Publications by authors named "Susanne Kneitz"

87 Publications

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

Mol Ecol Resour 2021 Feb 16. Epub 2021 Feb 16.

Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.
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http://dx.doi.org/10.1111/1755-0998.13360DOI Listing
February 2021

Crosstalk Between Retinoic Acid and Sex-Related Genes Controls Germ Cell Fate and Gametogenesis in Medaka.

Front Cell Dev Biol 2020 18;8:613497. Epub 2021 Jan 18.

University of Wuerzburg, Developmental Biochemistry, Biocenter, Wuerzburg, Germany.

Sex determination (SD) is a highly diverse and complex mechanism. In vertebrates, one of the first morphological differences between the sexes is the timing of initiation of the first meiosis, where its initiation occurs first in female and later in male. Thus, SD is intimately related to the responsiveness of the germ cells to undergo meiosis in a sex-specific manner. In some vertebrates, it has been reported that the timing for meiosis entry would be under control of retinoic acid (RA), through activation of . In this study, we used a fish model species for sex determination and lacking the gene, the Japanese medaka (), to investigate the connection between RA and the sex determination pathway. Exogenous RA treatments act as a stress factor inhibiting germ cell differentiation probably by activation of and . Disruption of the RA degrading enzyme gene induced precocious meiosis and oogenesis in embryos/hatchlings of female and even some males. Transcriptome analyzes of -/-adult gonads revealed upregulation of genes related to germ cell differentiation and meiosis, in both ovaries and testes. Our findings show that germ cells respond to RA in a independent model species. The responsiveness to RA is conferred by sex-related genes, restricting its action to the sex differentiation period in both sexes.
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http://dx.doi.org/10.3389/fcell.2020.613497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848095PMC
January 2021

Giant lungfish genome elucidates the conquest of land by vertebrates.

Nature 2021 02 18;590(7845):284-289. Epub 2021 Jan 18.

Developmental Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany.

Lungfishes belong to lobe-fined fish (Sarcopterygii) that, in the Devonian period, 'conquered' the land and ultimately gave rise to all land vertebrates, including humans. Here we determine the chromosome-quality genome of the Australian lungfish (Neoceratodus forsteri), which is known to have the largest genome of any animal. The vast size of this genome, which is about 14× larger than that of humans, is attributable mostly to huge intergenic regions and introns with high repeat content (around 90%), the components of which resemble those of tetrapods (comprising mainly long interspersed nuclear elements) more than they do those of ray-finned fish. The lungfish genome continues to expand independently (its transposable elements are still active), through mechanisms different to those of the enormous genomes of salamanders. The 17 fully assembled lungfish macrochromosomes maintain synteny to other vertebrate chromosomes, and all microchromosomes maintain conserved ancient homology with the ancestral vertebrate karyotype. Our phylogenomic analyses confirm previous reports that lungfish occupy a key evolutionary position as the closest living relatives to tetrapods, underscoring the importance of lungfish for understanding innovations associated with terrestrialization. Lungfish preadaptations to living on land include the gain of limb-like expression in developmental genes such as hoxc13 and sall1 in their lobed fins. Increased rates of evolution and the duplication of genes associated with obligate air-breathing, such as lung surfactants and the expansion of odorant receptor gene families (which encode proteins involved in detecting airborne odours), contribute to the tetrapod-like biology of lungfishes. These findings advance our understanding of this major transition during vertebrate evolution.
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http://dx.doi.org/10.1038/s41586-021-03198-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875771PMC
February 2021

Periodontal pathogens alter the synovial proteome. Periodontal pathogens do not exacerbate macroscopic arthritis but alter the synovial proteome in mice.

PLoS One 2020 31;15(12):e0242868. Epub 2020 Dec 31.

Core Facility for Cell Sorting & Cell Analysis, Laboratory for Clinical Immunology, University Medical Center Rostock, Rostock, Germany.

Rheumatoid arthritis (RA) and periodontitis (PD) are chronic inflammatory diseases that appear to occur in tandem. However, the mutual impact PD exerts on RA and vice versa has not yet been defined. To address this issue, we set up an animal model and analyzed how two prime inducers of periodontitis-Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa)-differ in their pathogenic potential. Our experimental setup included collagen induced arthritis (CIA) in the mouse, oral inoculation with Pg or Aa to induce alveolar bone loss and the combination of both diseases in inverted orders of events. Neither pathobiont impacted on macroscopic arthritis and arthritis did not exacerbate alveolar bone loss. However, there were subtle differences between Pg and Aa with the former inducing more alveolar bone loss if PD was induced before CIA. On a molecular level, Pg and Aa led to differential expression patterns in the synovial membranes that were reminiscent of cellular and humoral immune responses, respectively. The Pg and Aa specific signatures in the synovial proteomes suggest a role for oral pathogens in shaping disease subtypes and setting the stage for subsequent therapy response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242868PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774964PMC
January 2021

The Developmental and Genetic Architecture of the Sexually Selected Male Ornament of Swordtails.

Curr Biol 2021 Mar 3;31(5):911-922.e4. Epub 2020 Dec 3.

Institute of Neuroscience, University of Oregon, Eugene, OR 97401, USA. Electronic address:

Sexual selection results in sex-specific characters like the conspicuously pigmented extension of the ventral tip of the caudal fin-the "sword"-in males of several species of Xiphophorus fishes. To uncover the genetic architecture underlying sword formation and to identify genes that are associated with its development, we characterized the sword transcriptional profile and combined it with genetic mapping approaches. Results showed that the male ornament of swordtails develops from a sexually non-dimorphic prepattern of transcription factors in the caudal fin. Among genes that constitute the exclusive sword transcriptome and are located in the genomic region associated with this trait we identify the potassium channel, Kcnh8, as a sword development gene. In addition to its neural function kcnh8 performs a known role in fin growth. These findings indicate that during evolution of swordtails a brain gene has been co-opted for an additional novel function in establishing a male ornament.
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http://dx.doi.org/10.1016/j.cub.2020.11.028DOI Listing
March 2021

Infection With Attenuated Collagen-Induced Arthritis in Mice and Involved Mesenteric T and T Polarization.

Front Immunol 2020 30;11:571049. Epub 2020 Oct 30.

Laboratory for Clinical Immunology, Core Facility for Cell Sorting & Cell Analysis, University Medical Center Rostock, Rostock, Germany.

Objectives: Rheumatoid arthritis is an autoimmune disease with multifactorial etiopathogenesis. Among the environmental factors, mucosal infections and the inducing pathobionts are gaining increasing attention. We here set out to explore the gut-joint-axis and the impact of infection on subsequent arthritis.

Methods: We combined infection in DBA/1J × B10.Q F1 mice with collagen induced arthritis (CIA). Mice were infected oral gavage and infection was monitored by weight loss, colonic histology, and antibodies against bacteria. Scoring of arthritis was performed macroscopically. Intestinal microbiomes were analyzed and immune responses were monitored quantification of transcription factor-specific mRNA isolated from the inguinal and mesenteric lymph nodes.

Results: Infection with VPI 10463 resulted in significant weight loss and severe colitis yet accelerated the reversal towards the original microbiome after antibiotic treatment. Spontaneous clearance of VPI 10463 infection reduced the incidence of subsequent CIA and led to mesenteric T and T polarization. However, this attenuating effect was abrogated if VPI 10463 was eradicated vancomycin followed by fecal microbiota transplantation. Moreover, VPI 10463 infection following the onset of CIA lacked therapeutic potential.

Conclusion: Our results demonstrate that infection with VPI10463 induced an inflammation of the gut that protected from subsequent arthritis development in mice. Both, microbial changes to the gut and immune cell mobilization and/or polarization may have contributed to arthritis protection. The prospect of potential therapeutic benefits resulting from infections or some byproduct thereof call for further experiments that help elucidate exact mechanisms.
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http://dx.doi.org/10.3389/fimmu.2020.571049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662472PMC
October 2020

Oncogenic allelic interaction in highlights hybrid incompatibility.

Proc Natl Acad Sci U S A 2020 11 9;117(47):29786-29794. Epub 2020 Nov 9.

The Xiphophorus Genetic Stock Center, Texas State University, San Marcos, TX 78666;

Mixing genomes of different species by hybridization can disrupt species-specific genetic interactions that were adapted and fixed within each species population. Such disruption can predispose the hybrids to abnormalities and disease that decrease the overall fitness of the hybrids and is therefore named as hybrid incompatibility. Interspecies hybridization between southern platyfish and green swordtails leads to lethal melanocyte tumorigenesis. This occurs in hybrids with tumor incidence following progeny ratio that is consistent with two-locus interaction, suggesting melanoma development is a result of negative epistasis. Such observations make one of the only two vertebrate hybrid incompatibility examples in which interacting genes have been identified. One of the two interacting loci has been characterized as a mutant epidermal growth factor receptor. However, the other locus has not been identified despite over five decades of active research. Here we report the localization of the melanoma regulatory locus to a single gene, , which shows all expected features of the long-sought oncogene interacting locus. Our findings provide insights into the role of regulation in regard to cancer etiology. Finally, they provide a molecular explainable example of hybrid incompatibility.
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http://dx.doi.org/10.1073/pnas.2010133117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703647PMC
November 2020

The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression.

Oncogene 2020 10 25;39(44):6841-6855. Epub 2020 Sep 25.

Department of Physiological Chemistry, University of Würzburg, Würzburg, Germany.

The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by HO or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.
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http://dx.doi.org/10.1038/s41388-020-01477-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605435PMC
October 2020

Cxcl9l and Cxcr3.2 regulate recruitment of osteoclast progenitors to bone matrix in a medaka osteoporosis model.

Proc Natl Acad Sci U S A 2020 08 27;117(32):19276-19286. Epub 2020 Jul 27.

Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore;

Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka () osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits -positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.
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http://dx.doi.org/10.1073/pnas.2006093117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431079PMC
August 2020

The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization.

Nat Ecol Evol 2020 06 30;4(6):841-852. Epub 2020 Mar 30.

Developmental Biochemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

Sturgeons seem to be frozen in time. The archaic characteristics of this ancient fish lineage place it in a key phylogenetic position at the base of the ~30,000 modern teleost fish species. Moreover, sturgeons are notoriously polyploid, providing unique opportunities to investigate the evolution of polyploid genomes. We assembled a high-quality chromosome-level reference genome for the sterlet, Acipenser ruthenus. Our analysis revealed a very low protein evolution rate that is at least as slow as in other deep branches of the vertebrate tree, such as that of the coelacanth. We uncovered a whole-genome duplication that occurred in the Jurassic, early in the evolution of the entire sturgeon lineage. Following this polyploidization, the rediploidization of the genome included the loss of whole chromosomes in a segmental deduplication process. While known adaptive processes helped conserve a high degree of structural and functional tetraploidy over more than 180 million years, the reduction of redundancy of the polyploid genome seems to have been remarkably random.
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http://dx.doi.org/10.1038/s41559-020-1166-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269910PMC
June 2020

miR-221-3p Regulates VEGFR2 Expression in High-Risk Prostate Cancer and Represents an Escape Mechanism from Sunitinib In Vitro.

J Clin Med 2020 Mar 2;9(3). Epub 2020 Mar 2.

Department of Urology and Pediatric Urology, University Hospital Würzburg, 97080 Würzburg, Germany.

Downregulation of miR-221-3p expression in prostate cancer (PCa) predicted overall and cancer-specific survival of high-risk PCa patients. Apart from PCa, miR-221-3p expression levels predicted a response to tyrosine kinase inhibitors (TKI) in clear cell renal cell carcinoma (ccRCC) patients. Since this role of miR-221-3p was explained with a specific targeting of VEGFR2, we examined whether miR-221-3p regulated VEGFR2 in PCa. First, we confirmed VEGFR2/KDR as a target gene of miR-221-3p in PCa cells by applying Luciferase reporter assays and Western blotting experiments. Although VEGFR2 was mainly downregulated in the PCa cohort of the TCGA (The Cancer Genome Atlas) database, VEGFR2 was upregulated in our high-risk PCa cohort ( = 142) and predicted clinical progression. In vitro miR-221-3p acted as an escape mechanism from TKI in PC3 cells, as displayed by proliferation and apoptosis assays. Moreover, we confirmed that Sunitinib induced an interferon-related gene signature in PC3 cells by analyzing external microarray data and by demonstrating a significant upregulation of miR-221-3p/miR-222-3p after Sunitinib exposure. Our findings bear a clinical perspective for high-risk PCa patients with low miR-221-3p levels since this could predict a favorable TKI response. Apart from this therapeutic niche, we identified a partially oncogenic function of miR-221-3p as an escape mechanism from VEGFR2 inhibition.
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http://dx.doi.org/10.3390/jcm9030670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141373PMC
March 2020

miR-221 Augments TRAIL-Mediated Apoptosis in Prostate Cancer Cells by Inducing Endogenous TRAIL Expression and Targeting the Functional Repressors SOCS3 and PIK3R1.

Biomed Res Int 2019 14;2019:6392748. Epub 2019 Nov 14.

Department of Urology and Paediatric Urology, University Hospital Würzburg, Würzburg, Germany.

miR-221 is regarded as an oncogene in many malignancies, and miR-221-mediated resistance towards TRAIL was one of the first oncogenic roles shown for this small noncoding RNA. In contrast, miR-221 is downregulated in prostate cancer (PCa), thereby implying a tumour suppressive function. By using proliferation and apoptosis assays, we show a novel feature of miR-221 in PCa cells: instead of inducing TRAIL resistance, miR-221 sensitized cells towards TRAIL-induced proliferation inhibition and apoptosis induction. Partially responsible for this effect was the interferon-mediated gene signature, which among other things contained an endogenous overexpression of the TRAIL encoding gene TNFSF10. This TRAIL-friendly environment was provoked by downregulation of the established miR-221 target gene SOCS3. Moreover, we introduced PIK3R1 as a target gene of miR-221 in PCa cells. Proliferation assays showed that siRNA-mediated downregulation of SOCS3 and PIK3R1 mimicked the effect of miR-221 on TRAIL sensitivity. Finally, Western blotting experiments confirmed lower amounts of phospho-Akt after siRNA-mediated downregulation of PIK3R1 in PC3 cells. Our results further support the tumour suppressing role of miR-221 in PCa, since it sensitises PCa cells towards TRAIL by regulating the expression of the oncogenes SOCS3 and PIK3R1. Given the TRAIL-inhibiting effect of miR-221 in various cancer entities, our results suggest that the influence of miR-221 on TRAIL-mediated apoptosis is highly context- and entity-dependent.
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http://dx.doi.org/10.1155/2019/6392748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881584PMC
April 2020

Independent Origin of XY and ZW Sex Determination Mechanisms in Mosquitofish Sister Species.

Genetics 2020 01 8;214(1):193-209. Epub 2019 Nov 8.

Physiological Chemistry, Biocenter, University of Wuerzburg, 97074, Germany

Fish are known for the outstanding variety of their sex determination mechanisms and sex chromosome systems. The western () and eastern mosquitofish () are sister species for which different sex determination mechanisms have been described: ZZ/ZW for and XX/XY for Here, we carried out restriction-site associated DNA (RAD-) and pool sequencing (Pool-seq) to characterize the sex chromosomes of both species. We found that the ZW chromosomes of females and the XY chromosomes of males correspond to different linkage groups, and thus evolved independently from separate autosomes. In interspecific hybrids, the Y chromosome is dominant over the W chromosome, and X is dominant over Z. In , we identified a candidate region for the Y-linked melanic pigmentation locus, a rare male phenotype that constitutes a potentially sexually antagonistic trait and is associated with other such characteristics, , large body size and aggressive behavior. We developed a SNP-based marker in the Y-linked allele of (), which was linked to melanism in all tested populations. This locus represents an example for a color locus that is located in close proximity to a putative sex determiner, and most likely substantially contributed to the evolution of the Y.
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http://dx.doi.org/10.1534/genetics.119.302698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944411PMC
January 2020

Implications of the null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult .

J Exp Biol 2019 10 8;222(Pt 19). Epub 2019 Oct 8.

Institute of Clinical Neurobiology, University of Würzburg, 97078 Würzburg, Germany

The gene of encodes a highly abundant 47 kDa synaptic vesicle-associated protein. null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of or on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of but not reduces lifespan, whereas knockout of and , either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in and single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.
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http://dx.doi.org/10.1242/jeb.203505DOI Listing
October 2019

The Piranha Genome Provides Molecular Insight Associated to Its Unique Feeding Behavior.

Genome Biol Evol 2019 08;11(8):2099-2106

McDonnell Genome Institute, Washington University School of Medicine.

The piranha enjoys notoriety due to its infamous predatory behavior but much is still not understood about its evolutionary origins and the underlying molecular mechanisms for its unusual feeding biology. We sequenced and assembled the red-bellied piranha (Pygocentrus nattereri) genome to aid future phenotypic and genetic investigations. The assembled draft genome is similar to other related fishes in repeat composition and gene count. Our evaluation of genes under positive selection suggests candidates for adaptations of piranhas' feeding behavior in neural functions, behavior, and regulation of energy metabolism. In the fasted brain, we find genes differentially expressed that are involved in lipid metabolism and appetite regulation as well as genes that may control the aggression/boldness behavior of hungry piranhas. Our first analysis of the piranha genome offers new insight and resources for the study of piranha biology and for feeding motivation and starvation in other organisms.
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http://dx.doi.org/10.1093/gbe/evz139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681833PMC
August 2019

Expression Signatures of Cisplatin- and Trametinib-Treated Early-Stage Medaka Melanomas.

G3 (Bethesda) 2019 Jul;9(7):2267-2276

Physiological Chemistry, Biocenter, University of Wuerzburg, 97074 Wuerzburg, Germany.

Small aquarium fish models provide useful systems not only for a better understanding of the molecular basis of many human diseases, but also for first-line screening to identify new drug candidates. For testing new chemical substances, current strategies mostly rely on easy to perform and efficient embryonic screens. Cancer, however, is a disease that develops mainly during juvenile and adult stage. Long-term treatment and the challenge to monitor changes in tumor phenotype make testing of large chemical libraries in juvenile and adult animals cost prohibitive. We hypothesized that changes in the gene expression profile should occur early during anti-tumor treatment, and the disease-associated transcriptional change should provide a reliable readout that can be utilized to evaluate drug-induced effects. For the current study, we used a previously established medaka melanoma model. As proof of principle, we showed that exposure of melanoma developing fish to the drugs cisplatin or trametinib, known cancer therapies, for a period of seven days is sufficient to detect treatment-induced changes in gene expression. By examining whole body transcriptome responses we provide a novel route toward gene panels that recapitulate anti-tumor outcomes thus allowing a screening of thousands of drugs using a whole-body vertebrate model. Our results suggest that using disease-associated transcriptional change to screen therapeutic molecules in small fish model is viable and may be applied to pre-clinical research and development stages in new drug discovery.
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http://dx.doi.org/10.1534/g3.119.400051DOI Listing
July 2019

The Myb-MuvB Complex Is Required for YAP-Dependent Transcription of Mitotic Genes.

Cell Rep 2019 06;27(12):3533-3546.e7

Theodor Boveri Institute and Comprehensive Cancer Center Mainfranken, Biocenter, University of Wuerzburg, Wuerzburg 97074, Germany. Electronic address:

YAP and TAZ, downstream effectors of the Hippo pathway, are important regulators of proliferation. Here, we show that the ability of YAP to activate mitotic gene expression is dependent on the Myb-MuvB (MMB) complex, a master regulator of genes expressed in the G2/M phase of the cell cycle. By carrying out genome-wide expression and binding analyses, we found that YAP promotes binding of the MMB subunit B-MYB to the promoters of mitotic target genes. YAP binds to B-MYB and stimulates B-MYB chromatin association through distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. The cooperation between YAP and B-MYB is critical for YAP-mediated entry into mitosis. Furthermore, the expression of genes coactivated by YAP and B-MYB is associated with poor survival of cancer patients. Our findings provide a molecular mechanism by which YAP and MMB regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.
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http://dx.doi.org/10.1016/j.celrep.2019.05.071DOI Listing
June 2019

Periodontal treatment prevents arthritis in mice and methotrexate ameliorates periodontal bone loss.

Sci Rep 2019 05 31;9(1):8128. Epub 2019 May 31.

Institute for Immunology, University Medical Center Rostock, Rostock, Germany.

Recent studies indicate a causal relationship between the periodontal pathogen P. gingivalis and rheumatoid arthritis involving the production of autoantibodies against citrullinated peptides. We therefore postulated that therapeutic eradication P. gingivalis may ameliorate rheumatoid arthritis development and here turned to a mouse model in order to challenge our hypothesis. F1 (DBA/1 x B10.Q) mice were orally inoculated with P. gingivalis before collagen-induced arthritis was provoked. Chlorhexidine or metronidazole were orally administered either before or during the induction phase of arthritis and their effects on arthritis progression and alveolar bone loss were compared to intraperitoneally injected methotrexate. Arthritis incidence and severity were macroscopically scored and alveolar bone loss was evaluated via microcomputed tomography. Serum antibody titres against P. gingivalis were quantified by ELISA and microbial dysbiosis following oral inoculation was monitored in stool samples via microbiome analyses. Both, oral chlorhexidine and metronidazole reduced the incidence and ameliorated the severity of collagen-induced arthritis comparable to methotrexate. Likewise, all three therapies attenuated alveolar bone loss. Relative abundance of Porphyromonadaceae was increased after oral inoculation with P. gingivalis and decreased after treatment. This is the first study to describe beneficial effects of non-surgical periodontal treatment on collagen-induced arthritis in mice and suggests that mouthwash with chlorhexidine or metronidazole may also be beneficial for patients with rheumatoid arthritis and a coexisting periodontitis. Methotrexate ameliorated periodontitis in mice, further raising the possibility that methotrexate may also positively impact on the tooth supporting tissues of patients with rheumatoid arthritis.
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http://dx.doi.org/10.1038/s41598-019-44512-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544621PMC
May 2019

Expression Signatures of Cisplatin- and Trametinib-Treated Early-Stage Medaka Melanomas.

G3 (Bethesda) 2019 07 9;9(7):2267-2276. Epub 2019 Jul 9.

Physiological Chemistry, Biocenter, University of Wuerzburg, 97074 Wuerzburg, Germany

Small aquarium fish models provide useful systems not only for a better understanding of the molecular basis of many human diseases, but also for first-line screening to identify new drug candidates. For testing new chemical substances, current strategies mostly rely on easy to perform and efficient embryonic screens. Cancer, however, is a disease that develops mainly during juvenile and adult stage. Long-term treatment and the challenge to monitor changes in tumor phenotype make testing of large chemical libraries in juvenile and adult animals cost prohibitive. We hypothesized that changes in the gene expression profile should occur early during anti-tumor treatment, and the disease-associated transcriptional change should provide a reliable readout that can be utilized to evaluate drug-induced effects. For the current study, we used a previously established medaka melanoma model. As proof of principle, we showed that exposure of melanoma developing fish to the drugs cisplatin or trametinib, known cancer therapies, for a period of seven days is sufficient to detect treatment-induced changes in gene expression. By examining whole body transcriptome responses we provide a novel route toward gene panels that recapitulate anti-tumor outcomes thus allowing a screening of thousands of drugs using a whole-body vertebrate model. Our results suggest that using disease-associated transcriptional change to screen therapeutic molecules in small fish model is viable and may be applied to pre-clinical research and development stages in new drug discovery.
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http://dx.doi.org/10.1534/g3.119.400051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643878PMC
July 2019

Fgf3 is crucial for the generation of monoaminergic cerebrospinal fluid contacting cells in zebrafish.

Biol Open 2019 Jun 5;8(6). Epub 2019 Jun 5.

Department of Physiological Chemistry, Biocenter, Am Hubland, University of Würzburg, Germany

In most vertebrates, including zebrafish, the hypothalamic serotonergic cerebrospinal fluid-contacting (CSF-c) cells constitute a prominent population. In contrast to the hindbrain serotonergic neurons, little is known about the development and function of these cells. Here, we identify fibroblast growth factor (Fgf)3 as the main Fgf ligand controlling the ontogeny of serotonergic CSF-c cells. We show that positively regulates the number of serotonergic CSF-c cells, as well as a subset of dopaminergic and neuroendocrine cells in the posterior hypothalamus via control of proliferation and cell survival. Further, expression of the ETS-domain transcription factor is downregulated after impairment. Previous findings identified as critical for the proliferation of serotonergic progenitors in the hypothalamus, and therefore we now suggest that Fgf3 acts via during early development to ultimately control the number of mature serotonergic CSF-c cells. Moreover, our analysis of the developing hypothalamic transcriptome shows that the expression of is upregulated upon loss-of-function suggesting activation of a self-compensatory mechanism. Together, these results highlight Fgf3 in a novel context as part of a signalling pathway of critical importance for hypothalamic development.
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http://dx.doi.org/10.1242/bio.040683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602327PMC
June 2019

A novel evolutionary conserved mechanism of RNA stability regulates synexpression of primordial germ cell-specific genes prior to the sex-determination stage in medaka.

PLoS Biol 2019 04 4;17(4):e3000185. Epub 2019 Apr 4.

University of Wuerzburg, Physiological Chemistry, Biocenter, Wuerzburg, Germany.

Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1-acting as master sex-determining gene-has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within dmrt1 genes of metazoans-together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells-suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.
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http://dx.doi.org/10.1371/journal.pbio.3000185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448818PMC
April 2019

The genome of the arapaima (Arapaima gigas) provides insights into gigantism, fast growth and chromosomal sex determination system.

Sci Rep 2019 03 28;9(1):5293. Epub 2019 Mar 28.

University of Wuerzburg, Physiological Chemistry, Biocenter, 97074, Wuerzburg, Germany.

We have sequenced the genome of the largest freshwater fish species of the world, the arapaima. Analysis of gene family dynamics and signatures of positive selection identified genes involved in the specific adaptations and unique features of this iconic species, in particular it's large size and fast growth. Genome sequences from both sexes combined with RAD-tag analyses from other males and females led to the isolation of male-specific scaffolds and supports an XY sex determination system in arapaima. Whole transcriptome sequencing showed that the product of the gland-like secretory organ on the head surface of males and females may not only provide nutritional fluid for sex-unbiased parental care, but that the organ itself has a more specific function in males, which engage more in parental care.
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http://dx.doi.org/10.1038/s41598-019-41457-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439221PMC
March 2019

Application of the Transcriptional Disease Signature (TDSs) to Screen Melanoma-Effective Compounds in a Small Fish Model.

Sci Rep 2019 01 24;9(1):530. Epub 2019 Jan 24.

Xiphophorus Genetic Stock Center, Department of Chemistry and Biochemistry, 419 Centennial Hall, Texas State University, San Marcos, TX, USA.

Cell culture and protein target-based compound screening strategies, though broadly utilized in selecting candidate compounds, often fail to eliminate candidate compounds with non-target effects and/or safety concerns until late in the drug developmental process. Phenotype screening using intact research animals is attractive because it can help identify small molecule candidate compounds that have a high probability of proceeding to clinical use. Most FDA approved, first-in-class small molecules were identified from phenotypic screening. However, phenotypic screening using rodent models is labor intensive, low-throughput, and very expensive. As a novel alternative for small molecule screening, we have been developing gene expression disease profiles, termed the Transcriptional Disease Signature (TDS), as readout of small molecule screens for therapeutic molecules. In this concept, compounds that can reverse, or otherwise affect known disease-associated gene expression patterns in whole animals may be rapidly identified for more detailed downstream direct testing of their efficacy and mode of action. To establish proof of concept for this screening strategy, we employed a transgenic strain of a small aquarium fish, medaka (Oryzias latipes), that overexpresses the malignant melanoma driver gene xmrk, a mutant egfr gene, that is driven by a pigment cell-specific mitf promoter. In this model, melanoma develops with 100% penetrance. Using the transgenic medaka malignant melanoma model, we established a screening system that employs the NanoString nCounter platform to quantify gene expression within custom sets of TDS gene targets that we had previously shown to exhibit differential transcription among xmrk-transgenic and wild-type medaka. Compound-modulated gene expression was identified using an internet-accessible custom-built data processing pipeline. The effect of a given drug on the entire TDS profile was estimated by comparing compound-modulated genes in the TDS using an activation Z-score and Kolmogorov-Smirnov statistics. TDS gene probes were designed that target common signaling pathways that include proliferation, development, toxicity, immune function, metabolism and detoxification. These pathways may be utilized to evaluate candidate compounds for potential favorable, or unfavorable, effects on melanoma-associated gene expression. Here we present the logistics of using medaka to screen compounds, as well as, the development of a user-friendly NanoString data analysis pipeline to support feasibility of this novel TDS drug-screening strategy.
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http://dx.doi.org/10.1038/s41598-018-36656-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345854PMC
January 2019

Ras-Induced miR-146a and 193a Target Jmjd6 to Regulate Melanoma Progression.

Front Genet 2018 18;9:675. Epub 2018 Dec 18.

Cibio, University of Trento, Trento, Italy.

Ras genes are among the most commonly mutated genes in human cancer; yet our understanding of their oncogenic activity at the molecular mechanistic level is incomplete. To identify downstream events that mediate ras-induced cellular transformation , we analyzed global microRNA expression in three different models of Ras-induction and tumor formation in zebrafish. Six microRNAs were found increased in Ras-induced melanoma, glioma and in an inducible model of ubiquitous Ras expression. The upregulation of the microRNAs depended on the activation of the ERK and AKT pathways and to a lesser extent, on mTOR signaling. Two Ras-induced microRNAs (miR-146a and 193a) target Jmjd6, inducing downregulation of its mRNA and protein levels at the onset of Ras expression during melanoma development. However, at later stages of melanoma progression, levels were found elevated. The dynamic of Jmjd6 levels during progression of melanoma in the zebrafish model suggests that upregulation of the microRNAs targeting Jmjd6 may be part of an anti-cancer response. Indeed, triple transgenic fish engineered to express a microRNA-resistant Jmjd6 from the onset of melanoma have increased tumor burden, higher infiltration of leukocytes and shorter melanoma-free survival. Increased expression is found in several human cancers, including melanoma, suggesting that the up-regulation of Jmjd6 is a critical event in tumor progression. The following link has been created to allow review of record GSE37015: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jjcrbiuicyyqgpc&acc=GSE37015.
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http://dx.doi.org/10.3389/fgene.2018.00675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305343PMC
December 2018

Gene expression variation and parental allele inheritance in a Xiphophorus interspecies hybridization model.

PLoS Genet 2018 12 26;14(12):e1007875. Epub 2018 Dec 26.

The Xiphophorus Genetic Stock Center, Department of Chemistry and Biochemistry, Texas State University, San Marcos, Texas, United States of America.

Understanding the genetic mechanisms underlying segregation of phenotypic variation through successive generations is important for understanding physiological changes and disease risk. Tracing the etiology of variation in gene expression enables identification of genetic interactions, and may uncover molecular mechanisms leading to the phenotypic expression of a trait, especially when utilizing model organisms that have well-defined genetic lineages. There are a plethora of studies that describe relationships between gene expression and genotype, however, the idea that global variations in gene expression are also controlled by genotype remains novel. Despite the identification of loci that control gene expression variation, the global understanding of how genome constitution affects trait variability is unknown. To study this question, we utilized Xiphophorus fish of different, but tractable genetic backgrounds (inbred, F1 interspecies hybrids, and backcross hybrid progeny), and measured each individual's gene expression concurrent with the degrees of inter-individual expression variation. We found, (a) F1 interspecies hybrids exhibited less variability than inbred animals, indicting gene expression variation is not affected by the fraction of heterozygous loci within an individual genome, and (b), that mixing genotypes in backcross populations led to higher levels of gene expression variability, supporting the idea that expression variability is caused by heterogeneity of genotypes of cis or trans loci. In conclusion, heterogeneity of genotype, introduced by inheritance of different alleles, accounts for the largest effects on global phenotypical variability.
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http://dx.doi.org/10.1371/journal.pgen.1007875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324826PMC
December 2018

Long-term experimental hybridisation results in the evolution of a new sex chromosome in swordtail fish.

Nat Commun 2018 12 3;9(1):5136. Epub 2018 Dec 3.

Physiological Chemistry, Biozentrum, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.

The remarkable diversity of sex determination mechanisms known in fish may be fuelled by exceptionally high rates of sex chromosome turnovers or transitions. However, the evolutionary causes and genomic mechanisms underlying this variation and instability are yet to be understood. Here we report on an over 30-year evolutionary experiment in which we tested the genomic consequences of hybridisation and selection between two Xiphophorus fish species with different sex chromosome systems. We find that introgression and imposing selection for pigmentation phenotypes results in the retention of an unexpectedly large maternally derived genomic region. During the hybridisation process, the sex-determining region of the X chromosome from one parental species was translocated to an autosome in the hybrids leading to the evolution of a new sex chromosome. Our results highlight the complexity of factors contributing to patterns observed in hybrid genomes, and we experimentally demonstrate that hybridisation can catalyze rapid evolution of a new sex chromosome.
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http://dx.doi.org/10.1038/s41467-018-07648-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277394PMC
December 2018

Loss or oncogenic mutation of DROSHA impairs kidney development and function, but is not sufficient for Wilms tumor formation.

Int J Cancer 2019 03 3;144(6):1391-1400. Epub 2018 Dec 3.

Theodor-Boveri-Institute/Biocenter, Developmental Biochemistry, Wuerzburg University, Wuerzburg, Germany.

Wilms tumor (WT) is the most common kidney cancer in childhood. Mutations in the microprocessor genes DROSHA and DGCR8 have been identified as putative oncogenic drivers, indicating a critical role of aberrant miRNA processing in WT formation. To characterize the in vivo role of DROSHA mutations during kidney development and their oncogenic potential, we analyzed mouse lines with either a targeted deletion of Drosha or an inducible expression of human DROSHA carrying a tumor-specific E1147K mutation that acts in a dominant negative manner. Both types of mutation induce striking changes in miRNA patterns. Six2-cre mediated deletion of Drosha in nephron progenitors led to perinatal lethality with apoptotic loss of progenitor cells and early termination of nephrogenesis. Mosaic deletions via Wt1-cre resulted in a milder phenotype with viable offspring that developed proteinuria after 2-4 weeks, but no evidence of tumor formation. Activation of the DROSHA-E1147K transgene via Six2-cre, on the other hand, induced a more severe phenotype with apoptosis of progenitor cells, proteinuria and glomerular sclerosis. The severely growth retarded mice died within the first 2 months of life, confirming the predicted dominant-negative effect of DROSHA-E1147K in vivo. While our data underscores the importance of a viable self-renewing progenitor pool for kidney development, there was no evidence of tumor formation through impaired DROSHA function. This suggests that either additional alterations in mitogenic or antiapoptotic pathways are needed for malignant transformation, or premature loss of a susceptible target cell population and early lethality prevent WT formation.
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http://dx.doi.org/10.1002/ijc.31952DOI Listing
March 2019

Analysis of the putative tumor suppressor gene cdkn2ab in pigment cells and melanoma of Xiphophorus and medaka.

Pigment Cell Melanoma Res 2019 03 6;32(2):248-258. Epub 2018 Sep 6.

Physiological Chemistry, University of Würzburg, Biozentrum, Würzburg, Germany.

In humans, the CDKN2A locus encodes two transcripts, INK4A and ARF. Inactivation of either one by mutations or epigenetic changes is a frequent signature of malignant melanoma and one of the most relevant entry points for melanomagenesis. To analyze whether cdkn2ab, the fish ortholog of CDKN2A, has a similar function as its human counterpart, we studied its action in fish models for human melanoma. Overexpression of cdkn2ab in a Xiphophorus melanoma cell line led to decreased proliferation and induction of a senescence-like phenotype, indicating a melanoma-suppressive function analogous to mammals. Coexpression of Xiphophorus cdkn2ab in medaka transgenic for the mitfa:xmrk melanoma-inducing gene resulted in full suppression of melanoma development, whereas CRISPR/Cas9 knockout of cdkn2ab resulted in strongly enhanced tumor growth. In summary, this provides the first functional evidence that cdkn2ab acts as a potent tumor suppressor gene in fish melanoma models.
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http://dx.doi.org/10.1111/pcmr.12729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377863PMC
March 2019

RNA-seq analysis identifies different transcriptomic types and developmental trajectories of primary melanomas.

Oncogene 2018 11 11;37(47):6136-6151. Epub 2018 Jul 11.

Department of Physiological Chemistry, University of Würzburg, Biozentrum, Am Hubland, 97074, Würzburg, Germany.

Recent studies revealed trajectories of mutational events in early melanomagenesis, but the accompanying changes in gene expression are far less understood. Therefore, we performed a comprehensive RNA-seq analysis of laser-microdissected melanocytic nevi (n = 23) and primary melanoma samples (n = 57) and characterized the molecular mechanisms of early melanoma development. Using self-organizing maps, unsupervised clustering, and analysis of pseudotime (PT) dynamics to identify evolutionary trajectories, we describe here two transcriptomic types of melanocytic nevi (N1 and N2) and primary melanomas (M1 and M2). N1/M1 lesions are characterized by pigmentation-type and MITF gene signatures, and a high prevalence of NRAS mutations in M1 melanomas. N2/M2 lesions are characterized by inflammatory-type and AXL gene signatures with an equal distribution of wild-type and mutated BRAF and low prevalence of NRAS mutations in M2 melanomas. Interestingly, N1 nevi and M1 melanomas and N2 nevi and M2 melanomas, respectively, cluster together, but there is no clustering in a stage-dependent manner. Transcriptional signatures of M1 melanomas harbor signatures of BRAF/MEK inhibitor resistance and M2 melanomas harbor signatures of anti-PD-1 antibody treatment resistance. Pseudotime dynamics of nevus and melanoma samples are suggestive for a switch-like immune-escape mechanism in melanoma development with downregulation of immune genes paralleled by an increasing expression of a cell cycle signature in late-stage melanomas. Taken together, the transcriptome analysis identifies gene signatures and mechanisms underlying development of melanoma in early and late stages with relevance for diagnostics and therapy.
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http://dx.doi.org/10.1038/s41388-018-0385-yDOI Listing
November 2018