Zebrafish Model System Research Articles

Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.

Insights into the antibacterial and immunomodulatory functions of the antimicrobial peptide, epinecidin-1, against Vibrio vulnificus infection in zebrafish

In the present study, we used Vibrio vulnificus and a zebrafish model system to investigate the inhibitory effect of epinecidin-1 on acute bacterial infection and studied the impacts of pretreatment, co-treatment, and post-treatment with epinecidin-1 on its protective efficacy. In vivo experiments showed that co-treatment with epinecidin-1 and V. vulnificus achieved 78%–97% survival rates after 30 days. When epinecidin-1 and V. vulnificus were co-injected into zebrafish and zebrafish were re-challenged with V. vulnificus after 30 days, zebrafish had survival rates of 22%–47%. Pretreatment and post-treatment with epinecidin-1 obtained respective survival rates of 57% and 60%. In addition, epinecidin-1 modulated the expressions of immune-responsive genes like interleukin (IL)-10, IL-1b, tumor necrosis factor-α, and interferon-γ as analyzed by a microarray and qPCR approach. This study demonstrates the use of epinecidin-1 to develop inactivated material for fish bacterial infections which can provide guidelines for the future design of epinecidin-1-bacterial formulations for various in vivo applications.

The search for evolutionary developmental origins of aging in zebrafish: A novel intersection of developmental and senescence biology in the zebrafish model system

Senescence may be considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena during the process of aging. We investigated whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We conducted experiments to isolate zebrafish mutants expressing an apparent senescence phenotype during embryogenesis (embryonic senescence). Some of the genes we thereby identified had already been associated with cellular senescence and chronological aging in other organisms, but many had not yet been linked to these processes. Complete loss-of-function of developmentally essential genes induce embryonic (or larval) lethality, whereas it seems like their partial loss-of-function (i.e., decrease-of-function by heterozygote or hypomorphic mutations) still remains sufficient to go through the early developmental process because of its adaptive plasticity or rather heterozygote advantage. However, in some cases, such partial loss-of-function of genes compromise normal homeostasis due to haploinsufficiency later in adult life having many environmental stress challenges. By contrast, any heterozygote-advantageous genes might gain a certain benefit(s) (much more fitness) by such partial loss-of-function later in life. Physiological senescence may evolutionarily arise from both genetic and epigenetic drifts as well as from losing adaptive developmental plasticity in face of stress signals from the external environment that interacts with functions of multiple genes rather than effects of only a single gene mutation or defect. Previously uncharacterized developmental genes may thus mediate the aging process and play a pivotal role in senescence. Moreover, unexpected senescence-related genes might also be involved in the early developmental process and regulation. We wish to ascertain whether we can identify such genes promptly in a comprehensive manner. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes and small molecular compounds that can be linked to the senescence phenotype and thereby facilitates searching for the evolutionary and developmental origins of aging in vertebrates.

The Effect of Stocking Densities on Reproductive Performance in Laboratory Zebrafish (Danio rerio)

Despite the growing popularity of the zebrafish model system, the optimal husbandry conditions for this animal are not well defined. The aim of this study was to examine the effect of stocking density on reproductive performance in zebrafish. In this study, undertaken by eight different zebrafish facilities, clutches of at least 200 wild-type zebrafish embryos from a single pairwise mating were produced at each participating institution and subsequently reared according to "in-house protocols" until they were 14 weeks old. Fish were then randomly assigned into treatment groups with balanced sex ratios and densities of 3, 6, or 12 fish/L. After a 1-month acclimation period, fish were spawned in pair crosses every 2 weeks for 3 months, for a total of six spawning dates. The number of viable and nonviable embryos produced in each clutch were counted at 1 day postfertilization. Although there was a great deal of variability in clutch size and percent spawning success among laboratories, there were no significant differences in average clutch size, spawning success, or percent viable among the treatment densities. These data suggest that using stocking densities as high as 12 fish/L does not have a negative impact on performance, when measured by reproductive performance.

A transgenic zebrafish model of targeted oocyte ablation and de novo oogenesis

We describe here a novel transgenic zebrafish, Tg(zpc:G4VP16/UAS:nfsB-mCherry) that effectively demonstrates the targeted oocyte ablation in the adult zebrafish ovary. This transgenic line expresses bacterial nitroreductase enzyme (nfsB) under the control of the oocyte-specific zona pellucida C (zpc) gene promoter. Adult transgenic females exposed to the prodrug metronidazole demonstrated near-complete ablation of growing oocytes, resulting in ovarian degeneration and complete cessation of reproductive function. Within 4 weeks of prodrug removal, treated fish demonstrated complete anatomical regeneration of the ovary and, within 7 weeks, ovarian function (fertility) was fully restored. Together, these results demonstrate functional renewal of the oocyte pool in the adult zebrafish ovary. Accordingly, this transgenic zebrafish model system provides a novel means to investigate ovarian growth dynamics in a genetically tractable vertebrate, and may be useful for evaluating signaling interactions that regulate gonadal development processes such as de novo oogenesis.

Identifying an uptake mechanism for the antiepileptic and bipolar disorder treatment valproic acid using the simple biomedical modelDictyostelium

Valproic acid (VPA) is the most highly prescribed epilepsy treatment worldwide and is also used to prevent bipolar disorder and migraine. Surprisingly, very little is known about its mechanisms of cellular uptake. Here, we employ a range of cellular, molecular and genetic approaches to characterize VPA uptake using a simple biomedical model, Dictyostelium discoideum. We show that VPA is taken up against an electrochemical gradient in a dose-dependent manner. Transport is protein-mediated, dependent on pH and the proton gradient and shows strong substrate structure specificity. Using a genetic screen, we identified a protein homologous to a mammalian solute carrier family 4 (SLC4) bicarbonate transporter that we show is involved in VPA uptake. Pharmacological and genetic ablation of this protein reduces the uptake of VPA and partially protects against VPA-dependent developmental effects, and extracellular bicarbonate competes for VPA uptake in Dictyostelium. We further show that this uptake mechanism is likely to be conserved in both zebrafish (Danio rerio) and Xenopus laevis model systems. These results implicate, for the first time, an uptake mechanism for VPA through SLC4-catalysed activity.

A high-throughput drug-screen for new therapies in cystic kidney disease using a zebrafish model system

A high-throughput drug-screen for new therapies in cystic kidney disease using a zebrafish model system

Zebrafish: A Model System to Study Heritable Skin Diseases

Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in ∼500 different genes. A number of model systems have been developed to advance our understanding of the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5-6 days post-fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphological and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provide a useful model system to study the molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases.

Intrahepatic Biliary Anomalies in a Patient With Mowat‐Wilson Syndrome Uncover a Role for the Zinc Finger Homeobox Gene zfhx1b in Vertebrate Biliary Development

zfhz1b is the causative gene for Mowat-Wilson syndrome, in which patients demonstrate developmental delay and Hirschsprung disease, as well as other anomalies. We identified a patient with Mowat-Wilson syndrome who also developed cholestasis and histopathologic features consistent with biliary atresia, suggesting that mutations involving zfhz1b may lead to biliary developmental anomalies or injury to the biliary tract. We used the zebrafish model system to determine whether zfhx1b has a role in vertebrate biliary development. Using zebrafish we determined that zfhx1b was expressed in the developing liver during biliary growth and remodeling, and that morpholino antisense oligonucleotide-mediated knockdown of zfhx1b led to defects in biliary development. These findings were associated with decreased expression of vhnf1, a transcription factor known to be important in biliary development in zebrafish and in mammals. Our studies underscore the importance of genetic contributions in the etiology of infantile hepatobiliary disorders, including biliary atresia.

Functional analysis of BBS3 A89V that results in non-syndromic retinal degeneration

Bardet–Biedl syndrome (BBS) is a syndromic form of retinal degeneration. Recently, homozygosity mapping with a consanguineous family with isolated retinitis pigmentosa identified a missense mutation in BBS3, a known BBS gene. The mutation in BBS3 encodes a single amino acid change at position 89 from alanine to valine. Since this amino acid is conserved in a wide range of vertebrates, we utilized the zebrafish model system to functionally characterize the BBS3 A89V mutation. Knockdown of bbs3 in zebrafish alters intracellular transport, a phenotype observed with knockdown of all BBS genes in the zebrafish, as well as visual impairment. Here, we find that BBS3 A89V is sufficient to rescue the transport delays induced by the loss of bbs3, indicating that this mutation does not affect the function of BBS3 as it relates to syndromic disease. BBS3L A89V, however, was unable to rescue vision impairment, highlighting a role for a specific amino acid within BBS3 that is necessary for visual function, but dispensable in other cell types. These data aid in our understanding of why patients with the BBS3 A89V missense mutation only present with isolated retinitis pigmentosa.

Shared and Unique G Alpha Proteins in the Zebrafish Versus Mammalian Senses of Taste and Smell

Chemosensory systems in vertebrates employ G protein-coupled receptors as sensors. In mammals, several families of olfactory and gustatory receptors as well as specific G alpha proteins coupling to them have been identified, for example, gustducin for taste. Orthologous receptor families have been characterized in fish, but the corresponding G alpha genes have not been well investigated so far. We have performed a comprehensive search of several lower vertebrate genomes to establish the G alpha protein family in these taxa and to identify those genes that may be involved in chemosensory signal transduction in fish. We report that gustducin is absent from the genomes of all teleost and amphibian species analyzed, presumably due to independent gene losses in these lineages. However, 2 other G alpha genes, Gi1b and G14a, are expressed in zebrafish taste buds and 4 G proteins, Go1, Go2, Gi1b, and Golf2, were detected in the olfactory epithelium. Golf2, Gi1b, and G14a are expressed already shortly after hatching, consistent with the physiological and behavioral responses of larvae to odorants and tastants. Our results show general similarity to the mammalian situation but also clear-cut differences and as such are essential for using the zebrafish model system to study chemosensory perception.

Cleavage and Polyadenylation Specificity Factor 1 Is Required for Definitive Hematopoietic Stem Cell Survival In Zebrafish.

AbstractAbstract 1606Hematopoiesis is a tightly regulated process requiring the differential activation of specific genetic programs. Inactivating mutations of genes involved in normal hematopoiesis have been implicated in bone marrow failure syndromes, myelodysplastic syndromes and hematopoietic neoplasms. Despite recent advances, many genes regulating normal hematopoiesis are still unknown and innovative gene-discovery approaches can benefit our understanding of the molecular mechanisms underlying this complex developmental process. The combined genetic and embryologic strengths of the zebrafish model system are ideal for studying factors regulating hematopoiesis, which is highly conserved with mammals. We have used ENU-mutated zebrafish lines to screen for genes whose inactivation results in the loss of definitive hematopoiesis. Here, we report the identification and analysis of the zebrafish mutant grechetto, caused by a lethal, recessive, inactivating mutation (grcl8a12) of the cleavage and specificity factor 1 (cpsf1) gene. Cpsf1 encodes a protein required for processing the 3'UTR of a subset of pre-mRNAs. Cpsf1 is maternally expressed ubiquitously through the first five days of development including primitive myeloid and erythroid cells and definitive hematopoietic stem cells (HSC). Although grechetto mutants fail to express cspf1 already at 24 hpf, they appear to develop normally until 72 hpf, when they first become morphologically distinct from wild-type siblings with a smaller head and the absence of a protruding jaw. Importantly, primitive hematopoiesis appears normal, as shown by expression of the myeloid and erythroid markers mpx and band3, respectively, at 24 and 48 hpf by whole mount RNA in situ hybridization (WISH). By 120 hpf the phenotype is more marked and embryos have a curved body, cardiac edema, development defects in the jaw and gut and a reduction in the number of iridophores. While many of these affected tissues represent derivatives of the neural crest (NC), the expression of markers of NC specification (sox10 and foxd3 at 15,5 hpf, crestin at 24 hpf), migration (dlx2 at 36 and 48 hpf) and differentiation (pax9a at 48 hpf) are normal. When definitive hematopoiesis was assayed for markers of mature blood cells by WISH at 120 hpf, grechetto mutants exhibit a loss of myeloid (mpx, lysC, l-plastin), erythroid (band3, gata1) and lymphoid cells (rag1, lck). Since myeloid and erythroid cells are normal at 24 hpf, but severely reduced at 120 hpf, we investigated whether the absence of HSC could explain the loss of definitive hematopoiesis in grechetto mutants. Interestingly, WISH staining for the HSC marker c-myb showed that in mutant embryos HSCs are normally specified and represented in the ventral wall of the dorsal aorta at 36 and 48 hpf. Nevertheless, at 72 hpf the numbers of HSCs are reduced in grechetto mutants upon their migration to the caudal hematopoietic tissue (CHT) and by 120 hpf very few remain. To investigate the fate of these HSCs we crossed the grechetto line with the Tg(c-myb:EGFP) reporter line, that expresses EGFP under the control of the c-myb promoter. By whole mount immunostaining, we were able to find increased co-localization of activated caspase3 and TUNEL staining in the c-myb:EGFP positive cells in the CHT in grechetto mutants compared to WT siblings at 72 and 96 hpf, suggesting that HSCs undergo apoptosis during this stage. Furthermore, PI cell cycle profile of sorted c-myb:EGFP+ cells from 96 hpf mutants shows a sub-G1 peak, representing pyknotic cell nuclei undergoing advanced stages of apoptosis. Importantly, both activated caspase3 staining and TUNEL assay did not show increased apoptosis in the tissue immediately surrounding the CHT. These studies show that cpsf1 is not required for primitive hematopoiesis or definitive HSC specification, however, it is essential for HSC survival in the CHT. The fact that grechetto embryos do not undergo generalized apoptotic cell death suggests that, despite ubiquitous cpsf1 expression and the general development defects of grechetto mutants, the prosurvival pathways in the HSC compartment are particularly dependent on Cpsf1 activity. Disclosures:Zon:FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Identification and Modulation of Voltage-Gated Ca2+Currents in Zebrafish Rohon-Beard Neurons

Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca(2+) channels (VGCCs) are especially important as Ca(2+) serves as both a charge carrier and second messenger. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, (Isl2b:EGFP)(ZC7), were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca(2+) current (HVA- and LVA-I(Ca), respectively). Ni(+)-sensitive LVA-I(Ca) occur in the minority of R-B neurons (30%) and ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels underlie the vast majority (90%) of HVA-I(Ca). To identify G protein coupled receptors (GPCRs) that modulate HVA-I(Ca), a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of I(Ca) appears to be transduced primarily via a cholera toxin-sensitive Gα subunit. These results provide the basis for using the zebrafish model system to understanding Ca(2+) channel function, and in turn, how Ca(2+) channels contribute to mechanosensory function.

Dystrophin-deficient zebrafish feature aspects of the Duchenne muscular dystrophy pathology

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene. As in humans, zebrafish dystrophin is initially expressed at the peripheral ends of the myofibres adjacent to the myotendinous junction and gradually shifts to non-junctional sites. Dystrophin-deficient zebrafish larvae are characterised by abundant necrotic fibres being replaced by mono-nucleated infiltrates, extensive fibrosis accompanied by inflammation, and a broader variation in muscle fibre cross-sectional areas. Muscle progenitor proliferation cannot compensate for the extensive skeletal muscle loss. Live imaging of dystrophin-deficient zebrafish larvae documents detaching myofibres elicited by muscle contraction. Correspondingly, the progressive phenotype of dystrophin-deficient zebrafish resembles many aspects of the human disease, suggesting that specific advantages of the zebrafish model system, such as the ability to undertake in vivo drug screens and real time analysis of muscle fibre loss, could be used to make novel insights relevant to understanding and treating the pathological basis of dystrophin-deficient muscular dystrophy.

Administration of recombinant IFN1 protects zebrafish ( Danio rerio) from ISKNV infection

In the present study, we used the infectious spleen and kidney necrosis virus (ISKNV) and zebrafish model system to investigate the inhibitory effect of recombinant zebrafish interferon 1 (zfrIFN1) on acute viral infection and the impact of time of zfrIFN1 administration on its protective efficacy. In vivo experiments showed that administration of recombinant zfrINF1 up-regulated expression of several IFN-stimulated genes within 24 h of injection, and expression levels of these genes dropped to normal levels similar to those in control fish within three days. However, the transcriptions of two viral genes, the major capsid protein and virus protein 48 genes, were significantly inhibited for at least three days, indicating a longer duration of the zfrIFN1-mediated innate immune effect. To evaluate the protective efficacy of zfrIFN1 against ISKNV infection, we compared the relative percentage survival (RPS) of ISKNV-infected zebrafish by intraperitoneally (IP) injecting the fish with zfrIFN1 at different time points before or after infection. IP injection with 1 μg zfrIFN1/g fish body weight at 24, 6 or 0 h before virus infection or 6 h after virus infection significantly improved fish survival. However, IP injection with an equal dose of zfrIFN1 24 h post-infection did not provide significant protection to the fish. Our results suggest that zfrIFN1 is potent in inhibiting ISKNV acute infection and initiating the innate immune response in zebrafish, but its efficiency depends on the time of administration. This study shows the protective effects of interferon against a DNA-virus in fish for the first time and provides information about the efficacy of fish interferon that will prove useful in possible therapeutic applications.

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.

Identification and characterization of zebrafish Rpl22 and its role in T cell development in vivo (36.66)

Abstract Mutations in a number of ribosomal proteins (RP) disrupt hematopoiesis in such syndromes as Diamond Blackfan Anemia; however, RP mutations had not previously been reported to disrupt T lymphocyte development. We recently reported that germline ablation of Rpl22 in mice caused a selective block in development of αβ lineage T cells. To gain insight into the basis by which Rpl22 mutations disrupt T cell development, we are exploiting the zebrafish model system. We identified the zebrafish ortholog of rpl22 (zgc:123327). Whole mount in situ hybridization showed that zebrafish rpl22 is expressed both maternally and zygotically throughout early development, with relative enrichment in the aorta-gonad mesonephros (AGM) and intermediate cellular mass (ICM) which are the sites of primitive (erythroid and myeloid) and definitive (lymphoid) hematopioesis in zebrafish. Despite being highly expressed in the sites of primitive and early definitive hematopoiesis, knocking down Rpl22 using antisense morpholinos did not block either of these processes. However, morpholino knockdown of Rpl22 did block T cell development. Therefore, unlike other ribosomal protein (e.g., Rps19) whose loss primarily impairs erythropoiesis, Rpl22 function appears to be dispensable for erythroid development and is instead selectively important for T cell development. Efforts to exploit the zebrafish system to address the mechanism underlying rpl22 function in T cell development and thymopoiesis are ongoing.

Characterization of the six zebrafish clade B fibrillar procollagen genes, with evidence for evolutionarily conserved alternative splicing within the pro-α1(V) C-propeptide

Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I–III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-α1(V), pro-α3(V), pro-α1(XI) and pro-α2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-α1(V), pro-α3(V)a and b, pro-α1(XI)a and b, and pro-α2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH2-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-α1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system.

Genome-wide loss-of-function analysis of deubiquitylating enzymes for zebrafish development

BackgroundDeconjugation of ubiquitin and/or ubiquitin-like modified protein substrates is essential to modulate protein-protein interactions and, thus, signaling processes in cells. Although deubiquitylating (deubiquitinating) enzymes (DUBs) play a key role in this process, however, their function and regulation remain insufficiently understood. The "loss-of-function" phenotype studies can provide important information to elucidate the gene function, and zebrafish is an excellent model for this goal.ResultsFrom an in silico genome-wide search, we found more than 90 putative DUBs encoded in the zebrafish genome belonging to six different subclasses. Out of them, 85 from five classical subclasses have been tested with morpholino (MO) knockdown experiments and 57 of them were found to be important in early development of zebrafish. These DUB morphants resulted in a complex and pleiotropic phenotype that, regardless of gene target, always affected the notochord. Based on the huC neuronal marker expression, we grouped them into five sets (groups I to V). Group I DUBs (otud7b, uchl3 and bap1) appear to be involved in the Notch signaling pathway based on the neuronal hyperplasia, while group IV DUBs (otud4, usp5, usp15 and usp25) play a critical role in dorsoventral patterning through the BMP pathway.ConclusionWe have identified an exhaustive list of genes in the zebrafish genome belonging to the five established classes of DUBs. Additionally, we performed the corresponding MO knockdown experiments in zebrafish as well as functional studies for a subset of the predicted DUB genes. The screen results in this work will stimulate functional follow-up studies of potential DUB genes using the zebrafish model system.

The Human AF9 Homologue in Zebrafish Is Involved in Primitive Hematopoietic Development.

Abstract 3653Poster Board III-589 IntroductionHuman AF9 (alias MLLT3) gene was initially identified as one of the most common translocation partners of MLL associated with acute myeloid leukemia. More recently it has been demonstrated that AF9 can be a positive regulator of early erythroid/megakaryocytic lineage decisions. However, many biological functions of AF9 and its role in leukemia remain unclear. Here, we aimed to identify the zebrafish homologue of human AF9 and to analyze its function using the zebrafish model system. Methods and ResultsA single zebrafish af9 gene was identified by homology searching at zebrafish genome database (www.ncbi.nlm.nih.gov/). The putative af9 protein showed 60% identity and 73% similarity to human counterpart. These results from the perspective of phylogenetic and synteny analyses indicated that zebrafish af9 was the likely homologue of human AF9. We used whole-mount in situ hybridization to analyzed its spatial and temporal expression patterns during embryonic development. Af9 was expressed specifically in the posterior-lateral mesoderm at the 5-somite stage and in the intermediate cell mass (ICM, site of primitive hematopoiesis) between 18 to 24 hours post fertilization (hpf). In order to characterize the role of af9 in early hematopoietic development, we performed over-expression and loss-of-function experiments in zebrafish embryos, and then analyzed various hematopoietic markers by reverse transcriptase quantitative PCR (RT-qPCR). Inducing ectopic af9 expression by mRNA injection we found an increased expression of gata1 and hbae1 (erythroid markers) and reduced expression of runx1, scl, lmo2, c-myb (stem cell/precursor associated markers) as well as pu.1 and l-plastin (myeloid markers) at 22 hpf-old embryos. Conversely af9 loss-of-function obtained using anti-sense morpholinos showed decreased expression of gata1 and hbae1 mRNA, while pu.1 and l-plastin increased their expression in morphant embryos at 22 hpf. The same morphants did not show any appreciable change in stem cell/precursor marker expression. ConclusionThe finding that gata1 expression is controlled by af9 suggests that this gene might promote the erythroid development in zebrafish primitive hematopoiesis. As the genetic programs of AF9 are evolutionary conserved between human and zebrafish, these findings provide new insights and opening further perspectives in leukemia research and hematopoiesis studies. Disclosures:No relevant conflicts of interest to declare.