CENP-E is an essential kinetochore motor in maturing oocytes and is masked during Mos-dependent, cell cycle arrest at metaphase II

CENP-E, a kinesin-like protein that is known to associate with kinetochores during all phases of mitotic chromosome movement, is shown here to be a component of meiotic kinetochores as well. CENP-E is detected at kinetochores during metaphase I in both mice and frogs, and, as in mitosis, is relocalized to the midbody during telophase. CENP-E function is essential for meiosis I because injection of an antibody to CENP-E into mouse oocytes in prophase completely prevented progression of those oocytes past metaphase I. Beyond this, CENP-E is modified or masked during the natural, Mos-dependent, cell cycle arrest that occurs at metaphase II, although it is readily detectable at the kinetochores in metaphase II oocytes derived from mos-deficient (MOS−/−) mice that fail to arrest at metaphase II. This must reflect a masking of some CENP-E epitopes, not the absence of CENP-E, in meiosis II because a different polyclonal antibody raised to the tail of CENP-E detects CENP-E at kinetochores of metaphase II-arrested eggs and because CENP-E reappears in telophase of mouse oocytes activated in the absence of protein synthesis.

Germ-line transmission of transgenes in Xenopus laevis

Adult Xenopus laevis frogs made transgenic by restriction enzyme-mediated integration were bred to test the feasibility of establishing lines of frogs that express transgenes. All of the 19 animals raised to sexual maturity generated progeny that expressed the transgene(s). The patterns and levels of expression of green fluorescent protein transgenes driven by a viral promoter, rat promoter, and four X. laevis promoters were all unaffected by passage through the germ line. These results demonstrate the ease of establishing transgenic lines in X. laevis.

Transient “deafness” accompanies auditory development during metamorphosis from tadpole to frog

During metamorphosis, ranid frogs shift from a purely aquatic to a partly terrestrial lifestyle. The central auditory system undergoes functional and neuroanatomical reorganization in parallel with the development of new sound conduction pathways adapted for the detection of airborne sounds. Neural responses to sounds can be recorded from the auditory midbrain of tadpoles shortly after hatching, with higher rates of synchronous neural activity and lower sharpness of tuning than observed in postmetamorphic animals. Shortly before the onset of metamorphic climax, there is a brief “deaf” period during which no auditory activity can be evoked from the midbrain, and a loss of connectivity is observed between medullary and midbrain auditory nuclei. During the final stages of metamorphic development, auditory function and neural connectivity are restored. The acoustic communication system of the adult frog emerges from these periods of anatomical and physiological plasticity during metamorphosis.

Prolactin is not a juvenile hormone in Xenopus laevis metamorphosis

Prolactin (PRL) is widely considered to be the juvenile hormone of anuran tadpoles and to counteract the effects of thyroid hormone (TH), the hormone that controls amphibian metamorphosis. This putative function was concluded mainly from experiments in which mammalian PRL was injected into tadpoles or added to cultured tadpole tissues. In this study, we show that overexpression of ovine or Xenopus laevis PRL in transgenic X. laevis does not prolong tadpole life, establishing that PRL does not play a role in the life cycle of amphibians that is equivalent to that of juvenile hormone in insect metamorphosis. However, overexpression of PRL produces tailed frogs by reversing specifically some but not all of the programs of tail resorption and stimulating growth of fibroblasts in the tail. Whereas TH induces muscle resorption in tails of these transgenics, the tail fibroblasts continue to proliferate resulting in a fibrotic tail that is resistant to TH.

Mitochondria as ATP consumers: Cellular treason in anoxia

In anoxia, mitochondria change from being ATP producers to potentially powerful ATP consumers. This change occurs, because the mitochondrial F1F0-ATPase begins to hydrolyze ATP to avoid the collapse of the proton motive force. Species that can survive prolonged periods of O2 lack must limit such ATP use; otherwise, this process would dominate glycolytic metabolism and threaten ATP delivery to essential ATP-consuming processes of the cell (e.g., ion-motive ATPases). There are two ways to limit ATP hydrolysis by the F1F0-ATPase, namely (i) reduction of the proton conductance of the mitochondrial inner membrane and (ii) inhibition of the enzyme. We assessed these two possibilities by using intact mitochondria isolated from the skeletal muscle of anoxia-tolerant frogs. Our results show that proton conductance is unaltered between normoxia and anoxia. However, ATP use by the F1F0-ATPase is limited in anoxia by a profound inhibition of the enzyme. Even so, ATP use by the F1F0-ATPase might account for ≈9% of the ATP turnover in anoxic frog skeletal muscle.

The evolution of coloration and toxicity in the poison frog family (Dendrobatidae)

The poison frogs (family Dendrobatidae) are terrestrial anuran amphibians displaying a wide range of coloration and toxicity. These frogs generally have been considered to be aposematic, but relatively little research has been carried out to test the predictions of this hypothesis. Here we use a comparative approach to test one prediction of the hypothesis of aposematism: that coloration will evolve in tandem with toxicity. Recently, we developed a phylogenetic hypothesis of the evolutionary relationships among representative species of poison frogs, using sequences from three regions of mitochondrial DNA. In our analysis, we use that DNA-based phylogeny and comparative analysis of independent contrasts to investigate the correlation between coloration and toxicity in the poison frog family (Dendrobatidae). Information on the toxicity of different species was obtained from the literature. Two different measures of the brightness and extent of coloration were used. (i) Twenty-four human observers were asked to rank different photos of each different species in the analysis in terms of contrast to a leaf-littered background. (ii) Color photos of each species were scanned into a computer and a computer program was used to obtain a measure of the contrast of the colors of each species relative to a leaf-littered background. Comparative analyses of the results were carried out with two different models of character evolution: gradual change, with branch lengths proportional to the amount of genetic change, and punctuational change, with all change being associated with speciation events. Comparative analysis using either method or model indicated a significant correlation between the evolution of toxicity and coloration across this family. These results are consistent with the hypothesis that coloration in this group is aposematic.

On the origin of and phylogenetic relationships among living amphibians

The phylogenetic relationships among the three orders of modern amphibians (Caudata, Gymnophiona, and Anura) have been estimated based on both morphological and molecular evidence. Most morphological and paleontological studies of living and fossil amphibians support the hypothesis that salamanders and frogs are sister lineages (the Batrachia hypothesis) and that caecilians are more distantly related. Previous interpretations of molecular data based on nuclear and mitochondrial rRNA sequences suggested that salamanders and caecilians are sister groups to the exclusion of frogs. In an attempt to resolve this apparent conflict, the complete mitochondrial genomes of a salamander (Mertensiella luschani) and a caecilian (Typhlonectes natans) were determined (16,656 and 17,005 bp, respectively) and compared with previously published sequences from a frog (Xenopus laevis) and several other groups of vertebrates. Phylogenetic analyses of the mitochondrial data supported with high bootstrap values the monophyly of living amphibians with respect to other living groups of tetrapods, and a sister group relationship of salamanders and frogs. The lack of phylogenetically informative sites in the previous rRNA data sets (because of its shorter size and higher among-site rate variation) likely explains the discrepancy between our results and those based on previous molecular data. Strong support of the Batrachia hypothesis from both molecule- and morphology-based studies provides a robust phylogenetic framework that will be helpful to comparative studies among the three living orders of amphibians and will permit better understanding of the considerably divergent vertebral, brain, and digit developmental patterns found in frogs and salamanders.

Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).

Bombesin is a tetradecapeptide originally isolated from frog skin and demonstrated to have a wide range of actions in mammals. Based on structural homology and similar biological activities, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of bombesin. We previously reported that frogs have both GRP and bombesin, which therefore are distinct peptides. We now report the cloning of a bombesin receptor subtype (BB4) that has higher affinity for bombesin than GRP. PCR was used to amplify cDNAs related to the known bombesin receptors from frog brain. Sequence analysis of the amplified cDNAs revealed 3 classes of receptor subtypes. Based on amino acid homology, two classes were clearly the amphibian homologs of the GRP and neuromedin B receptors. The third class was unusual and a full-length clone was isolated from a Bombina orientalis brain cDNA library. Expression of the receptor in Xenopus oocytes demonstrated that the receptor responded to picomolar concentrations of [Phe13]-bombesin, the form of bombesin most prevalent in frog brain. The relative rank potency of bombesin-like peptides for this receptor was [Phe13]bombesin > [Leu13]bombesin > GRP > neuromedin B. In contrast, the rank potency for the GRP receptor is GRP > [Leu13]bombesin > [Phe13]bombesin > neuromedin B. Transient expression in CHOP cells gave a Ki for [Phe13]bombesin of 0.2 nM versus a Ki of 2.1 nM for GRP. Distribution analysis showed that this receptor was expressed only in brain, consistent with the distribution of [Phe13]-bombesin. Thus, based on distribution and affinity, this bombesin receptor is the receptor for [Phe13]bombesin. Phylogenetic analysis suggests that this receptor separated prior to separation of the GRP and neuromedin B receptors; thus, BB4 receptors and their cognate ligands may also exist in mammals.

A conserved family of elav-like genes in vertebrates.

A large family of genes encodes proteins with RNA recognition motifs that are presumed to bind RNA and to function in posttranscriptional regulation. Neural-specific members of this family include elav, a gene required for correct differentiation and maintenance of neurons in Drosophila melanogaster, and a related gene, HuD, which is expressed in human neuronal cells. I have identified genes related to elav and HuD in Xenopus laevis, zebrafish, and mouse that define a family of four closely related vertebrate elav-like genes (elrA, elrB, elrC, and elrD) in fish, frogs, and mammals. In addition to protein sequence conservation, a segment of the 3'-untranslated sequence of elrD is also conserved, implying a functional role in elrD expression. In adult frogs, elrC and elrD are exclusively expressed in the brain, whereas elrB is expressed in brain, testis, and ovary. During Xenopus development, elrC and elrD RNAs are detected by late gastrula and late neurula stages, respectively, whereas a nervous system-specific elrB RNA species is expressed by early tadpole stage. Additional elrB transcripts are detected in the ovary and early embryo, demonstrating a maternal supply of mRNA and possibly of protein. These expression patterns suggest a role for different elav-like genes in early development and neuronal differentiation. Surprisingly, elrA is expressed in all adult tissues tested and at all times during development. Thus, the widely expressed elrA is expected to have a related function in all cells.

Efeito do estresse hídrico sobre a locomoção e morfologia de girinos de Leptodactylus fuscus e Physalaemus nattereri

Diversas espécies de anuros da família Leptodactylidae se reproduzem em corpos dágua sazonais, temporários e mantidos exclusivamente por chuvas. Em períodos de estiagem prolongada a poça pode secar completamente, ocasionando elevadas taxas de mortalidade de ovos e girinos dessas espécies, podendo exercer forte pressão seletiva na evolução de mecanismos de resistência e sobrevivência nas fases iniciais do desenvolvimento. Algumas espécies de girinos conseguem sobreviver cerca de cinco dias fora dágua o que pode proporcionar uma adaptação vantajosa, porque possibilita a sobrevivência dos girinos por um período que pode ser suficiente para a reincidência de novas chuvas e restabelecimento do corpo dágua. Apesar dessa capacidade de sobrevivência, pouco se sabe sobre as possíveis modificações que a desidratação pode causar na locomoção e na morfologia durante o desenvolvimento desses animais. O presente trabalho teve como objetivo avaliar o efeito do estresse hídrico: (1) no nível de sobrevivência e perda de massa corpórea; (2) no desempenho locomotor; (3) na morfologia externa (morfometria linear) e interna, analisando tanto o volume total quanto o volume visceral (estereologia); e (4) no tempo até metamorfose após o estresse. Utilizamos girinos de duas espécies de anuros, Leptodactylus fuscus (Leptodactylinae) e Physalaemus nattereri (Leiuperinae), ambas as espécies se reproduzem em corpos dágua temporários, em áreas com estação seca definida estando, portanto sujeitas as mesmas pressões seletivas. Além disso, as duas espécies apresentam modos reprodutivos diferentes, podendo apresentar diferentes graus de resistência ao estresse hídrico. Os girinos das duas espécies foram divididos em dois grupos, os que ficaram em água (grupo controle) e os que foram submetidos ao estresse hídrico (grupo tratamento), por três períodos de tempo (12, 24 e 72 horas). Houve diferenças significativas para valores de perda de massa entre os grupos controle e tratamento em ambas as espécies, sendo o grupo tratamento que mais perdeu massa corpórea em todos os períodos, além disso, quase metade dos girinos de P. nattereri morreram em 36 horas de estresse. Não houve diferenças significativas para os dados de desempenho locomotor e volume total entre os grupos testado para girinos de L. fuscus, mas houve diferenças morfometricas significantes nos componentes relacionados a cauda e no volume visceral, onde, o intestino do grupo tratamento foi menor que do controle. Já em P. nattereri, houve diferenças significativas entre os grupos testados para desempenho locomotor, volume total, morfometria da cauda e volume visceral, sendo o estomago e anexo do tratamento maior que do controle. Nossos resultados sugerem que a exposição ao estresse hídrico não afeta significativamente a morfologia e o desempenho locomotor dos girinos de L. fuscus. No entanto, girinos de P. nattereri apresentaram uma sensibilidade ao estresse hídrico prolongado, principalmente sobre o seu desempenho locomotor.

Amphibian Hotspots and Conservation Priorities in Eastern Cuba Identified by Species Distribution Modeling

The high rate of amphibian endemism and the severe habitat modification in the Caribbean islands make them an ideal place to test if the current protected areas network might protect this group. In this study, we model distribution and map species richness of the 40 amphibian species from eastern Cuba with the objectives of identify hotspots, detect gaps in species representation in protected areas, and select additional areas to fill these gaps. We used two modeling methods, Maxent and Habitat Suitability Models, to reach a consensus distribution map for each species, then calculate species richness by combining specific models and finally performed gap analyses for species and hotspots. Our results showed that the models were robust enough to predict species distributions and that most of the amphibian hotspots were represented in reserves, but 50 percent of the species were incompletely covered and Eleutherodactylus rivularis was totally uncovered by the protected areas. We identified 1441 additional km2 (9.9% of the study area) that could be added to the current protected areas, allowing the representation of every species and all hotspots. Our results are relevant for the conservation planning in other Caribbean islands, since studies like this could contribute to fill the gaps in the existing protected areas and to design a future network. Both cases would benefit from modeling amphibian species distribution using available data, even if they are incomplete, rather than relying only in the protection of known or suspected hotspots.