in a developmentally controlled program.

Purification of a high-mobility-group 1 sea-urchin protein and cloning of cDNAs

The isolation of the sea urchin high-mobility-group 1 (HMG1) protein, the cloning of corresponding cDNA clones and the similarity to the human homologue are described. Sea urchin HMG1 was purified as one of the nuclear embryonic proteins which associate with an upstream regulatory element (E1) of the Strongylocentrotus purpuratus (Sp) CyIIIb actin-encoding gene. Using a synthetic oligodeoxyribonucleotide (oligo) which includes the E1 cis-acting element in a DNA affinity chromatography purification, the most prominent of the binding proteins was isolated and the N terminus sequenced. cDNA clones were isolated by screening an embryonic cDNA library with a synthetic oligo derived from the amino acid (aa) sequence. Comparison of the cDNAs ORF to known proteins revealed a 50% aa identity to the mammalian HMG1 and all the structural characteristics of this group of proteins. The sea urchin protein, SpHMG1, was synthesized in bacteria, as well as translated in vitro. Binding assays carried out with the recombinant SpHMG1 protein did not produce specific in vitro complexes with E1.

Sex-specific gene expression in distinct tissues of the shrimp Penaeus vannamei.

Abstract 1. 1. Soluble proteins extracted from male and female Penaeus vannamei tissues such as eyes, eyestalks, brain, nerve cord, hemolymph, heart, muscle, hepatopancreas, hepatopancreas membrane and cuticular epidermis were analyzed and compared by high-resolution mini-two-dimensional polyacrylamide gel electrophoresis (mini-2D-PAGE). 2. 2. In each shrimp tissue a large number of discrete polypeptides was observed. 3. 3. The polypeptide patterns from the same tissue of female and male shrimp were mostly similar but both qualitative and quantitative differences were noted, suggesting the presence of sex-specific gene products in various shrimp tissues. 4. 4. Future applications of these results are discussed.

Differential expression of cuticle-epidermis proteins in the shrimp Penaeus vannamei during molting

High-resolution mini-two-dimensional polyacrylamide gel electrophoresis (mini-2D-PAGE) was used to analyze silver-stained, soluble proteins from the cuticle-epidermis of Penaeus vannamei during molting. The 2D-PAGE patterns of epidermis polypeptides from metecdysis and anecdysis/proecdysis molt stages demonstrated similarities as well as several quantitative and qualitative differences. Quantitative modulation in polypeptide expression was noted in at least seven prevalent polypeptides during molting. A 50 kDa protein is specifically expressed in anecdysis/proecdysis tissue samples. Quantitative and qualitative differences were also noted in proteins migrating mainly in the molecular mass ranges of 26-32 kDa. An overall increase in polypeptide expression was noted in this molecular mass range at metecdysis as compared to anecdysis/proecdysis epidermis tissues. These results indicate modulation of cuticle-epidermis proteins in Penaeus vannamei shrimps during molting.

STRUCTURE AND EXPRESSION OF THE SEA URCHIN SPEC GENES

Publisher Summary This chapter elaborates the structure and the expression of the sea urchin Spec genes. The Spec genes comprise a family of 10 to 12 related genes whose expression is restricted to embryonic and larval aboral ectoderm cells of the sea urchin, Strongylocentrotus purpuratus. The Spec proteins are members of the troponin C superfamily of calcium-binding proteins and bind calcium ions in vitro as assayed by shifts on SDS polyacrylamide gels in the presence or the absence of EGTA. Because it is likely that proteins involved in activating the Spec genes are maternal or very early zygotic gene products, knowledge of the mechanisms by which the Spec genes are specifically activated in aboral ectoderm cells will necessarily yield information on how these embryonic cells are determined. Spec1 upstream sequences are compatable with promoter elements present on the deleted cyIIIa-CAT plasmid and that the position of the Spec1 upstream sequences is not critical to the enhancement of CAT activity.