Eugenia M. del Pino

DEVELOPMENTAL DIVERSITY OF AMPHIBIANS

The current model amphibian, Xenopus laevis, develops rapidly in water to a tadpole which metamorphoses into a frog. Many amphibians deviate from the X. laevis developmental pattern. Among other adaptations, their embryos develop in foam nests on land or in pouches on their mothers back or on a leaf guarded by a parent. The diversity of developmental patterns includes multinucleated oogenesis, lack of RNA localization, huge non‐pigmented eggs, and asynchronous, irregular early cleavages. Variations in patterns of gastrulation highlight the modularity of this critical developmental period. Many species have eliminated the larva or tadpole and directly develop to the adult. The wealth of developmental diversity among amphibians coupled with the wealth of mechanistic information from X. laevis permit comparisons that provide deeper insights into developmental processes. WIREs Dev Biol 2012, 1:345–369. doi: 10.1002/wdev.23

A comparative analysis of frog early development

The current understanding of Xenopus laevis development provides a comparative background for the analysis of frog developmental modes. Our analysis of development in various frogs reveals that the mode of gastrulation is associated with developmental rate and is unrelated to egg size. In the gastrula of the rapidly developing embryos of the foam-nesting frogs Engystomops coloradorum and Engystomops randi, archenteron and notochord elongation overlapped with involution at the blastopore lip, as in X. laevis embryos. In embryos of dendrobatid frogs and in the frog without tadpoles Eleutherodactylus coqui, which develop somewhat more slowly than X. laevis, involution and archenteron elongation concomitantly occurred during gastrulation; whereas elongation of the notochord and, therefore, dorsal convergence and extension, occurred in the postgastrula. In contrast, in the slow developing embryos of the marsupial frog Gastrotheca riobambae, only involution occurred during gastrulation. The processes of archenteron and notochord elongation and convergence and extension were postgastrulation events. We produced an Ab against the homeodomain protein Lim1 from X. laevis as a tool for the comparative analysis of development. By the expression of Lim1, we were able to identify the dorsal side of the G. riobambae early gastrula, which otherwise was difficult to detect. Moreover, the Lim1 expression in the dorsal lip of the blastopore and notochord differed among the studied frogs, indicating variation in the timing of developmental events. The variation encountered gives evidence of the modular character of frog gastrulation.

Embryogenesis and laboratory maintenance of the foam-nesting túngara frogs, genus Engystomops (= Physalaemus).

The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non‐model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of túngara frogs (genus Engystomops, also called Physalaemus). The frogs Engystomops pustulosus, Engystomops coloradorum, and Engystomops randi construct floating foam‐nests with small eggs. We define a table of 23 stages for the developmental period in the foam‐nest. Embryos were immunostained against Lim1, neural, and somite‐specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life‐cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development. Developmental Dynamics 238:1444–1454, 2009.

Bidder's organ in the toad Bufo marinus: effects of orchidectomy on the morphology and expression of lamina-associated polypeptide 2.

The Bidders organ is an undeveloped ovary located anterior to the testis in male true toads (Bufonidae). The presence of sperm and oocytes, derived from the primordial germ cells of the male toad, provides an exceptional condition for the study of germ cell differentiation in vertebrates. In this study, the effects of orchidectomy on morphology and on lamina‐associated polypeptide 2 (LAP2) expression were investigated in the Bidders organ of Bufo marinus. To characterize bidderian oocytes, oogenesis in the ovary was divided into six stages. It was found that ovarian and bidderian oocytes were morphologically identical. To determine the expression of LAP2 isoforms, oocytes from the ovary and the Bidders organ were examined by sodium dodecylsulfate–polyacrylamide gel electrophoresis and immunoblotting. In amphibians, LAP2β is expressed in somatic cells, whereas LAP2ω is expressed in oocytes. The Bidders organ normally contained previtellogenic oocytes of less than 150 µm in diameter that expressed LAP2β. However, the organ in some male toads contained a few larger oocytes, resulting in the faint detection of LAP2ω. After orchidectomy, bidderian oocytes grew and strongly expressed LAP2ω. Moreover, as in the ovary, LAP2ω was upregulated in bidderian oocytes of 240 µm in diameter. This work represents the first demonstration of the molecular similarity between ovarian and bidderian oocytes.

Expression of Brachyury during development of the dendrobatid frog Colostethus machalilla.

The expression of Brachyury (Bra) during development of Colostethus machalilla was analyzed with a polyclonal antibody. The observed molecular mass of Bra was of 48 kDa, as in Xenopus laevis. During cleavage, low levels of Bra were expressed. In contrast, in the blastula Bra became up‐regulated, and Bra protein was present in a wide ring of surface cells. The surface expression of Bra disappeared in the gastrula, and a new ring of Bra‐positive nuclei was detected in deep cells around the closing blastopore. The C. machalilla external and internal rings of Bra‐positive nuclei apparently mark the prospective mesoderm in the blastula and gastrula, respectively. The two Bra expression rings were dissociated in time in the fairly slow developing embryos of this frog. Brachyury expression in the notochord became visible only after the blastopore closed, in contrast with X. laevis. In addition, Bra expression in the notochord indicated that dorsal convergence and extension occurred after blastopore closure. The C. machalilla Bra‐positive notochord was originally exposed on the gastrocoel roof, in agreement with a superficial component of the prospective mesoderm.

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 × C to more than 8 × C. The C value for this species is 1.7 × 10−12g 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 1.2mm 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.

Comparison of Lim1 expression in embryos of frogs with different modes of reproduction

A polyclonal antibody was used to detect the expression of the homeodomain protein Lim1 (Lhx1) in embryos of Xenopus laevis, Engystomops randi, Colostethus machalilla and Gastrotheca riobambae. These frogs belong to four separate families, and have differences in their modes of reproduction and developmental rates. The expression of Lim1 in embryos of these frogs resembled the X. laevis expression pattern. Thus, the dorsal blastopore lip, axial mesoderm, pronephros and certain cells of the central nervous system were Lim1-positive in embryos of all frogs. There were, however, time differences; thus, in the mid-gastrula of the rapidly developing embryos of X. laevis and E. randi, the Lim1 protein was simultaneously detected in the prechordal plate (head organizer) and notochord (trunk organizer). In contrast, only the prechordal plate was Lim1-positive during gastrulation in the slow developing embryos of C. machalilla. The notochord elongated and became Lim1-positive after closure of the blastopore in C. machalilla and G. riobambae embryos. The prechordal plate of G. riobambae embryos could not be clearly detected, as the Lim1-signal remained around the blastopore during gastrulation. These observations indicate that the timing of gene expression at the dorsal blastopore lip in embryos of slow developing frogs differs from that of X. laevis. Moreover, the comparison shows that the developmental processes of the head and trunk organizers are basically separable and become dissociated in embryos of the slow developing frog, C. machalilla.

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.

The Morphology of Prehatching Embryos of Caecilia orientalis (Amphibia: Gymnophiona: Caeciliidae)

The state of development of advanced embryos of the direct‐developing Ecuadorian caecilian Caecilia orientalis (Caeciliidae: Gymnophiona: Amphibia) was examined. Because it is established that development is correlated with reproductive modes in a number of features, we included comparison with taxa that represent the major reproductive modes and all of the modern normal tables and ossification sequences. The embryos of C. orientalis most closely resemble those of stage 47/48 Gegeneophis ramaswamii, an Indian caeciliid, and stage 47/48 Hypogeophis rostratus, a Seychellian caeciliid, both direct developers, in details of bone mineralization, chondrocranial degeneration, and vertebrogenesis. They are most like stage 45 H. rostratus in external features (gills, pigmentation, etc.). They are less similar to prehatchings of Ichthyophis kohtaoensis, an ichthyophiid with free‐living larvae, and to fetuses of the viviparous caeciliid Dermophis mexicanus and the viviparous typhlonectid Typhlonectes compressicauda at comparable total lengths in both skeletal development and external features. The similarity of developmental features among the direct‐developers suggests a correlation with mode of life history. A noteworthy feature is that C. orientalis has an armature of multiple rows of teeth on the lower jaw with tooth crowns that resemble the “fetal” teeth of viviparous taxa and that are covered with a layer of oral mucosal epithelium until full development and eruption, but the upper jaw bears a single row of widely spaced, elongate, slightly recurved teeth that resemble those of the adult. J. Morphol., 2009.

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.