Sumario:
The International Journal of Developmental Biology
Linking Development, Stem Cells and Cancer Research
Euskal Herriko Unibertsitateko Argitalpen Zerbitzua / Servicio Editorial de la Universidad del País Vasco / University of the Basque Country Press
Volume 58 - Numbers 10-11-12 (2014) / Pages 719-960 Editor-in-Chief: Juan Aréchaga
MORE INFORMATION [Abstract - FullText / FullText Open Access]
ISSN: 0214-6282 / ISSN-e: 1696-3547 www.intjdevbiol.com
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Special Issue: Developmental Herpetology
Guest Editors: Jacek Z. Kubiak and Malgorzata Kloc
ABSTRACTS
Preface
EHU/UPV/UBC - The International Journal of Developmental Biology 58: 719-721 (2014)
doi: 10.1387/ijdb.150228jk / © UBC Press ( www.a360grados.net )
Developmental Herpetology - state of the art of amphibian and reptile developmental biology
Jacek Z. Kubiak, 1 and Malgorzata Kloc, 2
1. Institute of Genetics and Development of Rennes, Rennes, France
2. Houston Methodist Research Institute, Houston, TX, USA
Abstract: The Special Issue which you are now reading is the offspring of a vivid backstage conversation during a scientific meeting with the Editor-in-Chief of The International Journal of Developmental Biology (Int. J. Dev. Biol.) on the importance of research on the developmental biology of Amphibians and Reptiles yesterday, today and in the future. As you can see, we managed to convince the Editor-in-Chief that the matter is indeed important. We hope you will enjoy the outcome.
History & Perspectives
EHU/UPV/UBC - The International Journal of Developmental Biology 58: 723-725(2014)
doi: 10.1387/ijdb.150111jg / © UBC Press ( www.a360grados.net )
A view of amphibian embryology during the last century
John B. Gurdon
Wellcome Trust/Cancer Research UK University of Cambridge, Cambridge, UK
Abstract: Having started working in the field of amphibian embryology over 50 years ago, I make some comments about the changes that seem to me to have taken place in this field over this period. Over the period 1885 to 1960, much of the highly regarded experimental embryology was conducted on amphibian eggs and embryos. Indeed, much of this work was conducted in Germany and Switzerland using eggs and embryos of European newts (salamanders) and frogs of the Rana group. Xenopus started to be used extensively after the 1950s because eggs and embryos could be obtained throughout the year by hormone injection and because sexually mature animals could be raised from an egg within one year. Since the 1960s, publications using Xenopus have exceeded those using other amphibian species by 100-fold. This short commentary highlights some of the major advances attributable to embryological work with Amphibia and exemplifies these advances by reference to those who have made conspicuous contributions in this area.
Keywords: Amphibian, history, Spemann, Hamburger, Brachet, Gurdon
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 727-732(2014)
doi: 10.1387/ijdb.150026dd / © UBC Press ( www.a360grados.net )
Snakes: hatching of a model system for Evo-Devo?
Isabel Guerreiro1 and Denis Duboule, 1,2
1. Department of Genetics and Evolution, University of Geneva
2. School of Life Sciences, Federal Institute of Technology, Lausanne, Switzerland
Abstract: Evo-Devo studies rely on a collection of animal model systems belonging to different phylogenetic branches to try and understand how organisms carrying a similar set of genes and pathways can develop into such a variety of shapes and sizes. The squamate clade, however, has only recently started to receive the attention it deserves in particular due to extreme morphological and metabolic aspects and, consequently, the important insights that it could bring in different fields. The recent sequencing of several squamate genomes as well as the generation of high quality trancriptomes for different snake tissues now provide the necessary tools to complement biological studies. Here, we briefly report on recent work involving developing snake embryos to illustrate their interest to assess vertebrate developmental mechanisms. We also discuss the relevance to use snake species as Evo-Devo model systems and potential ways to cross the important limitations intrinsically associated with developmental and genetic studies of these fascinating animals.
Keywords: Hox gene, snake, model organism, axial extension, vertebrae
Developmental features
EHU/UPV/UBC - The International Journal of Developmental Biology 58: 733-741(2014)
doi: 10.1387/ijdb.140192ag / © UBC Press ( www.a360grados.net )
Expression of aromatase in the embryonic brain of the olive ridley sea turtle (Lepidochelys olivacea), and the effect of bisphenol-A in sexually differentiated embryos
Patsy Gómez-Picos 1, Itzel Sifuentes-Romero 1, Horacio Merchant-Larios 2, Rubí Hernández-Cornejo 1, Verónica Díaz-Hernández 3 and Alejandra García-Gasca 1
1. Laboratory of Molecular Biology, Centro de Investigación en Alimentación y Desarrollo (CIAD), Mazatlán, Sinaloa, Mexico
2. Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
3. Departamento de Embriología, Facultad de Medicina, Universidad Nacional Autónoma de Mexico, Mexico City, Mexico
Abstract: Brain aromatase participates in several biological processes, such as regulation of the reproductive-endocrine axis, memory, stress, sexual differentiation of the nervous system, male sexual behavior, and brain repair. Here we report the isolation and expression of brain aromatase in olive ridley sea turtle (Lepidochelys olivacea) embryos incubated at male- and female-promoting temperatures (MPT and FPT, respectively), at the thermosensitive period (TSP) and the sex-differentiated period. Also, aromatase expression was assessed in differentiated embryos exposed to bisphenol-A (BPA) during the TSP. BPA is a monomer of polycarbonate plastics and is considered an endocrine-disrupting compound. Normal aromatase expression was measured in both forebrain and hindbrain, showing higher expression levels in the forebrain of differentiated embryos at both incubation temperatures. Although no significant differences were detected in the hindbrain, expression was slightly higher at MPT. BPA did not affect aromatase expression neither in forebrains or hindbrains from embryos incubated at MPT, whereas at FPT an inverted U-shape curve was observed in forebrains with significant differences at lower concentrations, whereas in hindbrains a non-significant increment was observed at higher concentrations. Our data indicate that both incubation temperature and developmental stage are critical factors affecting aromatase expression in the forebrain. Because of the timing and location of aromatase expression in the brain, we suggest that brain aromatase may participate in the imprinting of sexual trends related to reproduction and sexual behavior at the onset of sex differentiation, and BPA exposure may impair aromatase function in the female forebrain.
Keywords: aromatase, gene expression, Lepidochelys olivacea, temperature-dependent sex determination, bisphenol-A
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 743-750(2014)
doi: 10.1387/ijdb.140296jp / © UBC Press ( www.a360grados.net )
Comparative analysis of pleurodiran and cryptodiran turtle embryos depicts the molecular ground pattern of the turtle carapacial ridge
Juan Pascual-Anaya 1, Tatsuya Hirasawa 1, Iori Sato 1, Shigehiro Kuraku 2 and Shigeru Kuratani 1
1. Evolutionary Morphology Laboratory, RIKEN
2. Phyloinformatics Unit, RIKEN Center for Life Science Technologies, Kobe, Japan
Abstract: The turtle shell is a wonderful example of a genuine morphological novelty, since it has no counterpart in any other extant vertebrate lineages. The evolutionary origin of the shell is a question that has fascinated evolutionary biologists for over two centuries and it still remains a mystery. One of the turtle innovations associated with the shell is the carapacial ridge (CR), a bulge that appears at both sides of the dorsal lateral trunk of the turtle embryo and that probably controls the formation of the carapace, the dorsal moiety of the shell. Although from the beginning of this century modern genetic techniques have been applied to resolve the evolutionary developmental origin of the CR, the use of different models with, in principle, dissimilar results has hampered the establishment of a common mechanism for the origin of the shell. Although modern turtles are divided into two major groups, Cryptodira (or hidden-necked turtles) and Pleurodira (or side-necked turtles), molecular developmental studies have been carried out mostly using cryptodiran models. In this study, we revisit the past data obtained from cryptodiran turtles in order to reconcile the different results. We also analyze the histological anatomy and the expression pattern of main CR factors in a pleurodiran turtle, the red-bellied short-necked turtle Emydura subglobosa. We suggest that the turtle shell probably originated concomitantly with the co-option of the canonical Wnt signaling pathway into the CR in the last common ancestor of the turtle.
Keywords: turtle, evolution, shell, Wnt pathway, Evo-Devo
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 751-755(2014)
doi: 10.1387/ijdb.140264ya / © UBC Press ( www.a360grados.net )
zfp36 expression delineates both myeloid cells and cells localized to the fusing neural folds in Xenopus tropicalis
Maud Noiret, Serge Hardy and Yann Audic
CNRS UMR 6092, Gene Expression and Development Team, Université Rennes 1, Institut de Génétique et Développement de Rennes (IGDR), Rennes, France
Abstract: Regulatory RNA binding proteins allow for specific control of gene expression in a very dynamic manner. In mammals ZFP36, formerly known as Tristetraprolin, controls the inflammatory response by binding to an AU-rich element located in the 3’ untranslated region of its target mRNAs. The developping embryo relies on a population of primitive macrophages to ensure proper immunity. Although the role of zfp36 in adult immunity has been extensively studied, its expression in the developing immune system has been poorly documented. Here, we have used whole mount in situ hybridization with a 3’ UTR specific probe to address the expression of zfp36 in developing Xenopus tropicalis embryos. We have shown that zfp36 is expressed in two distinct cellular populations. First, it is a new marker of primititive myeloid cells, being coexpressed with the myeloid marker mpo. Therefore this early expression may suggest a role for zfp36 in macrophage differentiation and activation. In addition, a second cell population was found to transiently express zfp36, but not mpo, along the fusing neural folds and may correspond to cells undergoing autophagy during neural tube closure.
Keywords: RNA binding proteins, Xenopus tropicalis, embryonic immunity
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 757-766(2014)
doi: 10.1387/ijdb.140271jr / © UBC Press ( www.a360grados.net )
Mechanisms of amphibian macrophage development: characterization of the Xenopus laevis colony-stimulating factor-1 receptor
Leon Grayfer, Eva-Stina Edholm and Jacques Robert
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, USA
Abstract: Macrophage-lineage cells are indispensable to vertebrate homeostasis and immunity. In turn, macrophage development is largely regulated through colony-stimulating factor-1 (CSF1) binding to its cognate receptor (CSF1R). To study amphibian monopoiesis, we identified and characterized the X. laevis CSF1R cDNA transcript. Quantitative analysis revealed that CSF1R tissue gene expression increased with X. laevis development, with greatest transcript levels detected in the adult lung, spleen and liver tissues. Notably, considerable levels of CSF1R mRNA were also detected in the regressing tails of metamorphosing animals, suggesting macrophage involvement in this process, and in the adult bone marrow; corroborating the roles for this organ in Xenopus monopoiesis. Following animal infections with the ranavirus Frog Virus 3 (FV3), both tadpole and adult X. laevis exhibited increased kidney CSF1R gene expression. Conversely, while FV3-infected tadpoles increased their spleen and liver CSF1R mRNA levels, the FV3-challenged adults did not. Notably, FV3 induced elevated bone marrow CSF1R expression, and while stimulation of tadpoles with heat-killed E. coli had no transcriptional effects, bacterial stimulation of adult frogs resulted in significantly increased spleen, liver and bone marrow CSF1R expression. We produced the X. laevis CSF1R in recombinant form (rXlCSF1R) and determined, via in vitro cross-linking studies, that two molecules of rXlCSF1R bound the dimeric rXlCSF1. Finally, administration of rXlCSF1R abrogated the rXlCSF1-induced tadpole macrophage recruitment and differentiation as well as bacterial and FV3-elicited peritoneal leukocyte accumulation. This work marks a step towards garnering greater understanding of the unique mechanisms governing amphibian macrophage biology.
Keywords: Xenopus, monopoiesis, CSF1, macrophage development, ranavirus
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 767-774(2014)
doi: 10.1387/ijdb.140101jk / © UBC Press ( www.a360grados.net )
Control of timing of embryonic M-phase entry and exit is differentially sensitive to CDK1 and PP2A balance
Mohammed El Dika 1,2, Damian Dudka 1,2, Claude Prigent 1,2, Jean-Pierre Tassan 1,2, Malgorzata Kloc 3 and Jacek Z. Kubiak 1,2
1. CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Cell Cycle Group, Rennes, France
2. University Rennes 1, UEB, IFR 140, Faculty of Medicine, Rennes, France
3. The Houston Methodist Research Institute, Houston, TX, USA
Abstract: Harmonious embryo development requires precise coordination between the timing of the cell cycle and the developmental program. Cyclin accumulation determines the timing of the cell cycle M-phase entry and its degradation determines the timing of the M-phase exit. It is well known that CDK1 and PP2A also govern M-phase entry. However, it is unknown how this kinase and phosphatase regulate the precise timing of events at the beginning of the M-phase and how they cooperate with cyclin metabolism. Here we use Xenopus laevis one-cell embryo cell-free extract experiments to answer this question critical for understanding the regulation of embryo development. Using, separately, low concentrations of the chemical inhibitor of CDK1, RO3306 (RO), or the inhibitor of phosphatases, okadaic acid (OA), we show that moderately diminished CDK1 or PP2A activities results in a delay and an acceleration respectively, of M-phase entry. Simultaneous diminution of CDK1 and PP2A activities results in an intermediate timing of M-phase entry, prolongs the duration of M-phase and diminishes the dynamics of cyclin B2 degradation. We thus show, for the first time, that equilibrium between CDK1 and PP2A specifies the timing of M-phase entry and exit and regulates the dynamics of cyclin B degradation upon M-phase exit in Xenopus laevis first embryonic mitosis.
Keywords: cell cycle, cyclin, PP2A, CDK, M-phase entry
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 775-781(2014)
doi: 10.1387/ijdb.140277jt / © UBC Press ( www.a360grados.net )
Epithelial cell division in the Xenopus laevis embryo during gastrulation
Guillaume Hatte 1,2, Marc Tramier 1,2, Claude Prigent 1,2 and Jean-Pierre Tassan 1,2
1. CNRS UMR 6290
2. Université de Rennes 1, Institut de Génétique et Développement de Rennes, Rennes, France
Abstract: How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.
Keywords: cytokinesis, asymmetric furrowing, contractile ring, anillin, actin, myosin
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 783-791(2014)
doi: 10.1387/ijdb.140244kd / © UBC Press ( www.a360grados.net )
Activin ligands are required for the re-activation of Smad2 signalling after neurulation and vascular development in Xenopus tropicalis
Yuki Nagamori, Samantha Roberts, Marissa Maciej and Karel Dorey
The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, UK
Abstract: The importance of Transforming Growth Factor β (TGFβ) signalling during early development has been well established. In particular, Nodal ligands have been shown to play essential roles for the specification and the patterning of the mesendoderm, axes formation and organogenesis. Activin ligands, like Nodal, signal by inducing the phosphorylation of the intracellular signal transducers Smad2 and Smad3. However, the roles of Activins during embryonic development are much less understood. Here, we report that during Xenopus tropicalis development two waves of Smad2 phoshorylation can be observed, first during gastrulation and then a second one after neurulation. Using a knock-down approach, we show that the second wave of Smad2 phosphorylation depends on activinβa (actβa) and activinβb (actβb) expression. Knocking down the expression of actβa, or treating the embryos with a chemical inhibitor inhibiting TGFβ receptor I (TGFβRI) activity after neurulation result in a decrease of the expression of endothelial cell markers and a lack of blood flow in Xenopus tadpoles. Taken together these data suggest that Activin ligands play an important role during vascular development in Xenopus tropicalis embryos.
Keywords: Activin, vasculogenesis, TGFβ, Xenopus, Smad
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 793-798(2014)
doi: 10.1387/ijdb.140308ho / © UBC Press ( www.a360grados.net )
Intracellular localizations of the Dead End protein in Xenopus primordial germ cells
Ayaka Taguchi, Kenji Watanabe and Hidefumi Orii
Department of Life Science, University of Hyogo, Kamigori, Akou-gun, Hyogo, Japan
Abstract: We investigated the intracellular localization of Xenopus Dead end protein (Dnd1) in primordial germ cells during early development by expressing the tagged protein in transgenic Xenopus embryos, with the germ plasm visualized. Dnd1 initially localized to the germ plasm in the cortex, moved to the perinuclear region together with the germ plasm after the midblastula transition, and then entered the nucleus. Using Dnd1 deletion mutants, we identified two distinct but overlapping regions of Dnd1 that were responsible for localization to either the germ plasm or nucleus. These Dnd1 regions appeared to function in primordial germ cell- and stage-specific manners.
Keywords: Dnd1, germline, germ plasm, nuage, nuclear localization signal, RNA-binding protein
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 799-809(2014)
doi: 10.1387/ijdb.140215ml / © UBC Press ( www.a360grados.net )
Left-right patterning in Xenopus conjoined twin embryos requires serotonin signaling and gap junctions
Laura N. Vandenberg 1,2, Douglas J. Blackiston 1, Adam C. Rea 3, Timothy M. Dore 3,4, and Michael Levin 1
1. Biology Department, Center for Regenerative and Developmental Biology, Tufts University, Medford, MA, USA
2. Department of Public Health, Division of Environmental Health Sciences, University of Massachusetts – Amherst, Amherst, MA, USA
3. Department of Chemistry, University of Georgia, Athens, GA, USA
4. New York University Abu Dhabi, Abu Dhabi, United Arab Emirates
Abstract: A number of processes operating during the first cell cleavages enable the left-right (LR) axis to be consistently oriented during Xenopus laevis development. Prior work showed that secondary organizers induced in frog embryos after cleavage stages (i.e. conjoined twins arising from ectopic induced primary axes) correctly pattern their own LR axis only when a primary (early) organizer is also present. This instructive effect confirms the unique LR patterning functions that occur during early embryogenesis, but leaves open the question: which mechanisms that operate during early stages are also involved in the orientation of later-induced organizers? We sought to distinguish the two phases of LR patterning in secondary organizers (LR patterning of the primary twin and the later transfer of this information to the secondary twin) by perturbing only the latter process. Here, we used reagents that do not affect primary LR patterning at the time secondary organizers form to inhibit each of 4 mechanisms in the induced twin. Using pharmacological, molecular-genetic, and photo-chemical tools, we show that serotonergic and gap-junctional signaling, but not proton or potassium flows, are required for the secondary organizer to appropriately pattern its LR axis in a multicellular context. We also show that consistently-asymmetric gene expression begins prior to ciliary flow. Together, our data highlight the importance of physiological signaling in the propagation of cleavage-derived LR orientation to multicellular cell fields.
Keywords: ion flux, polarity, heterotaxia, drug screen, connexin, twinning, asymmetry, gap junction
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 811-818(2014)
doi: 10.1387/ijdb.140302gc / © UBC Press ( www.a360grados.net )
Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs
Valeria S. Mouguelar and Gabriela Coux
IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Argentina
Abstract: Recently, we have provided evidence for the involvement of a cytosolic tyrosine–phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura: Bufonidae) fertilization. The aim of the present work was to characterize its phosphorylation, determine the identity of this protein and establish its biological role during the fertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observed in eggs activated with the calcium ionophore A23187. Pretreatment of oocytes with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation of the 70 kDa protein. In order to identify this protein, we examined the presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members of the Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R. arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blot analysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studies using phospho-Syk specific antibodies showed that fertilization rapidly (less than 10 minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that are necessary for the activation of the enzyme. Finally, specific inhibition of Syk with the R406 compound provoked a diminished fertilization score, thereby confirming a functional role of the Syk protein during R. arenarum fertilization. To our knowledge this is the first time that Syk is described as a player in the signaling cascade activated after fertilization.
Keywords: tyrosine phosphorylation, amphibian, signaling
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 819-827(2014)
doi: 10.1387/ijdb.140147 / © UBC Press ( www.a360grados.net )
Bidder’s organ – structure, development and function
Rafal P. Piprek 1, Malgorzata Kloc 4,5 and Jacek Z. Kubiak 2,3
1. Department of Comparative Anatomy, Institute of Zoology, Jagiellonian University, Krakow, Poland
2. CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Cell Cycle Group, France
3. Université Rennes 1, UEB, UMS Biosit, Faculty of Medicine, Rennes, France
4. Department of Surgery, The Houston Methodist Hospital, Houston, USA
5. The Houston Methodist Research Institute, Houston, USA
Abstract: Bidder’s organ is an ovary-like structure, which develops from the anterior part of the gonadal ridge in anuran amphibians belonging to the Bufonidae family. Bidder’s organs form in both males and females. Because Bidder’s organ contains female germ cells (oocytes), the bufonid males are de facto hermaphrodites. Due to similarity with the undeveloped ovary, Bidder’s organ was, in early literature, described, inaccurately, as a structure present only in males. Due to the fact that Bidder’s organ is a unique structure present only in Bufonidae, it is not well studied and its function still remains a mystery. Here we describe the development and structure of Bidder’s organs, summarize the knowledge on gene expression and steroidogenic activity in these organs, and present hypotheses regarding Bidder’s organ function.
Keywords: Bidder’s organ, testis, ovary, oocytes, sex hormones
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 829-839(2014)
doi: 10.1387/ijdb.140325la / © UBC Press ( www.a360grados.net )
Transition from embryonic to adult epidermis in reptiles occurs by the production of corneous beta-proteins
Lorenzo Alibardi
Comparative Histolab and Dipartimento Bigea, University of Bologna, Italy
Abstract: The adaptation of the epidermis in amniote vertebrates to life on land took place by a drastic change from an embryonic epidermis made of two-four periderm layers to a terrestrial-proof epidermis. This transition occurred by the increase in types and number of specialized corneous proteins coded by genes of the Epidermal Differentiation Complex. The prevalent types of corneous proteins produced in the reptilian epidermis contain a beta-sheet region of high amino acid homology which allows their polymerization into a meshwork of filaments forming the hard corneous material of scales and claws. The present immunogold ultrastructural study shows that this transition occurs with the synthesis of glycine-rich corneous beta-proteins (formerly indicated as beta-keratins) that are added to the initial framework of acidic intermediate filaments produced in the embryonic epidermis of lizards, snake, alligator and turtle. These corneous beta-proteins are accumulated in the transitional and definitive layers of reptilian epidermis formed underneath the transitory two-four layered embryonic epidermis. In the more specialized reptiles capable of shedding the epidermis as a single unit, such as lizards and snakes, special glycine-cysteine rich beta-proteins are initially produced in a single layer immediately formed beneath the embryonic epidermis, the oberhautchen. The latter layer allows the in ovo shedding of the embryonic epidermis in preparation for hatching, and in the following shedding cycles of the adult epidermis. The production of specialized corneous-specific beta-proteins in addition to intermediate filament keratins was probably an essential addition for terrestrial life during the evolution of reptiles into different lineages, including birds. The increase of glycine and cysteine in epidermal proteins enhanced the hydrophobicity, insolubility and mechanical strength of the stratum corneum in these amniotes.
Keywords: reptiles, embryos, acidic keratins, corneous beta-proteins, immunocytochemistry
Application of bioinformatic tools and new methods
EHU/UPV/UBC - The International Journal of Developmental Biology 58: 841-849(2014)
doi: 10.1387/ijdb.140270sk / © UBC Press ( www.a360grados.net )
Comparative expression analysis of cysteine-rich intestinal protein family members crip 1, 2 and 3 during Xenopus laevis embryogenesis
Annemarie Hempel 1,2 and Susanne J. Kühl 1
1. Institute for Biochemistry and Molecular Biology
2. International Graduate School in Molecular Medicine Ulm, Ulm University, Ulm, Germany
Abstract: Members of the cysteine-rich intestinal protein (Crip) family belong to the group 2 LIM proteins. Crip proteins are widely expressed in adult mammals but their expression profile and function during embryonic development are still mostly unknown. In this study, we have described for the first time the spatio-temporal expression pattern of the three family members crip1, crip2 and crip3 during Xenopus laevis embryogenesis by RT-PCR and whole mount in situ hybridization approaches. We observed that all three genes are expressed in the pronephros, branchial arches and the eye. Furthermore, crip1 transcripts could be visualized in the developing cranial ganglia and neural tube. In contrast, crip2 could be detected in the cardiovascular system, the brain and the neural tube while crip3 was expressed in the cranial ganglions and the heart. Based on these findings, we suggest that each crip family member may play an important role during embryonic development.
Keywords: Xenopus laevis, cystein-rich protein, crip1-3, LIM
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 851-861(2014)
doi: 10.1387/ijdb.140207ml / © UBC Press ( www.a360grados.net )
Optogenetics in Developmental Biology: using light to control ion flux-dependent signals in Xenopus embryos
Dany Spencer Adams 1, Joan M. Lemire 1, Richard H. Kramer 2 and Michael Levin 1
1. Dept of Biology and Center for Regenerative and Developmental Biology, Tufts University, Medford, MA
2. Dept of Molecular and Cell Biology, University of California, Berkeley, CA, USA
Abstract: Developmental bioelectricity, electrical signaling among non-excitable cells, is now known to regulate proliferation, apoptosis, gene expression, and patterning during development. The extraordinary temporal and spatial resolution offered by optogenetics could revolutionize the study of bioelectricity the same way it has revolutionized neuroscience. There is, however, no guide to adapting optogenetics to patterning systems. To fill this gap, we used optogenetic reagents, both proteins and photochemical switches, to vary steady-state bioelectrical properties of non-spiking embryonic cells in Xenopus laevis. We injected mRNA for various proteins, including Channelrhodopsins and Archaerhodopsin, into 1-8 cell embryos, or soaked embryos in media containing photochemical switches, then examined the effect of light and dark on membrane voltage (Vmem) using both electrodes and fluorescent membrane voltage reporters. We also scored tadpoles for known effects of varying Vmem, including left-right asymmetry disruption, hyperpigmentation, and craniofacial phenotypes. The majority of reagents we tested caused a significant increase in the percentage of light-exposed tadpoles showing relevant phenotypes; however, the majority of reagents also induced phenotypes in controls kept in the dark. Experiments on this “dark phenotype” yielded evidence that the direction of ion flux via common optogenetic reagents may be reversed, or unpredictable in non-neural cells. When used in combination with rigorous controls, optogenetics can be a powerful tool for investigating ion-flux based signaling in non-excitable systems. Nonetheless, it is crucial that new reagents be designed with these non-neural cell types in mind.
Keywords: optogenetics, photochemical switch, bioelectricity, xenopus, embryo
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 863-871(2014)
doi: 10.1387/ijdb.140316kk / © UBC Press ( www.a360grados.net )
Reptile genomes open the frontier for comparative analysis of amniote development and regeneration
Marc Tollis, Elizabeth D. Hutchins and Kenro Kusumi
School of Life Sciences, Arizona State University, Tempe, AZ, USA
Abstract: Developmental genetic studies of vertebrates have focused primarily on zebrafish, frog and mouse models, which have clear application to medicine and well-developed genomic resources. In contrast, reptiles represent the most diverse amniote group, but have only recently begun to gather the attention of genome sequencing efforts. Extant reptilian groups last shared a common ancestor ?280 million years ago and include lepidosaurs, turtles and crocodilians. This phylogenetic diversity is reflected in great morphological and behavioral diversity capturing the attention of biologists interested in mechanisms regulating developmental processes such as somitogenesis and spinal patterning, regeneration, the evolution of “snake-like” morphology, the formation of the unique turtle shell, and the convergent evolution of the four-chambered heart shared by mammals and archosaurs. The complete genome of the first non-avian reptile, the green anole lizard, was published in 2011 and has provided insights into the origin and evolution of amniotes. Since then, the genomes of multiple snakes, turtles, and crocodilians have also been completed. Here we will review the current diversity of available reptile genomes, with an emphasis on their evolutionary relationships, and will highlight how these genomes have and will continue to facilitate research in developmental and regenerative biology.
Keywords: reptile, genomics, gene expression, somitogenesis, regeneration
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 873-879(2014)
doi: 10.1387/ijdb.140193aw / © UBC Press ( www.a360grados.net )
In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster
Akihiko Watanabe 1, and Eriko Takayama-Watanabe 2
1. Department of Biology, Faculty of Science
2. Institute of Arts and Sciences, Yamagata University, Kojirakawa, Yamagata, Japan
Abstract: A specific sperm-egg interaction in the oviductal matrix is crucial for internal fertilization of the red-bellied newt, Cynops pyrrhogaster. An understanding of the molecular basis of this interaction is expected to elucidate the evolutionary history of internal fertilization in amphibians. Recently, deep sequencing technology has provided global gene information even in nonmodel animals, allowing us to understand specific features of the molecular mechanisms underlying fertilization in C. pyrrhogaster. In the present study, we screened de novo assembled RNAseq from ovary, testis, and oviduct samples in C. pyrrhogaster and identified the base sequences encoding zona pellucida (ZP) proteins, voltage-dependent Ca2+ channels, and cysteine-rich secretory proteins (CRISPs), which respectively are sperm receptors for egg envelopes, major mediators of sperm intracellular signaling, and expected extracellular modulators for sperm function in the female reproductive tract. In the ovary, ZP homologues of all six subgroups were found, including a ZP1 homologue that was newly found in amphibians, a ZP4 homologue, and six ZPC homologues. The unique combination of ZP proteins suggests a new mechanism for sperm binding to egg envelopes in the internal fertilization of C. pyrrhogaster. In the testis, CaV1.1, 1.2, and 3.2, which are L- and T-type voltage-dependent Ca2+ channels, were found as potential mediators for the internal fertilization-specific sperm-egg interaction. We also found CRISP 2 in the oviduct, which is speculated to participate in the sperm-egg interaction. These results indicate that the de novo assembled RNAseq is a powerful tool allowing analysis of the specific sperm-egg interactions in C. pyrrhogaster.
Keywords: zp proteins, ca2+ channel, crisp, rnaseq, internal fertilization, urodele
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 881-888(2014)
doi: 10.1387/ijdb.150060at / © UBC Press ( www.a360grados.net )
The genome sequence of the corn snake (Pantherophis guttatus), a valuable resource for EvoDevo studies in squamates
Asier Ullate-Agote 1,2,3, Michel C. Milinkovitch 1,2,3 and Athanasia C. Tzika 1,2,3
1. Laboratory of Artificial & Natural Evolution (LANE), Dept. of Genetics & Evolution, University of Geneva
2. SIB Swiss Institute of Bioinformatics
3. Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, Geneva, Switzerland
Abstract: Squamates (snakes and lizards) exhibit a striking variety of phenotypes, with little known on their generative mechanisms. Studies aiming to understand the genetic basis of this wide diversity in morphology, physiology and ecology will greatly benefit from whole genome sequencing initiatives, as they provide the foundation for comparative analyses and improve our understanding of the evolution, development and diversification of traits. Here, we present the first draft genome of the corn snake Pantherophis guttatus, an oviparous snake that we promote as a particularly appropriate model species for evolutionary developmental studies in squamates. We sequenced 100-base paired-end reads from multiple individuals of a single family (parents and offspring) that produced a genome assembly of 1.53 gigabases (Gb), roughly covering 75% of the expected total genome size, and 297,768 scaffolds >1 Kb. We were able to fully retrieve 86, and partially another 106, of the 248 CEGMA core genes, indicating that a high genome completeness was achieved, even though the assembly is fragmented. Using MAKER2, we annotated 10,917 genes with high confidence (Annotation Edit Distance (AED)<1) and an additional 5,263 predicted genes matched with the species’ transcriptome. Numerous colour and colour pattern morphs exist in P. guttatus, making it an ideal model to study the genetic determinism, development, and evolution of adaptive colour traits in reptiles. Using our draft genome and a Single-Nucleotide Polymorphism (SNP) calling approach, we confirmed the interval with the causative mutation for the amelanistic phenotype, a result supported by a parallel exome-based study.
Keywords: Pantherophis guttatus, corn snake, reptile, genome, amelanistic, SNP calling
Evolutionary aspects
EHU/UPV/UBC - The International Journal of Developmental Biology 58: 889-894(2014)
doi: 10.1387/ijdb.140239db / © UBC Press ( www.a360grados.net )
Amniote yolk sacs: diversity in reptiles and a hypothesis on their origin
Richard P. Elinson 1, James R. Stewart 2, Laurie J. Bonneau 3 and Daniel G. Blackburn 3
1. 240 West Neck Road, Huntington, NY
2. Department of Biological Sciences, East Tennessee State University, Johnson City, TN
3. Department of Biology, and Electron Microscopy Center, Trinity College, Hartford, CT, USA
Abstract: Oviparous amniotes produce a large yolky egg that gives rise to a free-living hatchling. Structural characteristics and functional attributes of the egg are best known for birds, which have a large mass of fluid yolk surrounded by an extraembryonic yolk sac. Yolk nutrients are delivered to the embryo via the vascular yolk sac. This developmental pattern and nutrient transport mechanism is thought to be representative of all other lineages of amniotes. Recent discovery of a snake with cellularized yolk organized around a meshwork of blood vessels reveals an additional pattern for yolk mobilization, which may also occur in other squamate reptiles (lizards and snakes). This complex yolk sac raises interesting questions about developmental mechanisms and suggests a possible model for the transition between the egg of anamniotes and that of amniotes.
Keywords: cleavage, yolk, embryo maintenance, yolk sac, oviparity
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 895-908(2014)
doi: 10.1387/ijdb.140255jg / © UBC Press ( www.a360grados.net )
Digit evolution in gymnophthalmid lizards
Juliana G. Roscito 1, Pedro M.S. Nunes 2 and Miguel T. Rodrigues 1
1. Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo-SP
2. Departamento de Zoologia, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Brazil
Abstract: The tetrapod limb is a highly diverse structure, and reduction or loss of this structure accounts for many of the limb phenotypes observed within species. Squamate reptiles are one of the many tetrapod lineages in which the limbs have been greatly modified from the pentadactyl generalized pattern, including different degrees of reduction in the number of limb elements to complete limblessness. Even though limb reduction is widespread, the evolutionary and developmental mechanisms involved in the formation of reduced limb morphologies remains unclear. In this study, we present an overview of limb morphology within the microteiid lizard group Gymnophthalmidae, focusing on digit arrangement. We show that there are two major groups of limb-reduced gymnophthalmids. The first group is formed by lizard-like (and frequently pentadactyl) species, in which minor reductions (such as the loss of 1-2 phalanges mainly in digits I and V) are the rule; these morphologies generally correspond to those seen in other squamates. The second group is formed by species showing more drastic losses, which can include the absence of an externally distinct limb in adults. We also present the expression patterns of Sonic Hedgehog (Shh) in the greatly reduced fore and hindlimb of a serpentiform gymnophthalmid. Our discussion focuses on identifying shared patterns of limb reduction among tetrapods, and explaining these patterns and the morphological variation within the gymnophthalmids based on current knowledge of the molecular signaling pathways that coordinate limb development.
Keywords: limb reduction, reptiles, morphological evolution, limb development
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 909-916(2014)
doi: 10.1387/ijdb.150 / © UBC Press ( www.a360grados.net )
Evolution of maternal egg size effects in sister salamander species
Tobias Landberg
Biology Department, Arcadia University, Glenside, PA, USA
Abstract: Egg size varies genetically and with the maternal environment. It is correlated with and can act as a resource fueling variation in many other key life history traits. This study examined hypotheses about how plastic responses of offspring to yolk variation evolve (and contribute to phenotypic evolution) when maternal investment in egg size evolves. I used a split-clutch, controlled, surgical experiment with a longitudinal (repeated-measures) design to examine the effects of yolk removal on sister salamander species with distinct egg and larval phenotypes. Yolk removal had large effects in the derived larger-egged species, A. barbouri, and greatly reduced effects in A. texanum. Early hatching and smaller larval body size was only found in A. barbouri and survival rates decreased more in A. barbouri. These results provide strong experimental evidence that as female salamanders evolve greater yolk investment in each egg, offspring coevolve an increased magnitude of phenotypic plasticity in response to yolk variation across a suite of life history traits. Yolk therefore acts as an integrator of phenotypes that allows females to modify modules of life history traits together (facilitating adaptation). When organisms invade new environments, complex integrated phenotypes may evolve via correlated responses to increased maternal investment, yet individual traits can be coupled or decoupled to yolk quantity variation in different species.
Keywords: ovum, propagule size fitness function, experimental embryology, yolkectomy
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 917-927(2014)
doi: 10.1387/ijdb.150053cr / © UBC Press ( www.a360grados.net )
The importance of cartilage to amphibian development and evolution
Christopher S. Rose
James Madison University, Harrisonburg, VA, USA
Abstract: The duality of amphibians is epitomized by their pharyngeal arch skeletons, the larval and adult morphologies of which enable very different feeding and breathing behaviors in aquatic and terrestrial life. To accomplish this duality, amphibian pharyngeal arch skeletons undergo two periods of patterning: embryogenesis and metamorphosis, and two periods of growth: larval and postmetamorphic. Their extreme ontogenetic variation, however, is coupled with relatively limited phylogenetic variation. I propose that amphibians face an evolutionary tradeoff between their ontogenetic and phylogenetic diversification that stems from the need to grow and transform the pharyngeal arch skeleton in cartilage rather than bone. Cartilage differs fundamentally from bone in its histology, function, development and growth. Cartilage is also the first skeletal tissue to form embryonically and provides more cellular pathways for shape change than bone. This article combines morphological, histological and experimental perspectives to explore how pharyngeal arch cartilage shape is controlled in amphibian embryogenesis, growth and metamorphosis, and how amphibian skeletal ontogenies are impacted by using cartilage to evolve a complex life cycle and in evolving away from a complex life cycle.
Keywords: Amphibian, cartilage, metamorphosis, evo-devo, pharyngeal arch skeleton
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 929-934(2014)
doi: 10.1387/ijdb.140155rp / © UBC Press ( www.a360grados.net )
Evolutionary trend for metamery reduction and gonad shortening in Anurans revealed by comparison of gonad development
Rafal P. Piprek 1, Anna Pecio 1, Malgorzata Kloc 2,3, Jacek Z. Kubiak 4,5 and Jacek M. Szymura 1
1. Department of Comparative Anatomy, Institute of Zoology, Jagiellonian University, Krakow, Poland
2. Department of Surgery, The Houston Methodist Hospital, Houston, USA
3. The Houston Methodist Research Institute, Houston, USA
4. CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Cell Cycle Group, F-35043, France
5. Université Rennes 1, UEB, UMS Biosit, Faculty of Medicine, F-35043 Rennes, France
Abstract: The gonads develop as the metameric mesodermal structures at the ventral surface of the mesonephroi. To study the evolutionary trends for anuran gonads, we performed comparative analysis of three species from the basal paraphyletic group – Archaeobatrachia (Bombina bombina, Xenopus laevis, Pelobates fuscus) and five species from more derived monophyletic Neobatrachia group (Hyla arborea, Bufotes viridis, Rana dalmatina, Rana arvalis, Rana temporaria). Light and scanning electron microscopy analysis showed that in Archaeobtrachia the undifferentiated gonads and ovaries, but not the testes, have pronounced external metamery. In contrast, Neobatrachia lacked external gonadal metamery and the internal metamery was well pronounced in the ovaries, but only rudimentary in the testes. There was also a difference in the site of genital ridge formation between these two groups. In Archaeobatrachia, the genital ridges developed along almost the entire length of mesonephroi, while in Neobatrachia they were limited to the anterior parts of the mesonephroi. A transient form was observed in P. fuscus, where the gonads formed along the anterior half of the mesonephroi. There was also an evolutionary trend for gradual postero-anterior shortening of the gonadal anlagen. In summary, our study indicates that among anurans there is an evolutionary trend for the gradual loss of metamery and shortening of the gonad.
Keywords: Anura, testis, ovary, mesonephros, metamery
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 935-948(2014)
doi: 10.1387/ijdb.150087db / © UBC Press ( www.a360grados.net )
Evolution of viviparous reproduction in Paleozoic and Mesozoic reptiles
Daniel G. Blackburn 1 and Christian A. Sidor 2
1. Department of Biology and Electron Microscopy Center, Trinity College, Hartford, CT
2. Department of Biology and Burke Museum, University of Washington, Seattle, WA, USA
Abstract: Although viviparity (live-bearing reproduction) is widely distributed among lizards and snakes, it is entirely absent from other extant Reptilia and many extinct forms. However, paleontological evidence reveals that viviparity was present in at least nine nominal groups of pre-Cenozoic reptiles, representing a minimum of six separate evolutionary origins of this reproductive mode. Two viviparous clades (sauropterygians and ichthyopterygians) lasted more than 155 million years, a figure that rivals the duration of mammalian viviparity. Circumstantial evidence indicates that extinct viviparous reptiles had internal fertilization, amniotic fetal membranes, and placentas that sustained developing embryos via provision of respiratory gases, water, calcium, and possibly organic nutrients. Production of offspring via viviparity facilitated the invasion of marine habitats in at least five reptilian lineages. Thus, this pattern of embryonic development and reproduction was central to the ecology and evolution of these ancient animals, much as it is to numerous extant species of vertebrates.
Keywords: viviparity, reproductive patterns, paleontology, placentas
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EHU/UPV/UBC - The International Journal of Developmental Biology 58: 949-960(2014)
doi: 10.1387/ijdb.140322mt / © UBC Press ( www.a360grados.net )
Integrating developmental biology and the fossil record of reptiles
Tomasz Skawinski 1 and Mateusz Talanda 2
1. Department of Evolutionary Biology and Conservation of Vertebrates, Faculty of Biological Sciences, University of Wroclaw, Wroclaw, Poland
2. Department of Palaeobiology and Evolution, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland
Abstract: Numerous new discoveries and new research techniques have influenced our understanding of reptile development from a palaeontological perspective. They suggest for example that transition from mineralized to leathery eggshells and from oviparity to viviparity appeared much more often in the evolution of reptiles than was previously thought. Most marine reptiles evolved from viviparous terrestrial ancestors and had probably genetic sex determination. Fossil forms often display developmental traits absent or rare among modern ones such as polydactyly, hyperphalangy, the presence of ribcage armour, reduction of head ornamentation during ontogeny, extreme modifications of vertebral count or a wide range of feather-like structures. Thus, they provide an empirical background for many morphogenetic considerations.
Keywords: evo-devo, palaeontology, embryology, development, ontogeny
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