Neural development in the marsupial frog Gastrotheca riobambae.

The expression pattern of the tyrosine kinase gene Pag in whole-mount preparations of Gastrotheca riobambae embryos and the immunostaining of embryos against the proteins vimentin, NCAM, Pax-2, Hoxd9, and antigen 2G9 allowed detection of migrating streams of cranial neural crest (NC) cells, the isthmus, the hindbrain boundaries, rhombomeres, cranial nerves, and the developing spinal cord. Expression patterns of these genes and the basic neural morphology of Gastrotheca have been conserved in comparison with other vertebrates. However, as in Xenopus, a prominent stream of migrating cranial NC cells from rhombomere 5 was found in Gastrotheca embryos. By contrast, in chick embryos, premigratory NC cells from rhombomeres 3 and 5 undergo extensive apoptosis, which suggests that in anurans, apoptosis of the cranial NC may deviate from the chick pattern. The branchial-anterior and branchial-posterior masses of cranial NC cells, that populate the gill arches, are very large in G. riobambae. We cannot distinguish whether this feature corresponds to an anuran trait related to development of the tadpole pharyngeal skeleton, or is related to development of the peculiar external bell gills of this frog. This work provides the descriptive groundwork for lineage studies of the NC in G. riobambae embryos. Gastrotheca embryos are large and flat, have prominent streams of cranial NC cells, and develop very large external bell gills. These unique characteristics may facilitate future comparative analysis of the role of apoptosis in patterning the amphibian NC cell streams, and the contribution of the NC to development of the gills.

The expression of Brachyury (T) during gastrulation in the marsupial frog Gastrotheca riobambae.

Gastrulation in the marsupial frog Gastrotheca riobambae has been analyzed by the distribution of the Brachyury (T) protein. Comparison with other amphibians provides mechanistic insights, since G. riobambae develops slowly and has the most divergent mode of amphibian gastrulation, producing an embryonic disk. The T pattern indicates that the prospective mesoderm is superficial, as in many amphibians. The dorsal blastopore lip could not be identified by the expression of T, or by morphological criteria, thus it is unknown whether Gastrotheca embryos have a dorsal organizer before or after blastopore closure. The circumblastoporal and notochordal expression of T, which are temporally contiguous in Xenopus, are separated in Gastrotheca, implying that distinct regulatory mechanisms may control the expression of T in its two domains. The separation of the T pattern also indicates that involution at the blastopore is separate from notochord formation. In addition, extension of the archenteron and notochord occurs after blastopore closure, suggesting that dorsal convergence and extension have been delayed until after blastopore closure. Therefore, dorsal convergence and extension need not be the cause of blastopore closure in Gastrotheca. The separation of gastrulation events in embryos that have not been experimentally manipulated, such as those of Gastrotheca, helps in understanding the distinct nature of gastrulation processes.

Somitogenesis in the marsupial frog Gastrotheca riobambae.

This paper reports the first description of somitogenesis in a non-aquatic-developing amphibian, the Andean marsupial frog, Gastrotheca riobambae. This frog develops from an embryonic disk located on top of a large yolky egg, bearing some resemblance to the embryo of the chick. Besides the histological characterization of somite formation, we quantified cell number in the developing somites of Gastrotheca, Xenopus laevis and the chick. Gastrotheca was found to have a mode of somitogenesis which has previously been encountered in the aquatic-developing toad Bombina. Somitic cell number was found to be an order of magnitude higher than that of Xenopus, and approximately double that of the chick. We discuss the possible relation between mode of somitogenesis, somitic cell number, speed of development and egg size.

5S rRNA-encoding genes of the marsupial frog Gastrotheca riobambae.

The major 5S rRNA gene repeat of the marsupial frog, Gastrotheca riobambae, is 1052 bp in length. It contains a 5S rRNA gene similar to the Xenopus laevis somatic gene, two spacer regions, and a pseudogene. The G. riobambae haploid genome contains about 500 copies of this predominant repeat. This relatively low number of 5S rRNA genes is associated with a limited amplification of the 18S, 5.8S and 28S rRNA genes in oocytes and with a slow rate of early development.

Ureotelism as the prevailing mode of nitrogen excretion in larvae of the marsupial frog Gastrotheca riobambae (Fowler) (Anura, Hylidae).

1. Embryos from the pouch of the marsupial hylid frog Gastrotheca riobambae excrete urea. 2. Free-living tadpoles of G. riobambae excrete mainly urea, in comparison with tadpoles of other hylid frogs. 3. The activity of arginase is high in embryos from the pouch, and in tadpoles of G. riobambae. 4. The ureotelism of G. riobambae larvae is an adaptation for prolonged incubation in the pouch of the mother, and for development in limited amounts of water.

The pattern of early cleavage of the marsupial frog Gastrotheca riobambae.

Comparison of early development of the marsupial frog Gastrotheca riobambae with Xenopus laevis suggests that the cleavage pattern of Xenopus and the tight coupling of events at the midblastula transition are features of the accelerated development of small amphibian eggs with aquatic reproduction, rather than generalized features of amphibian development. The large eggs of the marsupial frog Gastrotheca riobambae not only display an atypical holoblastic pattern of cleavage and a very slow rate of development, but the events of the midblastula transition are uncoupled, suggesting that amphibians may have a diversity of developmental patterns. Early cleavage of the egg, which measures about 3 mm in diameter, occurs mostly by meridional and vertical furrows. The first cleavage cycle takes about 16 to 20 h and about four days may be required to reach the midblastula stage. Cleavage becomes asynchronous at about the third cleavage cycle evidenced by the formation of cleavage furrows. However, during cleavage (up to 342-cells), the majority of the nuclei divide synchronously and only 15 to 40% of the nuclei of a given embryo have a different cleavage schedule. At the 8-cell stage, nucleoli become visible (approximately 24 h after amplexus), signaling that transcription of rRNA has started at this early stage. Cell motility was detected in three- to four-day old embryos and seems to be associated with changes in cell shape and with expansion of the blastocoel at this stage.

A Practical Guide to the Developmental Biology of Terrestrial-Breeding Frogs.

Many frogs lay their eggs in water; the development of these frogs is well-known. However, many frogs reproduce on land; their eggs are large and have an altered early development. As examples, Gastrotheca riobambae broods its embryos in a pouch on the mother's back, and Eleutherodactylus coqui exhibits direct development with no tadpole stage. We provide practical information on obtaining eggs and embryos from these terrestrial-breeding species and on analyzing their development. Our aim is to make these species more accessible to researchers who are interested in the developmental and evolutionary consequences of terrestrial development.

Cleavage and gastrulation in the egg-brooding, marsupial frog, Gastrotheca riobambae.

The marsupial frog Gastrotheca riobambae has several reproductive adaptations, most prominent of which is the incubation of the embryo in a pouch on the mother's back. We have followed cleavage and gastrulation by microscopical observation and by vital staining, and have found several alterations in these processes which may reflect the reproductive adaptations. The large, yolky egg has a cap of yolk-poor cytoplasm at the animal pole which is incorporated into a translucent blastocoel roof consisting of a single cell layer. The epithelium of the yolk sac is derived from the roof. The inconspicuous blastoporal lips form near the vegetal pole from cells of the marginal region. Gastrulation movements include the epibolic stretching of the surface towards the blastopore and a contraction of the vegetal surface. The blastoporal lips close over a small archenteron, and the cells of the lips become the embryonic disc, a discrete group of small cells which give rise to most of the embryo's body. The great size difference between animal and vegetal blastomeres during cleavage, the single-celled blastocoel roof, the dissociation in time between archenteron formation and its expansion, the embryonic disc and the slow development distinguish G. riobambae embryos from those of other frogs. The importance of the marginal region which produces the embryonic disc and the unimportance of the most animal region whose fate is primarily yolk sac emphasizes the role of the marginal region in amphibian development.

Role of progesterone on oocyte maturation in the egg-brooding hylid frog Gastrotheca riobambae (Fowler).

In the egg-brooding frog Gastrotheca riobambae (Fowler), oocyte maturation is comparable to the situation of other frog species. In isolated follicles, progesterone induces only germinal vesicle breakdown (GVBD), while human chorionic gonadotropin (hCG) induces GVBD and ovulation. In addition, defolliculated oocytes respond with GVBD to the treatment with progesterone, while hCG has no effect. As in other frogs, oocyte maturation in vitro depends on hormonal action and on the presence of divalent cations. In this frog, progesterone or a similar hormone conditions the brooding pouch for reproduction and induces pouch closure. Follicles from frogs with closed pouches showed GVBD after 15-17 hours of incubation with progesterone, while those from frogs with open pouches took 19-24 hours for GVBD. These findings suggest that follicles become stimulated for maturation when the pouch is closed and that this stimulated condition is maintained for several weeks in advance of the process of oocyte maturation. In G. riobambae, the external appearance of the pouch aperture indicates the reproductive condition of the ovary.

Progesterone induces incubatory changes in the brooding pouch of the frog Gastrotheca riobambae (Fowler).

Progesterone induces closure, vascularization, and the formation of embryonic chambers around inert beads in the brooding pouch of the frog Gastrotheca riobambae (Fowler). These changes of the pouch resemble those occurring during normal reproduction. In contrast to progesterone, human chorionic gonadotropin induces pouch closure only in females with large ovaries. The results suggest that pituitary gonadotropin indirectly induces the incubatory changes of the pouch by stimulating the secretion of a progesterone-like hormone from the ovary.

Ribosomal gene amplification in multinucleate oocytes of the egg brooding hylid frog Flectonotus pygmaeus.

The multinucleate oocytes of Flectonotus pygmaeus begin as cysts containing 2,000 or more meiotic nuclei. Each nucleus amplifies its ribosomal DNA early in oogenesis. The level of amplification is widely different from one nucleus to another, and ranges from less than 0.1 x C to more than 8 x C. The C value for this species is 1.7 x 10(-12) g of DNA. In oocytes of about 0.5 mm diameter the nuclei sort themselves out into an outer shell of several hundred nuclei that swell up to become much larger than the nuclei that remain in the inner compact mass. Later the outer nuclei continue to swell and decrease in number while the inner nuclei disintegrate and disappear. By the time the oocyte reaches. 1.2 mm diameter there are only a few large nuclei left and each has many large nucleoli and a full set of lampbrush chromosomes. Eventually, only one germinal vesicle nucleus is left, and this has all the characteristics of the single germinal vesicles that are typical of oocytes from other amphibians. It is suggested that the sorting out of nuclei into the inner mass and the outer shell of larger nuclei in middle sized oocytes is a consequence of the positions the nuclei happen to be in at the time, but that the "contest" for the role of germinal vesicle may be won by the nucleus of the outer shell that has the highest ribosomal DNA content.

Embryonic stages of Gastrotheca riobambae (Fowler) during maternal incubation and comparison of development with that of other egg-brooding hylid frogs.

A table of development (25 stages) for the period of incubation in the pouch was constructed for Gastrotheca riobambae; it can be used to stage embryos of other egg-brooding hylids. Analysis of embryonic weights during incubation shows that the mother does not contribute nutrients, but gases and other factors are probably exchanged between mother and embryos. According to species, incubation on the back of the mother is carried to the froglet or to the tadpole stages. Development in these hylids is characterized by specialized gills, the bell gills derived from the brachial arches. In some species, the bell gills derive from the first branchial arch and cover less than 50% of the embryo, while in others, the bell gills come from both branchial arches I and II and cover from less than 50% to 100% of the embryo. The most complex bell gills derive from the fusion of the two branchial arches. The majority of egg-brooding hylids live in tropical forests and carry development to the froglet stage. Tadpoles are produced by species of Flectonotus, Fritziana, and Gastrotheca. Tadpole-producing species of Gastrotheca have the most complex reproductive adaptations among egg-brooding hylids Acceleration and retardation in development seem to have played important roles in the evolution of these frogs. The evolutionary trend has been toward direct development, i.e., disappearance of the free-living larval stages through maternal incubation, and later to a recovery of the free-living tadpole stages in species of Gastrotheca with the most complex reproductive adaptations.

Ovarian structure of the marsupial frog Gastrotheca riobambae (Fowler).

The mature ovary of Gastrotheca riobambae presents large oocytes (3 mm in diameter) of pale yellow color. After ovulation and the beginning of embryonic incubation, the empty postovulatory follicles can be recognized in the ovary for about 30 days. The granulosa of these follicles never fills the follicular lumen and this cavity becomes filled with fluid during the first five days of incubation. Later, at 18 days of incubation, the lumen is mostly empty and contains few cells of the granulosa. Shrinkage results in the disappearance of the follicular cavity by approximately the thirtieth day of incubation. The thecae are thick and become separated by a space. This space is filled progressively with cells, blood capillaries and fluid. After the thirtieth to fortieth day of incubation, these follicles become regressive and disappear. The postovulatory follicles of Gastrotheca may correspond to corpora lutea. The evidence suggests that pouch vascularization and the formation of embryonic chambers of pouch tissue may be under ovarian control. In addition, the process of vitellogenesis is influenced by incubation, as most growth of the ovarian oocyte occurs after birth of the tadpoles. Follicular atresia is common and is similar to that of other frogs.