The International Journal of Developmental Biology
Linking Development, Stem Cells and Cancer Research

Volume 60 - Numbers 4-5-6 (2016) / Pages 77-188          Editor-in-Chief: Juan Aréchaga

MORE INFORMATION                              [Abstract - FullText / Full Text Open Access]

ISSN: 0214-6282 / ISSN-e: 1696-3547                              www.intjdevbiol.com

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CONTENTS

Essay         --------------------------------------------------------------

EHU/UPV/UBC - The International Journal of Developmental Biology 60: 60-84 (2016)
doi: 10.1387/ijdb.160097ag   /   © UBC Press           ( www.a360grados.net )

Development vs. behavior: a role for neural adaptation in evolution?
Alain Ghysen and Christine Dambly-Chaudière
Inserm, U1198, Montpellier, France; Université de Montpellier, Montpellier, France and EPHE, Paris, France

Abstract:  We examine the evolution of sensory organ patterning in the lateral line system of fish. Based on recent studies of how this system develops in zebrafish, and on comparative analyses between zebrafish and tuna, we argue that the evolution of lateral line patterns is mostly determined by variations in the underlying developmental processes, independent of any selective pressure. Yet the development of major developmental innovations is so directly linked to their exploitation that it is hard not to think of them as selected for, i.e., adaptive. We propose that adaptation resides mostly in how the nervous system adjusts to new morphologies to make them functional, i.e., that species are neurally adapted to whatever morphology is provided to them by their own developmental program. We show that recent data on behavioral differences between cave forms (blind) and surface forms (eyed) of the mexican fish Astyanax fasciatus support this view, and we propose that this species might provide a unique opportunity to assess the nature of adaptation and of selection in animal evolution.

Keywords:  lateral line, plasticity, sensory system, Astyanax, Danio, Thunnus

Original Articles       ---------------------------------------------------------------------

EHU/UPV/UBC - The International Journal of Developmental Biology 60: 85-93 (2016)
doi: 10.1387/ijdb.160083yy   /   © UBC Press           ( www.a360grados.net )

Hypoxia promotes thyroid differentiation of native murine induced pluripotent stem cells
Yipeng Yang 1, Yunshu Lu 1, Tong Chen 1, Shenglai Zhang 1, Bingfeng Chu 1, Yurong Gong 1, Weixin Zhao 2, Jian Zhu 1 and Yingbin Liu 1
1. General Surgery Department, Xin Hua Hospital of Shanghai Jiao Tong University School of Medicine, Shanghai, China
2. Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA

Abstract: Hypothyroidism is a very common hormonal deficiency and the stem cell technology which developed in the recent years may offer a therapeutic strategy for treating this disorder. Hypoxia has been demonstrated to play an important role in embryonic formation and development and to modulate stem cell differentiation. However, the influence of oxygen tension on thyroid differentiation has not been studied. In this study, we used murine induced pluripotent stem (iPS) cells for thyroid cell differentiation under normoxic and hypoxic conditions and compared differentiation efficiency in morphology, function, gene and protein expression under both conditions. We found that hypoxia promoted adhesion and outgrowth of embryoid bodies (EBs) derived from murine iPS cells. Expression of endodermal markers (Foxa2 and Gata4) and thyroid transcription factors (Pax8 and Nkx2.1) was increased by hypoxia at both gene and protein levels during early-mid differentiation stages (p<0.05). And so were the thyroid specific markers NIS and TSHR at the end of the experiment (p<0.05). In addition, functional iodide uptake by differentiated cells was also increased after hypoxia. Thyroid differentiation from iPS cells is enhanced under hypoxia and this may involve hypoxia inducible factors (HIFs) and their downstream gene FGF2. Our data offer a foundation for understanding thyroid development and provide a potentially more efficient way to use cell therapy for treating thyroid deficiency.

Keywords:  differentiation, thyroid, hypoxia, promotion, iPS cell

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 95-102 (2016)
doi: 10.1387/ijdb.160010hn   /   © UBC Press           ( www.a360grados.net )

The effect of amniotic membrane stem cells as donor nucleus on gene expression in reconstructed bovine oocytes
Hassan Nazari 1,3, Abolfazl Shirazi 2,3,4, Naser Shams-Esfandabadi 5, Azita Afzali6 and Ebrahim Ahmadi 3
1. Faculty of Veterinary Medicine, Shahrekord University, Shahrekord-Iran
2. Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran-Iran
3. Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord-Iran
4. Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord-Iran
5. Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord-Iran
6. Faculty of Basic Sciences, Shahrekord University, Shahrekord-Iran

Abstract: Nuclear reprogramming of a differentiated cell in somatic cell nuclear transfer (SCNT) is a major concern in cloning procedures. Indeed, the nucleus of the donor cell often fails to express the genes which are a prerequisite for normal early embryo development. This study was aimed to evaluate the developmental competence and the expression pattern of some reprogramming related genes in bovine cloned embryos reconstructed with amniotic membrane stem cells (AMSCs) in comparison with those reconstructed with mesenchymal stem cells (MSCs) and adult fibroblasts (AF) as well as with in vitro fertilized (IVF) oocytes. In vitro matured abattoir-derived oocytes were considered as recipients and a hand-made cloning technique was employed for oocyte enucleation and nuclear transfer (NT) procedures. The expression pattern of genes involved in self-renewal and pluripotency (POU5F1, SOX2, NANOG), imprinting (IGF2, IGF2R), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), and apoptosis (BAX, BCL2) were evaluated in NT and IVF derived embryos. Despite the insignificant difference in cleavage rate between reconstructed and IVF oocytes, the blastocyst rate in the IVF group was higher than that of other groups. Among reconstructed oocytes, a higher blastocysts rate was observed in MSC-NT and AMSCs-NT derived embryos that were significantly higher than AF-NT derived ones. There were more similarities in the expression pattern of pluripotency and epigenetic modification genes between MSC-NT and IVF derived blastocysts compared with other groups. In conclusion, considering developmental competence, AMSCs, as alternative donors in SCNT procedure, like MSCs, were prone to have more advantage compared with AF.

Keywords:  nuclear transfer, reprogramming, bovine, imprinting, embryo

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 103-110 (2016)
doi: 10.1387/ijdb.160014id   /   © UBC Press           ( www.a360grados.net )

Comparative epigenetic evaluation of human embryonic stem and induced pluripotent cells
Raha Favaedi 1, Maryam Shahhoseini 1, Mahammad Pakzad 2, Sepideh Mollamohammadi 2 and Hossein Baharvand 2
1. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
2. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Abstract: Histone H3 lysine 9 methylation has been shown to be a critical barrier to efficient cell reprogramming. This discovery allows the assessment of the cell pluripotency state by considering the extent of H3K9 methylation vs. acetylation at the same position. A set of pluripotent and differentiated human cells including embryonic stem cells, their differentiated and reprogrammed counterparts, along with human fibroblasts and their derived reprogrammed cells, were used to evaluate the ratio of total H3K9 methylation over acetylation using a quantitative ELISA-based approach. Also, the occurrence of the H3K4me3 and H3K27me3 bivalent marks was evaluated. Additionally, using ChIP-qPCR the occurrence of these histone marks on the regulatory regions of stemness genes (Nanog, Oct4 and Sox2) as well as on genes indicating fibroblast differentiation (Vim, COL1A1 and THY1) was evaluated. We evidence remarkably high ratios of H3K9ac/K9me2 in ES and iPS cells vs. differentiated cells. In iPSCs, a direct relationship between the ratios of total H3K9ac/H3K9me2 and the ratios of these marks on pluripotency gene regulatory regions and their expression was observed. In differentiated cells, in contrast, the ratios of global H3K9ac/K9me2 is low but the active genes escape this general situation and bear higher amounts of H3K9ac vs. H3K9me. Total H3K4me3/K27me3 ratios presented the same trends, but with reduced amplitudes. We propose that the rapid quantitative measurements of relative amounts of H3K9ac and K9me2 in iPS cells compared to the parental differentiated cells constitute a reliable and convenient criterion to rapidly assess the cell pluripotency potentials and the efficiency of cell reprogramming.

Keywords:  ESC, iPSC, epigenetic, histone modification

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 111-117 (2016)
doi: 10.1387/ijdb.130253sg   /   © UBC Press          ( www.a360grados.net )

UV induced foot duplication in regenerating hydra is mediated by metalloproteinases and modulation of the Wnt pathway
Lakshmi-Surekha Krishnapati 1, Rohini Londhe 1, Vaishali Deoli 1, Apurva Barve 1, Saroj Ghaskadbi 2 and Surendra Ghaskadbi 1
1. Developmental Biology Group, MACS-Agharkar Research Institute
2. Department of Zoology, Savitribai Phule Pune University, Pune, India

Abstract: We have shown earlier that irradiation with UV induces duplication of foot in regenerating middle pieces of hydra. The present study was undertaken to elucidate the underlying mechanism(s) leading to this curious phenomenon. UV irradiation induced duplicated foot in about 30% of regenerating middle pieces. Metalloproteinases are important in foot formation, while Wnt pathway genes are important in head formation in hydra. The effect of UV irradiation on expression of these genes was studied by in situ hybridization and q-PCR. In whole polyps and middle pieces, UV irradiation led to up-regulation of HMP2 and HMMP, the two metalloproteinases involved in foot formation in hydra. HMP2 expression was significantly increased starting from 30 min post exposure to UV at 254 nm (500 J/m2), while HMMP showed significant up-regulation 6 h post UV exposure onwards. In middle pieces, increased expression of both metalloproteinases was observed only at 48 h. In whole polyps as well as in middle pieces, expression of Wnt3 and β-catenin was detected within 30 min of UV exposure and was accompanied by up-regulation of GSK3β, DKK3 and DKK1/2/4, inhibitors of the Wnt pathway. These conditions likely lead to inactivation of Wnt signaling. We therefore conclude that duplication of foot due to UV irradiation in regenerating middle pieces of hydra is a combined effect of up-regulation of metalloproteinases and inactivation of the Wnt pathway. Our results suggest that UV irradiation can be employed as a tool to understand patterning mechanisms during foot formation in hydra.

Keywords:  Hydra, metalloproteinase, pattern formation, UV-induced foot duplication, Wnt signaling

EHU/UPV/UBC - The International Journal of Developmental Biology 60: 119-126 (2016)
doi: 10.1387/ijdb.160136se   /   © UBC Press          ( www.a360grados.net )

Etiology of intracerebral hemorrhage (ICH): novel insights from Zebrafish embryos
Shahram Eisa-Beygi and Mohammad Rezaei
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

Abstract: Intracerebral hemorrhage (ICH) is the most severe subtype of stroke. Treatment options are scarce and given the high morbidity and mortality, relatively ineffective. Since patients with ICH may have an unknown heritable component, the need to identify potential risk factors necessitates the use of animal models to elucidate the genetic underpinnings of neurovascular development and, thereby, identify candidate regulatory pathways that are likely to be disrupted in patients with ICH. Zebrafish (Danio rerio) exhibits the anatomical and physiological complexity of a closed circulatory system observed in all vertebrates (with arteries, veins and capillaries). Moreover, studies over the last decade, aided by the application of chemical mutagenesis screens, morpholino mediated knockdown approaches and tissue-specific transgenic markers, have paved the way for the identification of several genes and signaling pathways that regulate developmental neurovascular stabilization. We hypothesize that mutations in these genes or pharmacological perturbations of these gene-products may account, at least in part, for the etiology of some forms of spontaneous ICH in humans.

Keywords:  intracerebral hemorrhage, neurovascular development, stroke etiology, zebrafish

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 127-139 (2016)
doi: 10.1387/ijdb.150400tc   /   © UBC Press           ( www.a360grados.net )

Alterations to retinal architecture prior to photoreceptor loss in a mouse model of retinitis pigmentosa
Sarah l. Roche, Alice C. Wyse-Jackson, Ashleigh M. Byrne, Ana M. Ruiz-Lopez and Thomas G. Cotter
Cell Development and Disease Laboratory, Biochemistry Department, Biosciences Institute, University College Cork, Cork, Ireland

Abstract: Mouse models of retinitis pigmentosa (RP) are essential tools in the pursuit to understand fully what cell types and processes underlie the degeneration observed in RP. Knowledge of these processes is required if we are to develop successful therapies to treat this currently incurable disease. We have used the rd10 mouse model of RP to study retinal morphology prior to photoreceptor loss, using immunohistochemistry and confocal microscopy on cryosections, since little is known about how the mutation affects the retina during this period. We report novel findings that the mutation in the rd10 mouse results in retinal abnormalities earlier than was previously thought. Defects in rod and cone outer segments, bipolar cells, amacrine cells and photoreceptor synapses were apparent in the retina during early stages of postnatal retinal development and prior to the loss of photoreceptors. Additionally, we observed a dramatic response of glial cells during this period. Microglia responded as early as postnatal day (P) 5; ?13 days before any photoreceptor loss is detected with Müller glia and astrocytes exhibiting changes from P10 and P15 respectively. Overall, these findings present pathological aspects to the postnatal development of the rd10 retina, contributing significantly to our understanding of disease onset and progression in the rd10 mouse and provide a valuable resource for the study of retinal dystrophies.

Keywords:  Pde6b, rd10, retinitis pigmentosa, postnatal retinal development, microglia

Developmental Expression Patterns       ----------------------------------------

EHU/UPV/UBC - The International Journal of Developmental Biology 60: 141-150 (2016)
doi: 10.1387/ijdb.160108jb   /   © UBC Press           ( www.a360grados.net )

Genome-wide identification of enhancer elements
Sarah Tulin 1, Julius C. Barsi 2, Carlo Bocconcelli 1 and Joel Smith 1
1. Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA
2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

Abstract: We present a prospective genome-wide regulatory element database for the sea urchin embryo and the modified chromosome capture-related methodology used to create it. The method we developed is termed GRIP-seq for genome-wide regulatory element immunoprecipitation and combines features of chromosome conformation capture, chromatin immunoprecipitation, and paired-end next-generation sequencing with molecular steps that enrich for active cis-regulatory elements associated with basal transcriptional machinery. The first GRIP-seq database, available to the community, comes from S. purpuratus 24 hpf embryos and takes advantage of the extremely well-characterized cis-regulatory elements in this system for validation. In addition, using the GRIP-seq database, we identify and experimentally validate a novel, intronic cis-regulatory element at the onecut locus. We find GRIP-seq signal sensitively identifies active cis-regulatory elements with a high signal-to-noise ratio for both distal and intronic elements. This promising GRIP-seq protocol has the potential to address a rate-limiting step in resolving comprehensive, predictive network models in all systems.

Keywords:  GRIP-seq, chromatin conformation capture, anti-Pol-II, sea urchin development

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 151-157 (2016)
doi: 10.1387/ijdb.150402am   /   © UBC Press           ( www.a360grados.net )

Noggin 1 overexpression in retinal progenitors affects bipolar cell generation
Andrea Messina 1, Simone Bridi 1,2, Angela Bozza 1,3, Yuri Bozzi 3,4, Marie-Laure Baudet 2 and Simona Casarosa 1,4
1. Developmental Neurobiology Laboratory, CIBIO, University of Trento, Italy
2. The Armenise-Harvard Laboratory of Axonal Biology, CIBIO, University of Trento, Italy
3. Molecular Neuropathology Laboratory, CIBIO, University of Trento
4. CNR Neuroscience Institute, Pisa, Italy

Abstract: Waves of Bone Morphogenetic Proteins (BMPs) and their antagonists are present during initial eye development, but their possible roles in retinogenesis are still unknown. We have recently shown that noggin 1, a BMP antagonist, renders pluripotent cells able to differentiate into retinal precursors, and might be involved in the maintenance of retinal structures in the adult vertebrate eye. Here, we report that noggin 1, differently from noggin 2 and noggin 4, is expressed during all phases of Xenopus laevis retinal development. Gain-of-function experiments by electroporation in the optic vesicle show that overexpression of noggin 1 significantly decreases the number of bipolar cells in the inner nuclear layer of the retina, without significantly affecting the generation of the other retinal cell types. Our data suggest that BMP signaling could be involved in the differentiation of retinal progenitors into specific retinal subtypes during late phases of vertebrate retinal development.

Keywords:  noggin 1, retinal differentiation, BMP inhibition

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 159-166 (2016)
doi: 10.1387/ijdb.160014id   /   © UBC Press           ( www.a360grados.net )

Genes regulated by potassium channel tetramerization domain containing 15 (Kctd15) in the developing neural crest
Thomas C.B. Wong 1, Martha Rebbert 2, Chengdong Wang 1, Xiongfong Chen 3, Alison Heffer 2, Valeria E. Zarelli 2, Igor B. Dawid 2 and Hui Zhao 1,4
1. School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, P. R. China
2. Section of Developmental Biology, DDB, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
3. Advanced Biomedical Computing Center, National Cancer Institute, National Institutes of Health, Frederick, MD, USA
4. Kunming Institute of Zoology Chinese Academy of Sciences-The Chinese University of Hong Kong Joint Laboratory of Bioresources and Molecular Research of Common Diseases

Abstract: Neural crest (NC) development is controlled precisely by a regulatory network with multiple signaling pathways and the involvement of many genes. The integration and coordination of these factors are still incompletely understood. Overexpression of Wnt3a and the BMP antagonist Chordin in animal cap cells from Xenopus blastulae induces a large number of NC specific genes. We previously suggested that Potassium Channel Tetramerization Domain containing 15 (Kctd15) regulates NC formation by affecting Wnt signaling and the activity of transcription factor AP-2. In order to advance understanding of the function of Kctd15 during NC development, we performed DNA microarray assays in explants injected with Wnt3a and Chordin, and identified genes that are affected by Kctd15 overexpression. Among the many genes identified, we chose Duf domain containing protein 1 (ddcp1), Platelet-Derived Growth Factor Receptor a (pdgfra), Complement factor properdin (cfp), Zinc Finger SWIM-Type Containing 5 (zswim5), and complement component 3 (C3) to examine their expression by whole mount in situ hybridization. Our work points to a possible role for Kctd15 in the regulation of NC formation and other steps in embryonic development.

Keywords:  Neural crest, Kctd15, DNA microarray, Xenopus, gene regulation, transcription factor AP-2

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 167-173 (2016)
doi: 10.1387/ijdb.150334sv   /   © UBC Press           ( www.a360grados.net )

In silico analysis of histone H3 gene expression during human brain development
Megan Ren and Steve Van Nocker
Program in Cell and Molecular Biology, Michigan State University, MI, USA

Abstract:  Precise regulation of chromatin structure is essential for proper development of higher eukaryotes, and methylation of histone H3 at lysine-27 (H3K27) by the Polycomb Repressive Complex 2 (PRC2) component EZH2 has emerged as an important and conserved mechanism to ensure silencing of developmentally regulated genes. Recurrent mutations within the histone H3 genes H3F3A and HIST1H3B that convert K27 to methionine (H3K27M) and disrupt the global H3K27 methylation landscape and PRC2-dependent silencing, have recently been identified in pediatric high-grade gliomas including Diffuse Intrinsic Pontine Glioma (DIPG) and Glioblastoma multiforme (GBM; Type IV glioma). These findings have generated renewed interest in the dynamics of histone genes and their expression, which have been difficult to study due to redundancy and high sequence homology within the H3 gene family. In this in silico study, we re-evaluated genomic organization of the human H3 gene family and expression of these genes in the human brain, utilizing public RNA-based sequence datasets for the human genome and brain development. We identified transcriptional activity from at least 17 protein-encoding H3 genes in the developing brain, comprising at least 14 canonical (H3.1)-like and 3 ‘replication-independent’ (H3.3)-like forms, and encoding six distinct H3 isoforms. Transcripts for H3.3 genes including H3F3A show gradual decrease in abundance associated with developmental progression, whereas H3.1 transcripts including HIST1H3B tend to be strongly downregulated at an early prenatal stage and remain essentially silent thereafter. Twelve genes, including members of both H3.1 and H3.3 classes, contain a K27-AAG codon that is mutable to that for M (ATG), whereas the remaining contain the alternative, AAA codon for K at this position. H3F3A is the only H3.3-like gene containing the K27-AAG codon, whereas HIST1H3B is among ten H3.1-like genes containing this codon. This data indicates that, in the early developing human brain, HIST1H3B constitutes the largest proportion of H3.1 transcripts among H3.1 isoforms. We suggest that the apparent overrepresentation of K27M mutations in H3F3A relative to other H3 isoforms may result from its uniqueness among H3.3s for the K27-AAG codon and the functional relationship between H3.3 and PRC2, whereas overrepresentation of K27M mutations in HIST1H3B may be a product of strong relative expression of this gene in the early developing brain.

Keywords:  histone H3, H3 variant, brain development, glioma, DIPG

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 175-179 (2016)
doi: 10.1387/ijdb.160045mo   /   © UBC Press           ( www.a360grados.net )

Platelet derived growth factor B gene expression in the Xenopus laevis developing central nervous system
Kety Giannetti 1, Debora Corsinovi 1, Cristina Rossino 1, Irene Appolloni 2,3, Paolo Malatesta 2,3 and Michela Ori 1
1. Dipartimento di Biologia, Università di Pisa
2. IRCCS-AOU San Martino-IST, Genoa
3. Department of Experimental Medicine (DiMES), University of Genoa, Italy

Abstract:  Platelet-derived growth factor B (PDGF-B) belongs to the mitogen and growth factor family and like the other members it has many roles in cell differentiation, proliferation and migration during development, adult life and in pathological conditions. Among them it has been observed that aberrant PDGF signalling is frequently linked to glioma development and progression, and Pdgf-b over-expression in mouse neural progenitors leads to the formation of gliomas. Despite this evidence, the mechanisms underlying PDGF-B driven tumorigenesis and its role during brain development are not fully understood. In order to contribute to clarifying possible new roles of pdgf-b signalling, we present here the embryonic gene expression pattern of pdgf-b, so far unknown in early vertebrate development. By using Xenopus laevis as a model system we performed qRT-PCR and whole mount in situ hybridization. Pdgf-b mRNA is expressed in discrete regions of the developing central nervous system, in the cranial nerve placodes and in the notochord. We also compared the gene expression of pdgf-b with that of its receptor pdgfr-α suggesting so far unsuspected roles for this signalling pathway during the development of specific embryonic structures.

Keywords:  PDGF-B, PDGF receptor α, central nervous system, neural crest, Xenopus laevis

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 181-188 (2016)
doi: 10.1387/ijdb.150317nr   /   © UBC Press           ( www.a360grados.net )

Nucleoporin gene expression in Xenopus tropicalis embryonic development
Nooreen Reza, Mustafa K. Khokha and Florencia Del Viso
Program in Vertebrate Developmental Biology. Department of Pediatrics and Genetics, Yale University School of Medicine, New Haven, CT, USA

Abstract:  Nucleoporins (nups) compose the structure of the nuclear pore complex (NPC) of all cells, but several studies have illuminated nucleoporins’ additional roles in development and the cell cycle. However, a comprehensive study of nup expression in embryonic development has not yet been reported. We synthesized antisense probes for all nup genes and used whole-mount in situ hybridization techniques to determine the expression pattern of all members of the nup family of genes at three different developmental stages in Xenopus tropicalis. We found that the expression of nups was not ubiquitous in embryos, but was localized to specific and distinguishable anatomical structures at all three stages tested. We also found that the expression patterns for nups within the same subcomplexes were not necessarily identical. Thus, nup expression is subject to a significant level of regulation during development. These results provide new information for functional studies of nups to unravel their roles in embryonic development.

Keywords:  nups, NPC, nuclear pore complex, in situ hybridization