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.

Adaptive cell invasion maintains lateral line organ homeostasis in response to environmental changes.

Mammalian inner ear and fish lateral line sensory hair cells (HCs) detect fluid motion to transduce environmental signals. Actively maintained ionic homeostasis of the mammalian inner ear endolymph is essential for HC function. In contrast, fish lateral line HCs are exposed to the fluctuating ionic composition of the aqueous environment. Using lineage labeling, in vivo time-lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. This invasion is adaptive as it is triggered by environmental fluctuations. Our discovery of Nm ionocytes challenges the notion of an entirely placodally derived lateral line and identifies Nm ionocytes as likely regulators of HC function possibly by modulating the ionic microenvironment. Nm ionocytes provide an experimentally accessible in vivo system to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

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.

scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling.

Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental trajectory identified self-renewing cells and genes required for hair cell differentiation. scRNA-Seq analyses of fgf3 mutants, in which hair cell regeneration is increased, demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals. The data is searchable and publicly accessible via a web-based interface.

Embryonic Development in the Peppermint Shrimp, Lysmata boggessi (Caridea: Lysmatidae).

There are a limited number of model species for decapod experimental embryology. To improve our understanding of developmental pattern evolution in the Decapoda, here we describe the early embryonic development of the caridean shrimp Lysmata boggessi, from immediately after fertilization to the hatching of the zoea larva, using fluorescence microscopy and whole-mount nuclear staining with 4',6-diamidino-2-phenylindole. Lysmata boggessi follows the standard caridean pattern of early development, with early holoblastic cleavage that will later become superficial, to form a blastoderm. We found no evidence of stereotypical cleavage and the formation of blastomere interlocking bands, which suggests there is diversity in developmental patterns within the Caridea. Gastrulation starts 37 hours after fertilization, and the embryonized nauplius is formed 2 days later. Enlarged headlobes, early retinal differentiation, and delayed pereopod development are characteristics of the post-naupliar stages in this species. To facilitate comparative studies with other crustacean species, we propose a staging method based on our findings. Lysmata boggessi is a protandric simultaneous hermaphrodite that is relatively easy to breed in captivity and amenable to laboratory experimentation in studies of embryonic development.

Regeneration of Sensory Hair Cells Requires Localized Interactions between the Notch and Wnt Pathways.

In vertebrates, mechano-electrical transduction of sound is accomplished by sensory hair cells. Whereas mammalian hair cells are not replaced when lost, in fish they constantly renew and regenerate after injury. In vivo tracking and cell fate analyses of all dividing cells during lateral line hair cell regeneration revealed that support and hair cell progenitors localize to distinct tissue compartments. Importantly, we find that the balance between self-renewal and differentiation in these compartments is controlled by spatially restricted Notch signaling and its inhibition of Wnt-induced proliferation. The ability to simultaneously study and manipulate individual cell behaviors and multiple pathways in vivo transforms the lateral line into a powerful paradigm to mechanistically dissect sensory organ regeneration. The striking similarities to other vertebrate stem cell compartments uniquely place zebrafish to help elucidate why mammals possess such low capacity to regenerate hair cells.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line.

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/ß-catenin signaling following hair cell death. We propose that Wnt/ß-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.

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.