WT Zebrafish Research Articles

The Visually Mediated Social Preference Test: A Novel Technique to Measure Social Behavior and Behavioral Disturbances in Zebrafish.

Zebrafish are an emerging model in behavioral neuroscience. They display a wide range of measurable behaviors such as locomotion, aggression, anxiety, learning and memory, and social behavior. In addition, the relative ease of genetic manipulation and the increasing availability of disease models mean that zebrafish have gained in popularity as an animal model for various neurological and psychiatric diseases including autism spectrum disorder (ASD). In order to better characterize social behavior and behavioral abnormalities in zebrafish, we have developed the visually mediated social preference (VMSP) test, a novel assay to measure social preference and social novelty in two consecutive 5-min sessions. Using recording and video tracking, the time spent in different areas of the tank, the time spent immobile, swimming speed, and distance moved can be easily measured and analyzed. Untreated experimentally naive AB WT zebrafish typically show a strong preference for spending time near and interacting with a compartment containing unfamiliar conspecifics over the empty compartments during session 1 and a stronger preference for a group of unfamiliar zebrafish over familiar conspecifics from session 1, during session 2 of the test. Research in our lab has shown that the VMSP is suitable to measure the social behavior of individual zebrafish, to uncover social phenotypes of mutant strains, and to better understand animal models of disease that include impaired sociability such as ASD. The current paper provides a step-by-step guide on how to implement and perform this test and highlights important considerations for data acquisition, analysis, and interpretation.

Progestin and Nuclear Progestin Receptor Are Essential for Upregulation of Metalloproteinase in Zebrafish Preovulatory Follicles.

Ovulation requires proteinases to promote the rupture of ovarian follicles. However, the identity of these proteinases remains unclear. In our previous studies using RNA-seq analysis of differential expressed genes, we found significant down-regulation of five metalloproteinases: adam8b (a disintegrin and metalloproteinase domain 8b), adamts8a (a disintegrin and metalloproteinase with thrombospondin motif 8a), adamts9, mmp2 (matrix metalloproteinase 2), and mmp9 in the nuclear progestin receptor knockout (pgr−/−) zebrafish that have failed to ovulate. We hypothesize that these metalloproteinases are responsible for ovulation and are regulated by progestin and Pgr. In this study, we first determined the expression of these five metalloproteinases and adamts1 in preovulatory follicles at different times within the spawning cycle in pgr−/− and wildtype (wt) zebrafish and under varying hormonal treatments. We found that transcripts of adam8b, adamts1, adamts9, and mmp9 increased drastically in the preovulatory follicular cells of wt female zebrafish, while changes of adamts8a and mmp2 were not significant. This increase of adam8b, adamts9, and mmp9 was significantly reduced in pgr−/−, whereas expression of adamts1 was not affected in pgr−/− zebrafish. Among upregulated metalloproteinases, adamts9 mRNA was found to be expressed specifically in follicular cells. Strong immunostaining of Adamts9 protein was observed in the follicular cells of wt fish, and this expression was reduced drastically in pgr−/−. Interestingly, about an hour prior to the increase of metalloproteinases in wt fish, both Pgr transcript and protein increased transiently in preovulatory follicular cells. The results from in vitro experiments showed that adamts9 expression markedly increased in a dose, time and Pgr-dependent manner when preovulatory follicles were exposed to a progestin, 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Taken together, our results provide the first evidence that upregulation of adamts9 occurs specifically in preovulatory follicular cells of zebrafish prior to ovulation. Progestin and its receptor (Pgr) are essential for the upregulation of metalloproteinases.

The CCM1-CCM2 complex controls complementary functions of ROCK1 and ROCK2 that are required for endothelial integrity.

Endothelial integrity relies on a mechanical crosstalk between intercellular and cell-matrix interactions. This crosstalk is compromised in hemorrhagic vascular lesions of patients carrying loss-of-function mutations in cerebral cavernous malformation (CCM) genes. RhoA/ROCK-dependent cytoskeletal remodeling is central to the disease, as it causes unbalanced cell adhesion towards increased cell-extracellular matrix adhesions and destabilized cell-cell junctions. This study reveals that CCM proteins directly orchestrate ROCK1 and ROCK2 complementary roles on the mechanics of the endothelium. CCM proteins act as a scaffold, promoting ROCK2 interactions with VE-cadherin and limiting ROCK1 kinase activity. Loss of CCM1 (also known as KRIT1) produces excessive ROCK1-dependent actin stress fibers and destabilizes intercellular junctions. Silencing of ROCK1 but not ROCK2 restores the adhesive and mechanical homeostasis of CCM1 and CCM2-depleted endothelial monolayers, and rescues the cardiovascular defects of ccm1 mutant zebrafish embryos. Conversely, knocking down Rock2 but not Rock1 in wild-type zebrafish embryos generates defects reminiscent of the ccm1 mutant phenotypes. Our study uncovers the role of the CCM1-CCM2 complex in controlling ROCK1 and ROCK2 to preserve endothelial integrity and drive heart morphogenesis. Moreover, it solely identifies the ROCK1 isoform as a potential therapeutic target for the CCM disease.

Spatial and temporal variations in hemodynamic forces initiate cardiac trabeculation.

Hemodynamic shear force has been implicated as modulating Notch signaling-mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function.

The occhiolino (occ) mutant Zebrafish, a model for development of the optical function in the biological lens.

Zebrafish visual function depends on quality optics. An F3 screen for developmental mutations in the Zebrafish nervous system was conducted in wild-type (wt) AB Zebrafish exposed to 3 mM of N-ethyl-N-nitrosourea (ENU). Mutant offspring, identified in an F3 screen, were characterized by a small pupil, resulting from retinal hypertrophy or hyperplasia and a small lens. Deficits in visual function made feeding difficult after hatching at approximately 5-6 days postfertilization (dpf). Special feeding conditions were necessary for survival of the occhiolino (occ) mutants after 6 dpf. Optokinetic response (OKR) tests measured defects in visual function in the occ mutant, although electroretinograms (ERGs) were normal in the mutant and wt. Consistent with the ERGs, histology found normal retinal structure in the occ mutant and wt Zebrafish. However, lens development was abnormal. Multiphoton imaging of the developmental stages of live embryos confirmed the formation of a secondary mass of lens cells in the developing eye of the mutant Zebrafish at 3-4 dpf, and laminin immunohistochemistry indicated the lens capsule was thin and disorganized in the mutant Zebrafish. The occ Zebrafish is a novel disease model for visual defects associated with abnormal lens development. Developmental Dynamics 246:915-924, 2017. © 2017 Wiley Periodicals, Inc.

Surviving low oxygen: exploring the role of AMP‐activated Kinase in anoxia‐tolerance

High altitude, stroke and cardiorespiratory disorders are damaging to mammalian tissues in that they cause oxygen (O2) deprivation in organs that are energy‐demanding. The zebrafish model provides unique insight into mechanisms of adaptation to O2 deprivation, as zebrafish embryos can adapt to low O2 by entering a state of developmental arrest that conserves cellular energy (ATP). The zebrafish model may therefore reveal targets for therapeutic treatment of hypoxia‐related injury. The Brewster lab aims to identify the signaling pathways that induce the arrested state in response to anoxia (zero O2).We hypothesize that AMPK (AMP‐activated kinase) is a key mediator of developmental arrest. AMPK is activated by anoxia and conserves ATP by inhibiting cell cycle progression and protein translation in various contexts. We predict that activation of AMPK under anoxia is necessary for the zebrafish embryo to enter developmental arrest. If our hypothesis is correct, we predict that loss of AMPK function under anoxia will lead to premature death. In our ongoing study, we are testing this prediction by comparing arrest and survival rates of Wt (wild‐type) zebrafish embryos to that of mutant zebrafish embryos that are unable to activate AMPK due to a mutation in LKB1, an upstream activator of AMPK.In order to test this prediction, we are comparing the survival of Wt and LKB1 mutant zebrafish embryos under anoxia. We report here on preliminary studies assessing the LD‐50, the duration of sustained anoxia at which 50% of embryos survive after reintroduction to normal O2‐levels for 24 hours, on Wt embryos at dome stage. We have found that the LD‐50 for Wt zebrafish embryos at dome stage is between 18 and 24 hours under our environmental conditions. Similar studies using LKB‐1 embryos at the dome stage are underway. We anticipate that these studies may reveal a novel role for AMPK in anoxia‐induced arrest and survival.Support or Funding InformationFunding: American Physiological Society STRIDE program. Acknowledgments: Ikenna Okafor (Graduate Mentor), Sharlene Brown (Graduate Mentor), Dr. Rachel Brewster (PI)

Abstract 12739: Gap Junction Protein Beta 4 Plays an Important Role In Cardiac Hypertrophy and Cardiac Function in Human and Zebrafish

Introduction: Gap junction protein beta 4 (GJB4) encodes a transmembrane connexin protein (Cx30.3) that is a component of gap junctions. Mutations in this gene remain unknown to be associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction. Hypothesis: Our hypothesis is that mutations in GJB4 can cause familial HCM, which can progresses into dilated-phase HCM (d-HCM). Methods: We have previously examined a patient and her elder brother who both presented with complicated severe HCM and whose parents are married cousins. The elder brother had died suddenly as a child and the autopsy demonstrated the development of HCM. The patient was diagnosed with HCM, which progressed to d-HCM during the 20-year follow-up. The patient has recently undergone cardiac transplantation. The parents are healthy. We hypothesized that an autosomal recessive mutation caused their familial HCM. The whole-exome sequencing was performed from each of the three participants. We generated knockout zebrafish of GJB4 and ELP2 by CRISPER/Cas9 system and analysis the volume and function of ventricle at five days post fertilization (dpf). Results: The gene exome analysis showed 34 single nucleotide polymorphisms (SNPs) that caused amino acid change for which the patient was homozygous and both parents were heterozygous after excluding all known common (>10%) SNP gene mutations in the dbSNP. Among them, GJB4 and ELP2 were the identified genes that are possibly associated with cardiac muscle. The PolyPhen-2 scores of mutations in GJB4 and ELP2 were 1.000 and 0.001 respectively. The identified missense mutation was a 981 A>C nucleotide transition in GJB4. The resultant E204A substitution replaces a polar carboxylic glutamate with a nonpolar alanine. The endodiastolic volume was significantly lower in GJB4-KO (94pl, p<0.01) and ELP2-KO (95pl, p<0.01) than in wild type (WT, 145pl) zebrafish at 5 dpf. Furthermore the ventricular ejection fraction was decreased in GJB4 KO (0.66, p<0.01) and unchanged in ELP2-KO (0.80) respectively compared with WT zebrafish (0.82). Conclusions: These results indicate both that mutations in GJB4 and ELP2 can cause a familial form of HCM and that GJB4 especially may be a new target for treatment of cardiac function and hypertrophy.

Essential Opposite Roles of ERK and Akt Signaling in Cardiac Steroid‐Induced Increase in Heart Contractility

Interaction of cardiac steroids (CS) with the Na+, K+‐ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as ERK and Akt. We hypothesized that the activities of these pathways are involved in CS‐induced increase in heart contractility. This hypothesis was tested using in‐vivo and ex‐vivo wt and SERCA1a deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in‐vivo, and in primary cardiomyocytes, in wt zebrafish larvae and acc mutant. Ca2+ transients were determined ex‐vivo, in and adult zebrafish hearts. CS dose‐dependently, augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca2+ transients, in wt, but not in acc mutant. CS in‐vivo, increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of wt zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS‐induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS‐induced increase in heart contractility both, in‐vivo and ex‐vivo without affecting CS‐induced Ca2+ transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS‐induced increase in heart contractility also without affecting Ca2+ transients. These results support the notion that the activity of MAPK pathway is obligatory for CS‐induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca2+ independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy.Support or Funding InformationThis work was supported by grants from the Ministry of Trade and Industry (NOFAR), Israel and the Szolts Foundation, The Hebrew University of Jerusalem, and the Walter and Greta Chair in Heart Studies (to D.L).

The zebrafish mutant lessen: an experimental model for congenital enteric neuropathies.

Congenital enteric neuropathies of the distal intestine (CEN) are characterized by the partial or complete absence of enteric neurons. Over the last decade, zebrafish has emerged as a leading model organism in experimental research. Our aim was to demonstrate that the mutant zebrafish, lessen, expressing CEN characteristics, is an equally valuable animal model alongside mammalian models for CEN, by studying its enteric phenotype. The effect of the lessen mutation on the development of the enteric nervous system (ENS), interstitial cells of Cajal (ICC), and intestinal motility in each intestinal region of mutant and wild-type (wt) zebrafish embryos at 3-6 dpf, was analyzed by immunofluorescent detection of neurochemical markers and motility assays. Development of intestinal motility in the mutant was delayed and the majority of the observed contractions were disturbed. A significant disturbance in ENS development resulted in a distal intestine that was almost free of neuronal elements, in reduced neuronal density in the proximal and mid-intestine, and in a defect in the expression of neurochemical markers. Furthermore, markedly disturbed development of ICC gave rise to a less dense network of ICC. The observed alterations in intestinal motility, intrinsic innervation and ICC network of the mutant in comparison with the wt zebrafish, are similar to those seen in the oligo- and aganglionic regions of the intestine of CEN patients. It is concluded that the zebrafish mutant lessen is an appropriate animal model to investigate CEN.

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth.

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg+/− offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg−/− mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg+/− and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg−/− animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg−/− animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg−/− mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.

Abstract 1578: Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype.

Abstract Hypoxia Inducible Factor (HIF) target genes are involved in several cellular processes including angiogenesis, erythropoiesis, metabolism and stem cell proliferation. The von Hippel-Lindau (VHL) tumor suppressor protein targets HIF for degradation in conditions of normal oxygen tension. Loss-of-VHL leads to constitutive expression of HIF. We have discovered small molecules that repress HIF-2α translation by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5’-UTR of HIF-2α mRNA and showed that these small molecules specifically down-regulate HIF-2α expression in vitro. To validate the HIF-2α inhibitors in vivo, first we tested their efficacy to inhibit acute hif induction in wild-type (wt) zebrafish embryos challenged with the hypoxia mimetic DMOG. Treatment of wt embryos with 100μM of DMOG up-regulated the expression of zebrafish HIF-target genes, such as vascular endothelial growth factor (vegf), erythropoietin (epo) and prolyl-hydroxylase 3 (phd3). Pre-treatment of embryos with the small molecule HIF-2α inhibitor 76 (10nM) significantly decreased the DMOG-induced up-regulation of HIF target genes and resulted in decrease of DMOG-induced erythrocytosis and blood vessel sprouting. Furthermore, we tested the efficacy of this HIF-2α inhibitor in a vhl zebrafish model (homozygougous mutants harbouring inactivating vhl mutations), which recapitulates aspects of human VHL disease (erythrocytosis, abnormal vascular proliferation in sites such as retina and brain, early lethality). Treatment with inhibitor 76 (10nM, 100nM and 1μM) down regulated the expression of the HIF-target genes phd3, epo and vegf in a dose response manner and rescued the erythrocytosis and sprouting phenotype of vhl embryos. To image and quantify the blood vessel abnormalities of vhl embryos, we generated a Fli-GFP::vhl+/- line and showed that compound 76 improves the abnormal connections between the intersegmental vessels of the Fli-GFP::vhl embryo tail, as well as their human hemangioblastoma-resembling brain vessel proliferation. Finally, we showed that treatment of vhl embryos with compound 76 rescues early lethality. Our data demonstrate that HIF-2α inhibitor 76 is active in vivo at low concentrations and encourage further development and preclinical testing of this HIF-2α inhibitor for treatment of VHL-associated lesions, such as renal cell carcinoma, hemangioblastoma and neuroendocrine tumors. Citation Format: Ana Metelo, Li Xiang, Rania Baker, Lee Kamentsky, Anne Carpenter, Randall Peterson. Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1578. doi:10.1158/1538-7445.AM2013-1578

Structure-based optimization of oxadiazole-based GSK-3 inhibitors

Inhibition of glycogen synthase kinase-3 (GSK-3) induces neuroprotective effects, e.g. decreases β-amyloid production and reduces tau hyperphosphorylation, which are both associated with Alzheimer's disease (AD). The two isoforms of GSK-3 in mammalians are GSK-3α and β, which share 98% homology in their catalytic domains. We investigated GSK-3 inhibitors based on 2 different scaffolds in order to elucidate the demands of the ATP-binding pocket [1]. Particularly, the oxadiazole scaffold provided potent and selective GSK-3 inhibitors. For example, the most potent inhibitor of the present series, the acetamide 26d, is characterized by an IC50 of 2 nM for GSK-3α and 17 nM for GSK-3β. In addition, the benzodioxane 8g showed up to 27-fold selectivity for GSK-3α over GSK-3β, with an IC50 of 35 nM for GSK-3α. Two GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay to evaluate simultaneously permeability and safety.

Identification of Glycogen Synthase Kinase-3 Inhibitors with a Selective Sting for Glycogen Synthase Kinase-3α

The glycogen synthase kinase-3 (GSK-3) has been linked to the pathogenesis of colorectal cancer, diabetes, cardiovascular disease, acute myeloid leukemia (AML), and Alzheimer's disease (AD). The debate on the respective contributions of GSK-3α and GSK-3β to AD pathology and AML is ongoing. Thus, the identification of potent GSK-3α-selective inhibitors, endowed with favorable pharmacokinetic properties, may elucidate the effect of GSK-3α inhibition in AD and AML models. The analysis of all available crystallized GSK-3 structures provided a simplified scheme of the relevant hot spots responsible for ligand binding and potency. This resulted in the identification of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest GSK-3α selectivity reported so far. In addition, compound 14d did not display significant inhibition of 48 out of 50 kinases in the test panel. The GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay.

Pulmonary Disease and Exercise Intolerance in Boys with Fabry Disease: a Pilot Study

The severe pediatric disorder, mucolipidosis II (ML-II; I-cell disease), is caused by defects in mannose 6-phosphate (Man-6-P) biosynthesis. Patients with ML-II exhibit multiple developmental defects including skeletal, craniofacial and joint abnormalities. To date, the molecular mechanisms that underlie these clinical manifestations are poorly understood. Taking advantage of a zebrafish model for ML-II, we previously showed that the cartilage morphogenesis defects in this model are associated with altered chondrocyte differentiation and excessive deposition of type II collagen, indicating that aspects of development that rely on proper extracellular matrix homeostasis are sensitive to decreases in Man-6-P biosynthesis. To further investigate the molecular bases for the cartilage phenotypes, we analyzed the transcript abundance of several genes in chondrocyte-enriched cell populations isolated from wild-type WT and ML-II zebrafish embryos. Increased levels of cathepsin and matrix metalloproteinase (MMP) transcripts were noted in ML-II cell populations. This increase in transcript abundance corresponded with elevated and sustained activity of several cathepsins (K, L and S) and MMP-13 during early development. Unlike MMP-13, in which higher levels of enzyme was also detected, sustained activity of cathepsin K appeared to result from abnormal processing and activation of this enzyme at later stages. Remarkably, inhibition of cathepsin K activity by pharmacological or genetic means not only reduced the activity of this enzyme but led to a broad reduction in additional protease activity, significant correction of the cartilage morphogenesis phenotype and reduced type II collagen staining in ML-II embryos. Our findings suggest a central role for excessive cathepsin K activity in the developmental aspects of ML-II cartilage pathogenesis and highlight the utility of the zebrafish system to address the biochemical underpinnings of metabolic disease.

Ferroportin1 is required for normal iron cycling in zebrafish

Missense mutations in ferroportin1 (fpn1), an intestinal and macrophage iron exporter, have been identified between transmembrane helices 3 and 4 in the zebrafish anemia mutant weissherbst (weh(Tp85c-/-)) and in patients with type 4 hemochromatosis. To explore the effects of fpn1 mutation on blood development and iron homeostasis in the adult zebrafish, weh(Tp85c-/-) zebrafish were rescued by injection with iron dextran and studied in comparison with injected and uninjected WT zebrafish and heterozygotes. Although iron deposition was observed in all iron-injected fish, only weh(Tp85c-/-) zebrafish exhibited iron accumulation in the intestinal epithelium compatible with a block in iron export. Iron injections initially reversed the anemia. However, 8 months after iron injections were discontinued, weh(Tp85c-/-) zebrafish developed hypochromic anemia and impaired erythroid maturation despite the persistence of iron-loaded macrophages and elevated hepatic nonheme iron stores. Quantitative real-time RT-PCR revealed a significant decrease in mean hepatic transcript levels of the secreted iron-regulator hepcidin and increased intestinal expression of fpn1 in anemic weh(Tp85c-/-) adults. Injection of iron dextran into WT or mutant zebrafish embryos, however, resulted in significant increases in hepcidin expression 18 hours after injection, demonstrating that hepcidin expression in zebrafish is iron responsive and independent of fpn1's function as an iron exporter.

Axon pathfinding and the floor plate factor Reissner's substance in wildtype, cyclops and one-eyed pinhead mutants of Danio rerio

The ventral median floor plate (FP) is a well-examined embryonic structure, which is involved in neuron differentiation and axon outgrowth. The FP of different vertebrates expresses the glycoprotein Reissner's substance (RS). This glycoprotein is also produced by the dorsal median subcommissural organ (SCO). We examined if the dorsal SCO and the ventral FP are interdependent for the expression of RS and looked for indications for a role of RS in axon outgrowth. Therefore, we examined zebrafish embryos of wildtype ( wt) and the mutants cyclops tf219 ( cyc) and one-eyed pinhead tz257 ( oep), which both lack the FP. Our studies demonstrate that the FP is not necessary in order to induce the expression of RS in the SCO. The pattern of the anti-RS immunolabelling in the mutants is, however, changed compared to wt zebrafish embryos. As a consequence of the lacking FP and the degenerated ventricle system in cyc and oep mutants, a Reissner's fibre (RF) is not formed. Our studies confirm earlier results about the axon growth in cyc mutants, and provide the first detailed data about the aberrant axon growth in oep mutants. The modified outgrowth of the medial longitudinal fascicle in both mutants could be associated with the lack of RS/RF in the rhombencephalon and spinal cord. The neurites of the posterior commissure follow the aberrant position of the SCO in oep mutants. Our results suggest that both the RS of the ventral FP/flexural organ (FO) and the RS of the dorsal SCO have an influence on the outgrowth of axons and formation of commissures.