In Vivo Tracking and 3D Mapping of Cell Death in Regeneration and Cancer Using Trypan Blue.

Tracking cell death in vivo can enable a better understanding of the biological mechanisms underlying tissue homeostasis and disease. Unfortunately, existing cell death labeling methods lack compatibility with in vivo applications or suffer from low sensitivity, poor tissue penetration, and limited temporal resolution. Here, we fluorescently labeled dead cells in vivo with Trypan Blue (TBlue) to detect single scattered dead cells or to generate whole-mount three-dimensional maps of large areas of necrotic tissue during organ regeneration. TBlue effectively marked different types of cell death, including necrosis induced by CCl4 intoxication in the liver, necrosis caused by ischemia-reperfusion in the skin, and apoptosis triggered by BAX overexpression in hepatocytes. Moreover, due to its short circulating lifespan in blood, TBlue labeling allowed in vivo "pulse and chase" tracking of two temporally spaced populations of dying hepatocytes in regenerating mouse livers. Additionally, upon treatment with cisplatin, TBlue labeled dead cancer cells in livers with cholangiocarcinoma and dead thymocytes due to chemotherapy-induced toxicity, showcasing its utility in assessing anticancer therapies in preclinical models. Thus, TBlue is a sensitive and selective cell death marker for in vivo applications, facilitating the understanding of the fundamental role of cell death in normal biological processes and its implications in disease.

Whole-Mount Histochemical Protocols in Zebrafish for a Hands-On Developmental Biology Practical Course in Ecuador.

Research-based education at the undergraduate level is ideal for fostering the training of future scientists. In an undergraduate Developmental Biology course, this learning strategy requires the availability of model species and enough research reagents, not only for technique training but also for the development of student original projects. This might be challenging in most countries, where resources are limited. Hence, there is a need to develop low-cost solutions for use in the classroom. In this study, we describe the optimization and use of two low-cost protocols in zebrafish embryos for hands-on practical sessions and project-based learning in a Developmental Biology undergraduate course in Ecuador. These protocols were designed for the practical and experimental learning of vertebrate meroblastic cleavage, gastrulation, and neural crest differentiation. The proposed protocols have been previously described in the literature and use silver nitrate and alcian blue, two relatively inexpensive reagents, to label cell membranes and cartilage. The silver nitrate protocol allows the study of cell contact formation during cleavage and the identification of cellular changes during gastrulation, including yolk internalization and epiboly. The alcian blue staining allows the analysis of cranial mesenchymal differentiation into cartilage. These protocols are ideal for practical sessions due to their ease of application, quick results, adaptability to the class schedule, and robustness in the hands of beginning researchers. Finally, these protocols are adaptable for research-based class projects.

Direct development or endotrophic tadpole? Morphological aspects of the early ontogeny of the plump toad Osornophryne occidentalis (Anura: Bufonidae).

Andean Toads of the genus Osornophryne are suspected to have direct development on the basis of clutch and egg features. In this work, we describe the morphology of Osornophryne occidentalis embryos from a nest collected in the field. Several traits are similar to those reported in brachycephaloid Eleutherodactylus coqui and other direct-developing lineages. These include the pattern of formation of the body wall and the absence of oral and buccopharyngeal structures. Other features indicate the retention of ancestral larval characters. The development of forelimbs occurs in part within the operculum as in species with biphasic ontogeny; this has been reported in other direct-developing species, and is similar to what is described in African viviparous bufonids. This salient feature, along with the long, low-finned tail and the long and folded intestine early differentiated, gives these embryos a rather tadpole-like appearance. Our data confirm that development in O. occidentalis occurs within terrestrial eggs until advanced stages, and this would satisfy current definitions of direct development. At the same time, morphological differences regarding other species with comparable breeding reinforce interpretations about the wide anatomical and ontogenetic variations associated with endotrophic nutrition.

Visual Signaling in the Semi-Fossorial Lizard Pholidobolus montium (Gymnophthalmidae).

It has been suggested that gymnophthalmids, like most semi-fossorial lacertoids, rely more in chemical cues to communicate, in comparison to other groups, like Iguanids, on which communication is mostly based on visual signaling. We present the first description of visual signaling in the Andean lizard Pholidobolusmontium (Gymnophthalmidae) and a complete ethogram based on ex situ observations (34 different types of behaviors including positions and simple movements). Through the design of conspecific stimulus experiments, we were able to recognize leg-waving as a visual signal, as it is only displayed in presence of conspecifics or in presence of a mirror and was one of first and most frequent displays in this context. We also detected other visual displays like neck-arching and tail-undulation which may also be relevant as visual signals. Based on our results, we propose that visual signaling is also possible in semi-fossorial lizards; however, further studies regarding chemical signal recognition and color detection are required to confirm our hypothesis.

A polymorphism in oocyte pigmentation in natural populations of the glass frog Espadarana prosoblepon (Centrolenidae).

The adaptive role of amphibian oocyte melanic pigmentation and its molecular control are still elusive. Here we present evidence of a polymorphism in egg pigmentation in the emerald glass frog Espadarana prosoblepon. In Ecuadorian natural populations of this species, females can lay dark brown or pale eggs that develop into normal pigmented tadpoles and adults. This trait is a sex-limited phenotype which is inherited like a recessive allele that we called pale eggs like (pel). The pel phenotype is exclusive of oocyte cortical melanic pigmentation, which is reduced in comparison to wild type (wt) dark pigmented oocytes. Consequently, pel early embryos are paler in appearance, with reduced melanic pigmentation distributed to early blastomeres and embryonic ectoderm. However, these embryos form normal melanocyte derived pigmentation. Finally, we discuss the origin of this polymorphism and propose the use of E. prosoblepon as a model to study the adaptive role of egg pigmentation.

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.

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.