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Desmin, a major muscle-specific intermediate filament protein, is widely regarded as a ubiquitous component of mature skeletal muscle fibers. However, recent observations in human extraocular and palatal muscles indicate that a subset of fibers lack or show very weak desmin expression. The aim of the study was to investigate the presence of desmin in 21 different adult, two toddler, and one fetal human head and neck muscles. The samples were analyzed by immunohistochemistry, and, in one muscle group, also by in situ hybridization for desmin mRNA. Two limb muscles, the biceps brachii and the vastus lateralis, served as controls. In limb muscles, all fibers consistently exhibited desmin immunoreactivity. In contrast, fibers with absent or very weak desmin immunoreactivity were identified across all examined head and neck muscles, with substantial variation between muscle groups. These fibers were predominantly associated with the slow MyHC1 phenotype. A similar pattern was also observed in infant and fetal samples. In longitudinal sections, some fibers showed segmental absence of desmin along their length. In situ hybridization demonstrated absent or markedly reduced desmin mRNA in fibers with absent or reduced desmin immunoreactivity. Taken together, this study provides the first evidence that subsets of myofibers in most human head and neck muscles lack desmin immunoreactivity, indicating that desmin is not ubiquitous in all myofibers in these muscles. These findings reveal previously unrecognized cytoskeletal heterogeneity within human head and neck musculature. The functional significance of fibers lacking or showing very weak desmin immunoreactivity remains to be determined.

Purpose: To investigate the longitudinal development of refraction and strabismus in a national cohort of children born extremely preterm (EPT) and find relations to perinatal events and neurodevelopmental outcomes.

Methods: All surviving children born <27-gestation weeks in Sweden during April 2004–March 2007, and full-term born matched controls were invited to participate in the 12-year ophthalmological follow-up of the Extremely Preterm Infants in Sweden Study (EXPRESS). Refractive error after cycloplegia and manifest strabismus were studied in relation to gestational age, intraventricular haemorrhage (IVH), retinopathy of prematurity (ROP), ROP treatment, cerebral palsy, cognitive disability and visual impairment.

Results: Refractive error was measured in 309 EPT children and 184 controls, and strabismus was assessed in 332 EPT and 188 controls. In the EPT group, 13.6% (n = 42) had significant myopia, 24.6% (n = 76) had significant hyperopia, and 27.5% (n = 85) had significant astigmatism. The development of spherical refractive error in EPT from 2.5 to 12 years was similar to controls, though the dispersion of values was broader in the EPT group. Myopia was related to ROP treatment; astigmatism was related to ROP treatment and severe IVH. Hyperopia was higher in EPT children without ROP treatment compared to controls. Strabismus was found in 18% (61 of 332) EPT children, 68% had esotropia and 32% exotropia, and was associated with ROP treatment and IVH.

Conclusion: EPT birth affects emmetropisation longitudinally, with most EPT children remaining hyperopic, except those treated for ROP. A rising trajectory of strabismus contributes to the complex ophthalmological disabilities challenging children born EPT.

Purpose: The Extremely Preterm Infants in Sweden Study (EXPRESS) followed a national cohort of extremely preterm born (EPT, i.e. <27 weeks) children until 12 years of age. This study aimed to investigate the longitudinal development of visual acuity (VA) in children born EPT, explore the predictive value of early visual assessments, and evaluate risk factors for visual impairment at the age of 12 years.

Methods: All 462 children born EPT in Sweden during April 2004–March 2007, and surviving to age 6.5 years, and full-term born matched controls were invited to participate in the 12-year follow-up. VA was assessed at 12 years and the results were compared with values at 2.5 and 6.5 years.

Results: At age 12, 332 (72%) EPT survivors and 189 controls were examined. The mean VA in the EPT group was lower than in the control group (1.15, 95%CI [1.12–1.19] vs. 1.33, 95% CI [1.29–1.37]). Fifteen (4.5%) EPT had visual impairment. The examination at age 2.5 failed to identify most of them, while the examination at 6.5 could predict the final visual outcome at 12. Risk factors for visual impairment were low gestational age, moderate and severe intraventricular haemorrhage, treatment-requiring retinopathy of prematurity, cerebral palsy, and cognitive disability.

Conclusion: In this national cohort, the VA outcome at age 12 was lower in children born EPT than full-term controls. As eye examination at 2.5 years did not reliably identify visual impairment, clinical risk factors should be considered in the screening of children born EPT to early identify the visually impaired.

The extraocular muscles have complex structural and physiological characteristics adapted to meet the motility requirements of fovea-based binocular vision and show unique responses to disease. They are remarkably able to remain intact in muscular dystrophies and motor neuron diseases, but they are preferentially affected in myasthenia gravis, mitochondrial myopathies, oculopharyngeal muscular dystrophy, thyroid associated orbitopathy, and the Miller Fisher syndrome. The reasons behind these unusual properties are only beginning to be revealed.

Purpose: We have previously described a novel type of multiterminal en plaque motor endplates in the human extraocular muscles (EOMs). This study aimed to investigate whether multiterminal en plaque motor endplates are conserved in EOMs among vertebrates.

Methods: The motor endplates were identified with α-bungarotoxin (α-BTx) and antibodies against synaptic proteins and neurofilament in the EOMs of zebrafish, rabbits and mice. Transcriptomic data were re-analyzed to identify acetylcholine receptor (AChR) subunits in EOMs and trunk muscles of wild-type zebrafish at five and 20 months of age.

Results: In addition to the two typical types of single en plaque and multiple en grappe motor endplates, the third type of multiterminal en plaque motor endplates were observed in the EOMs of zebrafish, rabbits, and mice. The EOMs of zebrafish showed a significantly higher proportion of myofibers containing multiterminal en plaque motor endplates compared to EOMs of rabbits and mice. RNA sequencing data revealed significantly higher AChR subunits in the zebrafish EOMs compared to trunk muscles.

Conclusions: Multiterminal en plaque motor endplates are not exclusive to human EOMs but are also present in the EOMs of other vertebrate species, suggesting a conserved feature of the EOMs.

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.

We aimed to study aniridia-related keratopathy (ARK) relevant cell signaling pathways [Notch1, Wnt/β-catenin, Sonic hedgehog (SHH) and mTOR] in normal human fetal corneas compared with normal human adult corneas and ARK corneas. We found that fetal corneas at 20 weeks of gestation (wg) and normal adult corneas showed similar staining patterns for Notch1; however 10–11 wg fetal corneas showed increased presence of Notch1. Numb and Dlk1 had an enhanced presence in the fetal corneas compared with the adult corneas. Fetal corneas showed stronger immunolabeling with antibodies against β-catenin, Wnt5a, Wnt7a, Gli1, Hes1, p-rpS6, and mTOR when compared with the adult corneas. Gene expression of Notch1, Wnt5A, Wnt7A, β-catenin, Hes1, mTOR, and rps6 was higher in the 9–12 wg fetal corneas compared with adult corneas. The cell signaling pathway differences found between human fetal and adult corneas were similar to those previously found in ARK corneas with the exception of Notch1. Analogous profiles of cell signaling pathway activation between human fetal corneas and ARK corneas suggests that there is a less differentiated host milieu in ARK.

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.

Purpose: The purpose of this study was to investigate the cytoskeletal composition of myotendinous junctions (MTJs) in the human extraocular muscles (EOMs). Desmin and other major cytoskeletal proteins are enriched at the MTJs of ordinary myofibers, where they are proposed to be of particular importance for force transmission and required to maintain myofiber integrity. Methods: EOM and limb muscle samples were analyzed with immunohistochemistry using antibodies against the intermediate filament proteins desmin, nestin, keratin 19, vimentin, and different myosin heavy chain (MyHC) isoforms. MTJs were identified by labeling with antibodies against laminin or tenascin. Results: In contrast to MTJs in lumbrical muscle where desmin, nestin, and keratin 19 were always present, approximately one-third of the MTJs in the EOMs lacked either desmin and/or nestin, and all MTJs lacked keratin 19. Approximately 6% of the MTJs in the EOMs lacked all of these key cytoskeletal proteins. Conclusions: The cytoskeletal protein composition of MTJs in human EOMs differed significantly from that of MTJs in limb muscles. These differences in cytoskeletal protein composition may indicate particular adaptation to meet the functional requirements of the EOMs.