Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevis

Abstract

Eleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3' untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui.

Transcription factors Mix1 and VegT, relocalization of vegt mRNA, and conserved endoderm and dorsal specification in frogs

“…In oocytes, Ec_vegt transcripts localize predominantly to the animal hemisphere. However, in 16-cell embryos, Ec_vegt transcripts were detected in equal proportions in animal and vegetal blastomeres (18). In addition, the presumptive nutritional endoderm expresses EcSox 17, activin b, derriere, Ecsmad2, and the proteins Smad2, Smad4 (23)(24)(25), VegT, and Mix1 (this report).…”

“…The vegt ORF has been sequenced in the amphibians E. coqui (Ec_vegt), Rana pipiens (Rp_vegt), and Ambystoma mexicanum (Am_vegt) (16)(17)(18) and partially sequenced in Epipedobates machalilla (Cm_vegt), Pipa pipa (Pp_vegt), and Gastrotheca riobambae (Gr_vegt) (8). A high degree of vegt conservation was detected in these anurans (8).…”

RNA localization mechanisms in oocytes

“…The majority of RNA localization studies in oocytes are limited to model organisms such as Drosophila and Xenopus, which are not necessarily reflective of other dipterans (Bullock et al, 2004) or amphibians. For example, in directly developing frog Eleutherodactylus coqui, in contrast to Xenopus, the mRNAs of the Vg1 and VegT orthologs are localized at the animal pole of the oocyte (Beckham et al, 2003). Findings like this indicate the necessity of widening studies to other, lesser-known species and taxa.…”

Nutritional endoderm in a direct developing frog: A potential parallel to the evolution of the amniote egg

“…RNA for EcVegT, the E. coqui orthologue, is greatly enriched near the animal pole of the E. coqui oocyte and cleaving embryo, unlike the localization of VegT RNA to the vegetal cortex of the X. laevis oocyte (Beckham et al, 2003). Nodal signaling in the E. coqui embryo, as assayed by mesoderm inducing activity, is restricted to the surface cells of the early gastrula (Ninomiya et al, 2001).…”

“…The RNA for EcVegT, the orthologue of the X. laevis endo-mesodermal determinant, is concentrated in the animal cytoplasm of the oocyte, rather than localized to the vegetal cortex (Beckham et al, 2003). Mesoderm-inducing activity is restricted to marginal surface in the E. coqui early gastrula and is absent from the deeper, yolky cells and the vegetal pole (Ninomiya et al, 2001).…”