Kimberly Nath

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

Summary 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.

Lbx1 expression and frog limb development

In order to identify prospective limb muscle cells in a frog, we cloned Lbx1 from the direct developing frog Eleutherodactylus coqui. Like in embryos of the frog Xenopus laevis but unlike in other vertebrates, EcLbx1 is expressed in all trunk somites. Like in embryos of chick, mouse, and zebrafish, cells expressing EcLbx1 are then found in limb buds, consistent with migration of those cells from somites. EcLbx1 is also expressed in the dorsal spinal cord as in other vertebrates.

Raldh expression in embryos of the direct developing frog Eleutherodactylus coqui and the conserved retinoic acid requirement for forelimb initiation

Embryos of the direct developing frog, Eleutherodactylus coqui, provide opportunities to examine frog early limb development that are not available in species with tadpoles. We cloned two retinaldehyde dehydrogenase genes, EcRaldh1 and EcRaldh2, to see which enzyme likely supplies retinoic acid for limb development. EcRaldh1 is expressed in the dorsal retina, otic vesicle, pronephros, and pronephric duct, but not in the limb. EcRaldh2 is expressed early at the blastoporal lip and then in the mesoderm in the neurula, so this expression could function in forelimb initiation. Later EcRaldh2 is expressed in the mesoderm at the base of the limbs and in the ventral spinal cord where motor neurons innervating the limbs emerge. These observations on a frog support the functional conservation of EcRaldh2 in forelimb initiation in Osteichthyans and in limb patterning and motor neuron specification in tetrapods.

Expression of a cardiac myosin gene in non-heart tissues of developing frogs

Direct developing frogs, like Eleutherodactylus coqui, provide opportunities to investigate limb early development in anuran amphibians that are less available in species with tadpoles. We have found that myosin heavy chain 6 (myh6), a myosin gene usually considered heart-specific in Xenopus and other animals, is expressed in limbs of E. coqui embryos. The gene for microRNA(miR)-208 is contained in an intron of the E. coqui myh6 gene as in mammals, and miR-208 was detected as a microRNA, more highly expressed in a microarray of E. coqui limb buds, compared to Xenopus laevis limb buds. Myh6 is also expressed in several muscles of tadpoles and froglets of Xenopus tropicalis. These connections raise the possibility of an involvement of myh6 and miR-208 in the thyroid dependent metamorphosis of anurans.

Expression of cyclin D1, cyclin D2, and N-myc in embryos of the direct developing frog Eleutherodactylus coqui, with a focus on limbs

Species of frogs that develop directly have removed the tadpole from their ontogeny and form adult structures precociously. To see whether cell cycle regulators could be involved in this altered embryogenesis, we examined the expression of ccnd1, ccnd2, and mycn in embryos of the direct developing frog, Eleutherodactylus coqui. Notable differences compared to embryos of Xenopus laevis, a species with a tadpole, included prominent expression of ccnd2 in the midbrain and ccnd1 in the mandibular neural crest. The former may contribute to the precocious appearance of the adult-type visual system and the latter to the adult-type jaw. Large domains of ccnd2 and mycn presage the early appearance of limb buds, and ccnd1 and mycn are implicated in digit development.