Adult Zebrafish as a Model System for Cutaneous Wound-Healing Research

Upon injury, the skin must quickly regenerate to regain its barrier function. In mammals, wound healing is rapid and scar free during embryogenesis, whereas in adults it involves multiple steps including blood clotting, inflammation, re-epithelialization, vascularization, and granulation tissue formation and maturation, resulting in a scar. We have established a rapid and robust method to introduce full-thickness wounds onto the flank of adult zebrafish, and show that apart from external fibrin clot formation, all steps of adult mammalian wound repair also exist in zebrafish. Wound re-epithelialization is extremely rapid and initiates with no apparent lag phase, subsequently followed by the immigration of inflammatory cells and the formation of granulation tissue, consisting of macrophages, fibroblasts, blood vessels, and collagen. The granulation tissue later regresses, resulting in minimal scar formation. Studies after chemical treatment or with transgenic fish further suggest that wound re-epithelialization occurs independently of inflammation and fibroblast growth factor signaling, whereas both are essential for fibroblast recruitment and granulation tissue formation. Together, these results demonstrate that major steps and principles of cutaneous wound healing are conserved among adult mammals and adult zebrafish, making zebrafish a valuable model for studying vertebrate skin repair

Production of sterile Atlantic salmon by germ cell ablation with antisense oligonucleotides

Cultivation of sterile-only fish in aquaculture offers multiple benefits of environmental, economical, and social value. A reliable method for efficient sterilization without affecting fish welfare and performance traits would have significant impact on fish production practices. Here, we demonstrate sterilization of Atlantic salmon embryos by targeting the dead end gene with antisense oligonucleotides. Successful gene knock down and sterilization was achieved only when using Gapmer oligonucleotides and not with morpholino oligos. Germ cell-depleted embryos developed into morphologically normal male and female salmon with rudimentary gonads devoid of gametes

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers

Genetic Analysis of Fin Development in Zebrafish Identifies Furin and Hemicentin1 as Potential Novel Fraser Syndrome Disease Genes

Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease

Molekulare Mechanismen zur Steuerung der Keimzellentwicklung in Zebrafisch und die Rolle dieser Zelllinie in der Geschlechtsdifferenzierung

Während der Embryonalentwicklung des Zebrafisch (Danio rerio) werden die Urkeimzellen sehr früh von den anderen Zelllinien durch eine maternale, spezifische Form des Cytoplasmas, dem so genannten Keimplasma, unterschieden. Bei den nachfolgenden Zellteilungen verteilt sich dieses Keimplasma asymmetrisch, so dass es jeweils nur in die späteren Keimzellen und nicht in die späteren somatischen Zellen gelangt. Das Keimplasma enthält Schlüsselfaktoren, die das Entwicklungspotential seiner Zellen auf das der Keimzellen festlegen. So ist z.B. die RNS verschiedener Keimzell-spezifischer Gene, wie vasa, nanos1 und dead end (dnd), im Keimplasma angereichert und wird in der Keimbahn expremiert.Das erste Kapitel dieser Arbeit untersucht die zelluläre Lokalisation der Proteine Dead End und Nanos1 während verschiedener Entwicklungsstadien im Zebrafisch. Zu diesem Zweck entwickelten wir polyklonale Antikörper, die diese beiden Proteine detektieren. Dead End und Nanos1 kolokalisieren in den perinukleären Granula, welches Keimzell-spezifische Organellen mit noch unbekannter Funktion sind. In diesen perinukleären Granula sind Proteine angereichert, die von Keimplasma-spezifischen Genen kodiert werden. Eine genauere Charakterisierung dieser Organellen zeigte, dass sie in unmittelbarer Nähe zu den Zellkernporen lokalisiert sind. Wir stellen daher die Hypothese auf, dass Komponenten der perinukleären Granula an der posttranskriptionalen Regulation der Genexpression, wie z.B. mRNS- Stabilisierung, - Transport und/oder - Abbau, beteiligt sein könnten. In Übereinstimmung mit dieser Hypothese konnten wir zeigen, dass loss-of-function Punktmutationen in dead end dazu führen, dass das Dead End Protein nicht mehr in den perinukleären Granula sondern im Zellkern lokalisiert ist.Das zweite Kapitel dieser Arbeit behandelt die Rolle von dead end in der Geschlechtsdeterminierung. Embryonen, in die ein Morpholino injiziert wurde, der die Translation von dead end blockiert, besitzen keine Keimzellen. Darüber hinaus entwickeln sich diese Embryonen ausschließlich zu sterilen Männchen. Dies lässt vermuten, dass für die Geschlechtsdeterminierung im Zebrafisch entweder funktionales Dead End Protein oder das Vorhandensein von Keimzellen essentiell ist. Um zwischen diesen beiden Möglichkeiten zu unterscheiden, wurde eine alternative Methode zur Entfernung von Keimzellen angewandt. Diese Methode basiert auf dem bicistronischen Toxin-Antidote-System, welches die Expression eines Giftes (Toxin) in den Keimzellen ermöglicht während die somatischen Zellen durch ein Gegengift (Antidote) geschützt sind. Keimzelllose Embryonen, die durch diese dead end unabhängige Methode erzeugt wurden, entwickelten sich ebenfalls überwiegend zu Männchen. Dies zeigt, dass die Existenz von Keimzellen wichtig für die Geschlechtsdeterminierung im Zebrafisch ist.Das letzte Kapitel dieser Arbeit beschäftigt sich mit der Rolle von RNS-Interferenz während der Keimzellentwicklung im Zebrafisch. RNS-Interferenz ist ein in vielen Organismen verbreiteter Mechanismus zum Ausschalten von Genen. Über die Existenz von RNS-Interferenz im Zebrafisch gibt es jedoch nur wenige und widersprüchliche Daten. Da das Ausschalten von Genen während der Keimzellentwicklung in verschiedenen Organismen sehr häufig beobachtet wird, untersuchten wir, ob die Funktion von small interfering RNAs (siRNAs) und micro RNAs (miRNAs) wichtig für die Spezifizierung und Entwicklung von Keimzellen im Zebrafisch ist.Unsere Ergebnisse zeigen, dass beiden Prozesse, die Hemmung von Translation durch miRNA und der siRNA vermittelter Abbau von mRNS, ein unterschiedliches Potential zur Ausschaltung von Genen besitzen. Die Anwendung von siRNAs führte gelegentlich zu einem Abbau von mRNS, wobei die Effizienz des Abbaus von Gen zu Gen variierte. Die miRNA Oligonukleotide könnten benutzt werden, um spezifische Gensequenzen an genau definierten Positionen auszuschalten. Die genauen Anforderungen an diese Zielsequenzen können jedoch bislang noch nicht genau definiert werden. Dieses beschränkte Wissen bezüglich der erfolgreichen Voraussage von miRNA-Bindungsstellen erschwert den Gebrauch dieser Technik im Zebrafisch. Dies gilt vor allem im Vergleich zu gut etablierten Techniken wie Morpholino-antisense-Oligonukleotiden, die zur Hemmung der Translation von mRNS eingesetzt werden. Da wir die Existenz von RNS-Interferenz zur Regulation von endogenen Genen nicht nachweisen konnten, weder in somatischen noch in Keimzellen, bleibt die Frage offen, ob RNS-Interferenz die Genregulation in einer dieser beiden Zellpopulationen regelt

A Complex of BBS1 and NPHP7 Is Required for Cilia Motility in Zebrafish

<div><p>Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) are hereditary autosomal recessive disorders, encoded by two families of diverse genes. BBS and NPH display several overlapping phenotypes including cystic kidney disease, retinitis pigmentosa, liver fibrosis, <i>situs inversus</i> and cerebellar defects. Since most of the BBS and NPH proteins localize to cilia and/or their appendages, BBS and NPH are considered ciliopathies. In this study, we characterized the function of the transcription factor Nphp7 in zebrafish, and addressed the molecular connection between BBS and NPH. The knockdown of zebrafish <i>bbs1</i> and <i>nphp7.2</i> caused similar phenotypic changes including convergent extension defects, curvature of the body axis, hydrocephalus, abnormal heart looping and cystic pronephros, all consistent with an altered ciliary function. Immunoprecipitation assays revealed a physical interaction between BBS1 and NPHP7, and the simultaneous knockdown of z<i>bbs1</i> and z<i>nphp7.2</i> enhanced the cystic pronephros phenotype synergistically, suggesting a genetic interaction between z<i>bbs1</i> and z<i>nphp7.2 in vivo</i>. Deletion of zBbs1 or zNphp7.2 did not compromise cilia formation, but disrupted cilia motility. Although NPHP7 has been shown to act as transcriptional repressor, our studies suggest a crosstalk between BBS1 and NPHP7 in regulating normal function of the cilium.</p></div

Depletion of zBbs1 and zNphp7.2 causes dose-dependent hydrocephalus and pronephric cysts.

<p>Zebrafish embryos injected with MOs at the 1-cell stage were evaluated for their phenotype at 55 hpf. (A) Control zebrafish embryo with magnified normal brain area. Scale bar = 500 µm. (B and C) z<i>bbs1</i> AUG MO-injected embryo and z<i>nphp7.2</i> SP1 MO-injected embryo with hydrocephalus. (D) The Tg(<i>WT1b:EGFP</i>) transgenic line shows normal pronephric glomerulus and tubules (Scale bar = 100 µm), (E and F) z<i>bbs1</i> AUG MO-injected embryo and z<i>nphp7.2</i> SP1 MO-injected embryo show pronephric cysts (asterisks). At 55 hpf, larval dysmorphy was categorized according to the visual scale (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072549#pone.0072549.s003" target="_blank">Fig. S3</a>). The degree of dysmorphy was increased with the amount of injected MOs (G, z<i>bbs1</i> AUG MO; H, z<i>nphp7.2</i> SP1 MO). (G and H) Cystic pronephric phenotypes were dose-dependent in zebrafish embryos injected with z<i>bbs1</i> and z<i>nphp7.2</i> MO respectively. The graphs show percentages of the number (n) of embryos that were examined.</p

Expression of z<i>bbs1</i> and z<i>nphp7</i>.

<p>(A) Identification of 2 NPHP7 homologues in zebrafish: Zebrafish Nphp7.1 (zNphp7.1) and zebrafish Nphp7.2 (zNphp7.2) consist of 446 and 489 amino acids respectively. Amino acid sequence alignment showed that zNphp7.1 shares 43.9% identity and 50.8% similarity with the human NPHP7/GLIS2 (hNPHP7); zNphp7.2 was 51.4% identical and 60.2% similar to the human homologue. The ZF domains of zNphp7.1 and zNphp7.2 were 89.3% and 91.3% identical with those of the human homologue, respectively. (<b>*</b>, completely conserved; <b>.</b>, identical in 2 sequences or belonging to same type of amino acid group in 2 or 3 sequences) (B) Semi-quantitative RT-PCR reveals maternal transcript expression for z<i>nphp7.1</i> and z<i>nphp7.2</i> whereas z<i>bbs1</i> is not expressed maternally nor at 6 hpf. 2 maternal splice products were identified for z<i>nphp7.2</i> (open arrowhead: Transcript 1; filled arrowhead: Transcript 2). The transcript 2 of z<i>nphp7.2</i> is expressed only maternally. Sequencing of the lower splice product revealed an excision of 18 bp corresponding to amino acid (aa 101–118) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072549#pone.0072549.s001" target="_blank">Fig. S1</a>). (C) Semi-quantitative RT-PCR with organ specific cDNA from adult zebrafish indicated that z<i>bbs1</i> is expressed in kidney, eye and testis. z<i>nphp7.1</i> and z<i>nphp7.2</i> are expressed in other organs including kidney, eye, heart, testis, gut and muscle.</p

Genetic interaction between z<i>bbs1</i> and z<i>nphp7.2 in vivo</i>.

<p>Injected zebrafish embryos were assessed for the incidence of pronephric cysts. (A) Cont MO injected embryos with normal pronephros. While the majority of z<i>bbs1</i> or z<i>nphp7.2</i> morphants (suboptimal dose) showed pronephros of normal morphology, those of combined knockdown exhibited pronephros with cysts. Cysts are marked by asterisks. (Black scale bar = 500 µm, White scale bar = 100 µm) (B) Pronephric cysts were detectable after individual injections of <i>znphp7.2</i> MO at 0.1 mM and <i>zbbs1</i> MO at 0.2 mM with the level of 17% and 10% respectively whereas the combined knockdown caused pronephric cysts in 59% of microinjected embryos. The final MO concentration for injection was 0.3 mM in all conditions to keep total MO dose constant. Therefore suboptimal doses of z<i>bbs1</i> and z<i>nphp7.2</i> MO were combined with Cont MO to obtain this final concentration of 0.3 mM. The numbers in the brackets (n) are the numbers of total embryos which were examined.</p

Knockdown of <i>znphp7.2</i> showed normal development of cilia.

<p>(A) Images of live zebrafish embryos at the 8–10 somite stage. Kupffer's vesicle is located in the dashed box. (B) Measurement of relative KV area with setting control as ‘1.00’. The knockdown of z<i>nphp7.2</i> did not significantly affect on KV development and area. (C) Images of cilia in the KV of 8–10 somite stage were stained for acetylated tubulin. Scale bar = 10 µm. (D) zNphp7.2-deficient embryos showed shortened length of cilia in KV compared to control embryos. (E) Staining of acetylated tubulin in the anterior pronephric tubule of morphants at 48 hpf displayed that the overall distribution of cilia remained unchanged compared to control (Scale bar = 10 µm) even though (F) the knockdown either of z<i>bbs1</i> showed longer and the knockdown of z<i>nphp7.2</i> showed shorter cilia. (PT, Pronephric Tubule) (G and H) The morphology and length of cilia in posterior pronephric tubule appeared unchanged in combined knockdown of z<i>bbs1</i> and z<i>nphp7.2</i> compared to single knockdown of z<i>bbs1</i> or z<i>nphp7.2</i> or compared to Cont MO injected embryos. (PT, Pronephric Tubule) (I) The morphants of z<i>bbs1</i> and z<i>nphp7.2</i> displayed normal ultrastructure of motile cilia in pronephric tubule without recognizable deficiency in dynein arms (outer dynein arm marked by arrowhead). The numbers (n) in the graphs are the number of total cilia which were examined. 4–6 individual embryos per group were examined.</p