The Role of Vasa Protein Accumulation in Sea Urchin Embryogenesis

What is the role of Vasa accumulation in sea urchin embryogenesis, and how does this selective accumulation take place?

Vasa protein overview

Vasa is an ATP-dependent DEAD box helicase (a family of proteins which unwind nucleic acids) present in numerous animals across many phylum. In many animals, including sea urchins, vasa is a marker of the germline (the line of cells that contains genetic information). Initially identified in Drosophila melanogaster as a part of the germ plasm, this component functions as a translational activator of the maternally inherited RNAs gurken and oskar (Juliano and Wessel, 2009).

Vasa accumulation in sea urchin embryos

In sea urchin embryogenesis, Vasa mRNA is uniformly distributed throughout blastula formation, and then is specifically expressed in the small micromere lineage during gastrulation. However, the fourth cell division causes the Vasa protein to be distributed unevenly throughout the cells. After the fourth cell division, the 16-cell embryo contains 8 mesomeres, 4 macromeres, and 4 micromeres. (The micromeres function as signaling centers that influence the fate of adjacent cells.) This uneven distribution eventually results in Vasa protein enrichment in the small micromeres, which remain at the vegetal plate of the blastula. These cells are then carried into the blastocoel during gastrulation, and are incorporated into the left and right coelomic pouches of the larva (which later become the site of adult rudiment formation) (Juliano and Wessel, 2009).

Fig. 1. Echinoderm phylogeny and small micromere evolution. (A) Micromeres are a derived feature among echinoderms. In the sea urchin embryo, four micromeres (purple) asymmetrically divide to give rise to four large micromeres (blue) and four small micromeres (red). Large micromeres give rise to the larval skeleton. Small micromeres reside at the vegetal plate of the blastula, travel at the tip of archenteron in the gastrula, and divide only once before being incorporated into the left and right coelomic pouches of the larva. (B) Members of the phylum Echinodermata are a sister-group to the chordates. There are five extant classes of echinoderms: Crinoidea (feather stars), Ophiuroidea (brittle stars), Asteroidea (sea stars), Holothuridea (sea cucumbers), and Echinoidea. Members of the class Echinoidea, which have a micromere lineage, are split into the subclasses Euechinoids (sea urchins and sand dollars) and Cidaroids (pencil urchins) (Ettensohn et al. 2004).

Since vasa mRNA is uniformly distributed in the sea urchin embryo but the Vasa protein accumulates selectively at the fourth cleavage division, an important post-transcriptional regulation must be taking place. This can either be due to specific control of the ubiquitous mRNA and/or stability of the Vasa protein in specific cell types (Juliano and Wessel, 2009).

A suggested method of selective Vasa accumulation

Recent studies suggest that the Vasa protein is subject to proteastome-mediated degradation, which drives selective accumulation in the small micromere lineage. Gustafson et al. demonstrated this by testing protein turnover and its role in regulating Vasa protein accumulation. They treated sea urchin embryos with the reversible proteasome inhibitor MG132. Vasa protein levels were assayed by immunofluorescence and Western blotting. Their results demonstrate that MG132-treated embryos showed an increase in the Vasa immunofluorescence signal throughout the embryo. Both the immunofluorescence and Western blotting show a dosage-dependent increase of approximately 2- to 3-fold after treatment with MG132. These data show that in wild-type embryos, the Vasa protein is subject to proteasome-dependent degradation in non-small micromeres (Gustavson et al., 2011).

Figure 2. Vasa protein accumulates in all cells of the embryo following proteasome inhibition. (A) Immunofluorescence localization of endogenous Vasa protein in embryos cultured in artificial seawater (ASW) alone, ASW containing 0.5% dimethyl sulfoxide (DMSO), or ASW containing 10 μM, 25 μM or 50 μM MG132. Images of Vasa protein staining are shown in grayscale next to their corresponding DIC images. (B) Quantitative immunoblot analysis of Vasa levels normalized to actin levels using protein extracts from each culture. (C) Quantitative analysis of Vasa fluorescence per embryo in (A, purple) and immunoblotting (B, red). Error bars correspond to the fluorescence intensity standard deviation within the individual embryos assayed (ASW, n=26; DMSO, n=29; 10 μM MG132 n=30; 25 μM MG132 n=27; 50 μM MG132 n=31) in immunofluorescence quantification and the standard deviation from 3 separate immunoblots in Western blotting quantification (Juliano and Wessel, 2009).

Gustavus binding plays a role in selective Vasa accumulation

Gustavus, an E3 ubiqiutin ligase specificity receptor, interacts with the Vasa protein through its B30.2/SPRY binding domain. Based on experimental data from a GST pull-down assay, Gustafson et al. showed that Gustavus has at least two interaction sites on the Vasa protein. They suggest that Gustavus binds to the N-terminal portion and the DEAD-box domain independently through two separate binding surfaces (Figure 3, Gustavson et al., 2011).

Figure 3. A bipartite Gustavus–Vasa binding model in S. purpuratus. The gustavus B30.2/SPRY domain is shown in blue with binding surface A and binding surface B indicated as predicted previously (Woo et al., 2006a). A schematic representation of the S. purpuratus vasa DEAD-box domain in orange, C-terminus domain in pink, N-terminus CCHC zinc knuckles in purple and unstructured glycine-rich flexible sequence in yellow (Gustavson et al., 2011).

Because of this specific binding, Gustafson et al. suggest two hypotheses for Gustavus regulation of Vasa: first, that Gustavus may function as a negative regulator of Vasa protein stability. They believe that Gustavus achieves this by targeting Vasa for polyubiquitylation and degradation by the proteasome in non-small micromere cells. Second, they suggest that Gustavus may function as a positive regulator of Vasa protein stability by competing for Vasa binding with other proteins that act to target Vasa for proteolysis. In both scenarios , Gustavus would most likely directly interact with the Vasa protein through its B30.2/SPRY binding domain and link it to the ubiquitin conjugating machinery (Gustavson et al., 2011).

In summary, Vasa is an important helicase protein that serves as a marker of the germ plasm in sea urchin embryogenesis. Selective accumulation and degradation of this protein drives micromere cell differentiation, and subsequent post-translational modification. Recent studies show that the protein Gustavus may serve as a regulator of the Vasa protein and link it to ubiquitin conjugating machinery.

Resources

Gustafson, E., Yajima, M., Juliano, C. and Wessel, G. (2011). Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis. Developmental Biology 349: 440–450.

Juliano, C.E. and Wessel, G.M. (2009). An evolutionary transition of vasa regulation in echinoderms. Evolution and Development 11(5): 560-573.

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