Prdm1a and miR-499 act sequentially to restrict Sox6 activity to the fast-twitch muscle lineage in the zebrafish embryo.

Sox6 has been proposed to play a conserved role in vertebrate skeletal muscle fibre type specification. In zebrafish, sox6 transcription is repressed in slow-twitch progenitors by the Prdm1a transcription factor. Here we identify sox6 cis-regulatory sequences that drive fast-twitch-specific expression in a Prdm1a-dependent manner. We show that sox6 transcription subsequently becomes derepressed in slow-twitch fibres, whereas Sox6 protein remains restricted to fast-twitch fibres. We find that translational repression of sox6 is mediated by miR-499, the slow-twitch-specific expression of which is in turn controlled by Prdm1a, forming a regulatory loop that initiates and maintains the slow-twitch muscle lineage.

Developing a bioink for single-step deposition and maturation of human epidermis.

Patients with severe burns, which cause extensive damage to their skin, require rapid intervention to prevent life-threatening hypothermia, infection, and fluid loss. Current treatments typically involve surgical excision of the burned skin and reconstruction of the wound with the aid of skin autografts. However, there is a lack of donor site in the most severe cases. While alternative treatments such as cultured epithelial autografts and "spray-on" skin can allow much smaller donor tissues to be used (and hence reduce donor site morbidity), they present their own challenges in terms of fragility of the tissues and control of the cell deposition, respectively. Recent advances in bioprinting technology have led researchers to explore its use to fabricate skin grafts, which depend on several factors, including appropriate bioinks, cell types, and printability. In this work, we describe a collagen-based bioink that allows the deposition of a contiguous layer of the keratinocytes directly onto the wound. Special attention was given to the intended clinical workflow. For example, since media changes are not feasible once the bioink is deposited onto the patient, we first developed a media formulation designed to permit a single deposition step and promote self-organization of the cells into the epidermis. Using a collagen-based dermal template populated with dermal fibroblasts, we demonstrated by immunofluorescence staining that the resulting epidermis recapitulates the features of natural skin in expressing p63 (stem cell marker), Ki67 and keratin 14 (proliferation markers), filaggrin and keratin 10 (keratinocyte differentiation and barrier function markers), and collagen type IV (basement membrane protein involved in adherence of the epidermis to the dermis). While further tests are still required to verify its utility as a burn treatment, based on the results we have achieved thus far, we believe that our current protocol can already produce donor-specific model for testing purposes.

Analysis of Pax7 expressing myogenic cells in zebrafish muscle development, injury, and models of disease.

The transcription factor Pax7 is a marker and regulator of muscle progenitors and satellite cells that contribute to the embryonic development and postembryonic growth of skeletal muscle in vertebrates, as well as to its repair and regeneration. Here, we identify Pax7(+ve) myogenic cells in the zebrafish and characterize their behavior in postembryonic stages. Mononucleate Pax7(+ve) cells can first be found associated with myofibers at 72 hours post fertilization (hpf). To follow the behavior of muscle progenitor cells in vivo, we generated transgenic lines expressing fluorescent proteins under the control of the pax7a or pax3a promoters. We established an injury model using cardiotoxin injection and monitored cell proliferation and myogenic regulatory factor expression in myogenic precursors cells and muscle fibers after injury using proliferation markers and the transgenic lines. We also analyzed Pax7(+ve) cells in animals with dystrophic phenotypes and found an increased number compared with wild-type.

Disrupting the LINC complex by AAV mediated gene transduction prevents progression of Lamin induced cardiomyopathy.

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.

The mammalian LINC complex component SUN1 regulates muscle regeneration by modulating drosha activity.

Here we show that a major muscle specific isoform of the murine LINC complex protein SUN1 is required for efficient muscle regeneration. The nucleoplasmic domain of the isoform specifically binds to and inhibits Drosha, a key component of the microprocessor complex required for miRNA synthesis. Comparison of the miRNA profiles between wildtype and SUN1 null myotubes identified a cluster of miRNAs encoded by a non-translated retrotransposon-like one antisense (Rtl1as) transcript that are decreased in the WT myoblasts due to SUN1 inhibition of Drosha. One of these miRNAs miR-127 inhibits the translation of the Rtl1 sense transcript, that encodes the retrotransposon-like one protein (RTL1), which is also required for muscle regeneration and is expressed in regenerating/dystrophic muscle. The LINC complex may therefore regulate gene expression during muscle regeneration by controlling miRNA processing. This provides new insights into the molecular pathology underlying muscular dystrophies and how the LINC complex may regulate mechanosignaling.

Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library.

Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model - the Tg(mpx:GFP)(i114) zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) - to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P3 (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration.

Striking denervation of neuromuscular junctions without lumbar motoneuron loss in geriatric mouse muscle.

Reasons for the progressive age-related loss of skeletal muscle mass and function, namely sarcopenia, are complex. Few studies describe sarcopenia in mice, although this species is the mammalian model of choice for genetic intervention and development of pharmaceutical interventions for muscle degeneration. One factor, important to sarcopenia-associated neuromuscular change, is myofibre denervation. Here we describe the morphology of the neuromuscular compartment in young (3 month) compared to geriatric (29 month) old female C57Bl/6J mice. There was no significant difference in the size or number of motoneuron cell bodies at the lumbar level (L1-L5) of the spinal cord at 3 and 29 months. However, in geriatric mice, there was a striking increase (by ∼2.5 fold) in the percentage of fully denervated neuromuscular junctions (NMJs) and associated deterioration of Schwann cells in fast extensor digitorum longus (EDL), but not in slow soleus muscles. There were also distinct changes in myofibre composition of lower limb muscles (tibialis anterior (TA) and soleus) with a shift at 29 months to a faster phenotype in fast TA muscle and to a slower phenotype in slow soleus muscle. Overall, we demonstrate complex changes at the NMJ and muscle levels in geriatric mice that occur despite the maintenance of motoneuron cell bodies in the spinal cord. The challenge is to identify which components of the neuromuscular system are primarily responsible for the marked changes within the NMJ and muscle, in order to selectively target future interventions to reduce sarcopenia.