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Joanna von Hofsten,1,2 Tomas Bergström,3 Madeleine Zetterberg1,4 1Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, SE-405 30, Sweden; 2Department of Ophthalmology, Halland Hospital Halmstad, Halmstad, SE- 301 85, Sweden; 3Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, SE-413 46, Sweden; 4Department of Ophthalmology, Sahlgrenska University Hospital, Mölndal, SE-431 30, SwedenCorrespondence: Joanna von Hofsten, Department of Ophthalmology, Halland Hospital Halmstad, Lasarettsvägen, Halmstad, S-301 85, Sweden, Fax +4635158205, Email joanna.von.hofsten@gu.sePurpose: To assess herpesvirus DNA detection in aqueous humor from a cohort of asymptomatic Scandinavian patients undergoing elective cataract surgery.Patients and Methods: Prospective case series. Aqueous samples were obtained from 30 patients undergoing elective cataract surgery. Polymerase chain reaction (PCR) analysis for herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), cytomegalovirus (CMV), Epstein Barr virus (EBV) was performed. Toxoplasma was added to the analysis due to its role as pathogen with ocular latency.Results: Mean age of participants was 75.3 years. Sixteen subjects (53%) had ocular comorbidities. Five subjects (17%) had endothelial dysfunction without known hereditary pattern. None of the samples were positive for herpesviruses or toxoplasma.Conclusion: None of the aqueous samples were positive, suggesting shedding does not frequently occur in the aqueous humor of asymptomatic patients.Keywords: shedding, aqueous humor, herpes simplex virus, varicella zoster virus

Joanna von Hofsten,1,2 Maria Egardt,2 Madeleine Zetterberg2–41Department of Ophthalmology, Halland Hospital Halmstad, Halmstad, 2Department of Ophthalmology, Sahlgrenska University Hospital, Mölndal, 3Department of Clinical Neuroscience and Rehabilitation, 4Department of Ophthalmology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, SwedenPurpose: The study aims to describe an intact cohort with mixed ocular surface disease (OSD) treated with autologous serum (AS) eye drops in a tertiary eye center.Patients and methods: All cases (n=32 eyes, 24 patients) treated with AS for OSD at the Department of Ophthalmology, Sahlgrenska University Hospital, Mölndal, between 2002 and 2013 were included and medical records were reviewed retrospectively.Results: Mean duration of treatment with 20% AS was 28.3±56.1 (median: 12, range: 3–217) days. The most common indication for AS treatment was a persistent epithelial defect (PED), which was seen in 16 eyes of 14 patients. Mean duration of PED prior to treatment was 19.3±18.9 (median: 10, range: 5–68) days. Complete or partial epithelial healing occurred in nine eyes (56.2%). The remaining seven eyes (44%) did not respond to treatment or data were missing. The second group consisted of nine eyes of five patients with superficial punctate keratitis (SPK) secondary to dry eye syndrome. Complete or partial healing of the epithelium occurred in five eyes (56%), and the remaining four eyes (44%) were lost to follow-up. A third group included five eyes with AS as an adjuvant treatment after corneal perforation, whereas a fourth group consisted of one patient with dry eye after laser-assisted in situ keratomileusis (LASIK).Conclusion: In this cohort, patients with PED or SPK responded well to treatment with AS. Standardized preparation protocols, defined optimal serum concentrations for various indications, and large randomized clinical trials are needed to fully comprehend the role of AS in the treatment of OSD.Keywords: dry eye syndrome, persistent epithelial defect, superficial punctate keratitis

Abstract Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.

Abstract Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.

Purpose: To investigate predisposing immunosuppressive conditions causing cytomegalovirus retinitis (CMVr) and risk factors for delayed diagnosis in patients diagnosed between 2008 and 2018 in the Swedish population of 10 million., Results: Sixty-three consecutive patients (100 eyes) were diagnosed with CMVr. The most common immunosuppressive state predisposing for CMVr was hematopoietic stem cell transplant recipients (27%) and hematological malignancy (24%). Two patients (3.2%) had no other predisposing factor than diabetes mellitus (DM). Patients with delayed diagnosis (≤30 days) were older than those with earlier diagnosis, mean age 68.7 (±9.8) and 48.8 (±17.6), respectively, p = .001. Signs of intraocular inflammation (IOI) were seen in 42 (70%) of cases and more common in delayed than early diagnosis, 16 (89%) and 21 (60%) respectively ( p = .03)., Conclusion: Delayed diagnosis was more common in older individuals with signs of IOI. DM may be a risk factor for CMVr, and clinical signs can be mistaken for diabetic retinopathy.

Autophagy is a degradational pathway with pivotal roles in cellular homeostasis and survival, including protection of neurons in the central nervous system (CNS). The significance of autophagy as antiviral defense mechanism is recognized and some viruses hijack and modulate this process to their advantage in certain cell types. Here, we present data demonstrating that the human neurotropic herpesvirus varicella zoster virus (VZV) induces autophagy in human SH-SY5Y neuronal cells, in which the pathway exerts antiviral activity. Productively VZV-infected SH-SY5Y cells showed increased LC3-I-LC3-II conversion as well as co-localization of the viral glycoprotein E and the autophagy receptor p62. The activation of autophagy was dependent on a functional viral genome. Interestingly, inducers of autophagy reduced viral transcription, whereas inhibition of autophagy increased viral transcript expression. Finally, the genotype of patients with severe ocular and brain VZV infection were analyzed to identify potential autophagy-associated inborn errors of immunity. Two patients expressing genetic variants in the autophagy genes ULK1 and MAP1LC3B2, respectively, were identified. Notably, cells of both patients showed reduced autophagy, alongside enhanced viral replication and death of VZV-infected cells. In conclusion, these results demonstrate a neuro-protective role for autophagy in the context of VZV infection and suggest that failure to mount an autophagy response is a potential predisposing factor for development of severe VZV disease., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: Desmin is a major cytoskeletal protein considered ubiquitous in mature muscle fibers. However, we earlier reported that a subgroup of muscle fibers in the soft palate of healthy subjects and obstructive sleep apnea patients (OSA) lacked immunoexpression for desmin. This raised the question of whether these fibers also lack messenger ribonucleic acid (mRNA) for desmin and can be considered a novel fiber phenotype. Moreover, some fibers in the OSA patients had an abnormal distribution and aggregates of desmin. Thus, the aim of the study was to investigate if these desmin protein abnormalities are also reflected in the expression of desmin mRNA in an upper airway muscle of healthy subjects and OSA patients., Methods: Muscle biopsies from the musculus uvulae in the soft palate were obtained from ten healthy male subjects and six male patients with OSA. Overnight sleep apnea registrations were done for all participants. Immunohistochemistry, in-situ hybridization, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) techniques were used to evaluate the presence of desmin protein and its mRNA., Results: Our findings demonstrated that a group of muscle fibers lacked expression for desmin mRNA and desmin protein in healthy individuals and OSA patients (12.0 ± 5.6% vs. 23.1 ± 10.8%, p = 0.03). A subpopulation of these fibers displayed a weak subsarcolemmal rim of desmin accompanied by a few scattered mRNA dots in the cytoplasm. The muscles of OSA patients also differed from healthy subjects by exhibiting muscle fibers with reorganized or accumulated aggregates of desmin protein (14.5 ± 6.5%). In these abnormal fibers, the density of mRNA was generally low or concentrated in specific regions. The overall quantification of desmin mRNA by RT-qPCR was significantly upregulated in OSA patients compared to healthy subjects (p = 0.01)., Conclusions: Our study shows evidence that muscle fibers in the human soft palate lack both mRNA and protein for desmin. This indicates a novel cytoskeletal structure and challenges the ubiquity of desmin in muscle fibers. Moreover, the observation of reorganized or accumulated aggregates of desmin mRNA and desmin protein in OSA patients suggests a disturbance in the transcription and translation process in the fibers of the patients., (© 2024. The Author(s).)

In muscular dystrophies, muscle fibers loose integrity and die, causing significant suffering and premature death. Strikingly, the extraocular muscles (EOMs) are spared, functioning well despite the disease progression. Although EOMs have been shown to differ from body musculature, the mechanisms underlying this inherent resistance to muscle dystrophies remain unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscles in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is increased in response to the knockout of desmin, plectin and obscurin, cytoskeletal proteins whose knockout causes different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival. Therefore, Fhl2 is a protective agent and a candidate target gene for therapy of muscular dystrophies., (© 2024. The Author(s).)

Purpose: The cytoskeleton of the extraocular muscles (EOMs) is significantly different from that of other muscles. We aimed to investigate the role of obscurin, a fundamental cytoskeletal protein, in the EOMs., Methods: The distribution of obscurin in human and zebrafish EOMs was compared using immunohistochemistry. The two obscurin genes in zebrafish, obscna and obscnb, were knocked out using CRISPR/Cas9, and the EOMs were investigated using immunohistochemistry, qPCR, and in situ hybridization. The optokinetic reflex (OKR) in five-day-old larvae and adult obscna-/-;obscnb-/- and sibling control zebrafish was analyzed. Swimming distance was recorded at the same age., Results: The obscurin distribution pattern was similar in human and zebrafish EOMs. The proportion of slow and fast myofibers was reduced in obscna-/-;obscnb-/- zebrafish EOMs but not in trunk muscle, whereas the number of myofibers containing cardiac myosin myh7 was significantly increased in EOMs of obscurin double mutants. Loss of obscurin resulted in less OKRs in zebrafish larvae but not in adult zebrafish., Conclusions: Obscurin expression is conserved in normal human and zebrafish EOMs. Loss of obscurin induces a myofiber type shift in the EOMs, with upregulation of cardiac myosin heavy chain, myh7, showing an adaptation strategy in EOMs. Our model will facilitate further studies in conditions related to obscurin.

Purpose: Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are neurotropic human alphaherpesviruses endemic worldwide. Upon primary infection, both viruses establish lifelong latency in neurons and reactivate intermittently to cause a variety of mild to severe diseases. Acute retinal necrosis (ARN) is a rare, sight-threatening eye disease induced by ocular VZV or HSV infection. The virus and host factors involved in ARN pathogenesis remain incompletely described. We hypothesize an underlying genetic defect in at least part of ARN cases., Methods: We collected blood from 17 patients with HSV-or VZV-induced ARN, isolated DNA and performed Whole Exome Sequencing by Illumina followed by analysis in Varseq with criteria of CADD score > 15 and frequency in GnomAD < 0.1% combined with biological filters. Gene modifications relative to healthy control genomes were filtered according to high quality and read-depth, low frequency, high deleteriousness predictions and biological relevance., Results: We identified a total of 50 potentially disease-causing genetic variants, including missense, frameshift and splice site variants and on in-frame deletion in 16 of the 17 patients. The vast majority of these genes are involved in innate immunity, followed by adaptive immunity, autophagy, and apoptosis; in several instances variants within a given gene or pathway was identified in several patients., Discussion: We propose that the identified variants may contribute to insufficient viral control and increased necrosis ocular disease presentation in the patients and serve as a knowledge base and starting point for the development of improved diagnostic, prophylactic, and therapeutic applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Heinz, Swagemakers, von Hofsten, Helleberg, Thomsen, De Keukeleere, de Boer, Ilginis, Verjans, van Hagen, van der Spek and Mogensen.)

Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Migrating muscle progenitors delaminate from the somite and subsequently form muscle tissue in distant anatomical regions such as the paired appendages, or limbs. In amniotes, this process requires a signaling cascade including the transcription factor paired box 3 (Pax3)., Results: In this study, we found that, unlike in mammals, pax3a/3b double mutant zebrafish develop near to normal appendicular muscle. By analyzing numerous mutant combinations of pax3a, pax3b and pax7a, and pax7b, we determined that there is a feedback system and a compensatory mechanism between Pax3 and Pax7 in this developmental process, even though Pax7 alone is not required for appendicular myogenesis. pax3a/3b/7a/7b quadruple mutant developed muscle-less pectoral fins., Conclusions: We found that Pax3 and Pax7 are redundantly required during appendicular myogenesis in zebrafish, where Pax7 is able to activate the same developmental programs as Pax3 in the premigratory progenitor cells., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Purpose: To study the medial rectus (MR) muscle of zebrafish ( Danio rerio ) with respect to the pattern of distribution of desmin and its correlation to distinct types of myofibers and motor endplates., Methods: The MRs of zebrafish were examined using confocal microscopy in whole-mount longitudinal specimens and in cross sections processed for immunohistochemistry with antibodies against desmin, myosin heavy chain isoforms, and innervation markers. Desmin patterns were correlated to major myofiber type and type of innervation. A total of 1382 myofibers in nine MR muscles were analyzed., Results: Four distinct desmin immunolabeling patterns were found in the zebrafish MRs. Approximately a third of all slow myofibers lacked desmin, representing 8.5% of the total myofiber population. The adult zebrafish MR muscle displayed en grappe, en plaque, and multiterminal en plaque neuromuscular junctions (NMJs) with intricate patterns of desmin immunolabeling., Conclusions: The MRs of zebrafish showed important similarities with the human extraocular muscles with regard to the pattern of desmin distribution and presence of the major types of NMJs and can be regarded as an adequate model to further study the role of desmin and the implications of heterogeneity in cytoskeletal protein composition., Translational Relevance: The establishment of a zebrafish model to study the cytoskeleton in muscles that are particularly resistant to muscle disease opens new avenues to understand human myopathies and muscle dystrophies and may provide clues to new therapies., Competing Interests: Disclosure: N. Dennhag, None; J.-X. Liu, None; H. Nord, None; J. von Hofsten, None; F. Pedrosa Domellöf, None, (Copyright 2020 The Authors.)

We report a case of acute retinal necrosis presenting with acute glaucoma preceding inflammatory signs by several days. High-throughput sequencing on aqueous humor revealed a low-level diversity in the viral genome comparable to diversity seen in cutaneous vesicles in contrast to high diversity in encephalitis., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts., (© 2019 Tran et al.)

The hepatocyte growth factor receptor C-met plays an important role in cellular migration, which is crucial for many developmental processes as well as for cancer cell metastasis. C-met has been linked to the development of mammalian appendicular muscle, which are derived from migrating muscle progenitor cells (MMPs) from within the somite. Mammalian limbs are homologous to the teleost pectoral and pelvic fins. In this study we used Crispr/Cas9 to mutate the zebrafish met gene and found that the MMP derived musculature of the paired appendages was severely affected. The mutation resulted in a reduced muscle fibre number, in particular in the pectoral abductor, and in a disturbed pectoral fin function. Other MMP derived muscles, such as the sternohyoid muscle and posterior hypaxial muscle were also affected in met mutants. This indicates that the role of met in MMP function and appendicular myogenesis is conserved within vertebrates., Competing Interests: The authors have declared that no competing interests exist.

Background: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of β-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling., Methods: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling., Findings: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of β-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes., Interpretation: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression. FUND: KAW 2012.0090, CAN 2017/544, Swedish Medical Research Council (2016-02513), Prostatacancerförbundet, Konung Gustaf V:s Frimurarestiftelse and Cancerforskningsfonden Norrland. The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To identify all patients tested positive for herpes viruses in intraocular samples between 2007 and 2016 in South-Western Sweden and evaluate which of these met the criteria of acute retinal necrosis (ARN). To compare viral load in intraocular samples and virus type with clinical outcome., Method and Analysis: Retrospective case series. Intraocular samples and serum were analysed with quantitative real-time PCR (qPCR) and presence of antibodies (IgG and IgM) were detected by ELISA in serum., Results: Between 2007 and 2016, 13 patients met the clinical criteria of ARN and were PCR-positive in aqueous or vitreous for herpes simplex virus 1 (HSV1; n=4), herpes simplex virus 2 (HSV2; n=3) and varicella zoster virus (VZV; n=6). None of the patients tested positive for cytomegalovirus (n=13) or Epstein Barr virus (n=2) met the criteria of ARN. All ARN patients had specific serum IgG and three patients exhibited virus DNA in serum. There was no correlation between high viral load and worse visual outcome. However, higher viral loads were seen in samples taken earlier in the disease process. Median age was higher (p=0.049) in VZV-ARN than for HSV-ARN patients (60.5 and 45.4 years, respectively) with a tendency of worse best corrected visual acuity at presentation (1.62 and 0.79 log MAR, respectively; p=0.079)., Conclusion: ARN is a reactivation of alpha herpes virus and presence of herpes DNA in serum may occur. VZV-ARN are older than HSV-ARN patients. High viral load does not appear to be a predictor of worse visual outcome, but rather indicates earlier sampling., Competing Interests: Competing interests: None declared.

Using Caenorhabditis elegans as an infection host model for Vibrio cholerae predator interactions, we discovered a bacterial cytotoxin, MakA, whose function as a virulence factor relies on secretion via the flagellum channel in a proton motive force-dependent manner. The MakA protein is expressed from the polycistronic makDCBA ( m otility- a ssociated ki lling factor) operon. Bacteria expressing makDCBA induced dramatic changes in intestinal morphology leading to a defecation defect, starvation and death in C. elegans . The Mak proteins also promoted V. cholerae colonization of the zebrafish gut causing lethal infection. A structural model of purified MakA at 1.9 Å resolution indicated similarities to members of a superfamily of bacterial toxins with unknown biological roles. Our findings reveal an unrecognized role for V. cholerae flagella in cytotoxin export that may contribute both to environmental spread of the bacteria by promoting survival and proliferation in encounters with predators, and to pathophysiological effects during infections., Competing Interests: The authors declare no competing interests.

The pigment pattern of many animal species is a result of the arrangement of different types of pigment-producing chromatophores. The zebrafish has three different types of chromatophores: black melanophores, yellow xanthophores, and shimmering iridophores arranged in a characteristic pattern of golden and blue horizontal stripes. In the zebrafish embryo, chromatophores derive from the neural crest cells. Using pax7a and pax7b zebrafish mutants, we identified a previously unknown requirement for Pax7 in xanthophore lineage formation. The absence of Pax7 results in a severe reduction of xanthophore precursor cells and a complete depletion of differentiated xanthophores in embryos as well as in adult zebrafish. In contrast, the melanophore lineage is increased in pax7a/pax7b double-mutant embryos and larvae, whereas juvenile and adult pax7a/pax7b double-mutant zebrafish display a severe decrease in melanophores and a pigment pattern disorganization indicative of a xanthophore- deficient phenotype. In summary, we propose a novel role for Pax7 in the early specification of chromatophore precursor cells., (© 2016 Nord et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Numerous muscle lineages are formed during myogenesis within both slow- and fast-specific cell groups. In this study, we show that six fast muscle-specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms.

Background: Previously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion., Results: Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels., Conclusions: These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.

In the embryonic zebrafish, skeletal muscle fibres are formed from muscle progenitors in the paraxial mesoderm. The embryonic myotome is mostly constituted of fast-twitch-specific fibres, which are formed from a fast-specific progenitor cell pool. The most lateral fraction of the fast domain in the myotome of zebrafish embryos derives from the Pax7-positive dermomyotome-like cells. In this study, we show that two genes, belonging to the sine oculus class 1 (six1) genes (six1a and six1b), are both essential for the regulation of Pax7(+) cell proliferation and, consequently, in their differentiation during the establishment of the zebrafish dermomyotome. In both six1a and six1b morphant embryos, Pax7(+) cells are initially formed but fail to proliferate, as detected by reduced levels of the proliferation marker phosphohistone3 and reduced brdU incorporation. In congruence, overexpression of six1a or six1b leads to increased Pax7(+) cell number and reduced or alternatively delayed fibre cell differentiation. Bone morphogenetic protein signalling has previously been suggested to inhibit differentiation of Pax7(+) cells in the dermomyotome. Here we show that the remaining Pax7(+) cells in six1a and six1b morphant embryos also have significantly reduced pSmad1/5/8 levels and propose that this leads to a reduced proliferative activity, which may result in a premature differentiation of Pax7(+) cells in the zebrafish dermomyotome. In summary, we show a mechanism for Six1a and Six1b in establishing the Pax7(+) cell derived part of the fast muscle and suggest new important roles for Six1 in the regulation of the Pax7(+) muscle cell population through pSmad1/5/8 signalling.

Although the function of the adult olfactory system has been thoroughly studied, the molecular mechanisms regulating the initial formation of the olfactory nerve, the first cranial nerve, remain poorly defined. Here, we provide evidence that both modulated Notch and bone morphogenetic protein (BMP) signaling affect the generation of neurons in the olfactory epithelium and reduce the number of migratory neurons, so called epithelioid cells. We show that this reduction of epithelial and migratory neurons is followed by a subsequent failure or complete absence of olfactory nerve formation. These data provide new insights into the early generation of neurons in the olfactory epithelium and the initial formation of the olfactory nerve tract. Our results present a novel mechanism in which BMP signals negatively affect Notch activity in a dominant manner in the olfactory epithelium, thereby regulating neurogenesis and explain why a balance of BMP and Notch activity is critical for the generation of neurons and proper development of the olfactory nerve.

The olfactory sensory epithelium and the respiratory epithelium are derived from the olfactory placode. However, the molecular mechanisms regulating the differential specification of the sensory and the respiratory epithelium have remained undefined. To address this issue, we first identified Msx1/2 and Id3 as markers for respiratory epithelial cells by performing quail chick transplantation studies. Next, we established chick explant and intact chick embryo assays of sensory/respiratory epithelial cell differentiation and analyzed two mice mutants deleted of Bmpr1a;Bmpr1b or Fgfr1;Fgfr2 in the olfactory placode. In this study, we provide evidence that in both chick and mouse, Bmp signals promote respiratory epithelial character, whereas Fgf signals are required for the generation of sensory epithelial cells. Moreover, olfactory placodal cells can switch between sensory and respiratory epithelial cell fates in response to Fgf and Bmp activity, respectively. Our results provide evidence that Fgf activity suppresses and restricts the ability of Bmp signals to induce respiratory cell fate in the nasal epithelium. In addition, we show that in both chick and mouse the lack of Bmp or Fgf activity results in disturbed placodal invagination; however, the fate of cells in the remaining olfactory epithelium is independent of morphological movements related to invagination. In summary, we present a conserved mechanism in amniotes in which Bmp and Fgf signals act in an opposing manner to regulate the respiratory versus sensory epithelial cell fate decision.

The zebrafish u-boot (ubo) gene encodes the transcription factor Prdm1, which is essential for the specification of the primary slow-twitch muscle fibres that derive from adaxial cells. Here, we show that Prdm1 functions by acting as a transcriptional repressor and that slow-twitch-specific muscle gene expression is activated by Prdm1-mediated repression of the transcriptional repressor Sox6. Genes encoding fast-specific isoforms of sarcomeric proteins are ectopically expressed in the adaxial cells of ubo(tp39) mutant embryos. By using chromatin immunoprecipitation, we show that these are direct targets of Prdm1. Thus, Prdm1 promotes slow-twitch fibre differentiation by acting as a global repressor of fast-fibre-specific genes, as well as by abrogating the repression of slow-fibre-specific genes.

Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT., (Copyright (c) 2007 Wiley-Liss, Inc.)

The developing oocyte is surrounded by an acellular envelope that is composed of 2-4 isoforms of zona pellucida (ZP) proteins. The ZP proteins comprise the ZP1, ZP2, ZP3, and ZPX isoforms. While ZP1 (ZPB) and ZP3 (ZPC) are present in all species, ZP2 (ZPA) is not found in teleost fish and ZPX is not found in mammals. In the present study, we identify and characterize the ZP1, ZP3 and ZPX isoforms of gilthead seabream. Furthermore, by analyzing the conserved domains, which include the external hydrophobic patch and the internal hydrophobic patch, we show that ZP2 and ZPX are closely related isoforms. ZP proteins are synthesized in either the liver or ovary of most teleosts. Only in rainbow trout has it been shown that zp3 has dual transcription sites. In gilthead seabream, all four mRNA isoforms are transcribed in both the liver and ovary, with zp1a, zp1b, and zp3 being highly expressed in the liver, and zpx being primarily expressed in the ovary. However, determination of the ZP proteins in plasma showed high levels of ZP1b, ZP3, and ZPX, with low or non-detectable levels of ZP1a. In similarity to other teleost ZPs, the hepatic transcription of all four ZP isoforms is under estrogenic control. Previously, we have shown that cortisol can potentiate estrogen-induced ZP synthesis in salmonids, and now we show that this is not the case in the gilthead seabream. The present study shows for the first time the endocrine regulation of a teleost ZPX isoform, and demonstrates the dual-organ transcriptional activities of all the ZP proteins in one species.

Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.

Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-beta subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.

Vibrio anguillarum is an invasive pathogen of fish causing a septicaemia called vibriosis. In this work, transparent zebrafish were immersed in water containing green fluorescent protein labelled V. anguillarum. The infection was visualised at the whole fish and single bacterium levels using microscopy. The gastrointestinal tract was the first site where the pathogen was detected. This enteric localisation occurred independently of the flagellum or motility. On the other hand, chemotactic motility was essential for association of the pathogen with the fish surface. In conclusion, the zebrafish infection model provides evidence that the intestine and skin represent sites of infection by V. anguillarum and suggests a host site where chemotaxis may function in virulence.

Fushi tarazu factor-1 (FTZ-F1) genes belong to the nuclear receptor family 5A (NR5A). The distribution pattern of NR5A genes in teleosts suggests that they control functions separate to, or in addition to, those of other vertebrates. In mammals NR5A1 genes, including steroidogenic factor-1 (SF-1), are primarily involved in steroidogenesis. NR5A2 contain the alpha-fetoprotein transcription factor (FTF) genes, which protect mammalian embryos against maternal estrogens, and are involved in cholesterol transfer and metabolism. In this study we have analysed the expression of two Arctic char FTZ-F1 forms belonging to the NR5A2 group. The expression starts during early development and the transcripts are present in embryonic liver/pancreas and gonadal regions. The genes are up-regulated during embryogenesis as the embryo develops towards hatch, as shown by increased mRNA and protein levels. In adult Arctic char the FTZ-F1 forms are primarily located to tissues involved in steroidogenesis as well as cholesterol metabolism. Thus, a division of NR5A into SF-1 (NR5A1) and FTF (NR5A2) specific functions does not appear to have occurred in teleosts.

Teleost fushi tarazu factor-1 (FTZ-F1) is a potential regulator of steroidogenesis. The present study shows sex-specific regulation of Arctic char fushi tarazu factor-1 (acFF1) and steroidogenic genes during reproductive maturation and in response to hormone treatment. A link between gonadal expression of acFF1, steroidogenic acute regulatory protein (StAR), and cytochrome P450-11A (CYP11A), was observed in the reproductive maturation process, as elevated acFF1 mRNA and protein levels preceded increased StAR and CYP11A transcription. Sex-specific differences were observed as estrogen treatment resulted in down-regulated levels of acFF1 mRNA in testis and male head kidney, whereas no significant effect was observed in females. 11-Ketotestosterone (11-KT) down-regulated CYP11A and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in head kidney and up-regulated CYP11A in testis. StAR remained unaffected by hormone treatment. This suggests that acFF1 is controlled by 17beta-estradiol, whereas the effects on CYP11A and 3betaHSD are mediated by 11-KT. Coexpression of acFF1, StAR, and CYP11A was observed in head kidney, in addition to gonads, indicating correlation between these steroidogenic genes. StAR and acFF1 were also coexpressed in liver, suggesting a potential role in cholesterol metabolism. Although these results indicate conserved steroidogenic functions for FTZ-F1 among vertebrates, they also raise the question of additional roles for FTZ-F1 in teleosts.

In zebrafish (Danio rerio) pigmentation is initiated during embryogenesis and begins in the retinal epithelium and in the melanophores. The pigment cells develop rapidly, and within hours they constitute a prominent feature of the embryo. In order to improve signal detection by whole mount in situ hybridization, confocal microscopy, or expression of GFP, embryos may be treated with 1-phenyl 2-thiourea (PTU) during embryogenesis. PTU inhibits melanogenesis by blocking all tyrosinase-dependent steps in the melanin pathway but can be toxic at high concentrations. The embryos remain transparent as long as the PTU treatment is continued. However, PTU treatment must be initiated before the initial pigmentation because it does not remove already formed pigment. Here we provide a protocol for generating transparent zebrafish while avoiding the toxic and teratogenic effects of PTU treatment.

The fushi tarazu factor 1 (FTZ-F1) gene family constitutes a subgroup of orphan nuclear receptors which can be divided into two groups (LRH/FTF- and SF-1/Ad4BP-like) based on sequence homology, function, and tissue distribution. Analysis of zebrafish FTZ-F1 homologues (zFF1 and ff1b) during embryogenesis indicated distinct expression patterns for both genes. Besides the previously observed expression in pituitary/hypothalamus and mandibular arch, zFF1 transcripts were also detected in domains corresponding to the pronephric duct, somites, liver, and hindbrain. Additionally, ff1b transcripts were detected at other developmental stages than earlier documented. Comparative sequence analysis showed that zFF1 exhibited higher sequence similarity to the LRH/FTF group than the SF-1/Ad4BP group, whereas ff1b was indistinguishable between the groups. These observations, coupled with obtained expression patterns, indicate that zebrafish FTZ-F1 homologues exhibit characteristics that are indicative of both LRH/FTF- and SF-1/Ad4BP-like genes., (Copyright 2001 Academic Press.)