Comprehensive Colony Health Management and Emerging Pathogens of the Annual Killifish Species Nothobranchius furzeri.

The Leibniz Institute on Aging has maintained killifish colonies for over 15 y. Our veterinarians, scientists, and animal technicians developed a fish health scoring system and routine colony health surveillance program for our colonies. Over a 4-y period, health data from the African turquoise killifish Nothobranchius furzeri colony were systematically collected and analyzed. The fish health assessment system facilitated categorization of clinical signs and differentiation of fish with mild clinical signs from fish that required euthanasia. This report provides new information on clinical signs and conditions that may occur in young and aged N. furzeri. To be comprehensive, a colony health surveillance program incorporates animal health at both the individual and the population levels. The quarterly routine health monitoring program identified Mycobacterium spp. as the most common agent in our facility and identified the killifish pathogen (Loma acerinae) for the first time. Taken together, these findings demonstrate the importance of a comprehensive colony health management system in a fish research facility. By improving the health and welfare of fish used for research, the scientific community will benefit from less variable and more reliably reproducible research results.

Wt1 transcription factor impairs cardiomyocyte specification and drives a phenotypic switch from myocardium to epicardium.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.

Zebrafish Wtx is a negative regulator of Wnt signaling but is dispensable for embryonic development and organ homeostasis.

BACKGROUND: The X-chromosomally linked gene WTX is a human disease gene and a member of the AMER family. Mutations in WTX are found in Wilms tumor, a form of pediatric kidney cancer and in patients suffering from OSCS (Osteopathia striata with cranial sclerosis), a sclerosing bone disorder. Functional data suggest WTX to be an inhibitor of the Wnt/ß-catenin signaling pathway. Deletion of Wtx in mouse leads to perinatal death, impeding the analysis of its physiological role. RESULTS: To gain insights into the function of Wtx in development and homeostasis we have used zebrafish as a model and performed both knockdown and knockout studies using morpholinos and transcription activator-like effector nucleases (TALENs), respectively. Wtx knockdown led to increased Wnt activity and embryonic dorsalization. Also, wtx mutants showed a transient upregulation of Wnt target genes in the context of caudal fin regeneration. Surprisingly, however, wtx as well as wtx/amer2/amer3 triple mutants developed normally, were fertile and did not show any anomalies in organ maintenance. CONCLUSIONS: Our data show that members of the zebrafish wtx/amer gene family, while sharing a partially overlapping expression pattern do not compensate for each other. This observation demonstrates a remarkable robustness during development and regeneration in zebrafish.

Kidney regeneration in fish.

Age-related diseases, such as kidney diseases, are becoming more prevalent in aging societies. Currently, patients with reduced kidney function require dialysis or organ transplants. Those who suffer from kidney disease would benefit from regenerative therapies. Thus, one of the ultimate goals of regeneration research is to enhance an individual's capacity of self-repairing damaged tissue; something that fish models can contribute towards. Kidney structures are conserved among vertebrates highlighting the opportunities for fish to act as human disease models. Here, different species can offer respective advantages. An understanding of the different modes of regeneration can help to visualize the differences in mammalian and fish regenerative capacity. The remarkable regenerative capacity of fish is well known, but kidney regeneration is an understudied area. The kinetics of kidney regeneration allows one to investigate early damage responses, as well as the initiation and completion of repair. Age-related reductions in regeneration are an additional societal problem; again an area where fish models can be of help. Age-matched experiments between varied vertebrate species will help us to learn from those that do or do not exhibit age-related phenotypes. The goal of such experiments is not only to outline important age-related factors and pathways, but, in addition, to see if age-related decreases in regenerative capacity can be reduced. Widening our knowledge of this very complex process will help to address many of the unanswered questions in the field.

Function and Regulation of the Wilms' Tumor Suppressor 1 (WT1) Gene in Fish.

The Wilms' tumor suppressor gene Wt1 is highly conserved among vertebrates. In contrast to mammals, most fish species possess two wt1 paralogs that have been named wt1a and wt1b. Concerning wt1 in fish, most work so far has been done using zebrafish, focusing on the embryonic kidney, the pronephros. In this chapter we will describe the structure and development of the pronephros as well as the role that the wt1 genes play in the embryonic zebrafish kidney. We also discuss Wt1 target genes and describe the potential function of the Wt1 proteins in the adult kidney. Finally we will summarize data on the role of Wt1 outside of the kidney.

Fluorescence-Activated Cell Sorting (FACS) Protocol for Podocyte Isolation in Adult Zebrafish.

Zebrafish is becoming a very important model for studying human diseases. The conserved structure of the nephrons in the kidney allows the user to answer questions relating to study human kidney disorders. Wt1a-expressing podocytes are the most important cells within the glomeruli of adult zebrafish. In order to understand the molecular characteristics of these cells, within damage models, we have established a method for isolating them.

Expression and evolution of the non-canonically translated yeast mitochondrial acetyl-CoA carboxylase Hfa1p.

The Saccharomyces cerevisiae genome encodes two sequence related acetyl-CoA carboxylases, the cytosolic Acc1p and the mitochondrial Hfa1p, required for respiratory function. Several aspects of expression of the HFA1 gene and its evolutionary origin have remained unclear. Here, we determined the HFA1 transcription initiation sites by 5' RACE analysis. Using a novel "Stop codon scanning" approach, we mapped the location of the HFA1 translation initiation site to an upstream AUU codon at position -372 relative to the annotated start codon. This upstream initiation leads to production of a mitochondrial targeting sequence preceding the ACC domains of the protein. In silico analyses of fungal ACC genes revealed conserved "cryptic" upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δhfa1 baker's yeast mutant phenotype rescue studies using the protoploid Kluyveromyces lactis ACC confirmed functionality of the cryptic upstream mitochondrial targeting signal. These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in S. cerevisiae have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms. Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5' upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.

Mycophenolate mofetil in combination with recombinant interferon alfa-2a in interferon-nonresponder patients with chronic hepatitis C.

BACKGROUND/AIMS: Since ribavirin was able to improve the antiviral efficacy of interferon alfa in patients with chronic hepatitis C, several other adjuncts have been studied. It has been shown that mycophenolate mofetil (MMF) is a more potent inhibitor of the inosine 5'-monophosphate-dehydrogenase (IMPDH) than ribavirin. The present study is a pilot study evaluating the efficacy and safety of combination therapy with interferon alfa-2a and MMF in interferon alfa nonresponder patients. METHODS: Thirty-eight adult patients with chronic hepatitis C who did not respond to a previous interferon alfa monotherapy were enrolled to receive 6 million units of interferon alfa-2a tiw in combination with MMF (1 week 500 mg/day, 1 week 1000 mg/day, 22 weeks 2000 mg/day). RESULTS: An interim analysis of 29 patients after 12 weeks of therapy showed that only one patient had negative hepatitis C virus-RNA at this time point. There was no significant reduction of the viral load during therapy. Due to inefficacy the study was discontinued. CONCLUSIONS: Combination therapy of interferon alfa-2a and MMF is ineffective in improving virological response rates in nonresponder patients with chronic hepatitis C. These data suggest that inhibition of the IMPDH seems not to be the major mechanism of ribavirin in enhancing the antiviral effect of interferon alfa in chronic hepatitis C.

Impact of HLA-compatibilities in patients undergoing liver transplantation for HBV-cirrhosis.

Liver transplantation (OLT) for end-stage chronic hepatitis-B-virus (HBV) infection is frequently complicated by HBV recurrence. In the present study we investigated whether human leucocyte antigen (HLA)-matching influences the outcome after OLT. In a retrospective analysis we reviewed 84 recipients of liver transplants for end-stage HBV-cirrhosis and complete HLA-typing for outcome after OLT. Follow-up ranges from 1 to 110 months (median = 55.6 months). Immunosuppression consisted of Cyclosporin A (CsA)-based quadruple induction therapy or Tacrolimus-based induction protocols. Immunoprophylaxis with hepatitis B immunoglobulin was started at OLT and continued long-term. Actuarial 1- and 5-yr graft survival figures were 90.5 and 80.4%, respectively. Hepatitis-B recurrence was responsible for 15 of 20 (75%) graft failures. We observed a significantly improved graft survival in patients with more HLA-A, -B compatibilities (p = 0.02), whereas the degree of HLA-DR compatibilities did not influence the outcome. The occurrence of HBV-reinfection was significantly lower in HLA-A, -B matched grafts (p < 0.05). Additionally, graft survival was prolonged in patients with HBV-reinfection and 1 or 2 HLA-B compatibilities when compared with patients with HBV-reinfection and a complete HLA-B mismatch (p = 0.02). In conclusion, this retrospective analysis shows that more HLA-A, -B compatibilities seems to be associated with an improved graft survival in patients after OLT for end-stage HBV infection.