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.

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.

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

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.

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.

Comparative analysis of Xenopus VegT, the meso-endodermal determinant, identifies an unusual conserved sequence.

The transcription factor, VegT, is the meso-endodermal determinant in Xenopus laevis. We examined VegT orthologs from several anuran amphibians and the urodele amphibian, the Mexican axolotl. In addition to the conserved T-box, the DNA-binding domain, the orthologs share a conserved 57 amino acid domain at the C-terminal. Most striking is a 17-nucleotide (nt) sequence near the 3' end of the open reading frame. The 17 nts are absolutely conserved among the anurans, whose last common ancestor lived 200 million years ago. As an initial test of the function of the 17 nts, 27 or 49 amino acids, which include the six amino acids coded by the 17 (+1) nts, were deleted from the C-terminal of VegT. These truncated VegT's retained some transcriptional activity, indicating that the 17 nts are not absolutely required for this function. The function of the highly conserved 17 nts is unknown. Two possibilities are that the conserved 17 nts interact with the cytoskeleton or that they are a target for regulation by a microRNA.

RNA of AmVegT, the axolotl orthologue of the Xenopus meso-endodermal determinant, is not localized in the oocyte.

The transcription factor, VegT, is the meso-endodermal determinant in Xenopus laevis, and the localization of VegT RNA to the vegetal cortex of the oocyte is an important starting point for embryonic patterning. We have cloned the VegT orthologue from the urodele amphibian, Ambystoma mexicanum, the Mexican axolotl. Axolotl VegT (AmVegT) is expressed zygotically in the presumptive mesoderm and Rohon-Beard neurons as in X. laevis, and its expression persists at the tip of the tail as with zebrafish and chicken orthologues. Unlike in X. laevis, AmVegT RNA is not localized in the oocyte. This result highlights a major difference in the molecular organization of oocytes of these two amphibian orders.

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui.

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.

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

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.