Elinson RP et al. (2011), Germ plasm in Eleutherodactylus coqui, a direct...

XB-ART-44164

Evodevo 2011 Oct 06;2:20. doi: 10.1186/2041-9139-2-20.

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Germ plasm in Eleutherodactylus coqui, a direct developing frog with large eggs.

Elinson RP , Sabo MC , Fisher C , Yamaguchi T , Orii H , Nath K .

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BACKGROUND: RNAs for embryo patterning and for germ cell specification are localized to the vegetal cortex of the oocyte of Xenopus laevis. In oocytes of the direct developing frog Eleutherodactylus coqui, orthologous RNAs for patterning are not localized, raising the question as to whether RNAs and other components of germ plasm are localized in this species. METHODS: To identify germ plasm, E. coqui embryos were stained with DiOC6(3) or examined by in situ hybridization for dazl and DEADSouth RNAs. The cDNAs for the E. coqui orthologues were cloned by RT-PCR using degenerate primers. To examine activity of the E. coqui orthologues, RNAs, made from constructs of their 3'UTRs with mCherry, were injected into X. laevis embryos. RESULTS: Both DiOC6(3) and dazl and DEADSouth in situs identified many small islands at the vegetal surface of cleaving E. coqui embryos, indicative of germ plasm. Dazl was also expressed in primordial germ cells in the genital ridge. The 3'UTRs of E. coqui dazl and DEADSouth directed primordial germ cell specific protein synthesis in X. laevis. CONCLUSIONS: E. coqui utilizes germ plasm with RNAs localized to the vegetal cortex to specify primordial germ cells. The large number of germ plasm islands suggests that an increase in the amount of germ plasm was important in the evolution of the large E. coqui egg.

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Species referenced: Xenopus laevis
Genes referenced: dazl ddx25 pgc

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	Figure 1. Migrating PGCs. The skin was removed from this TS8 embryo to reveal white, yolk-filled PGCs in the posterior part of the dorsal mesentery (arrow). The inset shows an enlargement of this region.
	Figure 2. PGCs in embryo sections. (A) PGCs are present in the dorsal mesentery (arrow) which attaches the yolky endoderm to the rest of the body of this TS8 embryo. The inset shows an enlargement with three PGCs in the mesentery. (B) At TS9, yolk-filled PGCs are clumped in the prospective genital ridges (arrow), ventral to the dorsal aorta. The inset shows an enlargement of this region.
	Figure 3. DiOC6(3) staining of cleaving embryos. (A) Only one furrow (bright green line) has reached the vegetal pole in this eight-cell embryo. A field of small DiOC6(3) stained islands is present on the vegetal surface, with most of the islands on one side of the furrow. Furrows stain brightly as they represent two closely-apposed new membranes. (B) This photograph of a different eight-cell embryo shows a cleavage furrow (bright green line) and the boundary between vegetal regions with DiOC6(3) stained islands and with no stained islands. (C, D) Vegetal (C) and animal (D) views of the same approximately 16-cell embryo. Although seven cells are present near the animal pole (D), only two furrows are present in the vegetal region (C). DiOC6(3) stained islands are present primarily in three of the four vegetal cells (C). (E, F) Vegetal (E) and equatorial (F) views of the same morula stage embryo. Although the region near the animal pole is divided into many cells, a limited number of cleavage furrows have reached the vegetal pole. DiOC6(3) islands remain on the vegetal surface, with islands lined up in the forming furrows (E). Embryos are about 3.5 mm in diameter, with (C) enlarged approximately 30% relative to (A) (D) (E) (F).
	Figure 4. Ecdazl expression in oocytes. In this ovarian fragment, in situ hybridization showed Ecdazl RNA throughout small oocytes, just beginning vitellogenesis, and localized to one side of vitellogenic oocytes. The vitellogenic oocytes in this preparation are about 1 mm in diameter. Large oocytes, up to 3.5 mm, did not show clear staining and were removed prior to processing of the ovarian fragment.
	Figure 5. Ecdazl and EcDEADSouth RNA in cleaving embryos. (A) In this vegetal view, many islands containing Ecdazl RNA are visible near the surface of a four-cell embryo. Cleavage furrows have not reached the vegetal half. (B) In this side view of a 16-cell embryo, islands of Ecdazl RNA are present near the vegetal pole. Cleavage furrows have reached the vegetal half. (C) In this vegetal view, many EcDEADSouth islands are visible despite some surface damage. (D) In this 16-cell embryo with two furrows reaching the vegetal pole, most of the surface surrounding the vegetal pole has been lost, revealing the whiter underlying cytoplasm. The surface loss permits several observations. First, EcDEADSouth islands are visible on the intact surface. Second, the islands are located asymmetrically relative to the vegetal pole, marked by the intersecting furrows. Third, EcDEADSouth islands are present in the cleavage furrow, where internal cytoplasm is exposed.
	Figure 6. Ecdazl expression in primordial germ cells. Yolk-filled cells with Ecdazl RNA (arrows) are present in the genital ridges of this TS14 embryo.
	Figure 7. Activity of the EcDazl3'UTR. A X. laevis egg was injected with RNAs of both Venus-XlDS3'UTR and mCHERRY-EcDazl3'UTR. The genital ridge region of the embryo (A) was examined at stage 40 with different light sources to detect Venus (B, green) and mCHERRY (C, red) proteins. The merged image (D, yellow) shows that these two proteins co-localized. The co-localization indicates that the EcDazl 3'UTR directed PGC-specific protein expression in X. laevis.

References [+] :

Bachvarova, Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes. 2009, Pubmed