Transcription factors Mix1 and VegT, relocalization of vegt mRNA, and conserved endoderm and dorsal specification in frogs.

Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin-insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs.

Analysis of Cell Size in the Gastrula of Ten Frog Species Reveals a Correlation of Egg with Cell Sizes, and a Conserved Pattern of Small Cells in the Marginal Zone.

We investigated the relationship between egg and cell sizes in the early gastrula of ten species of frogs with eggs of 1,100-3,500 µm diameters. We asked whether differences in cell size of the vegetal region, blastocoel roof, and marginal zone of the early gastrula were associated with egg size. Alternatively, we proposed that cell size differences may associate with gastrulation characteristics. The analyzed species were as follows: Xenopus laevis, Engystomops randi, Engystomops coloradorum, Espadarana callistomma, Epipedobates machalilla, Epipedobates anthonyi, Epipedobates tricolor, Dendrobates auratus, Gastrotheca riobambae, and Eleutherodactylus coqui. A positive correlation between egg and cell size was detected in the three regions of the gastrula. The correlation was strong in the vegetal region and blastocoel roof, and weak in the marginal zone. Large eggs allowed the evolution of frog terrestrial reproductive modes by storing nourishment for the developing embryos. Large cells, laden with yolk, occur in the vegetal region. However, small cell size characterized the marginal zone and blastocoel roof. We proposed that small cells of the marginal zone are required for involution and blastopore formation. The evolution pressure toward small cells in the marginal zone contributed to maintain the blastopore as a universal feature of frog gastrulation in eggs of different sizes and gastrulation modes. Our comparative analysis revealed two fundamental and conserved properties of the frog early gastrula, the correlation of egg with cell sizes, and the general small size of cells in the marginal zone.

Variation in the schedules of somite and neural development in frogs.

The timing of notochord, somite, and neural development was analyzed in the embryos of six different frog species, which have been divided into two groups, according to their developmental speed. Rapid developing species investigated were Xenopus laevis (Pipidae), Engystomops coloradorum, and Engystomops randi (Leiuperidae). The slow developers were Epipedobates machalilla and Epipedobates tricolor (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Blastopore closure, notochord formation, somite development, neural tube closure, and the formation of cranial neural crest cell-streams were detected by light and scanning electron microscopy and by immuno-histochemical detection of somite and neural crest marker proteins. The data were analyzed using event pairing to determine common developmental aspects and their relationship to life-history traits. In embryos of rapidly developing frogs, elongation of the notochord occurred earlier relative to the time point of blastopore closure in comparison with slowly developing species. The development of cranial neural crest cell-streams relative to somite formation is accelerated in rapidly developing frogs, and it is delayed in slowly developing frogs. The timing of neural tube closure seemed to be temporally uncoupled with somite formation. We propose that these changes are achieved through differential timing of developmental modules that begin with the elongation of the notochord during gastrulation in the rapidly developing species. The differences might be related to the necessity of developing a free-living tadpole quickly in rapid developers.

The gastrocoel roof plate in embryos of different frogs.

The morphology of the gastrocoel roof plate and the presence of cilia in this structure were examined in embryos of four species of frogs. Embryos of Ceratophrys stolzmanni (Ceratophryidae) and Engystomops randi (Leiuperidae) develop rapidly, provide comparison for the analysis of gastrocoel roof plate development in the slow-developing embryos of Epipedobates machalilla (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Embryos of the analyzed frogs develop from eggs of different sizes, and display different reproductive and developmental strategies. In particular, dorsal convergence and extension and archenteron elongation begin during gastrulation in embryos of rapidly developing frogs, as in Xenopus laevis. In contrast, cells that involute during gastrulation are stored in the large circumblastoporal collar that develops around the closed blastopore in embryos of slow-developing frogs. Dorsal convergence and extension only start after blastopore closure in slow-developing frog embryos. However, in the neurulae, a gastrocoel roof plate develops, despite the accumulation of superficial mesodermal cells in the circumblastoporal collar. Embryos of all four species develop a ciliated gastrocoel roof plate at the beginning of neurulation. Accordingly, fluid-flow across the gastrocoel roof plate is likely the mechanism of left-right asymmetry patterning in these frogs, as in X. laevis and other vertebrates. A ciliated gastrocoel roof plate, with a likely origin as superficial mesoderm, is conserved in frogs belonging to four different families and with different modes of gastrulation.

A display of Developmental Biology in Ibero-America.

This issue of the International Journal of Developmental Biology is dedicated to Ibero-America, and includes research articles from Argentina, Brazil, Chile, Colombia, Ecuador, Mexico, Panama, Puerto Rico, and Uruguay. It also describes the history of developmental biology in several Ibero-American countries. Moreover, the volume contains interviews with scientists living in the region and abroad. Other articles highlight The Latin American Society for Developmental Biology (LASDB), and the International Courses. The main purpose of this volume is to stimulate interest and reseach in developmental biology in Ibero-America.

Experiences with the marsupial frogs: reminiscences of a developmental biologist.

This article provides a brief account of the career of Eugenia M. del Pino. Casual events and serendipity played important roles in modeling her career as a developmental biologist. In collaboration with colleagues and students, she analyzed the biology and development of the marsupial frog Gastrotheca riobambae (family: Hemiphractidae) in comparison with Xenopus laevis and tropical frogs. The emphasis was placed on oogenesis and the early stages of development. Topics include the mono- and multi-nucleated modes of oogenesis. She described two modes of gastrulation in frogs, gastrulation modes one and two, according to the timing of notochord elongation. She was able to establish a pioneer laboratory for the comparative analysis of frog development in Ibero America at the Pontifical Catholic University of Ecuador, in Quito. Her contributions to society include her influence in the establishment of the National Academy of Sciences of Ecuador, and efforts toward the conservation of the Galápagos Archipelago. She is part of a pioneer group of professors that placed Biology as an academic discipline in Ecuador. The experiences of her career reveal that we all face difficulties in our jobs. However, nothing is impossible when we follow a passion. Her work reveals that the key to success is to turn obstacles into opportunities.

From egg to embryo in marsupial frogs.

Marsupial frogs (Hemiphractidae) evolved exceptional mechanisms for the conquest of terrestrial life. These adaptations include very large eggs. In some species eggs reach 10mm in diameter, and are considered to be the largest in frogs. Females have reproductive modifications for the incubation of embryos in their bodies. Modifications of embryos include adaptations for development inside the body of the mother, and changes in the developmental pattern. Moreover, in some species, oocytes are multinucleated instead of having a single germinal vesicle as in most vertebrates. This chapter provides an overview of the adaptations of marsupial frogs associated with terrestrial life, with a discussion of gastrulation and multinucleated oogenesis.

Embryogenesis of Marsupial Frogs (Hemiphractidae), and the Changes that Accompany Terrestrial Development in Frogs.

The developmental adaptations of the marsupial frogs Gastrotheca riobambae and Flectonotus pygmaeus (Hemiphractidae) are described and compared with frogs belonging to seven additional families. Incubation of embryos by the mother in marsupial frogs is associated with changes in the anatomy and physiology of the female, modifications of oogenesis, and extraordinary changes in embryonic development. The comparison of early development reveals that gene expression is highly conserved. However, the timing of gene expression varies between frog species. There are two modes of gastrulation according to the onset of convergent extension. In gastrulation mode 1, convergent extension is an intrinsic mechanism of gastrulation. This gastrulation mode occurs in frogs with aquatic reproduction, such as Xenopus laevis. In gastrulation mode 2, convergent extension occurs after the completion of gastrulation movements. Gastrulation mode 2 occurs in frogs with terrestrial reproduction, such as the marsupial frog, G. riobambae. The two modes of frog gastrulation resemble the two transitions toward meroblastic cleavage of ray-finned fishes (Actinopterygii). The comparison indicates that a major event in the evolution of frog terrestrial development is the separation of convergent extension from gastrulation.

The extraordinary biology and development of marsupial frogs (Hemiphractidae) in comparison with fish, mammals, birds, amphibians and other animals.

The study of oogenesis and early development of frogs belonging to the family Hemiphractidae provide important comparison to the aquatic development of other frogs, such as Xenopus laevis, because reproduction on land characterizes the Hemiphractidae. In this review, the multinucleated oogenesis of the marsupial frog Flectonotus pygmaeus (Hemiphractidae) is analyzed and interpreted. In addition, the adaptations associated with the incubation of embryos in the pouch of the female marsupial frog Gastrotheca riobambae (Hemiphractidae) and the embryonic development of this frog are summarized. Moreover, G. riobambae gastrulation is compared with the gastrulation modes of Engystomops randi and Engystomops coloradorum (Leptodactylidae); Ceratophrys stolzmanni (Ceratophryidae); Hyalinobatrachium fleischmanni and Espadarana callistomma (Centrolenidae); Ameerega bilinguis, Dendrobates auratus, Epipedobates anthonyi, Epipedobates machalilla, Epipedobates tricolor, and Hyloxalus vertebralis (Dendrobatidae); Eleutherodactylus coqui (Terrarana: Eleutherodactylidae), and X. laevis (Pipidae). The comparison indicated two modes of frog gastrulation. In X. laevis and in frogs with aquatic reproduction, convergent extension begins during gastrulation. In contrast, convergent extension occurs in the post-gastrula of frogs with terrestrial reproduction. These two modes of gastrulation resemble the transitions toward meroblastic cleavage found in ray-finned fishes (Actinopterygii). In spite of this difference, the genes that guide early development seem to be highly conserved in frogs. I conclude that the shift of convergent extension to the post-gastrula accompanied the diversification of frog egg size and terrestrial reproductive modes.

Developmental biology in Ecuador: a 30-year teaching experience.

Developmental biology has been taught at the Pontificia Universidad Católica del Ecuador, in Quito for 30 years by the author. The experience of teaching development is described within the broader context of science in Latin America. It is recognized that developmental biology is poorly represented in research and teaching in this part of the world. The teaching of developmental biology to Ecuadorian students contributes to their intellectual training, by helping them to integrate concepts derived from various branches of biology. Moreover, the highly conserved molecular mechanisms of development provide extraordinary examples of the unity of biology, and many complex biological processes can be more easily grasped when studying embryos.

Lamina-associated polypeptide 2 (LAP2) expression in fish and amphibians.

Somatic and germinal cells of 15 fish and 33 amphibian species were examined by SDS-PAGE followed by immunoblotting to determine the expression of LAP2 (lamina-associated polypeptide 2). LAP2 expression in frogs, salamanders and fish does not vary with the mode of reproduction. In fish and frog cells, a rim-like LAP2 positive region was detected around the nucleus by indirect immunofluorescence microscopy. The cell distribution and expression patterns of LAP2 in fish, frogs and salamanders are comparable with those found in Xenopus and zebrafish. The mammalian somatic cell pattern, which may also occur in gymnophione amphibians, includes LAP2alpha, beta and gamma as major isoforms, whereas LAP2alpha does not occur in cells of fish, frogs and salamanders. In fish, LAP2gamma is the major isoform of somatic cells, suggesting that LAP2gamma may be ancestral. However, in the rainbow trout, as in frogs and salamanders, LAP2beta was the major somatic isoform. Fish and frog sperm only express low molecular weight polypeptides. In contrast, fish and frog oocytes express an oocyte-specific LAP2 isoform of high molecular weight. In the toad Bufo marinus this isoform becomes upregulated in pre-vitellogenic oocytes of 150-200 microm in diameter. The absence of LAP2alpha and the differential expression of LAP2 isoforms in somatic and germ cells, as found in fish and frogs, may be ancestral vertebrate characters. In spite of differences in developmental time, the LAP2 isoforms of somatic cells are upregulated during gastrulation, suggesting that LAP2 may be implicated in the early development of fish and frog.

Development of the dendrobatid frog Colostethus machalilla.

To provide a developmental correlate with other frogs, we prepared a normal table of development for the dendrobatid, Colostethus machalilla and analyzed the morphology of its early development. This frog reproduces in captivity and deposits moderately sized eggs (1.6 mm in diameter) in terrestrial nests. The father guards the embryos until tadpole hatching. We divided development until hatching into 25 stages and implemented methods for in vitro culture of the embryos. The external and internal morphology of embryos were evaluated by observations in whole mount and in sections. Neural, notochord and somite specific antibodies were used to analyze gene expression patterns by immunostaining of embryos. Embryonic development of C. machalilla is slow and deviates from Xenopus laevis. In C. machalilla the elongation of the notochord, neural plate and somite formation occur after blastopore closure, possibly due to a delay in the dorsal convergence and extension movements. The gastrula of C. machalilla also deviates from X. laevis. The archenteron remains small until blastopore closure, where small cells accumulate at the blastopore lips. Simultaneously, the blastocoel roof thins until it becomes a monolayer of cells. Although C. machalilla does not form an embryonic disk, its thick blastopore lips resemble the embryonic disk of the marsupial frog Gastrotheca riobambae and represent an interesting deviation from the gastrulation pattern observed in X. laevis.

Urea is Necessary for the Culture of Embryos of the Marsupial Frog Gastrotheca riobambae, and is Tolerated by Embryos of the Aquatic Frog Xenopus laevis: (ureotelism/urea concentration in frog blood/culture of frog embryos/mesoderm induction).

Urea was found in the capsular fluid that bathes Gastrotheca riobambae embryos during incubation in the maternal pouch. The urea concentration in this fluid is higher than in blood from the mother, indicating that urea is accumulated by the embryo during the period of maternal incubation. Gastrotheca tadpoles tolerate up to 500 mM urea with 86% survival after 24 hours and die in solutions of 0.5 mM ammonia. These findings suggest that urea plays a role in the adaptation of G. riobambae embryos to the conditions of water stress within the maternal pouch. To improve the in vitro culture conditions of early embryos taken from the maternal pouch, a saline solution that contains urea was designed (GRS). GRS plus 30 mM urea was used for the culture of cleavage to the neurula stage embryos of G. riobambae. During organogenesis, the urea concentration was raised to 60 mM. Early embryos of Xenopus laevis tolerate urea, and in addition, no inducing effects of urea have been detected in animal cap explants of Xenopus.

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 mother's 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.

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.

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.

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. (c) 2009 Wiley-Liss, Inc.