Stable Transgenic Zebrafish Lines Research Articles

In situ assessment of statins' effect on autophagic activity in zebrafish larvae cardiomyocytes.

Improving the survival rate of cardiomyocytes is the key point to treat most of the heart diseases, and targeting autophagy is a potential advanced therapeutic approach. Monitoring autophagic activity in cardiomyocytes in situ will be useful for studying autophagy-related heart disease and screening autophagy-modulating drugs. Zebrafish, Danio rerio, has been proven as an animal model for studying heart diseases in situ. Taken the advantage of zebrafish, especially the imaging of intact animals, here we generated two stable transgenic zebrafish lines that specifically expressed EGFP-map1lc3b or mRFP-EGFP-map1lc3b in cardiomyocytes under the promoter of myosin light chain 7. We first used a few known autophagy-modulating drugs to confirm their usefulness. By quantifying the density of autophagosomes and autolysosomes, autophagy inducers and inhibitors showed their regulatory functions, which were consistent with previous studies. With the two lines, we then found a significant increase in the density of autophagosomes but not autolysosomes in zebrafish cardiomyocytes at the early developmental stages, indicating the involvement of autophagy in early heart development. To prove their applicability, we also tested five clinical statins by the two lines. And we found that statins did not change the density of autophagosomes but reduced the density of autolysosomes in cardiomyocytes, implying their regulation in autophagic flux. Our study provides novel animal models for monitoring autophagic activity in cardiomyocytes in situ, which could be used to study autophagy-related cardiomyopathy and drug screening.

Bone morphogenetic protein signaling regulates Id1-mediated neural stem cell quiescence in the adult zebrafish brain via a phylogenetically conserved enhancer module.

In the telencephalon of adult zebrafish, the inhibitor of DNA binding 1 (id1) gene is expressed in radial glial cells (RGCs), behaving as neural stem cells (NSCs), during constitutive and regenerative neurogenesis. Id1 controls the balance between resting and proliferating states of RGCs by promoting quiescence. Here, we identified a phylogenetically conserved cis-regulatory module (CRM) mediating the specific expression of id1 in RGCs. Systematic deletion mapping and mutation of conserved transcription factor binding sites in stable transgenic zebrafish lines reveal that this CRM operates via conserved smad1/5 and 4 binding motifs under both homeostatic and regenerative conditions. Transcriptome analysis of injured and uninjured telencephala as well as pharmacological inhibition experiments identify a crucial role of bone morphogenetic protein (BMP) signaling for the function of the CRM. Our data highlight that BMP signals control id1 expression and thus NSC proliferation during constitutive and induced neurogenesis.

New insights into organ-specific oxidative stress mechanisms using a novel biosensor zebrafish

BackgroundReactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. ObjectivesOur aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. MethodsTo achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. ResultsWe produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. DiscussionOur model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.

A novel transgenic zebrafish line allows for in vivo quantification of autophagic activity in neurons

ABSTRACT The pathophysiology of most neurodegenerative diseases includes aberrant accumulation of protein aggregates. Recent evidence highlights the role of protein degradation pathways in neurodegeneration. Concurrently, genetic tools have been generated to enable zebrafish, Danio rerio, to be used as an animal model to study neurodegenerative processes. In addition to optical clarity and fast ex utero development, the zebrafish brain is relatively small and has conserved structures with its mammalian counterparts. To take advantage of this model organism and to aid further studies on autophagy and neurodegeneration, we created a stable transgenic zebrafish line that expresses eGFP-Map1lc3b specifically in post-mitotic neurons under the elavl3 promoter. This line is useful for indirectly monitoring autophagic activity in neurons in vivo and screening for macroautophagy/autophagy-modulating compounds. We determined the applicability of this transgenic line by modulating and quantifying the number of autophagosomes via treatment with a known autophagy inducer (rapamycin) and inhibitors (3-methyladenine, protease inhibitors). Additionally, we proposed an in vivo method for quantifying rates of autophagosome accumulation, which can be used to infer occurrence of autophagic flux. Last, we tested two FDA-approved drugs currently undergoing clinical studies for Parkinson disease, isradipine and nilotinib, and found that isradipine did not modulate autophagy, whereas nilotinib induced both autophagosome number and autophagic flux. It is hoped that others will find this line useful as an in vivo vertebrate model to find or validate autophagy modulators that might be used to halt the progression of neurodegenerative diseases. Abbreviations 3MA: 3-methyladenine; BafA: bafilomycin A1; dd: dorsal diencephalon; dpf: days post fertilization; e: eye; eGFP: enhanced green fluorescent protein; Elavl3: ELAV like neuron-specific RNA binding protein 3; FDA: Food and Drug Administration; hb: habenula; hpt, hours post treatment; Map1lc3b: microtubule-associated protein 1 light chain 3 beta; nt: neural tube; ot, optic tectum; P/E: pepstatin A and E64d; PD: Parkinson disease; PMTs: photomultiplier tubes; PTU: 1-phenyl-2-thiourea; Ta: annealing temperature; Tel, telencephalon

Using the Zebrafish to Model the Tumour-Suppressor Effects of NUP98 in NUP98-NSD1 mediated AML

The NUP98-NSD1 (NND1) translocation is a fusion oncogene that causes high-risk acute myeloid leukemia (AML) in ~16% of pediatric AML patients. The presence of NND1 predicts a dismal prognosis, with a 4-year event-free survival of less than 10%. These high-risk patients often do not respond to current chemotherapy treatments, and would benefit from a targeted therapy approach. The zebrafish has served as a versatile in vivo model of human leukemia both for elucidating underlying disease pathogenesis and for preclinical drug discovery.We previously generated a transgenic zebrafish model expressing the human NUP98-HOXA9 fusion oncogene, which developed a myeloproliferative neoplasm and revealed DNA methyltransferase 1 as a novel downstream target amenable to epigenetic therapy (Forrester et al., BJH 2011; Deveau et al., Leukemia 2015). Like NUP98-HOXA9, NND1 is thought to induce AML through activation of HOXA9, as well as downstream epigenetic modification. In the case of NND1, sustained NSD1 expression induces tri-methylation of the activating H3K36 locus (Wang et al., Nat. Cell Biol. 2007). While the contribution of these NUP98 partner genes to leukemogenesis has been explored, the loss of normal NUP98 function that occurs in the context of these oncogenic fusions remains understudied. As NUP98 is a potential tumour suppressor (Singer et al., Mol. Cell 2012), further exploration of its role in leukemogenesis is required. With the advent of CRISPR/Cas9 technology, the zebrafish is ideally suited to interrogate both elements of NUP98 gene modification in AML.Plasmid constructs containing the human NND1 oncogene driven by either the ubiquitous ubi promoter or the pan-leukocyte cd45 promoter have been incorporated into the zebrafish using the Tol2 system. Expression of NND1 is visualized via co-expression of green fluorescent protein (GFP) at 24-48 hours post-fertilization. GFP expression appears strongest in the regions of embryonic hematopoiesis, such as the posterior blood island. Whole-mount in situ hybridization of plasmid-injected embryos expressing NND1 show a decrease in red blood cells and an increase in myeloid cells, indicating a disruption in hematopoiesis, suggesting a pre-leukemic state. Stable transgenic zebrafish lines are currently being screened. Simultaneously, we are generating transgenic zebrafish expressing a catalytically inactive Cas9 (dCas9) and two guide RNAs (sgRNAs) under the cd45 promoter, to create a leukocyte-specific knockdown of endogenous nup98. This novel dCas9 system will result in selective knockdown and reduce toxicity caused by off-target effects in other tissues.Our unique zebrafish model in which the NND1 oncogene is expressed and wild type NUP98 is functionally inhibited will exhibit greater fidelity to that which occurs in patients with NND1-induced AML, providing an unprecedented opportunity to determine the relative contributions of these lesions to disease pathogenesis. Moreover, given that NUP98-fusion oncogenes are common in multiple hematologic malignancies including AML, chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS), broader insights into leukemogenesis may be realized and new therapeutic targets identified. DisclosuresNo relevant conflicts of interest to declare.

Apurinic/Apyrimidinic Endonuclease 1 Induced Genomic Instability Causes T-Cell Acute Lymphoblastic Leukemia in Zebrafish

Genomic instability is not only a hallmark of cancer, but potentially a primary mechanism for its occurrence. DNA repair mechanisms play a protective role during DNA damage induced by both normal metabolic activities and environmental factors such as reactive oxygen species (ROS), UV light and γ-irradiation. Genes related to DNA repair are usually considered as tumor suppressors. However, incomplete repair may induce severe genomic instability, leading eventually to transformation.Apurinic/apyrimidinic endonuclease 1 (APEX1), a gene involved in DNA repair with an important role in the base excision repair pathway, leads to transformation of normal cells in vitro. To investigate the role of APEX1 in tumor initiation in vivo, we generated a novel transgenic zebrafish model to overexpress APEX1 in fish. Specifically, pDestTol2A2_ubi:loxP-EGFP-loxP-APEX1-mCherry plasmid was injected into single cell embryos derived from the TP53 mutant line Tp53M214K/M214K to generate a stable conditional inducible transgenic zebrafish line: Tg:APEX1fl/- mCherry Tp53M214K/M214K. To activate APEX1 expression in vivo, this line was mated with Tg:HSP70-Cre+/+ fish. Compound zebrafish Tg: APEX1fl/- mCherry,Tp53+/M214K ,HSP70-Cre+/- carrying a Cre-activatable APEX1 knock-in allele were heated at 24hpf, and induction of APEX1 expression was monitored by downstream reporter - mCherry expression. Ten to twelve months post-fertilization, Tg:APEX1fl/- mCherry,Tp53+/M214K ,HSP70-Cre+/- fish developed abnormal swelling. Flow cytometry analysis of fish kidney marrow and peripheral blood showed dramatically increased precursor populations in scatter analysis. Histopathologic analysis showed that multiple organs were infiltrated with malignant lymphoblastic cells. None of the control fish Tg: GFP,Tp53+/M214K ,HSP70-Cre+/- developed tumors during their life span. Zebrafish with T-ALL have heterozygous Tp53+/M214K background, but the expression of p21, mdm2 and bax in Tp53+/M214K fish is exactly the same as in Tp53+/+ fish; and Tp53+/M214K zebrafish themselves have not developed tumors during their life span. RNA from lymphoblastic cells was evaluated by qRT-PCR and showed increased expression of CD3, LCK and Tal indicating a T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). We have performed whole genomic DNA sequencing in extracted DNA from fish tumor cells and compared it with their normal counterpart and observed multiple copy number changes and mutations. We have now begun to see the development of other tumors in other organs including the eye and the testis. To uncover the molecular mechanism of tumorigenesis induced by APEX1, we have performed Mass Spectrometry analysis on APEX1 pulled down from 293T and AG08498 cells ectopically expressing APEX1. Besides verified binding proteins, such as PCNA, we also identified Ku70 and Ku80 binding to APEX1 directly. Further immunoflurescent staining and confocal microscopy of 293T cells also found co-localization of APEX1 and Ku70/Ku80. Those two proteins initiate Non-Homologous End Joining (NHEJ) repair and start the error-prone double strand repair and DNA damage. These results indicate that excessive repair activity may induce DNA damage and genomic instability.In summary, this is the first demonstration where overexpression of a DNA repair gene is responsible for induction of genomic instability leading to malignant transformation. It provides new insight into the process of tumorigenesis and development of both therapy as well as preventive strategies. DisclosuresZon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Abstract A04: Metabolic reprogramming of MYCN amplified neuroblastoma

Abstract Neuroblastoma (NB) is a highly malignant embryonic tumor of neural crest origin and accounts for 15% of all cancer deaths in children. Amplification of the MYCN oncogene, which occurs in ~25% of patients, is the most reliable marker of metastatic disease and predicts poor outcome. Recent findings show MYCN reprograms NB cell metabolism to promote glycolysis and shunting of glutamate into the TCA cycle, causing dependence on increased levels of TCA intermediates, such as a-KG and succinate. Allelic loss of the short arm of chromosome 1, (1p36) is associated with the vast majority of MYCN amplified NB, and a candidate tumor suppressor gene (TSG) in this region is succinate dehydrogenase subunit B,SDHB, which encodes a core component of the succinate dehydrogenase (SDH) complex, converting succinate to fumarate. We show that MYCN-amplified NB cells, with 1p deletion, have reduced SDHB protein levels and elevated succinate levels. Thus, we are now testing a model that the combined loss of SDH activity with MYCN amplification causes dramatic accumulation of cytosolic succinate, which in turn stabilizes HIF1α to accelerate NB pathogenesis in vivo. To test this model, we have generated mutations in zebrafish sdhb to determine if 1) it is normally required for sympathetic neuron differentiation and 2) cooperates with MYCN to accelerate zebrafish NB in vivo (Zhu et. al, Cancer Cell, 2012). As HIF1a is predicted to be upregulated in SDH-deficient cells, as well as MYCN-amplified NB, we have also generated stable HIF1α transgenic zebrafish lines to directly determine if HIF1α alone is sufficient to drive NB or cooperates with MYCN to accelerate it. The results from our studies of sdhb loss and hif1α gain on peripheral sympathetic nervous system development and NB formation in vivo will be discussed as well as potential therapeutic approaches to target SDH/MYCN-dependent tumors. Citation Format: Monique Morrison, Scott Khuu, Joshua Schiffman, Bradley Cairns, Jared Rutter, Dennis Winge, Frank Speleman, Rodney Stewart. Metabolic reprogramming of MYCN amplified neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A04.

Dynamic visualization of transcription and RNA subcellular localization in zebrafish.

Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at ∼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.

Two types of transgenic lines for doxycycline-inducible, cell-specific gene expression in zebrafish ultraviolet cone photoreceptors

Temporal and spatial control of gene expression is important for studying the molecular and cellular mechanisms of development, physiology, and disease. We used the doxycycline (Dox)-inducible, Tet-On system to develop transgenic zebrafish for inducible, cell specific control of gene expression in the ultraviolet (UV) cone photoreceptors. Two constructs containing the reverse tetracycline-controlled transcriptional transactivator (rtTA) gene driven by the UV opsin-specific promoter (opn1sw1) were used to generate stable transgenic zebrafish lines using the Tol2-based transgenesis method. One construct included a self-reporting GFP (opn1sw1:rtTA, TRE:GFP) and the other incorporated an epitope tag on the rtTA protein (opn1sw1:rtTAflag). UV cone-specific expression of TRE-controlled transgenes was induced by Dox treatment in larvae and adults. Induction of gene expression was observed in 96% of all larval UV cones within 16h of Dox treatment. UV cone-specific expression of two genes from a bidirectional TRE construct injected into one-cell Tg(opn1sw1:rtTAflag) embryos were also induced by Dox treatment. In addition, UV cone-specific expression of Crb2aIntraWT was induced by Dox treatment in progeny from crosses of the TRE-response transgenic line, Tg(TRE:HA-Crb2aIntraWT), to the Tg(opn1sw1:rtTA, TRE:GFP) line and the Tg(opn1sw1:rtTAflag) line. These lines can be used in addition to the inducible, rod-specific gene expression system from the Tet-On Toolkit to elucidate the photoreceptor-specific effects of genes of interest in photoreceptor cell biology and retinal disease.

Angiopoietin-Like Proteins Stimulate HSC Development Through Direct Interaction With Notch

The angiopoietin-like proteins (angptls) are novel growth factors that are capable of stimulating expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs) ex vivo. Although the receptor for some of the angptl family members is known, their molecular mechanism of action is undefined. Here, we show that overexpression of angptl2 in a stable heatshock inducible transgenic zebrafish line, Tg(hsp70:zangptl2), is sufficient to increase cmyb- and runx1-positive HSPCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis. Anti-sense morpholino knockdown of angptl1 and 2 resulted in a significant decrease in cmyb- and runx1-positive HSPCs in the AGM, suggesting that angptls are required for definitive hematopoiesis. These double morphants also displayed severe disruption in vascular development and differentiation prior to the defects observed in the AGM, indicating that angptl regulation of HSPC development occurs through an early specification of the hemogenic endothelium. The loss of function phenotypes in the developing aorta is reminiscent of mutant fish with defective notch signaling (mindbomb) and a tight genetic interaction occurs between angptl and notch signaling. Knocking down angptl1 and 2 decreased notch signaling in a transgenic notch reporter line while overexpression of a constitutively active intracellular notch rescues the angptl double morphant phenotype. These data imply that angptls function upstream of notch signaling. Interestingly, the absence of HSPCs and notch signaling in the mindbomb mutants are rescued by overexpression of angptl2 in Tg(hsp70:zangptl2), suggesting that angptls can regulate notch signaling. To examine the molecular mechanism of Angptl-mediated Notch activation, we stimulated cultured human CD34+ cells or endothelial cells with purified Angptl2 and observed a rapid increase in Notch receptor cleavage indicating that Angptl2 can induce Notch activation. Furthermore, we found through endogenous co-immunoprecipitation experiments that the Angptl receptor, LILRB2, interacted with Notch receptor. This strongly points to a direct regulation of Notch activation/signaling by Angptls through physical interactions between Notch and Angptl receptors. Previously, we found that angptl-mediated akt activation is important for HSPC formation in the AGM. To examine the role of Akt during Notch activation, we immunoprecipitated all Akt-phosphorylated substrates in Angptl-stimulated cells and found the presence of ADAM17/TACE, one of the cleavage enzymes that is crucial to Notch receptor activation. Together these results suggest a model in which Angptl-binding of the LILRB2 receptor enables recruitment of downstream molecules such as Akt proximal to Notch, allowing for subsequent cleavage and activation of Notch receptor. Finally, to examine downstream signaling of Angptl-mediated Notch activation, we performed chromatin immunoprecipitation for Notch followed by sequencing in Angptl2-stimulated CD34+ cells and found enrichment for Myc binding elements. Independent microarrays also revealed a strong Myc signature through gene set enrichment analysis. Thus, to confirm results from our bioinformatics analyses, we overexpressed zebrafish myc in angptl1 and 2 double morphants or mindbomb mutants, and found a significant rescue in HSPCs formation in the AGM. Collectively with these results, we propose that angptl can regulate notch signaling through receptor interaction, leading to activation of myc target genes during definitive hematopoiesis. Our data provide new insights to the previously uncharacterized Angptl signaling during HSPC development and present a novel mechanism of action for Notch activation. Disclosures: Aster: Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria. White:N-of-One: Consultancy, Equity Ownership. Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator (rtTA-M2). Southern blotting analysis and high-throughput genome sequencing verified that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline (Dox), a strong green fluorescent protein (GFP) was seen in rtTA-transgenic eggs injected with pTRE–EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox-induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 (DKK1) in pTRE-DKK1-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.

Fluorescent activated cell sorting (FACS) combined with gene expression microarrays for transcription enrichment profiling of zebrafish lateral line cells

Transgenic lines carrying fluorescent reporter genes like GFP have been of great value in the elucidation of developmental features and physiological processes in various animal models, including zebrafish. The lateral line (LL), which is a fish specific superficial sensory organ, is an emerging organ model for studying complex cellular processes in the context of the whole living animal. Cell migration, mechanosensory cell development/differentiation and regeneration are some examples. This sensory system is made of superficial and sparse small sensory patches called neuromasts, with less than 50 cells in any given patch. The paucity of cells is a real problem in any effort to characterize those cells at the transcriptional level. We describe here a method which we applied to efficiently separate subpopulation of cells of the LL, using two distinct stable transgenic zebrafish lines, Tg(cldnb:gfp) and Tg(tnks1bp1:EGFP). In both cases, the GFP positive (GFP+) cells were separated from the remainder of the animal by using a Fluorescent Activated Cell Sorter (FACS). The transcripts of the GFP+ cells were subsequently analyzed on gene expression microarrays. The combination of FACS and microarrays is an efficient method to establish a transcriptional signature for discrete cell populations which would otherwise be masked in whole animal preparation.

Tissue and cell-specific transcriptional activity of the human cytomegalovirus immediate early gene promoter (UL123) in zebrafish

The human cytomegalovirus (CMV) is a member of the herpesvirus superfamily and causes different diseases including encephalitis, gastrointestinal diseases, pneumonitis, hepatitis, and retinitis. The immediate early (IE) gene of the human cytomegalovirus is essential to the viral replication. The proximal promoter region of this gene behaves as a strong enhancer and was commonly used to overexpress genes in vitro and in vivo in numerous cell types and species. However, there was no detailed report on the spatial and temporal transcriptional activity of the human CMV-IE gene promoter in zebrafish. In the present study, we generated stable transgenic zebrafish lines carrying the eGFP reporter gene under the control of the human CMV-IE gene promoter (−602/−14). We demonstrated that the hCMV-IE:eGFP transgene was expressed in numerous tissues but transgene expression was either regionalized or restricted to specific cell types as embryo and larval development progressed. In adult, the global expression pattern was similar but not identical to that described for the simian CMV-IE gene promoter in stable zebrafish with high transgene expression in the spinal cord, olfactory organs, central nervous system, neuromasts, retina, and skeletal muscles. However, we describe additional major expression sites in the hepatocytes, the epithelial cells of the intestine, the epithelial cells of the renal tubules, and the oocytes. Interestingly, our study shows that the tissue and cell specific expression pattern of the human CMV-IE gene promoter is rather well conserved in stable transgenic zebrafish compared to that observed in mouse. The major expression sites described in zebrafish are in agreement with the targeted cells and symptoms resulting from CMV infections in human. Finally, the hCMV:eGFP transgenic lines described in the present study will be valuable tools to trace specific cell lineages in adult zebrafish.

Neuromuscular effects of G93A-SOD1 expression in zebrafish

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal disorder involving the degeneration and loss of motor neurons. The mechanisms of motor neuron loss in ALS are unknown and there are no effective treatments. Defects in the distal axon and at the neuromuscular junction are early events in the disease course, and zebrafish provide a promising in vivo system to examine cellular mechanisms and treatments for these events in ALS pathogenesis.ResultsWe demonstrate that transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in early defects in motor neuron outgrowth and axonal branching. This is consistent with previous reports on motor neuron axonal defects associated with familial ALS genes following knockdown or mutant protein overexpression. We also demonstrate that upregulation of growth factor signaling is capable of rescuing these early defects, validating the potential of the model for therapeutic discovery. We generated stable transgenic zebrafish lines expressing G93A-SOD1 to further characterize the consequences of G93A-SOD1 expression on neuromuscular pathology and disease progression. Behavioral monitoring reveals evidence of motor dysfunction and decreased activity in transgenic ALS zebrafish. Examination of neuromuscular and neuronal pathology throughout the disease course reveals a loss of neuromuscular junctions and alterations in motor neuron innervations patterns with disease progression. Finally, motor neuron cell loss is evident later in the disease.ConclusionsThis sequence of events reflects the stepwise mechanisms of degeneration in ALS, and provides a novel model for mechanistic discovery and therapeutic development for neuromuscular degeneration in ALS.

Visualization and exploration of Tcf/Lef function using a highly responsive Wnt/β-catenin signaling-reporter transgenic zebrafish

Evolutionarily conserved Tcf/Lef transcription factors (Lef1, Tcf7, Tcf7l1, and Tcf7l2) mediate gene expression regulated by Wnt/β-catenin signaling, which has multiple roles in early embryogenesis, organogenesis, adult tissue homeostasis, and tissue regeneration. However, the spatiotemporal dynamics of Tcf/Lef activity during these events remain poorly understood. We generated stable transgenic zebrafish lines carrying a new Wnt/β-catenin signaling reporter, Tcf/Lef-miniP:dGFP. The reporter revealed the transcriptional activities of four Tcf/Lef members controlled by Wnt/β-catenin signaling, which were expressed in known Wnt/β-catenin signaling-active sites during embryogenesis, organ development and growth, and tissue regeneration. We used the transgenic lines to demonstrate the contribution of Tcf/Lef-mediated Wnt/β-catenin signaling to the development of the anterior lateral line, dorsal and secondary posterior lateral lines, and gill filaments. Thus, these reporter lines are highly useful tools for studying Tcf/Lef-mediated Wnt/β-catenin signaling-dependent processes.

Identification and Analysis of Conserved cis-Regulatory Regions of the MEIS1 Gene

Meis1, a conserved transcription factor of the TALE-homeodomain class, is expressed in a wide variety of tissues during development. Its complex expression pattern is likely to be controlled by an equally complex regulatory landscape. Here we have scanned the Meis1 locus for regulatory elements and found 13 non-coding regions, highly conserved between humans and teleost fishes, that have enhancer activity in stable transgenic zebrafish lines. All these regions are syntenic in most vertebrates. The composite expression of all these enhancer elements recapitulate most of Meis1 expression during early embryogenesis, indicating they comprise a basic set of regulatory elements of the Meis1 gene. Using bioinformatic tools, we identify a number of potential binding sites for transcription factors that are compatible with the regulation of these enhancers. Specifically, HHc2:066650, which is expressed in the developing retina and optic tectum, harbors several predicted Pax6 sites. Biochemical, functional and transgenic assays indicate that pax6 genes directly regulate HHc2:066650 activity.

Genetic Interaction Between Angiopoietin-Like Proteins 1 and 2 and Notch Signaling During HSC Development

AbstractAbstract 573Members of the angiopoietin-like protein (angptl) family have recently been shown to stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptls for HSCs during development and their mechanism of action have yet to be determined. To first recapitulate the effects of exogenous angptl on HSC expansion, we made a stable transgenic zebrafish line, Tg(hsp70:angptl2), that overexpresses angptl2 upon heatshock induction. Heatshocked Tg embryos showed a significant increase in cmyb- and runx1-positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf), suggesting that angptl2 can sufficiently expand definitive HSCs during development. We then assessed the effects of angptl1 and/or angptl2 morpholino (MO) knockdown. Single MO knockdown resulted in a decrease in cmyb- and runx1-positive HSCs in the AGM whereas embryos injected with combined angptl1 and 2 MOs (double morphants) exhibited more severe phenotype in which HSCs were completely absent. Angptl regulation of HSC development may occur by stimulating the production or specification of a patent hemogenic endothelium, as the double morphants displayed a disruption of vascular specification at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4 in the dorsal aorta. These double morphants also exhibited disrupted intersegmental blood vessel sprouting. Because proper patterning of the developing blood vessels is a prerequisite for subsequent HSC formation, these results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium specification. To dissect the mechanism of angptl signaling, we asked whether there is a genetic interaction between notch and angptl signaling since the observed phenotype in the angptl double morphants resembled that in the notch mutant mindbomb, mib. We first monitored notch signaling using a transgenic notch reporter zebrafish line that expresses EGFP when the active component of notch signaling, notch intracellular domain (NICD), is expressed. MO knockdown of angptls 1 and 2 resulted in an absence of notch signaling, particularly in the vasculature. Interestingly, overexpressing angptl2 by crossing Tg(hsp70:angptl2) into the mib mutant showed a significant rescue of the HSC phenotype, implying that angptl2 acts downstream of mib signaling. When MOs against angptls 1 and 2 were then injected into the NICD overexpressing Tg embryos, we found that NICD restored HSC formation in the angptl double morphants. Together, these data corroborate the hypothesis that angptls can regulate notch signaling by acting downstream of notch ligand-receptor interaction and upstream of NICD during definitive hematopoiesis. To further probe at the downstream signaling of angptls since their receptor is currently unknown, we found that both angptls can stimulate activation of akt in cultured endothelial cells. To investigate whether akt is involved in angptl signaling during development, we injected the constitutively active myr-akt into angptl double morphants and observed rescue of both HSC and vascular defects. This strongly indicates that akt is the key signaling component downstream of angptl signaling during these biological processes. Injection of myr-akt can also rescue HSC defects in mib, suggesting that notch signaling also requires akt activation for definitive HSC formation. Taken together, our data demonstrate that the angptls are required for HSC development through notch and akt signaling, by coordinating the production of a functional hemogenic endothelium. 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.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Deceptively simple (Wnt Won the War: Antagonistic Role of Wnt Over Shh Controls Dorsoventral Patterning of the Vertebrate Neural Tube by Fausto Ulloa and Elisa Martí, Dev Dyn 239:69–76) Dorsoventral (D/V) patterning in the neural tube (nt) appears to operate on principles of straightforward design. In addition to other molecules, opposing signals from ventrally localized Sonic hedgehog (Shh) and dorsally localized Wnts combine to produce a readout of cell types localized to specific points along the D/V axis. Although mechanisms of patterning by Shh are relatively well understood, those of Wnts are not. In this review, Ulloa and Marti argue that one of the main functions of Wnts in this context is to promote dorsal identities while inhibiting ventral ones. They present evidence that at the dorsal nt, Wnt and/or its downstream effector β-catenin directly activates expression of Gli3, the repressor form of which inhibits expression of Shh target genes. Wnt/β-cat also appears to specify fates by mechanisms independent of the Shh/Gli pathway. Whether in the latter case Wnt/β-cat affects patterning directly, or by means of cross-talk with other molecular pathways, remains to be determined. The data presented reinforce the notion that a good design looks simple but masks the complexity beneath it. Diffusing the destruction complex (Xenopus Axin-Related Protein: A Link Between Its Centrosomal Localization and Function in the Wnt/β-Catenin Pathway by Evguenia M. Alexandrova and Sergei Y. Sokol, Dev Dyn 239:261–270) Wnt pathway's “destruction complex” is merciless. In the absence of Wnt ligand, the complex marks β-catenin—the transcriptional activator of Wnt targets—for destruction. How does the cell keep this killing machine at bay? Alexandrova and Sokol find that a destruction complex component, Xenopus axin-related protein (XARP) localizes to centrosomes and basal bodies—modified centrosomes that initiate and maintain cilia growth. Furthermore, overexpressed XARP lacking most of its DIX domain, or adjacent conserved, charged residues, leaves its centrosomal post and becomes distributed throughout the cytoplasm. These mutants also display reduced binding to Dishevelled (Dsh), a protein that promotes β-catenin stability and thus activation of Wnt targets. Accordingly, Wnt reporter assays show that XARP DIX domain mutants have increased inhibitory activity. The results illuminate the possibility that association with centrosomes may attenuate the destruction complex. This is an intriguing finding particularly in light of other work suggesting that cilia, the assembly of which depends on centrosomal centrioles, are important for normal Wnt activity. Future studies will gauge the relative contributions of subcellular localization and Dsh binding in keeping destruction complex activity in check. Demistifying iguana (The Iguana/DZIP1 Protein Is a Novel Component of the Ciliogenic Pathway Essential for Axonemal Biogenesis by Shang Yew Tay, Xianwen Yu, Kangli Noel Wong, Pallavi Panse, Chee Peng Ng, and Sudipto Roy, Dev Dyn 239:527–534) The iguana (igu) mystery first crept out from the shadows 6 years ago. Zebrafish lacking Dzip1, the zinc finger protein encoded by igu, display altered Sonic hedgehog (Shh) signaling. Yet it remained a puzzle as to how igu fit into the Shh pathway. Inspired by work demonstrating that immotile, primary cilia are a repository for Shh pathway components, Tay and colleagues postulated that igu might have a primary function in ciliogenesis. Confirming their hypothesis, they present here that igu mutants lack primary cilia. Furthermore, in igu cells that normally bear primary cilia, basal bodies—modified centrosomes from which cilia are derived—are present in normal numbers and are correctly localized. However, characteristic ciliary pits and axonemes—ciliary inner cytoskeletal structures—are absent. Agreeing with a role in cilia growth, stable transgenic zebrafish lines bearing gfp-igu display puncta near or colocalized with basal bodies. Of interest phenotypes in igu motile cilia, reduced cilia numbers and/or length, are less penetrant. These effects could be due to functional redundancy of igu/dzip1 with a paralogous gene, dzip1-like. With the original iguana mystery solved, a new one rears its head. How does igu/dzip1 regulate ciliary pit formation and axoneme growth?

Cis-acting elements responsible for dopaminergic neuron-specific expression of zebrafish slc6a3 (dopamine transporter) in vivo are located remote from the transcriptional start site

The purpose of this study was to analyze the transcriptional regulation of the zebrafish solute carrier family 6 member 3 gene (slc6a3, dopamine transporter, dat), as a first step towards isolating regulatory sequences useful for driving transgene expression within dopaminergic neurons of the zebrafish CNS in vivo. We found that the 3.0 kb slc6a3 mRNA is expressed in each of the major groups of dopaminergic neurons previously identified in the zebrafish CNS. The slc6a3 gene spans >20 kb of genomic DNA and contains 15 exons. The genomic organization of slc6a3 is highly conserved with respect to its human orthologue, including the presence of an untranslated first exon. The promoter lacks a canonical TATA box and there are multiple transcriptional start sites. Functional analysis of cis-acting elements responsible for the expression pattern of slc6a3 was carried out by generating stable transgenic zebrafish lines expressing fluorescent reporters under transcriptional control of fragments of slc6a3 genomic sequence. The region between −2 kb and +5 kb with respect to the transcriptional start site contains the core slc6a3 promoter, in addition to neuronal enhancers and/or non-neuronal repressors that restrict expression to the CNS, but this region lacks cis-acting elements responsible for slc6a3 expression in dopaminergic neurons. The upstream sequence between −6 kb and −2 kb contains an enhancer element that drives slc6a3 expression in dopaminergic neurons of the pretectal region, and additional sequences that partially repress expression in non-dopaminergic neurons. However, expression of slc6a3 in dopaminergic neurons of the ventral diencephalon and telencephalon is dependent on elements that lie outside the region −6 kb to +5 kb. These data provide a detailed analysis of the slc6a3 gene and show that its expression in different populations of dopamine neurons is driven by discrete enhancers, rather than a single target sequence for a terminal factor involved in specifying neurochemical phenotype.

Zebrafish Dorsal Root Ganglia Neural Precursor Cells Adopt a Glial Fate in the Absence ofNeurogenin1

The proneural transcription factor neurogenin 1 (neurog1) has been shown to be a key regulator of dorsal root ganglion (DRG) neuron development. Here we use a novel transgenic zebrafish line to demonstrate that the neural crest population that gives rise to DRG neurons becomes fate restricted to a neuronal/glial precursor before the onset of neurog1 function. We generated a stable transgenic zebrafish line that carries a modified bacterial artificial chromosome that expresses green fluorescent protein (GFP) under the control of the neurog1 promoter [Tg(neurog1:EGFP)]. In contrast to previously described neurog1 transgenic lines, Tg(neurog1:EGFP) expresses GFP in DRG neuronal precursors cells as they migrate ventrally and after their initial differentiation as neurons. Using this line, we are able to track the fate of DRG neuronal precursor cells during their specification. When Neurog1 function is blocked, either by neurog1 morpholino antisense oligonucleotide injection or in neurog1 mutants, GFP expression initiates in neural crest cells, although they fail to form DRG neurons. Rather, these cells take on a glial-like morphology, retain proliferative capacity, and express glial markers and become associated with the ventral motor root. These results suggest that, within the zebrafish neural crest, there is a fate-restricted lineage that is limited to form either sensory neurons or glia in the developing DRG. Neurog1 acts as the key factor in this lineage to direct the formation of sensory neurons.