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

Volume 60 - Numbers 1-2-3 (2016) / Pages 1-76                    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 

EHU/UPV/UBC - The International Journal of Developmental Biology 60: 1-3 (2016)
doi: 10.1387/ijdb.160014id   /   © UBC Press           ( www.a360grados.net )

In Memoriam - Prof. G. Barry Pierce (1925-2015)
Ivan Damjanov
Kansas City, Kansas, USA

Abstract:  Gordon Barry Pierce, my great mentor and long-time friend died in November 2015 at the age of 90 years. We will all miss him. What we are left with, however, are reminiscences of moments we spent with him, his jokes and stories to be retold and passed along, titbits of advice, and pearls of his common-sense Canadian wisdom. A vision of a better world to which he contributed so much. Scientific contributions too numerous to list, many of which had major impact on us who were interested in the same problems as he was. Seminal discoveries that impacted the progress in several fields of scientific endeavor. Major new concepts of oncology and developmental biology that opened new vistas and revolutionized our thinking about the crucial problems of biology and medicine. Unforgettable seminars and lectures. Unquenchable love for science. And much more that, nevertheless, can be summarized in two wondrous exclamations: What a man! What a life!

Keywords:  Gordon Barry Pierce, In Memoriam

Original articles

EHU/UPV/UBC - The International Journal of Developmental Biology 60: 5-12(2016)
doi: 10.1387/ijdb.150222cl   /   © UBC Press           ( www.a360grados.net )

Live imaging reveals spatial separation of parental chromatin until the four-cell stage in Caenorhabditis elegans embryos
Jitka Bolková and Christian Lanctôt
Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic

Abstract: The parental genomes are initially spatially separated in each pronucleus after fertilization. Here we have used green-to-red photoconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their spatial distribution in living Caenorhabditis elegans embryos starting shortly after fertilization. Intermingling of the parental chromatin did not occur until after the division of the AB and P1 blastomeres, at the 4-cell stage. Unexpectedly, we observed that the intermingling of chromatin did not take place during mitosis or during chromatin decondensation, but rather ∼ 3-5 minutes into the cell cycle. Furthermore, unlike what has been observed in mammalian cells, the relative spatial positioning of chromatin domains remained largely unchanged during prometaphase in the early C. elegans embryo. Live imaging of photoconverted chromatin also allowed us to detect a reproducible 180° rotation of the nuclei during cytokinesis of the one-cell embryo. Imaging of fluorescently-labeled P granules and polar bodies showed that the entire embryo rotates during the first cell division. To our knowledge, we report here the first live observation of the initial separation and subsequent mixing of parental chromatin domains during embryogenesis.

Keywords:  Caenorhabditis elegans, parental chromatin, live imaging, embryogenesis, photoconversion

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Coordinate involvement of Nodal-dependent inhibition and Wnt-dependent activation in the maintenance of organizer-specific bmp2b in zebrafish
Yu Xue¹, Cencan Xing^2,3, Wenjuan Zhang¹, Canbin Chen¹, Jingjin Xu¹, Anming Meng³ and Yutian Pan^1
1 The Engineering Technological Center of Mushroom Industry, Minnan Normal University, Zhangzhou, Fujian
² Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing
³ State Key Laboratory of Biomembrane and Membrane Engineering, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

Abstract:  A vertebrate signaling center, known in zebrafish as the organizer, is essential for axis patterning and formation and is regulated by multiple cell signaling pathways, including Wnt, Nodal, and Bmp. Organizer-specific Bmp2b plays important roles in the maintenance of the Bmp activity gradient and dorsal-ventral patterning. However, it is unknown how transcription of bmp2b in the organizer is regulated. In this study, we generated a bmp2b transgenic line Tsg(-2.272bmp2b:gfp) that reproduced organizer-specific bmp2b expression. Dissection analysis revealed that a 0.273-kb minimal promoter was indispensable for bmp2b expression in the dorsal organizer. Reporter assays showed that organizer-specific bmp2b is negatively regulated by the Nodal signal and positively regulated by the Wnt signal in both embryos and cell lines. Promoter analysis and chromatin-immunoprecipitation (ChIP) indicated that one consensus Smad-binding element (SBE) (CAGAC) and one Lef/Tcf-binding element (LBE) (AGATAA) were present in the 0.273-kb promoter, and could be directly bound by Smad2 and β-catenin proteins. Together, these results suggest that maintenance of organizer-specific bmp2b expression involves opposite and concerted regulation by Nodal and Wnt signaling.

Keywords:  zebrafish, organizer-specific bmp2b, transcriptional regulation, Nodal, Wnt

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 21-28(2016)
doi: 10.1387/ijdb.160040mk   /   © UBC Press           ( www.a360grados.net )

Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells
Sumiyo Mimura^1,2, Mika Suga¹, Kaori Okada¹, Masaki Kinehara^1,3, Hiroki Nikawa² and Miho K. Furue^1
1 Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan
² Department of Oral Biology & Engineering Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
³ Department of Cellular and Molecular Biology, Basic Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan

Abstract:  Neural crest (NC) cells are a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. Cranial NC cells migrate to the branchial arches and give rise to the majority of the craniofacial region, whereas trunk and tail NC cells contribute to the heart, enteric ganglia of the gut, melanocytes, sympathetic ganglia, and adrenal chromaffin cells. Positional information is indispensable for the regulation of cranial or trunk and tail NC cells. However, the mechanisms underlying the regulation of positional information during human NC induction have yet to be fully elucidated. In the present study, supplementation of bone morphogenetic protein (BMP) 4 in defined serum-free culture conditions including fibroblast growth factor-2 and Wnt3a from day 8 after NC specification induced the expression of cranial NC markers, AP2alpha, MSX1, and DLX1, during NC cell differentiation from human pluripotent stem cells. On the other hand, the proportion of cells expressing p75^NTR or HNK1 decreased compared with that of cells cultured without BMP4, whereas gene expression analysis demonstrated that the expression levels of cranial NC-associated genes increased in BMP4-treated NC cells. These BMP4-treated NC cells were capable of differentiation into osteocytes and chondrocytes. The results of the present study indicate that BMP4 regulates cranial positioning during NC development.

Keywords:  cranial neural crest, BMP signaling, homeobox gene, human embryonic stem cell, defined culture condition

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 29-38(2016)
doi: 10.1387/ijdb.150132rh   /   © UBC Press           ( www.a360grados.net )

SDF-1 controls the muscle and blood vessel formation of the somite
Aisha Abduelmula^1, Ruijin Huang^2,3, Qin Pu¹, Hirokazu Tamamura⁴, Gabriela Morosan-Puopolo¹ and Beate Brand-Saberi^1
1 Institute of Anatomy, Department of Anatomy and Molecular Embryology, Ruhr-University Bochum, Bochum, Germany
² Institute of Anatomy, Department of Neuroanatomy, Medical Faculty Bonn, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany
³ Department of Molecular Embryology, Institute of Anatomy and Cell Biology, Albert-Ludwig-University of Freiburg, Freiburg, Germany
⁴ Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Department of Medicinal Chemistry, Chiyoda-ku, Tokyo, Japan

Abstract:  Stromal-cell-derived factor-1 (SDF-1), the only ligand of the chemokine receptor CXCR4, is involved in skeletal muscle development. However, its role in the proliferation, differentiation and migration of somite cells is not well understood. Here, we investigated its function during somite development in chicken embryos by using gain-of-function and loss-of-function experiments. Overexpression of SDF-1 was performed by electroporating SDF-1 constructs into the ventrolateral part of the somite, or by injecting SDF-1-expressing cells into the somites of stages HH14-16 chicken embryos. We found that enhanced SDF-1 signaling induced cell proliferation in the somite. This resulted in an increase in number of both myotomal and endothelial cells. In contrast, inhibition of SDF-1/CXCR4 signaling led to a reduction of myotomal cells. Injection of SDF-1 producing cells into the somite induced ectopic localization of myotomal cells in the sclerotome. Although many SDF-1-expressing somite cells colonized the limb, only a few of them developed into muscle cells. This resulted in a reduction of the limb muscle mass. This means that most myogenic progenitors were stopped on their migration towards the limb due to the high concentration of the SDF-1 signal in the somite. Most of the SDF-1-expressing somite cells found in the limb were of endothelial cell fate and they contributed to the increase in limb blood vessels. These results reveal that SDF-1 promotes the proliferation of both myogenic and angiogenic progenitor cells of the somite and controls myotome formation. Furthermore, SDF-1 controls muscle and blood vessel formation in the limb in different ways.

Keywords:  chicken embryo, SDF-1, CXCR4, proliferation, myogenesis, angiogenesis

Developmental Expression Patterns

EHU/UPV/UBC - The International Journal of Developmental Biology 60: 39-51(2016)
doi: 10.1387/ijdb.150364nn   /   © UBC Press           ( www.a360grados.net )

Evolution of the vertebrate claudin gene family: insights from a basal vertebrate, the sea lamprey
Christian Mukendi¹, Nicholas Dean¹, Rushil Lala¹, Jeramiah Smith², Marianne E. Bronner³ and Natalya V. Nikitina^1
1 School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa
² Department of Biology, University of Kentucky, Lexington, KY, USA
³ Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

Abstract:  Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of this gene family. Expression analysis revealed that claudins are expressed in discrete and specific domains, many of which represent vertebrate-specific innovations, such as in cranial ectodermal placodes and the neural crest; whereas others represent structures characteristic of chordates, e.g. pronephros, notochord, somites, endostyle and pharyngeal arches. By comparing the embryonic expression of claudins in the lamprey to that of other vertebrates, we found that ancestral expression patterns were often preserved in higher vertebrates. Morpholino mediated loss of Cldn3b demonstrated a functional role for this protein in placode and pharyngeal arch morphogenesis. Taken together, our data provide novel insights into the origins and evolution of the claudin gene family and the significance of claudin proteins in the evolution of vertebrates.

Keywords:  claudin, lamprey, vertebrate primordia, embryo

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 53-56(2016)
doi: 10.1387/ijdb.150307rs   /   © UBC Press           ( www.a360grados.net )

Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos
Supriya Borah^1,2, Praveen Barrodia^1,2 and Rajeeb K. Swain^1
1 Institute of Life Sciences, Nalco Square, Chandrasekhar Pur, Bhubaneswar, Odisha, India
² Manipal University, Manipal, Karnataka, India

Abstract:  The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut.

Keywords:  diencephalon, telencephalon, trigeminal ganglia, spinal cord neuron, pronephros

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 57-63(2016)
doi: 10.1387/ijdb.150381ld   /   © UBC Press           ( www.a360grados.net )

pdzrn3 is required for pronephros morphogenesis in Xenopus laevis
Silvia Marracci, Alberto Vangelisti, Vittoria Raffa, Massimiliano Andreazzoli and Luciana Dente
Laboratory of Cell and Developmental Biology, Dept. of Biology, University of Pisa, Pisa, Italy

Abstract:  Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.

Keywords:  Pdzrn3, pronephros anlagen, tubulogenesis, Xenopus embryogenesis

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 65-69(2016)
doi: 10.1387/ijdb.150409th   /   © UBC Press           ( www.a360grados.net )

Expressional characterization of mRNA (guanine-7) methyltransferase (rnmt) during early development of Xenopus laevis
Ashwin Lokapally, Sanjeeva Metikala and Thomas Hollemann
Martin-Luther-University Halle-Wittenberg, Institute for Physiological Chemistry, Halle (Saale), Germany

Abstract:  Methylation of the guanosine cap structure at the 5’ end of mRNA is essential for efficient translation of all eukaryotic cellular mRNAs, gene expression and cell viability and promotes transcription, splicing, polyadenylation and nuclear export of mRNA. In the current study, we present the spatial expression pattern of the Xenopus laevis rnmt homologue. A high percentage of protein sequence similarity, especially within the methyltransferase domain, as well as an increased expression in the cells of the transcriptionally active stages, suggests a conserved RNA cap methylation function. Spatial expression analysis identified expression domains in the brain, the retina, the lens, the otic vesicles and the branchial arches.

Keywords:  rnmt, methylation, 5’ cap structure

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EHU/UPV/UBC - The International Journal of Developmental Biology 60: 71-76(2016)
doi: 10.1387/ijdb.150365jb   /   © UBC Press           ( www.a360grados.net )

Matrix Gla Protein expression pattern in the early avian embryo
Elizabeth Correia^1,2, Natércia Conceição^1,3, M. Leonor Cancela^1,3and José A. Belo^1,2,4
1 Department of Biomedical Sciences and Medicine, University of Algarve, Faro
² Center for Biomedical Research (CBMR), Universidade do Algarve, Campus de Gambelas, Faro
³ Centre of Marine Sciences, University of Algarve
⁴ CEDOC, NOVA Medical School / Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal

Abstract:  MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell–matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

Keywords:  MGP, angiogenesis, heart development, brain development, yolk sac