BECKHAM, Y.M.; NATH, K.; ELINSON, R.P.; Univ. of Toronto, Toronto; Duquense Univ., Pittsburgh: Eleutherodactylus coqui homologues of VegT and Vg1 and their localization during early development

Eleutherodactylus coqui has a large 3.5 mm oocyte which allows it to develop directly to a frog without a 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 homologues of VegT and Vg1, due to the large egg size. Through a combination of degenerate RT-PCR and RACE we cloned 1747 bp of EcVegT and 1314 bp of EcVg1. Northern blot analysis shows that the lengths of these mRNAs are 2.5 kb and 1.3 kb, respectively, indicating that we have the complete EcVg1 mRNA. The EcVg1 mRNA has an extremely short 3′ UTR in comparison to X. laevis, 252 bp and 1240 bp respectively. 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 EcVegT and EcVg1 are present in both animal and vegetal blastomeres after the first horizontal cleavage. Thus, the animal blastomeres inherit half of the EcVegT and EcVg1 mRNAs although they are only one percent of the embryo volume. 3’RACE of EcVegT also resulted in a fragment of 1311 bp containing the VegT T-box and 3′ sequence of latent TGFb binding protein. The unusual nature of this mRNA, with a DNA binding domain and an extracellular domain, is not a cloning artifact, as demonstrated by RT-PCR and sequencing. The relative importance and functions of these two EcVegT mRNAs remain to be determined.