From the Belousov-Zhabotinsky reaction to biochemical clocks, traveling waves and cell cycle regulation.

In the last 20 years, a growing army of systems biologists has employed quantitative experimental methods and theoretical tools of data analysis and mathematical modeling to unravel the molecular details of biological control systems with novel studies of biochemical clocks, cellular decision-making, and signaling networks in time and space. Few people know that one of the roots of this new paradigm in cell biology can be traced to a serendipitous discovery by an obscure Russian biochemist, Boris Belousov, who was studying the oxidation of citric acid. The story is told here from an historical perspective, tracing its meandering path through glycolytic oscillations, cAMP signaling, and frog egg development. The connections among these diverse themes are drawn out by simple mathematical models (nonlinear differential equations) that share common structures and properties.

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.

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.

Parallel evolution of direct development in frogs - Skin and thyroid gland development in African Squeaker Frogs (Anura: Arthroleptidae: Arthroleptis).

BACKGROUND: Cases of parallel evolution offer the possibility to identify adaptive traits and to uncover developmental constraints on the evolutionary trajectories of these traits. The independent evolution of direct development from the ancestral biphasic life history in frogs is such a case of parallel evolution. In frogs, aquatic larvae (tadpoles) differ profoundly from their adult forms and exhibit a stunning diversity regarding their habitats, morphology and feeding behaviors. The transition from the tadpole to the adult is a climactic, thyroid hormone (TH)-dependent process of profound and fast morphological rearrangement called metamorphosis. One of the organ systems that experiences the most comprehensive metamorphic rearrangements is the skin. Direct-developing frogs lack a free-swimming tadpole and hatch from terrestrial eggs as fully formed froglets. In the few species examined, development is characterized by the condensed and transient formation of some tadpole-specific features and the early formation of adult-specific features during a "cryptic" metamorphosis. RESULTS: We show that skin in direct-developing African squeaker frogs (Arthroleptis) is also repatterned from a tadpole-like to an adult-like histology during a cryptic metamorphosis. This repatterning correlates with histological thyroid gland maturation. A comparison with data from the Puerto Rican coqui (Eleutherodactylus coqui) reveals that the evolution of direct development in these frogs is associated with a comparable heterochronic shift of thyroid gland maturation. CONCLUSION: This suggests that the development of many adult features is still dependent on, and possibly constrained by, the ancestral dependency on thyroid hormone signaling.

Tadpoles of hybridising fire-bellied toads (B. bombina and B. variegata) differ in their susceptibility to predation.

The role of adaptive divergence in the formation of new species has been the subject of much recent debate. The most direct evidence comes from traits that can be shown to have diverged under natural selection and that now contribute to reproductive isolation. Here, we investigate differential adaptation of two fire-bellied toads (Anura, Bombinatoridae) to two types of aquatic habitat. Bombina bombina and B. variegata are two anciently diverged taxa that now reproduce in predator-rich ponds and ephemeral aquatic sites, respectively. Nevertheless, they hybridise extensively wherever their distribution ranges adjoin. We show in laboratory experiments that, as expected, B. variegata tadpoles are at relatively greater risk of predation from dragonfly larvae, even when they display a predator-induced phenotype. These tadpoles spent relatively more time swimming and so prompted more attacks from the visually hunting predators. We argue in the discussion that genomic regions linked to high activity in B. variegata should be barred from introgression into the B. bombina gene pool and thus contribute to gene flow barriers that keep the two taxa from merging into one.

Introduction of Developmental Biology at Utkal University, (Odisha, India).

The paper deals with the background and the establishment of a Developmental Biology Laboratory in Utkal University in Odisha state. It describes the process from a humble beginning with limited facilities into a leading research centre, initially for amphibians and later for the endangered olive ridley (Lepidochelys olivacea) turtle. Starting from the biology, reproduction and development in many anurans, the laboratory took up research on regeneration, especially on super-regeneration in tadpoles under the influence of morphogens such as vitamin A (retinoids). Treatment with vitamin A after amputation of the tail inhibited tail regeneration but unexpectedly induced homeotic transformation of tails into limbs in many anurans, starting with the marbled balloon frog Uperodon systoma. This was the first observation of homeotic transformation in any vertebrate. The laboratory continues research on histological and molecular aspects of this phenomenon. In addition, taking advantage of the largest rookery of olive ridley sea turtles in Gahirmatha, in the same state the laboratory has contributed significantly to the biology, breeding patterns, development and especially the temperature-dependent sex determination phenomenon (TSD). This research was extended to biochemical and ultrastructural aspects during development for the first time for any sea turtle. The laboratory has contributed significantly to the conservation of olive ridleys as well as the saltwater crocodile (Crocodylus porosus). Recognition and awards for the laboratory have been received from both national and international bodies.

Studies on tail regeneration and homeotic transformation in anuran tadpoles.

Anuran tadpoles are excellent models for regeneration studies. The tail, an organ essential for swimming for the aquatic tadpole, regenerates completely following injury or amputation. However, treatment with the morphogen, vitamin A or retinoic acid inhibits normal tail regeneration and induces homeotic transformation of tail to limbs. This phenomenon was discovered for the first time in the Indian marbled balloon frog Uperodon systoma in the Developmental Biology laboratory of Utkal University (Odisha, India) in the year 1992. In this paper, we present the results of morphological, histological, biochemical and molecular (immonohistochemistry) investigations of vitamin A induced homeotic transformation in different anuran species. In addition, we discuss the putative role of fibroblast growth factor 1 during spinal cord regeneration in the tadpoles of the Indian tree frog, Polypedates maculatus, an ideal model for regeneration studies in an Indian context.

Ontogeny of the anuran urostyle and the developmental context of evolutionary novelty.

Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan.

Embryonic ontogeny of three species of Horned Frogs, with a review of early development in Ceratophryidae.

Horned Frogs of the family Ceratophryidae are conspicuous anurans represented by three endemic South American genera. Most ceratophryids inhabit semiarid environments, but three species of Ceratophrys occupy tropical or temperate humid areas. Several morphological and behavioral characters of larvae and adults are conserved across the family. Based on examination of specimens and accounts in the literature, the embryonic development of C. ornata, C. cranwelli, and the monotypic genus Chacophrys are described and compared with that of species of Lepidobatrachus. Ceratophryid embryos share a suite of morphological features and heterochronic shifts during development. Most features, such as gill structure, ciliation, early hatching, and precocious differentiation of the gut and hind limbs, are shared by all the species regardless the differences in the habitats that occupy. This is consistent with previous observations of some adult characters, and likely supports the hypothesis of an early diversification of ceratophryids in semiarid environments. Other embryonic features, such as the morphology and ontogeny of the oral disc and digestive tract, are correlated with larval feeding habits and vary within the family. The evolutionary and ecological significance of some conserved characters (e.g., gastrulation pattern, Type-A adhesive glands) and other taxon-specific features (e.g., nasal appendix) remain to be explored in the group.

How do red-eyed treefrog embryos sense motion in predator attacks? Assessing the role of vestibular mechanoreception.

The widespread ability to alter timing of hatching in response to environmental cues can serve as a defense against threats to eggs. Arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, can hatch up to 30% prematurely to escape predation. This escape-hatching response is cued by physical disturbance of eggs during attacks, including vibrations or motion, and thus depends critically on mechanosensory ability. Predator-induced hatching appears later in development than flooding-induced, hypoxia-cued hatching; thus, its onset is not constrained by the development of hatching ability. It may, instead, reflect the development of mechanosensor function. We hypothesize that vestibular mechanoreception mediates escape-hatching in snake attacks, and that the developmental period when hatching-competent embryos fail to flee from snakes reflects a sensory constraint. We assessed the ontogenetic congruence of escape-hatching responses and an indicator of vestibular function, the vestibulo-ocular reflex (VOR), in three ways. First, we measured VOR in two developmental series of embryos 3-7 days old to compare with the published ontogeny of escape success in attacks. Second, during the period of greatest variation in VOR and escape success, we compared hatching responses and VOR across sibships. Finally, in developmental series, we compared the response of individual embryos to a simulated attack cue with their VOR. The onset of VOR and hatching responses were largely concurrent at all three scales. Moreover, latency to hatch in simulated attacks decreased with increasing VOR. These results are consistent with a key role of the vestibular system in the escape-hatching response of A. callidryas embryos to attacks.

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.

Early ontogeny and sequence heterochronies in Leiuperinae frogs (Anura: Leptodactylidae).

The study of early development in Neotropical Leiuperinae frogs (Anura, Leptodactylidae) has been addressed by several works in recent times. However, a comparative developmental approach under a phylogenetic context was not available. Herein we analyzed the morphological and ontogenetic diversity of embryos belonging to 22 species of the three largest genera in Leiuperinae. We find that in most cases, variations fit with the phylogeny at the inter- and intrageneric levels. Embryo kyphosis and whitish color are synapomorphies for the clade grouping Physalaemus and Engystomops. The presence of a third lower tooth row on the oral disc is plesiomorphic for Leiuperinae, only changing in derived clades. The configurations and developmental trajectories of the lower lip are exceptionally diverse. The developmental sequences optimized on the phylogenetic tree recover an early differentiated first lower tooth row a synapomorphy of Pseudopaludicola and Physalaemus, and an early differentiated second row as synapomorphy of Pleurodema. On the other hand, few features are highly conserved in the subfamily, such as the adhesive glands universally present in a type-C configuration. Our results also suggest that the morphology and ontogeny of embryos is in some cases associated to the environment where they develop. A large body size, poorly developed transient respiratory structures, large yolk provision and delayed development of the digestive tract occur convergently in embryos inhabiting cold, oxygenated environments. Embryos that develop in warmer water bodies in xeric environments show more complex and persistent transient respiratory structures and an early onset of hind limbs development. Our survey highlights that morphology and early development of anurans can be a valuable source of information for phylogenetic studies, and provide fundamental bases to explore and discuss how evolutionary changes can be shaped by environmental conditions.

Genotoxic effects of 4-nonylphenol and Cyproterone Acetate on Rana catesbeiana (anura) tadpoles and juveniles.

Genotoxic analyses are commonly used in ecotoxicological studies as early biomarkers to investigate the potential effects of environmental contaminants on biological models. Several pollutants can induce DNA damage and, therefore, counting micronuclei and other nuclear abnormalities are efficient tools to evaluate genotoxicity. Some pollutants such as 4-nonylphenol (NP), a detergent used mainly in industries, and Cyproterone Acetate (CPA), an antiandrogenic medicine, have already shown genotoxic effects on some vertebrates. However, although amphibians are considered bioindicators of environmental quality and their populations are declining worldwide, the effects of these compounds on anurans are not yet known and, therefore, we believe that it is important to investigate such effects on anurans. Since water contamination is one of the ultimate causes of amphibian decline, ecotoxicological studies are important to discuss the appropriate solutions to avoid species extinction. Thus, this study investigates the genotoxic effects on Rana catesbeiana tadpoles and juveniles after being exposed to 1, 10 and 100 µg/L NP and 0.025, 0.25 and 2.5 ng/L CPA, by counting the nuclear abnormalities after exposure. The laboratory experiments lasted 28 days. The experimental conditions were the same except for the water volume since tadpoles and juveniles exhibit different habits at different developmental stages. Compared to juveniles, tadpoles were more susceptible to both compounds as indicated by the increased nuclear abnormalities observed in the highest NP concentration and all tested CPA concentrations. The juveniles, on the other hand, responded only to the two highest CPA concentrations. We concluded that CPA, even at very low concentrations, is extremely harmful to both anuran developmental stages and, particularly, to tadpoles. The significant effects observed on tadpoles is an important outcome of this study since 100 µg/L or higher NP concentrations are frequently detected in the environment.

Neural crest streaming as an emergent property of tissue interactions during morphogenesis.

A fundamental question in embryo morphogenesis is how a complex pattern is established in seemingly uniform tissues. During vertebrate development, neural crest cells differentiate as a continuous mass of tissue along the neural tube and subsequently split into spatially distinct migratory streams to invade the rest of the embryo. How these streams are established is not well understood. Inhibitory signals surrounding the migratory streams led to the idea that position and size of streams are determined by a pre-pattern of such signals. While clear evidence for a pre-pattern in the cranial region is still lacking, all computational models of neural crest migration published so far have assumed a pre-pattern of negative signals that channel the neural crest into streams. Here we test the hypothesis that instead of following a pre-existing pattern, the cranial neural crest creates their own migratory pathway by interacting with the surrounding tissue. By combining theoretical modeling with experimentation, we show that streams emerge from the interaction of the hindbrain neural crest and the neighboring epibranchial placodal tissues, without the need for a pre-existing guidance cue. Our model suggests that the initial collective neural crest invasion is based on short-range repulsion and asymmetric attraction between neighboring tissues. The model provides a coherent explanation for the formation of cranial neural crest streams in concert with previously reported findings and our new in vivo observations. Our results point to a general mechanism of inducing collective invasion patterns.

Lethal and Sublethal Effects of the Herbicide Atrazine in the Early Stages of Development of Physalaemus gracilis (Anura: Leptodactylidae).

Water sources used as reproductive sites by crying frog, Physalaemus gracilis, are extensively associated with agroecosystems in which the herbicide atrazine is employed. To evaluate the lethal and sublethal effects of atrazine commercial formulation, acute and chronic toxicity tests were performed in the embryonic phase and the beginning of the larval phase of P. gracilis. Tests were started on stage 19 of Gosner (Herpetologica 16:183-190, 1960) and performed in 24-well cell culture plates. Acute tests had a duration of 96 h with embryo mortality monitoring every 24 h. Chronic assays contemplated the transition from the embryonic to larval stages and lasted 168 h. Every 24 h the embryos/larvae were observed for mortality, mobility, and malformations. The LC50 of atrazine determined for P. gracilis embryos was 229.34 mg L-1. The sublethal concentrations did not affect the development of the larvae but were observed effects on mobility and malformations, such as spasmodic contractions, reduced mobility, malformations in mouth and intestine, and edema arising. From 1 mg L-1 atrazine, the exposed larvae began to have changes in mobility and malformations. The atrazine commercial formulation has caused early life effects of P. gracilis that may compromise the survival of this species but at higher concentrations than recorded in the environment, so P. gracilis can be considered tolerant to this herbicide at environmentally relevant concentrations.

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.

The hatching process and mechanisms of adaptive hatching acceleration in hourglass treefrogs, Dendropsophus ebraccatus.

Environmentally cued hatching is well documented in anurans, enabling embryos to escape diverse threats. However, knowledge of anuran hatching mechanisms is limited and based largely on aquatic-breeding species without known plasticity in hatching timing. Generally, hatching gland cells produce a hatching enzyme that degrades the vitelline membrane. We investigated hatching and its regulation in terrestrial embryos of hourglass treefrogs, Dendropsophus ebraccatus, which accelerate hatching to escape dehydration. We specifically tested if changes in hatching gland cell development or hatching enzyme gene expression are associated with accelerated hatching. We measured perivitelline chamber size of well-hydrated eggs over development as an indicator of breakdown of the vitelline membrane and found that the size of the perivitelline chamber increased steadily until hatching, suggesting gradual hatching enzyme release and vitelline membrane degradation. Hatching gland cells peaked in abundance and began regression substantially prior to hatching, but we found no developmental differences in the abundance or surface area of hatching gland cells between dry and well-hydrated embryos. Hatching enzyme gene expression also peaked early in development then declined, with no difference between hydration treatments. In D. ebraccatus breakdown of the vitelline membrane appears gradual, mediated by hatching enzyme release starting long before hatching. However, hatching acceleration is not associated with ontogenetic changes in hatching gland cell development or hatching enzyme gene expression. This hatching process contrasts with that of red-eyed treefrogs, Agalychnis callidryas, which appear to release enzyme acutely at hatching, yet both species are capable of hatching to escape acute threats.

Bayesian Unidimensional Scaling for visualizing uncertainty in high dimensional datasets with latent ordering of observations.

BACKGROUND: Detecting patterns in high-dimensional multivariate datasets is non-trivial. Clustering and dimensionality reduction techniques often help in discerning inherent structures. In biological datasets such as microbial community composition or gene expression data, observations can be generated from a continuous process, often unknown. Estimating data points' 'natural ordering' and their corresponding uncertainties can help researchers draw insights about the mechanisms involved. RESULTS: We introduce a Bayesian Unidimensional Scaling (BUDS) technique which extracts dominant sources of variation in high dimensional datasets and produces their visual data summaries, facilitating the exploration of a hidden continuum. The method maps multivariate data points to latent one-dimensional coordinates along their underlying trajectory, and provides estimated uncertainty bounds. By statistically modeling dissimilarities and applying a DiSTATIS registration method to their posterior samples, we are able to incorporate visualizations of uncertainties in the estimated data trajectory across different regions using confidence contours for individual data points. We also illustrate the estimated overall data density across different areas by including density clouds. One-dimensional coordinates recovered by BUDS help researchers discover sample attributes or covariates that are factors driving the main variability in a dataset. We demonstrated usefulness and accuracy of BUDS on a set of published microbiome 16S and RNA-seq and roll call data. CONCLUSIONS: Our method effectively recovers and visualizes natural orderings present in datasets. Automatic visualization tools for data exploration and analysis are available at: https://nlhuong.shinyapps.io/visTrajectory/ .

Induction of Hypoxia in Living Frog and Zebrafish Embryos.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h. To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1α) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into the DNA. HIF-1α levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos. Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.