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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.

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.

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.

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.

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.

Rhabdomyosarcoma is a soft tissue cancer that arises in skeletal muscle due to mutations in myogenic progenitors that lead to ineffective differentiation and malignant transformation. The transcription factors Pax3 and Pax7 and their downstream target genes are tightly linked with the fusion positive alveolar subtype, whereas the RAS pathway is usually involved in the embryonal, fusion negative variant. Here, we analyse the role of Pax3 in a fusion negative context, by linking alterations in gene expression in pax3a/pax3b double mutant zebrafish with tumour progression in kRAS-induced rhabdomyosarcoma tumours. Several genes in the RAS/MAPK signalling pathway were significantly down-regulated in pax3a/pax3b double mutant zebrafish. Progression of rhabdomyosarcoma tumours was also delayed in the pax3a/pax3b double mutant zebrafish indicating that Pax3 transcription factors have an unappreciated role in mediating malignancy in fusion negative rhabdomyosarcoma.

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.

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 wild-type 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 wildtype hearts.

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. Cmet 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.

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.