Zebrafish Symposium Compiled Abstracts 2015 - [email protected]/zebrafish/files/2015/04/Zebrafish-Symposium-Abstracts.pdf · (TPHP; also abbreviated TPP), tetrabromobisphenol

2015 Duke Zebrafish Research Symposium Abstracts

Session 1: Environment

Talk 1: 1000-1015

Andrey Massarsky Postdoctoral Fellow Di Giulio Lab

Impacts of total particulate matter from cigarette smoke on early development of zebrafish (Danio rerio)

Cigarette smoke has been associated with a number of pathologies; however mechanisms leading to developmental effects are yet to be fully understood. This study examined the effects of Total Particulate Matter (TPM) from 3R4F reference cigarettes on the early development of zebrafish (Danio rerio). Zebrafish embryos were exposed to two concentrations of TPM, corresponding to 0.4 and 1.4 mg/mL equi-nicotine units. The exposures (single and acute) began at 2 h post fertilization (hpf) and lasted until 96 hpf. Several physiological parameters were assessed during or after the exposure. We show for the first time in a zebrafish model that TPM increased mortality, delayed hatching, and increased the incidence of pericardial edema and other deformities (assessed by microscopy). TPM exposure also increased the incidence of cranial hemorrhage (microscopic observations and o-Dianisidine staining) and disrupted the proper angiogenesis of the major vessels in the brain (alkaline phosphatase staining). Moreover, TPM exposure reduced the larval body length, and decreased the heart rate. Several oxidative stress parameters were also affected, including glutathione levels and activities of several antioxidant enzymes, suggesting that oxidative stress contributes to the toxicity of TPM. TPM-exposed zebrafish also differed behaviorally: at 24 hpf the embryos had a higher frequency of spontaneous contractions (microscopic observations) and at 144 hpf the larvae displayed hyperactivity, as assessed by monitoring the swimming using Danio Vision. It is important to note that the effects reported for TPM are not attributable to nicotine, since embryos treated with nicotine alone did not differ from the control embryos. This study demonstrates that TPM disrupts early development in zebrafish.

Talk 2: 1015-1030 Stephanie Padilla Faculty U.S. Environmental Protection Agency

Neurodevelopmental Behavioral Effects of Ten Flame Retardants and

Related Chemicals in Zebrafish Larvae As the older flame retardants are phased out, new chemical flame retardants are being adopted for use in consumer and electronic products. Little is known, however, about the developmental neurotoxicity of these replacements. This study evaluated the neurobehavioral effects of developmental exposure to t-butylphenyl diphenyl phosphate (BPDP), 2-ethylhexyl diphenyl phosphate (EHDP), isodecyl diphenyl phosphate (IDDP), isopropylated phenyl phosphate (IPP), tricresyl phosphate (TMPP; also abbreviated TCP), triphenyl phosphate (TPHP; also abbreviated TPP), tetrabromobisphenol A (TBBPA), tris (2-chloroethyl) phosphate (TCEP), tris (1,3-dichloroisopropyl) phosphate (TDCIPP; also abbreviated TDCPP), tri-o-cresyl phosphate (TOCP), or 2,2-,4,4’-tetrabromodiphenyl ether (BDE-47) in zebrafish (Danio rerio) larvae. Larvae (n≈24 per dose per compound) were exposed to test compounds (0.4 - 120 µM) at sub-teratogenic concentrations developmentally, and locomotor activity was assessed at 6 days post fertilization using a 2 hour test of light/dark alterations employing different levels of light. The positive control chemicals BDE-47, chlorpyrifos or heptachlor all produced effects on zebrafish behavior at 6 days post fertilization. All the test flame retardant chemicals except BPDP, IDDP and TBBPA also produced behavioral effects after developmental exposure, with some of them eliciting effects at the submicromolar level. Moreover, the pattern of effects after developmental exposure was distinct from the pattern of acute pharmacological effects of the same chemicals, indicating that our experimental design was able to distinguish between developmental and pharmacological mechanisms. The results indicate that these replacement flame retardants may have neurodevelopmental effects in a vertebrate model system. This abstract may not necessarily reflect official Agency policy.

Talk 3: 1030-1045 Amanda Cass Postdoctoral Fellow Roberts - North Carolina State University

Danio in a comparative context: the developmental basis of dietary adaptation in the cichlid fishes of Lake Malawi

The zebrafish has a simple gut without obvious rostro-caudal regionalization; it has no stomach, and no morphological distinction between small and large intestine. This particularly derived state makes a direct comparison to the generalized tetrapod condition challenging. However, molecular data have shown that the zebrafish gut is regionalized, providing phylogenetically robust markers for identifying gut functional regions in species with cryptic gut regionalization, and a mechanism for nesting the zebrafish condition in a larger comparative framework. Species adapted to different diets can provide a unique insight into the molecular, morphological, and microbial characteristics of trophic differentiation. Through experimental feeding regimes, these species can also help to elucidate the effect of maladaptive diets on phenotype and development of disease states. The cichlid fishes of East Africa have rapidly evolved an exceptional degree of trophic diversity, making them an excellent system for studying dietary adaptation. Thus far, we have shown that there are species-specific differences in gut length and microbial community between species adapted to different diets, and using markers developed in zebrafish we have shown that the relative proportion of small and large intestinal length also varies across species. Due to their recent divergence, our study species species can be readily hybridized for gene mapping, facilitating the identification of individual genetic variations underlying the described gut phenotypes and trophic adaptation. These data will directly inform studies of dietary disease, provide candidates for better understanding the unusual zebrafish condition, and help us to better understand the mechanism for an important axis of evolutionary diversification among cichlids and across vertebrates.

Session 2: Human Disease and Tissue Repair

Talk 4: 1055-1115 Blair Anderson Graduate Student CHDM - Ashley-Koch

Functional annotation of APOL1 risk alleles for Sickle Cell Nephropathy and possible genetic interaction with MYH9

African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed largely to coding variants (G1 and G2) in apolipoprotein L1 (APOL1); however, there is little direct functional evidence supporting the role of this protein in renal function. Here, we combined genetics and in vivo modeling to examine the role of apol1 in glomerular development and pronephric filtration and to test the pathogenic potential of APOL1 G1 and G2. Suppression of apol1 in zebrafish embryos results in podocyte loss and generalized edema, likely due to glomerular filtration defects. Complementation of apol1 morphants with wild-type human APOL1 mRNA rescues the observed defects. However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G). In vivo complementation studies of the G2 risk allele also indicate that the variant is deleterious to protein function. Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein. In Sickle Cell Disease (SCD) patients, we reported previously a genetic interaction between APOL1 and MYH9. Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects. However, upon induction of anemia, we observed a significantly exacerbated nephropathy phenotype. Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9. Our data indicate a critical role for APOL1 in renal function that is compromised by nephropathy-risk encoding variants. Moreover, our interaction studies indicate that the MYH9 locus is also relevant to the phenotype in a stressed microenvironment and suggest that consideration of the context-dependent functions of both proteins will be required to develop therapeutic paradigms.

Talk 5: 1115-1130 Maria Kousi Postdoctoral Fellow Center for Human Disease Modeling

ZNHIT3 is defective in PEHO syndrome, an inherited severe encephalopathy

Progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO syndrome) is a severe autosomal recessively inherited progressive encephalopathy presenting in early childhood and characterized by significant loss of cerebellar granule neurons. We report that PEHO syndrome is caused by a homozygous missense mutation in ZNHIT3 encoding a zinc finger HIT domain-containing protein 3, previously implicated as a transcriptional co-regulator. The p.Ser31Leu PEHO mutation affects a highly conserved residue of ZNHIT3 and renders the mutant protein unstable when transiently expressed. Knockdown of znhit3 in zebrafish embryos resulted in morphant embryos that recapitulate a range of disease symptoms seen in PEHO patients, including cerebellar defects, microcephally and edema. The observed defects could be rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the p.S31L allele is damaging and sufficient to cause disease. In mouse cerebellum ZNHIT3 is prominently expressed in granule cells. Knockdown of mouse Znhit3 in cultured granule neurons and in ex vivo cerebellar slices indicated that ZNHIT3 is indispensable for granule neuron survival and migration, compatible with the neuropathology in human patients.

Talk 6: 1130-1145 Jordan Ferguson Technician/Manager NC State University College of Veterinary Medicine-Population Health and Pathobiology

Assessment of BRCA2-associated genomic instability in cancer with a brca2-mutant zebrafish model

Heritable mutations in known cancer-associated genes, such as BRCA2, are strongly linked to cancer risk. However, numerous collaborating genetic mutations contribute significantly to cancer development, and often occur via amplification and deletion events (copy number alterations; CNA). Defining recurrent cancer-associated CNA in a genetically tractable model such as the zebrafish will facilitate identification and assessment of novel cancer-associated genes. To assess the impact of heritable BRCA2 mutation on genomic instability in zebrafish cancer, we performed a pilot analysis of cancers from a brca2-mutant zebrafish model. We directly compared cancers from zebrafish with and without brca2 mutation on a tp53-mutant background, as we have previously shown that brca2 mutation status impacts several features of carcinogenesis on this background. Zebrafish cancers were analyzed by histology to determine tumor type and by flow cytometry to determine ploidy. A subset of cancers were analyzed by high resolution oligonucleotide array comparative genomic hybridization (oaCGH) to assess for specific CNA linked to brca2 mutation status. Zebrafish cancers were histologically consistent with malignant peripheral nerve sheath tumor or undifferentiated sarcoma, as we reported previously. Patterns of ploidy in brca2-associated zebrafish cancers were similar to BRCA2-associated human cancers and different from non-brca2-associated zebrafish cancers. oaCGH analysis identified multiple discrete CNA linked to brca2 mutation status in zebrafish cancers. These findings suggest that brca2 mutation exerts a specific effect on genomic instability in zebrafish cancers that is (1) comparable to BRCA2-associated human cancers, and (2) distinct from the effects of concurrent tp53 mutation. These data additionally support the feasibility of oaCGH as a method for detecting discrete CNA linked to brca2 mutation in zebrafish cancers. This approach will allow us to define recurrently disrupted genomic regions in brca2-associated zebrafish cancers and identify conserved candidate genes that can be efficiently assessed in this model for their functional contributions to carcinogenesis.

Talk 7: 1145-1200 Junsu Kang Postdoctoral Fellow Ken Poss lab

Session 3: Novel Tools

Talk 8: 1300-1315 Rebecca Beerman Postdoctoral Fellow David Tobin Lab

Visualization and Manipulation of Calcium Dynamics in Inflammation and Infection

Innate immune cells are known to utilize calcium signaling during chemotaxis, phagocytosis and phagolysosome maturation. Pathogens can manipulate each of these processes to promote their own survival. Studies of immune cell calcium dynamics have been largely restricted to ex vivo cell culture systems. Here we combine light sheet microscopy with genetically encoded calcium indicators to analyze whole-animal calcium dynamics in live animals during inflammation and infection. We have developed multiple transgenic zebrafish lines to provide an unprecedented window into calcium signaling in neutrophils and macrophages as they migrate toward a wound site during the first steps of a sterile inflammatory response and in the context of infection with diverse bacterial pathogens. We find that migrating neutrophils display unique calcium flux depending on the distance to inflammatory cues at the wound site. To assess the role of these patterns in vivo, we developed a broadly applicable approach to pharmacologically reprogram calcium levels in individual cell types. We demonstrate a functional requirement for calcium signaling in neutrophils in vivo in the context of wound healing and bacterial infection. Finally, we observe differential calcium signaling in macrophages infected with Mycobacterium marinum compared with other bacterial species. These findings represent the first in vivo analysis of immune cell calcium dynamics in whole animals during mycobacterial infection.

Talk 9: 1315-1330 James Minchin Postdoctoral Fellow John Rawls Lab

Modeling pancreatic steatosis in zebrafish  Pancreatic  disease  represents  a  cluster  of  highly  related  and  interacting  disorders,  including  pancreatitis,  pancreatic  cancer,  exocrine  pancreatic  insufficiency  and  diabetes  mellitus.  Cumulatively,  pancreatic  disease  effects  over  9.3%  of  the  US  population,  and  substantially  increases  risk  for  cardiovascular  disease  and  death.  Fatty  infiltration  of  the  pancreas  (pancreatic  steatosis)  is  associated  with  all  forms  of  pancreatic  disease.  However,  remarkably  little  is  understood  regarding  the  etiology  and  physiological  effect  of  pancreatic  steatosis,  in  part  due  to  the  lack  of  a  tractable  experimental  model.  Here,  we  undertake  initial  experiments  to  establish  zebrafish  as  a  model  for  pancreatic  steatosis.  First,  we  utilize  in  vivo  imaging  to  document  the  progressive  accumulation  of  lipid  within  the  zebrafish  exocrine  pancreas.  Second,  we  undertake  a  small-­‐scale  forward  genetic  screen  to  identify  a  recessive  zebrafish  mutant  resistant  to  pancreatic  steatosis.  Although  preliminary,  our  results  indicate  that  unbiased  forward  genetics  in  zebrafish  will  provide  a  useful  tool  to  understanding  the  etiology  and  physiological  consequences  of  pancreatic  steatosis.  

Talk 10: 1330-1345 Eric Walton Graduate Student David Tobin Lab High Resolution Melting Analysis: A rapid, reliable method for genotyping

mutant zebrafish larvae Genome editing via site-directed nucleases (i.e., ZFNs, TALENs, CRISPR/Cas9) has become an invaluable tool for facilitating reverse genetics studies in the zebrafish. However, the high degree of mosaicism within founders, in both lesion frequency and type, postpones the full utility of the system until a useful allele can be isolated and propagated. Crossing F1s heterozygous for the same mutant allele is the most reliable way to generate a population of larvae in which experiments can be carried out in an unbiased, internally controlled manner with respect to genotype. For the results to be meaningful, however, each larva must be genotyped at the conclusion of the experiment. In order to retain the numerical advantage afforded by use of zebrafish larvae, a fast, accurate method for genotyping must be implemented. The T7 endonuclease assay, which results in DNA cleavage at sites of mismatched base pairing, has been widely employed for determining the presence of a mutant allele. However, this method is cumbersome and time-consuming when involving a large number of samples. Furthermore, it cannot distinguish homozygous wild-type from homozygous mutant alleles containing small indels, which are the most common mutations observed following repair of the nuclease-induced lesions. Here, I present an alternative: High Resolution Melting Analysis (HRMA). HRMA takes advantage of the melting temperature (Tm) differences among small PCR amplicons containing either only wild-type sequence, only mutant sequence, or a combination of both. Following a rapid, easily scalable gDNA extraction, the PCR and Tm measurements are carried out using a standard qPCR instrument. In this way, dozens of larvae may be genotyped in parallel in just over 2 hours, and several hundred within a single day. Thus, the sensitivity, reproducibility, and speed of HRMA compared to other genotyping methods make it particularly well suited to experiments using the zebrafish model system.

Talk 11: 1345-1405 Jared D. Chrispell Postdoctoral Fellow Weiss Lab - University of North Carolina at Chapel Hill

Role of GRK Expression and cAMP in the Recovery of the Cone Photoresponse in Zebrafish Larvae

In the vertebrate retina, termination of the photoresponse is essential for continued function of photoreceptor cells in response to repeated visual stimuli Abrogation of visual signaling requires phosphorylation of photoactivated visual pigments in rods and cones by the photoreceptor cell-specific G protein-coupled receptor kinases (GRKs), GRK1 and GRK7. Previous work by our laboratory demonstrated that both GRK1 and GRK7 are expressed in cones in humans and other vertebrates including zebrafish. In zebrafish, cones mature prior to rods. Electroretinogram (ERG) analyses detect cone photoresponses as early as 4 days post fertilization (dpf), whereas responses in rods appear between 15 and 21 dpf. Therefore, the zebrafish retina at 4-7 dpf serves functionally as an all-cone retina. To determine the contribution of these GRKs to recovery in cones, we created Grk1b-/- and Grk7a-/- zebrafish and analyzed larvae using ERG analysis. Deletion of either Grk1b or Grk7a leads to decreased recovery to successive stimuli compared to wild type larvae. Therefore, our results indicate the participation of both GRKs in the termination of signaling. Our laboratory has also demonstrated that these GRKs are substrates for cAMP-dependent protein kinase (PKA) in vivo, and that phosphorylation reduces the ability of GRK1 and GRK7 to phosphorylate their substrates. GRK phosphorylation is high in the dark and low in the light, corresponding to changing levels of photoreceptor cAMP. To evaluate the influence of increasing cAMP on the cone photoresponse, we incubated zebrafish in forskolin and measured the effect on cone recovery by ERG analysis of zebrafish larvae. Larvae display a decreased rate of recovery to successive stimuli when exposed to forskolin compared to vehicle. These observations indicate a potentially critical role for cAMP in the regulation photoresponse recovery in cones. Future studies will determine whether the observed decrease in recovery occurs through phosphorylation of GRKs.

Session 4: Skeleton

Talk 12: 1415-1435 Mayssa Mokalled Postdoctoral Fellow Ken Poss lab

Talk 13: 1435-1450 AtLee Watson Graduate Student North Carolina State University Kullman Laboratory

An AOP Approach to Modeling Alterations in Axial Bone Development: A Case Study with TCDD and Japanese Medaka

Studies from mammalian, fish, and in vitro models have identified bone and cartilage development as sensitive targets for dioxin and other aryl hydrocarbon receptor ligands. Teleost fish models have gained considerable traction recently as alternative in vivo models of human skeletal development. In this study, we assess how embryonic TCDD exposure impacts axial osteogenesis in the teleost Japanese medaka (Oryzias latipes). Embryos from three transgenic medaka lines (twist:EGFP, osx:mCherry, col10a1:nlGFP) were exposed to a range of TCDD concentrations (ppt-ppb) and reared to 10 dpf (eleutheroembryo) and 20 dpf (larval). Individuals were stained for mineralized bone matrix and imaged in vivo to assess skeletal alterations within medial and rostral vertebrae in relation to qualitative spatio-temporal analysis of osteoblast and osteoblast precursor cell populations. Exposure to 100 ppt TCDD impacted axial bone development through an overall attenuation of skeletal mineralization resulting in truncated centra, and reduced neural and hemal arch lengths. This effect was more pronounced at the larval stage. Effects on mineralization were consistent with modifications in cell number and cell localization of transgene-labeled osteoblast and osteoblast progenitor cells. Through targeted RT-PCR, we confirmed altered transgene expression (twist, osx, and col10a1), and identified a significant reduction in osc, a marker of differentiated osteoblasts. A global transcriptomic analysis using RNA-Seq revealed reveal additional genes mediating inflammatory disease, connective tissue disorders, and musculoskeletal disorders. Interestingly, Il-1beta was heavily enriched within these disease pathways, suggesting that inflammation may also mediate deficits in early bone formation. Current in vivo and in vitro studies are underway to investigate the molecular initiating events involving Il-1beta. Taken together, our results indicate that early sublethal TCDD exposure impacts axial bone development at apical endpoints through proliferation and/or differentiation of osteoblasts and osteoclasts, which may be mediated in part by an inflammatory response.

Talk 14: 1450-1505 Valerie Tornini Graduate Student Ken Poss Lab

Poster abstracts

Poster 1 Mark Cronan Postdoctoral Fellow David Tobin lab

A macrophage-epithelial transition underlies tuberculous granuloma formation

Mycobacterium tuberculosis infection in humans triggers the formation of granulomas, tightly organized immune structures that are the hallmark of tuberculosis. For more than a century, pathologists have noted the characteristic epithelioid appearance of granuloma macrophages. However, the molecular architecture of these cells and their impact on granuloma structure and infection are unclear. Here we show that induction of bona fide epithelial markers by granuloma macrophages is a defining feature of the tuberculous granuloma. Using live imaging and lineage-tracing in a zebrafish-Mycobacterium marinum model, we find that macrophages entering the granuloma undergo a phenotypic transformation in which they induce adherens junctions between macrophages, marked by macrophage-specific expression of the classic epithelial marker E-cadherin. Cell-specific disruption of E-cadherin function causes large, disordered granulomas, compromised macrophage adherens junctions, ragged borders, and multifocal disseminated infections. Finally, we show in clinical specimens that macrophages in Mycobacterium tuberculosis-induced granulomas undergo this same transition in human disease. The phenotypic transformation of mycobacterial granuloma macrophages closely parallels mesenchymal-to-epithelial transitions in development and cancer, E-cadherin-dependent processes that profoundly regulate cell fate and function.

Poster 2 Chen-Hui Chen Postdoctoral Fellow Poss lab

Poster 3 Matt Foglia Graduate Student Poss Lab

Poster 4 I-Chun Tsai Postdoctoral Fellow Center for Human Disease Modeling

Genome-wide siRNA Screening identifies the functional interaction between ciliopathies and proteasome

The ciliopathies are a group of phenotypically overlapping disorders caused by structural or functional defects in the primary cilium or its anchoring structure, the basal body. Since cilia are present on almost all vertebrate cell types, disruption of ciliary function has been shown to cause numerous human genetic disorders, including Polycystic Kidney Disease (PKD), Nephronophthisis (NPHP) and Bardet-Biedl Syndrome (BBS). Although improved sequencing techniques have facilitated improved diagnostics, these disorders remain untreatable. To date, the primary cilium has been linked to several signaling pathways crucial for proper morphologic development, including Wnt, Shh, and Notch. Therefore, we hypothesized that the identification of the genes whose suppression rescues the aberrant signaling transduction caused by ciliary dysfunction might identify potential therapeutic targets. We executed a genome-wide RNAi screen, aiming to identify suppressor genes of hyper Wnt/β-cat signaling caused by loss of BBS4, a basal body protein that when mutated causes BBS. We have identified thirteen genes as the suppressors of Bbs4 depletion. Among them, USP35, is a ubiquitin specific peptidase that acts as a negative regulator in ubiquitin proteasome system-mediated protein degradation; this finding is consistent with our previous observation that bbs4 depletion perturbs proteasome-mediated degradation, leading to the accumulation of signal effectors. We suspected that the inhibition of USP35 could facilitate the clearance of signaling components, thereby ameliorating the phenotypes caused by bbs4 depletion. To test this hypothesis, we asked whether suppression of usp38 (the zebrafish ortholog of USP35) could rescue bbs4-depletion induced phenotypes in zebrafish. We found that Usp38 suppression ameliorated hallmark ciliopathy defects including impaired convergent extension, cerebellum development and renal tubule convolution. Together, our findings demonstrate the efficiency of usp38 suppression to rescue the ciliopathy phenotypes in a zebrafish model, and open a novel potential avenue to improve the prognosis for ciliopathies through attenuated USP35 activity in patients.

Poster 5 Steven Canon Brodar Technician/Manager Center for Human Disease Modeling

PCM1 is necessary for the maintenance of focal ciliary integrity and dopamine signaling in the postnatal brain

The neuronal primary cilium and the pericentriolar matrix (PCM) have established roles in neurogenesis. However, the role of this subcellular region in the postnatal brain is not clear. Here we show that perturbation of the PCM alters brain anatomical and behavioral patterns in a spatial and temporal manner. Ablation of the pericentriolar material 1 gene (Pcm1) in the mouse leads to enlarged lateral ventricles and sensorimotor gating deficits in adult, but not juvenile animals. Cytoanatomical analyses revealed progressive shortening of the primary cilium in discrete brain regions, most prominently the CA1 region of the hippocampus, the amygdala, and the prelimbic cortex. These were associated with an aggregation of dopamine D2 receptors (D2R) at the PCM and failure to fully ameliorate adult sensorimotor gating defects by treatment with antipsychotic drugs. Given the previous association of PCM1 with schizophrenia (SZ) and our present observations, we sequenced the locus in a cohort of SZ patients unresponsive to antipsychotics. In doing so, we identified a significant enrichment for rare alleles in cases, the effects of which were established by systematic modeling of all rare variants in a zebrafish pcm1 model. Together, our data highlight a role for the PCM in the postnatal brain; they suggest that a progressive degenerative ciliary/PCM phenotype can induce behavioral defects in adults, and intimate a role for PCM1 in severe psychosis.

Poster 6 Molly Matty Graduate Student MGM Duke University David Tobin Lab

Host-Directed Therapies for Mycobacterial Disease Mycobacterium tuberculosis (Mtb) infections kill over one million people annually worldwide. The current course of treatment has resulted in drug-resistant selection in bacterial populations. Multi-drug resistant Mtb is a global problem that cannot be solved using the current antibiotic therapies alone. We have used Mycobacterium marinum in zebrafish as a model for human:Mtb infection in an in vivo chemical screen for host-directed and anti-mycobacterial drugs using a chemical library of FDA-approved compounds. The hits from this screen represent a diverse set of novel molecules that reduce bacterial load in a whole animal host. Further investigation of these drugs and their targets is currently in progress.

Poster 7

Stefan Oehlers Postdoctoral Fellow MGM Duke University David Tobin Lab

Interception of host angiogenic signalling limits mycobacterial growth Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularisation of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model characterised by organised granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule VEGFA. Pharmacological inhibition of the VEGF pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergise with the first-line anti-tubercular antibiotic rifampicin as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data suggest that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting TB therapy, particularly in extensively drug-resistant disease where current antibiotic regimens are largely ineffective.

Poster 8

Rachel Shaffer Graduate Student Toxicology, UNC-Chapel Hill Padilla Lab

Acute Neurobehavioral Toxicity of Flame Retardant Replacement Compounds in Zebrafish Larvae

As polybrominated diphenyl ethers (PBDEs) are phased out, numerous compounds are emerging as potential replacement flame retardants for use in consumer and electronic products. Little is known, however, about the neurobehavioral toxicity of these replacements. This study evaluated the acute neurobehavioral effects of t-butylphenyl diphenyl phosphate (BPDP), 2-ethylhexyl diphenyl phosphate (EHDP), isodecyl diphenyl phosphate (IDDP), isopropylated phenyl phosphate (IPP), tricresyl phosphate (TMPP), triphenyl phosphate (TPHP), tetrabromobisphenol A (TBBPA), tris (2-chloroethyl) phosphate (TCEP), tris (1,3-dichloroisopropyl) phosphate (TDCIPP), tri-o-cresyl phosphate (TOCP), and 2,2-,4,4’-tetrabromodiphenyl ether (BDE-47) in zebrafish (Danio rerio) larvae. Larvae were exposed to test compounds (dose-response; varying doses between 0.4 - 120 µM ; n≈24 larvae per dose per compound) at 6 days post fertilization, and locomotor activity was assessed using automated video tracking in 96-well microtiter plates during three sequential dark/light/dark test periods beginning 30 minutes after chemical exposure. All compounds produced changes in activity, usually manifested as hypoactivity in the dark periods and hyperactivity in the light periods. Across all doses and time periods, PBDE-47 and TCEP produced the weakest acute effects, while EHDP, IPP, EPHP, and TBBPA produced the strongest effects. These data indicate that acute exposure to selected flame retardant replacement candidates alters swimming behavior in young zebrafish. This abstract may not necessarily reflect official Agency policy.

Poster 9

Alexander Merriman Undergraduate Student Department of Cell Biology, Duke University Ken Poss Lab

Poster 10 Shad Mosher Postdoctoral Fellow USEPA NCCT

Toxicity Screening of the ToxCast Phase II Chemical Library Using a Zebrafish Developmental Assay

As part of the chemical screening and prioritization research program of the US EPA, the ToxCast Phase II chemicals were assessed using a vertebrate screen for developmental toxicity. Zebrafish embryos (Danio rerio) were exposed in 96-well plates from late-blastula stage (6hr post fertilization, pf) through day 5pf (1-2 days post-hatch). All exposures were by immersion and renewed daily. The 775 chemicals included food additives, consumer use product ingredients, pesticides, failed pharmaceuticals, and “green” plasticizers (http://epa.gov/ncct/toxcast/chemicals.html). Intra- and inter-plate replicates were included for quality control. Developmental toxicity was initially assessed using a single nominal concentration of 80 µM. On day 5pf, larvae were moved from exposure solution to a control solution without chemical, and on day 6pf were assessed for overt toxicity (i.e., death, non-hatching and dysmorphology; n=4 embryos per chemical). Dysmorphology was a combined score using both in-life observation and Brightfield, high-content image analysis. All chemicals positive at the single concentration (361) and a selection of negatives (55) were confirmed and further assessed for potency using a Dose-Response Study (8-point, semi-log concentration curve: n=3 embryos per concentration). Overt toxicity was noted with 40% of the chemicals tested, compared to 62% positive chemicals when the ToxCast Phase I library, consisting of mostly pesticide active ingredients, was previously tested. As with the Phase I library, the octanol-water partition coefficient (logkow ) of the Phase II library chemicals was positively correlated with overt toxicity: ranging from 4% positive chemicals with logkow <0, to 70% positive chemicals with logkow of 4 to 6. These data demonstrate the utility of zebrafish in medium-throughput chemical screening for detection of adverse developmental outcomes. This abstract may not necessarily reflect official Agency policy.

Poster 11 Anne Knecht Lab Manager Cell Biology, Duke University Ken Poss Lab

Poster 12 Leigh Ann Samsa Graduate Student UNC-CH – Cell Biology and Physiology Jiandong Liu Lab

Heartbeat activates endocardial Notch signaling to modulate cardiac chamber maturation

Congenital heart disease often features structural abnormalities that emerge during development. Accumulating evidence indicates a critical role for cardiac contraction, and the resulting fluid forces, in shaping the heart, yet the molecular basis of this function is largely unknown. Using zebrafish as a model of early heart development, we investigated the role of cardiac contraction in chamber maturation, focusing on the formation of muscular protrusions called trabeculae. By genetic and pharmacological ablation of cardiac contraction in Notch reporter fish, we show that cardiac contraction is required for trabeculation through its role in initiating notch1b transcription in the ventricular endocardium. Close examination of Notch reporter embryos revealed that Notch1 signaling is detectable throughout the ventricular endocardium shortly after initiation of contraction, but becomes restricted to the endocardial cushions during trabeculation. We further demonstrate that Notch1 activation induces the expression of its downstream effectors ephrinb2a (efnb2a) and neuregulin 1 (nrg1) in the endocardium to promote trabeculation, and that forced Notch activation in the absence of cardiac contraction rescued efnb2a and nrg1 expression. Interestingly, using in an in vitro system to isolate the effect of shear stress from other effects of cardiac contraction, endothelial cells upregulated Notch1, Efnb2, and Nrg1 in response to flow in a primary cilia-dependent manner. Primary cilia-deficient morphant embryos exhibited dramatically reduced Notch reporter levels, notch1b and nrg1 expression in the heart. Together, our findings indicate that heartbeat-responsive transcriptional changes through epistasis of notch1b/efnb2/nrg1 in ventricular endocardium are important for cardiac chamber maturation.

Poster 13 Colin Lickwar Technician/Manager MGM – Duke University John Rawls Lab

Ancient Elements: Identifying conserved regulatory regions and master regulators that drive microbially-responsive expression in the intestinal

epithelium

Defining similarity between related objects requires assumptions based on function and form. DNA sequence provides a common 27nformatics metric of comparison and identity between different species and individuals. However, non-coding regulatory regions typically are less conserved as they require shorter sequence tracts composed mostly of modular, degenerate transcription factor (TF) binding sites to function. Under the assumption that regulatory sequences that are conserved are foundational for organismal biology across species, we have profiled the intestinal epithelium of four species (Zebrafish, Stickleback, Mouse, and Human) using RNA-seq and open chromatin (FAIRE-seq and Dnase-seq) assays. By 400 million years of differences that have arisen in the molecular and functional organization of the intestine, differences in diet, microbial composition and structure, we find evidence of a conserved regulatory architecture that maintains functional specification along the length of the gut, as well as a response to environmental stimuli such as the microbiota. Specifically, aspects of lipid metabolism appear to be commonly regulated along the length of the gut and in response to microbes. However, only a limited number of conserved non-coding elements (CNEs), that we can detect, are specifically open in the intestinal epithelium, suggesting intestinal specificity is dictated by multi-purpose regulatory regions or regions that are not highly conserved by traditional metrics. Currently, we are functionally testing the capacity of conserved regulatory elements to maintain intestinal expression as well as response to environmental stimuli. We have identified candidate transcription factors that may define segment specificity and a response to microbes.

Poster 14 Sungjin Moon Postdoctoral Fellow Center for Human Disease Modeling – Duke University

In vivo dissection and network analysis of copy number variants associated with autism

Copy number variants (CNVs) are frequent lesions involved in both rare and complex human traits. This has raised the challenge of identifying which genes within a CNV drive clinical traits. We have shown previously how the combinatorial use of surrogate phenotypes in zebrafish embryos and genomic studies can help dissect CNVs on 8q24.3, and 16p11.2. However, although successful in identifying primary drivers, these data indicated that each of the primary dosage-sensitive transcripts were not sufficient to drive the CNV-associated pathology. We extended our study to model cis-epistasis within CNVs and to determine global transcriptional changes and dissect likely contributory transcripts. We focused on the 1q21.1 CNV, encompassing a nine-gene region, deletion of which is associated with microcephaly, while the reciprocal duplication is associated with macrocephaly. Systematic overexpression and suppression of all genes in the CNV showed that dosage perturbation of the chromodomain-helicase-DNA-binding protein CHD1L, gave significant head size changes: overexpression of humanCHD1L mRNA led to macrocephaly, while suppression of chd1l lead to a significant decrease in head size. However, given that human genetic data suggested that CHD1L might not be sufficient to explain causality of this CNV, we asked whether cis-CNV and trans-CNV genetic interactions might contribute to pathogenesis. To test this possibility, we tested the pairwise interaction of CHD1L with each of the other 8 genes within the CNV, though which we identified several epistatic partners that modulate the expressivity of micro- and macrocephaly. We also generated RNA-seq data from heads of control, CHD1L RNA-, and chd1l MO-injected embryos; we identified ~700 differentially expressed transcripts; strikingly, the top 20 genes in this cohort have been implicated in autism. Epistatic analysis of a subset of candidates whose dosage is perturbed reciprocally by the CNV showed complementation, indicating that their transcriptional misregulation is likely relevant to the observed neurodevelopmental phenotypes.

Poster 15 Caitlin Murdoch Graduate Student Molecular Genetics and Microbiology – Duke University John Rawls Lab Microbiota-Induced Serum Amyloid A (Saa) mediates host innate immune

responses Intestinal microbial communities play critical roles in maintaining host physiologic homeostasis and contribute to inflammatory disorders such as the inflammatory bowel diseases (IBD). This is achieved in part through microbial regulation of host gene expression. However, the mechanisms by which host cells integrate signals from microbiota to modulate gene expression and downstream physiologies remain unresolved. This is underscored by the fact that most of the human SNPs linked to IBD and other inflammatory diseases by genome-wide association studies occur in noncoding regions that include potential cis-regulatory elements (CREs). Serum amyloid A (Saa) is a highly conserved multifunctional apolipoprotein that is induced by the microbiota, and is a salient biomarker for IBD and other inflammatory conditions. Using a gnotobiotic zebrafish model, we found that the zebrafish saa ortholog is potently induced by microbiota in the intestine and other tissues. We further demonstrated that systemic neutrophil migration induced by microbiota is attenuated by saa knockdown. These data suggest that microbiota-dependent expression of saa directs host innate immune response and may play a role in inflammatory pathologies such as IBD. To identify putative CREs at saa and other loci, we used Dnase-seq and FAIRE-seq to define the open chromatin landscape in the intestines of mice and zebrafish, respectively. By combining this information with transgenic reporter assays in gnotobiotic zebrafish, we are determining the capacity of putative zebrafish, mouse and human CREs at saa loci to mediate microbiota-responsive expression. Preliminary results suggest a conserved CRE architecture confers intestinal expression of saa. These studies are uncovering novel regulatory mechanisms underlying microbiota-dependent transcription of saa, and may ultimately provide insights into the biological significance of saa induction in intestinal inflammation.

Poster 16 Joan Hedge Technician/Manager ORD/NHEERL/ISTD – US EPA Padilla Lab

Effect of X-ray Contrast Media, Chlorination, and Chloramination on Zebrafish Development

Little is known about the vertebrate developmental toxicity of chlorinated or chloraminated drinking water (DW), iodinated X-ray contrast media (ICM, a common contaminate of DW) or how the combination modifies the toxicity of the ICM. Four ICM (iopamidol, iopromide, diatrizoate, or iohexol) at concentrations of 5 µM were added to source water collected from a DW treatment plant prior to disinfection. Source water alone and source water with one of the four ICM added were disinfected with chlorine or preformed monochloramine for 3 days. All samples were extracted on cleaned conditioned XAD resins, concentrated by evaporation and solvent-exchanged to DMSO. Extracts were analyzed for disinfection byproducts (DBPs) by GC-MS. Vertebrate developmental toxicity using zebrafish, Danio rerio, was used to assess toxicity of the concentrated samples. At 5-7 hours post fertilization, individual embryos were treated in 96-well glass vial plates with the test material (8 doses, 40 to 0.7x times concentrated, ¼-log spacing, n=5-10 per condition per dose). Positive and negative controls were included on each plate. Overt toxicity (lethality, dysmorphology, and hatching) was assessed via visual inspection at 5 days post fertilization. In general, formation of the investigated DBPs was favored during the chlorination process. ICM alone did not lead to the formation of any of the investigated DBPs. The chloraminated water did not appear to elicit toxicity in a dose dependent manner, whereas chlorinated water did at 40x. All the ICM caused death, non-hatching or significant dysmorphology at 40x concentration, except iopamidol. Chlorination or chloramination of water containing ICM showed no change in developmental toxicity compared to ICM alone, except for diatrizoate, which increased toxicity 5 fold over chlorination or diatrizoate alone. This abstract does not necessarily reflect U.S. EPA policy.

Poster 17 Karine Ferri Lagneau Postdoctoral Fellow BBRI – NCCU Leung Lab

Ginger rescues the specification of arteries and definitive hematopoietic stem cells in plcγ1 mutant

Effective stimulation of arteriogenesis and production of hematopoietic stem cells (HSCs) have long been important therapeutic aims in clinical and regenerative medicine. Here, we show that the natural product ginger can rescue phospholipase C gamma 1 (plcγ1) zebrafish mutants which are defective in arterial fate and hematopoietic stem cell specifications. Plcγ1 function is required downstream of VEGF to drive arterial development and hematopoietic stem cell formation. Exposure of plcγ1 embryos to ginger extract or 10-gingerol rescues the expression of the arterial marker efnb2a in the dorsal aorta and the HSC marker cmyb in the ventral wall of the dorsal aorta (the hemogenic endothelium) as well as in the forming caudal hematopoietic tissue (CHT). During definitive hematopoiesis, HSCs emerge from the dorsal aorta at day 1 and migrate in the CHT where they expand and differentiate from day 2 onwards. Ginger can also rescue the circulation of blood in a fraction of plcγ1mutant embryos. Indeed, ginger rescues the lumenization of the axial vasculature as well as the primary sprouting of inter-segmental vessels (angiogenesis) in plcγ1 mutants. In addition, ginger re-establishes the formation of lumenized sinusoids in their CHT thus favoring the homing of circulating blood progenitors and providing the microenvironment for the development of HSCs. Finally, plcγ1 embryos treated with ginger show ectopic expression of scl in the ventral part of the tail at day 2. Scl, the master hematopoietic transcriptional regulator, was recently shown to prime the hemogenic endothelium for endothelial-to-hematopoietic transition. Thus, ginger might also induce the hemogenic endothelium fate ectopically in the CHT of plcγ1 mutants. These effects of ginger are dependent on downstream pathways including Bmp, Hh, Notch, Mek1/2, PI3K/Akt and nitric oxide. Taken together, our results show that ginger is able to rescue the specification of arterial and HSC identities in embryos defective for the VEGF signaling.

Poster 18 Jingli Cao Postdoctoral Fellow Cell Biology – Duke University Poss Lab

Poster 19 Ted Espenschied Graduate Student The Department of Molecular Genetics and Microbiology – Duke University John Rawls Lab

The Intestinal Microbiota Regulate the Development and Function of the

Innate Immune System in Zebrafish The intestinal microbiota influence a diverse array of host physiological processes, yet the mechanisms by which the host integrates signals from the commensal microbiota are poorly understood. Numerous studies have implicated a role for the composition of the microbiota in a variety of pathological conditions including response to infection and metabolic syndrome. Furthermore, recent studies have identified that transient perturbations in the commensal microbiota during early life have lasting consequences on host metabolism and immunity. Zebrafish are an attractive model system for investigating the relationship between commensal microbiota and host systemic immune function as they can be reared in gnotobiotic conditions, and are amenable to in vivo imaging as well as genetic and pharmacological manipulation. Previously our lab has demonstrated that the microbiota modulate the number, distribution and behavior of neutrophils in larval zebrafish. It remains unknown whether other leukocyte lineages are similarly affected. Here we provide evidence of conserved phenotypes in macrophages using transgenic zebrafish expressing mCherry under the control of the mpeg1 promoter. These results suggest that microbial signals influence leukocyte progenitor cells, perhaps hematopoietic stem cells. Further, we present data from pilot studies for developing protocols to interrogate how the transcriptional profile and chromatin landscape of leukocytes and their parental lineages are influenced by colonization status. Collectively, these data provide additional evidence for the role of commensal microbiota in modulating systemic innate immune function. Future studies will determine mechanisms by which microbial colonization influences innate immune response to various inflammatory stimuli.

Poster 20 Yangfan Liu Postdoctoral Fellow Center for Human Disease Modeling - Duke University

Candidate Gene Screening for CAKUT and DiGeorge Syndrome Congenital anomalies of the kidney and urinary tract (CAKUT) are early onset inheritable renal diseases observed in about 1 in 500 live births, leading to neonatal death in about 1 in 2000 births. CAKUT comprise a broad spectrum of malformations in kidney and urinary tract, from asymptomatic anomalies to complete renal agenesis. Though DSTYK has been identified as a causal gene, considering the incomplete penetrance and variable expressivity, CAKUT should be heterogenic or multigenic. Because CAKUT can be part of the symptoms in some syndromes, one possible way to screen for CAKUT candidate genes is to examine the (candidate) causal genes of the related syndromes. Here we studied a cohort of DiGeorge Syndrome with nearly only CAKUT symptoms, finding a recurrent deletion in Chromosome 22q11.21, in which there are seven genes with zebrafish homologs. To identify the causal gene in this region, we suppressed the expression of each homolog gene in zebrafish, quantified the total length of pronephros by NaK ATPase immunostaining, and found two genes (aifm3 and snap29) that contribute to normal pronephro development, especially the development of the convoluted tubule. These two genes are strong candidate genes for CAKUT and may also be the drivers of the renal defects in patients with DiGeorge Syndrome.

Poster 21 Kelly McKnight Postdoctoral Fellow Center for Human Disease Modeling - Duke University PTCH1 is a major contributor to ocular developmental defects and extends

the SOX2 regulatory network Ocular developmental anomalies (ODA) such as Anophthalmia/Microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10.000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes, and targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Consistent with a role of PTCH1 in ODA, functional analysis using physiologically relevant phenotypes in a zebrafish in vivo complementation model showed that all six patient missense mutations affect SHH signaling. We confirmed the relevance of PTCH1 alteration to microphthalmia phenotypes using quantitative measurements of eye development in transient suppressant and CRISPR/Cas9 ptch1 mutant embryos. Further, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression in CCE-Rx cells. Finally, we show that SOX2 regulates ptch1 expression in vivo; suppression of SOX2 in zebrafish embryos results in augmented expression of downstream SHH targets, including PTCH1. Together, these results identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, suggest that either an increase or a decrease of PTCH1 can result in similar ocular phenotypes, and demonstrate that PTCH1 contributes mutations to as much as 10% of ODA.

Poster 22 Jordan Cocchiaro Postdoctoral Fellow Molecular Genetics and Microbiology - Duke University John Rawls Lab

Identifying bacterial products that stimulate fatty acid uptake in the

zebrafish intestinal epithelium Jordan L. Cocchiaro (1), Susan Grass (2), Sena Bae (1,3), Olaf Mueller (4), Raphael H. Valdivia (1,4), and John F. Rawls (1,4) (1) Dept. of Molecular Genetics and Microbiology, Duke University, Durham, NC; (2) Dept. of Pediatrics, Duke University, Durham, NC; (3) Dept. of Biomedical Engineering, Duke University, Durham, NC; (4) Center for the Genomics of Microbial Systems, Duke University, Durham, NC The capacity of the intestinal microbiota to influence host nutrition has emerged as a major theme in host-microbe commensalism; however, the impact of microbiota on dietary fat metabolism remains unresolved. Previous work in our lab showed that microbial colonization stimulates uptake and storage of dietary fatty acids in cytoplasmic lipid droplets within intestinal enterocytes of zebrafish larvae. This phenotype was specifically reproduced when germ-free (GF) fish were monoassociated or treated with media conditioned by a bacterial isolate from the Firmicutes phylum, Exiguobacterium indicum. We hypothesize that a molecule being secreted or shed by the bacteria is sufficient to increase enterocyte lipid uptake. The objective of this study is to identify the E. indicum factor(s) that affect lipid uptake in enterocytes. Basic biochemical characterization of E. indicum conditioned media (EiCM) revealed that the bioactive factor is heat labile and may be lipid or lipid-soluble. Since these data suggested a potential protein or lipid of bacterial origin, HPLC methods were developed to fractionate the components of EiCM for activity screening in vivo. To support these approaches, we sequenced and assembled the E. indicum genome into a 3.2 Mb chromosome and four plasmids (135 kb, 4.4 kb, 3.7 kb, 1.6 kb) encoding approximately 3448 genes. These combined methodologies provide resources for identifying microbial factors that can control dietary fat assimilation. Increased knowledge of the dynamic relationships between microbiota, diet, and host physiology will potentially lead to new therapeutic approaches for human diseases such as obesity and malnutrition.

Poster 23 James Davison Graduate Student MGM - Duke University John Rawls Lab

Zebrafish gut microbiota control expression of angptl4 by mediating nuclear receptor binding to the in3.4 enhancer module

The gut microbiota are capable of controlling transcription of key host genes in a tissue specific manner. For instance, the presence of gut microbiota in zebrafish and mice leads to significant reductions in angiopotein-like protein 4 (angptl4/fiaf) expression in the intestinal epithelium (Bäckhed et al 2004 and Rawls et al 2004). In the mouse, this microbial suppression of intestinal Angptl4 increases peripheral lipoprotein lipase activity and as a result increases fat storage in adipose tissues (Bäckhed et al 2004). Recent studies have demonstrated that a discrete DNA cis-regulatory module (CRM) at the zebrafish angptl4 locus named in3.4 mediates microbial control of transcription in the intestinal epithelium (Camp et al 2012). This 316bp enhancer module is enriched for nuclear receptor binding sites, and using an unbiased approach, we have identified candidate nuclear receptors that specifically bind in3.4. These data support other findings that suggest the gut microbiota regulate nuclear receptor activity and expression

Poster 24 Stefan Oehlers Postdoctoral Fellow MGM Duke University David Tobin Lab

Live imaging of Cryptococcus neoformans pathogenesis in the zebrafish embryo model

Traditional mammalian models of cryptococcal pathogenesis are limited by their accessibility to real-time analysis. Conversely, in vitro models of cryptococcal pathogenesis fail to recapitulate complex host cellular dynamics. Here we have utilized the zebrafish embryo infection model to non-invasively study C. neoformans pathogenesis in a whole animal in real time. The zebrafish model organism has emerged as a powerful platform for investigating host-microbe interactions due to the availability of fluorescently labeled host cell lines. Following intravenous infection we observe that C. neoformans is rapidly and preferentially phagocytosed by zebrafish macrophages. C. neoformans is able to divide inside zebrafish macrophages and the zebrafish innate immune system is essential for containing cryptococcal burden. Furthermore, we find C. neoformans readily homes to the brain of infected fish and is able to cross the blood brain barrier. Having established these parameters of cryptococcal infection in zebrafish, we will utilize the live imaging accessibility of the zebrafish model to investigate cryptococcal determinants of pathogenesis.

Poster 25 Aaron Goldman Postdoctoral Fellow Ken Poss Lab