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1.
Development ; 145(9)2018 05 08.
Article in English | MEDLINE | ID: mdl-29650589

ABSTRACT

Zebrafish mutants with increased retinoic acid (RA) signaling due to the loss of the RA-inactivating enzyme Cyp26b1 develop a hyper-mineralized spine with gradually fusing vertebral body precursors (centra). However, the underlying cellular mechanisms remain incompletely understood. Here, we show that cells of the notochord epithelium named chordoblasts are sensitive to RA signaling. Chordoblasts are uniformly distributed along the anteroposterior axis and initially generate the continuous collagenous notochord sheath. However, subsequently and iteratively, subsets of these cells undergo further RA-dependent differentiation steps, acquire a stellate-like shape, downregulate expression of the collagen gene col2a1a, switch on cyp26b1 expression and trigger metameric sheath mineralization. This mineralization fails to appear upon chordoblast-specific cell ablation or RA signal transduction blockade. Together, our data reveal that, despite their different developmental origins, the activities and regulation of chordoblasts are very similar to those of osteoblasts, including their RA-induced transition from osteoid-producing cells to osteoid-mineralizing ones. Furthermore, our data point to a requirement for locally controlled RA activity within the chordoblast layer in order to generate the segmented vertebral column.


Subject(s)
Calcification, Physiologic/physiology , Gene Expression Regulation, Developmental/physiology , Notochord/embryology , Spine/embryology , Tretinoin/metabolism , Zebrafish/embryology , Animals , Collagen/biosynthesis , Collagen/genetics , Notochord/cytology , Retinoic Acid 4-Hydroxylase/genetics , Retinoic Acid 4-Hydroxylase/metabolism , Spine/cytology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
2.
Elife ; 72018 02 06.
Article in English | MEDLINE | ID: mdl-29405914

ABSTRACT

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments.


Subject(s)
Nerve Regeneration , Neuroglia/chemistry , Neuroglia/physiology , Notochord/injuries , WT1 Proteins/analysis , Wound Healing , Animals , Cell Movement , Zebrafish
3.
Gene Expr Patterns ; 25-26: 159-166, 2017 11.
Article in English | MEDLINE | ID: mdl-28826993

ABSTRACT

It has been well established that many types of rapidly dividing normal and diseased cells require an increased amount of folate for DNA replication and repair as well as cellular metabolism. Thus one of folate's cognate receptors, Folate Receptor 1 (FOLR1) is usually up-regulated in rapidly dividing cells, including many types of cancerous tumors. Because zebrafish have become a model organism for understanding conserved vertebrate cellular pathways and human disease, there has been an increased need to identify and elucidate orthologous zebrafish genes that are central to known human maladies. The cells of all early animal embryos go through a phase of rapid division (cleavage) where particular cell cycle checkpoints are skipped until a specification event occurs directing these embryonic stem cells to their fated germ layer cell type. Interestingly, this rapid cell division that ignores cell cycle checkpoints is also observed in many cancers. Developing blastula and tumor cells both require folr1 expression to obtain folate. In this report we have identified the expression pattern of the zebrafish gene zgc:165502, located on chromosome 15. Using computational and comparative methods and molecular biology techniques such as reverse transcription polymerase chain reaction (RT-PCR) and whole mount in situ hybridization (WISH) during embryogenesis, we demonstrate that zgc:165502 is the zebrafish orthologue of the human FOLR1 gene. Understanding when and where FOLR1 orthologues are expressed in different biomedical model organisms such as the zebrafish will help researchers design better experiments to study the endogenous FOLR1 activity.


Subject(s)
Folate Receptor 1/genetics , Folate Receptor 1/metabolism , Gene Expression Profiling/methods , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Computational Biology , Conserved Sequence , Embryonic Development , Folate Receptor 1/chemistry , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Phylogeny , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/chemistry
4.
Mech Dev ; 138 Pt 3: 279-90, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26459057

ABSTRACT

The Wnt/Planar Cell Polarity (PCP) pathway controls cell morphology and behavior during animal development. Several zebrafish mutants were identified as having perturbed Wnt/PCP signaling. Many of these mutants have defects in craniofacial formation. To better understand the role that Wnt/PCP plays in craniofacial development we set out to identify which of the mutants, known to be associated with the Wnt/PCP pathway, perturb head cartilage formation by disrupting chondrocyte morphology. Here we demonstrate that while vang-like 2 (vangl2), wnt11 and scribbled (scrib) mutants have severe craniofacial morphogenesis defects they do not display the chondrocyte stacking and intercalation problems seen in glypican 4 (gpc4) and wnt5b mutants. The function of Gpc4 or Wnt5b appears to be important for chondrocyte organization, as the neural crest in both mutants is specified, undergoes migration, and differentiates into the same number of cells to compose the craniofacial cartilage elements. We demonstrate that Gpc4 activity is required cell autonomously in the chondrocytes and that the phenotype of single heterozygous mutants is slightly enhanced in embryos double heterozygous for wnt5b and gpc4. This data suggests a novel mechanism for Wnt5b and Gpc4 regulation of chondrocyte behavior that is independent of the core Wnt/PCP molecules and differs from their collaborative action of controlling cell movements during gastrulation.


Subject(s)
Chondrocytes/metabolism , Chondrogenesis/genetics , Glypicans/genetics , Wnt Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Zebrafish/genetics , Animals , Animals, Genetically Modified , Branchial Region/embryology , Branchial Region/metabolism , Cell Count , Cell Movement/genetics , Cell Size , Chondrocytes/cytology , Gastrulation/genetics , Gene Expression Regulation, Developmental , Glypicans/deficiency , Mutation , Neural Crest/embryology , Neural Crest/metabolism , Phenotype , Wnt Proteins/deficiency , Wnt Signaling Pathway/genetics , Wnt-5a Protein , Zebrafish/metabolism , Zebrafish Proteins/deficiency
5.
Zebrafish ; 11(1): 17-25, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24266790

ABSTRACT

For many years, zebrafish have been the prototypical model for studies in developmental biology. In recent years, zebrafish has emerged as a powerful model system to study infectious diseases, including viral infections. Experiments conducted with herpes simplex virus type-1 in adult zebrafish or in embryo models are encouraging as they establish proof of concept with viral-host tropism and possible screening of antiviral compounds. In addition, the presence of human homologs of viral entry receptors in zebrafish such as 3-O sulfated heparan sulfate, nectins, and tumor necrosis factor receptor superfamily member 14-like receptor bring strong rationale for virologists to test their in vivo significance in viral entry in a zebrafish model and compare the structure-function basis of virus zebrafish receptor interaction for viral entry. On the other end, a zebrafish model is already being used for studying inflammation and angiogenesis, with or without genetic manipulations, and therefore can be exploited to study viral infection-associated pathologies. The major advantage with zebrafish is low cost, easy breeding and maintenance, rapid lifecycle, and a transparent nature, which allows visualizing dissemination of fluorescently labeled virus infection in real time either at a localized region or the whole body. Further, the availability of multiple transgenic lines that express fluorescently tagged immune cells for in vivo imaging of virus infected animals is extremely attractive. In addition, a fully developed immune system and potential for receptor-specific knockouts further advocate the use of zebrafish as a new tool to study viral infections. In this review, we focus on expanding the potential of zebrafish model system in understanding human infectious diseases and future benefits.


Subject(s)
Herpesvirus 1, Human/pathogenicity , Zebrafish/virology , Animals , Herpes Simplex , Models, Animal , Sulfotransferases/metabolism , Virus Internalization , Zebrafish Proteins/metabolism
6.
Dev Biol ; 386(1): 72-85, 2014 Feb 01.
Article in English | MEDLINE | ID: mdl-24333517

ABSTRACT

Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue.


Subject(s)
Collagen Type VIII/physiology , Gene Expression Regulation, Developmental , Spine/abnormalities , Zebrafish/embryology , Alleles , Animals , Collagen Type VIII/genetics , Crosses, Genetic , In Situ Hybridization , Meiosis , Microscopy, Confocal , Microscopy, Electron, Transmission , Mutation , Notochord/abnormalities , Osteoblasts/cytology , Osteoblasts/metabolism , Protein-Lysine 6-Oxidase/metabolism , Time Factors , Zebrafish/genetics
7.
Organogenesis ; 7(4): 255-9, 2011.
Article in English | MEDLINE | ID: mdl-22134372

ABSTRACT

Out of the several signaling pathways controlling craniofacial development, the role of planar cell polarity (PCP) signaling is relatively poorly understood. This pathway, originally identified as a mechanism to maintain cell polarity within the epithelial cells of the Drosophila wing, has been linked to the proper development of a wide variety of tissues in vertebrates and invertebrates. While many of the pathway members are conserved, it appears that some of the members of the pathway act in a tissue-specific manner. Here, we discuss the role of this pathway in vertebrate craniofacial development, highlighting cranial neural crest migration, skull and palate formation and the role of non-traditional modulators of PCP signaling within this developmental process.


Subject(s)
Cell Polarity , Face/embryology , Signal Transduction , Skull/cytology , Skull/embryology , Animals , Neural Crest/cytology , Wnt Proteins/metabolism
8.
Dev Biol ; 357(2): 518-31, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21723274

ABSTRACT

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5' of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue.


Subject(s)
Collagen Type II/genetics , Conserved Sequence/genetics , Evolution, Molecular , Regulatory Sequences, Nucleic Acid/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Animal Fins/embryology , Animal Fins/metabolism , Animals , Cartilage/embryology , Cartilage/metabolism , Collagen Type II/metabolism , Down-Regulation/genetics , Ear/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Genes, Reporter , Genome/genetics , Intervertebral Disc/embryology , Intervertebral Disc/metabolism , Larva/genetics , Notochord/embryology , Notochord/metabolism , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Sequence Homology, Amino Acid , Synteny/genetics , Zebrafish/embryology , Zebrafish Proteins/metabolism
9.
Zebrafish ; 6(1): 9-14, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19250029

ABSTRACT

Over the last two decades zebrafish has been an excellent model organism to study vertebrate development. Mutant analysis combined with gene knockdown and other manipulations revealed an essential role of Wnt signaling, independent of beta-catenin, during development. Especially well characterized is the function of Wnt/planar cell polarity (PCP) signaling in the regulation of gastrulation movements and neurulation, described in other reviews within this special issue. Here, we set out to highlight some of the new and exciting research that is being carried out in zebrafish to elucidate the role that Wnt/PCP signaling plays in the formation of specific organs, including the lateral line, craniofacial development, and regeneration. We also summarized the emerging connection of the Wnt/PCP pathway with primary cilia function, an essential organelle in several organ activities.


Subject(s)
Organogenesis , Signal Transduction , Wnt Proteins/metabolism , Zebrafish/embryology , Animals , Zebrafish/metabolism
10.
Biochem Biophys Res Commun ; 357(3): 683-7, 2007 Jun 08.
Article in English | MEDLINE | ID: mdl-17445768

ABSTRACT

The FOG family of transcriptional co-factors is composed of two members in mammals: FOG-1 and FOG-2. Both have been shown to bind to GATA factors and function as transcriptional co-repressors in specific cell and promoter contexts. We have previously defined a novel repression domain localized to the N-terminus of each FOG family member, the FOG repression motif, which is necessary for FOG-mediated transcriptional repression. In this report, we describe the identification and characterization of a novel isoform of FOG-2 lacking the FOG repression motif. In contrast to full-length FOG-2, this isoform is expressed predominately in the embryonic and adult heart. It can bind GATA4 avidly, but is unable to repress GATA4-mediated activation of cardiac-restricted gene promoters. Together, these results suggest that FOG-2 repressive activity may be modulated by the generation of isoforms of FOG-2 lacking the FOG repression motif.


Subject(s)
Alternative Splicing , DNA-Binding Proteins/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Animals , Binding Sites , Blotting, Western , DNA-Binding Proteins/metabolism , GATA4 Transcription Factor/genetics , GATA4 Transcription Factor/metabolism , Gene Expression Profiling , Male , Mice , Molecular Sequence Data , NIH 3T3 Cells , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolism , Transcriptional Activation , Transfection
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