Ameliorating pathogenesis by removing an exon containing a missense mutation: a potential exon-skipping therapy for laminopathies.

Exon skipping, as a therapy to restore a reading frame or switch protein isoforms, is under clinical trial. We hypothesised that removing an in-frame exon containing a mutation could also improve pathogenic phenotypes. Our model is laminopathies: incurable tissue-specific degenerative diseases associated with LMNA mutations. LMNA encodes A-type lamins, that together with B-type lamins, form the nuclear lamina. Lamins contain an alpha-helical central rod domain composed of multiple heptad repeats. Eliminating LMNA exon 3 or 5 removes six heptad repeats, so shortens, but should not otherwise significantly alter, the alpha-helix. Human Lamin A or Lamin C with a deletion corresponding to amino acids encoded by exon 5 (Lamin A/C-Δ5) localised normally in murine lmna-null cells, rescuing both nuclear shape and endogenous Lamin B1/emerin distribution. However, Lamin A carrying pathogenic mutations in exon 3 or 5, or Lamin A/C-Δ3, did not. Furthermore, Lamin A/C-Δ5 was not deleterious to wild-type cells, unlike the other Lamin A mutants including Lamin A/C-Δ3. Thus Lamin A/C-Δ5 function as effectively as wild-type Lamin A/C and better than mutant versions. Antisense oligonucleotides skipped LMNA exon 5 in human cells, demonstrating the possibility of treating certain laminopathies with this approach. This proof-of-concept is the first to report the therapeutic potential of exon skipping for diseases arising from missense mutations.

Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells.

Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34(+ve) myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34(+ve) cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express beta-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34(-ve), Myf5(-ve) minority may be involved in maintaining the lineage-committed majority.

Multiple transcriptional domains, with distinct left and right components, in the atrial chambers of the developing heart.

During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.

Cardiosensor mice and transcriptional subdomains of the vertebrate heart.

Differential regulation of cardiac gene expression in vertebrates has been extensively documented in the context of atrial and ventricular morphogenesis. Recent data, largely from the analysis of transgene and endogenous gene expression patterns, have revealed transcriptional differences between left and right cardiac chambers which suggest that the heart is composed of a series of distinct transcriptional domains. Such phenomena provide regional markers (cardiosensors) for the fine analysis of normal and abnormal heart development. Regional subdivisions and transcriptional heterogeneity within the myocardium emerge in response to patterning of the precardiac mesoderm and early heart tube on the anterior-posterior axis, and to embryonic left-right signals which dictate the direction of cardiac looping. Several families of transcription factors have been characterized which may be implicated in the regionalization of myocardial gene expression.

Macrophage response during axonal regeneration in the axolotl central and peripheral nervous system.

We have used a monoclonal antibody (5F4) and Griffonia lectin to study the recruitment of macrophages after crushing axolotl central and peripheral axons. In both cases axonal regeneration begins within one to two days and, in the CNS, proceeds at a rate of about 0.05 mm per day. However, in the spinal cord, macrophage entry is restricted to the lesion site whilst in peripheral nerves macrophages rapidly enter the distal nerve stump after injury. These results suggest that the role (if any) played by macrophages during axonal regeneration may differ in these two situations.

Alveolar rhabdomyosarcoma-associated proteins PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target genes in muscle stem cells.

Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma in childhood and is characterized by expression of myogenic proteins, including the transcription factors MyoD and myogenin. There are two main subgroups, embryonal RMS and alveolar RMS (ARMS). Most ARMS are associated with chromosomal translocations that have breakpoints in introns of either PAX3 or PAX7, and FOXO1A. These translocations create chimeric transcription factors termed PAX3/FOXO1A and PAX7/FOXO1A respectively. Upon ectopic PAX3/FOXO1A expression, together with other genetic manipulation in mice, both differentiating myoblasts and satellite cells (the resident stem cells of postnatal muscle) can give rise to tumours with ARMS characteristics. As PAX3 and PAX7 are part of transcriptional networks that regulate muscle stem cell function in utero and during early postnatal life, PAX3/FOXO1A and PAX7/FOXO1A may subvert normal PAX3 and PAX7 functions. Here we examined how PAX3/FOXO1A and PAX7/FOXO1A affect myogenesis in satellite cells. PAX3/FOXO1A or PAX7/FOXO1A inhibited myogenin expression and prevented terminal differentiation in murine satellite cells: the same effect as dominant-negative (DN) Pax3 or Pax7 constructs. The transcription of MyoD-target genes myogenin and muscle creatine kinase were suppressed by PAX3/FOXO1A or PAX7/FOXO1A in C2C12 myogenic cells again as seen with Pax3/7DN. PAX3/FOXO1A or PAX7/FOXO1A did not inhibit the transcriptional activity of MyoD by perturbing MyoD expression, localization, phosphorylation or interaction with E-proteins. Chromatin immunoprecipitation on the myogenin promoter showed that PAX3/FOXO1A or PAX7/FOXO1A did not prevent MyoD from binding. However, PAX3/FOXO1A or PAX7/FOXO1A reduced occupation of the myogenin promoter by RNA polymerase II and decreased acetylation of histone H4, but did not directly bind to the myogenin promoter. Together, these observations reveal that PAX3/FOXO1A and PAX7/FOXO1A act to prevent myogenic differentiation via suppression of the transcriptional activation of MyoD-target genes.

BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells.

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.

Restoration of motor unit properties and fiber type distribution in reinnervated axolotl skeletal muscle.

The contractile properties of functionally isolated motor units and the muscle fiber type distribution of reinnervated iliotibialis posterior muscles were examined in axolotls (Ambystoma mexicanum) 7 to 12 months after complete transection of the hind limb nerve trunks. Motor units were continuously distributed with respect to size and contractile speeds and there was a positive correlation between motor unit size and twitch:tetanus ratio. The degree of overlap between motor units was positively correlated with motor unit size and inversely correlated with similarity of contractile speeds. In addition, the muscle fiber type distribution within the reinnervated muscles, as revealed by immunocytochemical localization of tonic, slow, and fast myosin isoforms, was indistinguishable from that in the contralateral unoperated muscles, with no evidence of fiber type grouping. Thus, the motor organization after reinnervation was very similar to that of normal muscles examined previously. The only difference found was that a small number of motor units contracted unusually rapidly in reinnervated muscles. The absence of muscle fiber type redistribution and the essentially normal motor unit characteristics suggest that the original pattern of synaptic connections was reestablished following reinnervation. These observations are consistent with the returning motor neurons selectively reinnervating their original muscle fiber types.

Effects on fluid and Na+ flux of varying luminal hydraulic resistance in rat colon in vivo.

1. A new method of measuring fluid and ionic movements and the dehydrating power of the colon in vivo is described. A range of agarose gel cylinders, with calibrated hydraulic conductivities (Lp), were inserted into the lumen of the descending colon of anaesthetized rats. Fluxes of fluid, Na+ and K+ out of the gels were measured over a period of 60-110 min. 2. Fluid absorption by the colon from 2.5% agarose gels was not slower than from solution without gel. Fluid absorption was inhibited by 66% when the agarose concentration was raised to 10%. In contrast 2.5% agarose gels caused a 73% (P < 0.001) reduction in water flow from rat ileum. 3. Increasing gel concentration to 10% or above caused the absorbate from the gels to become hypertonic (P < 0.001). 4. The measured suction pressure applied by the colonic hypertonic absorbate to the gels increased from 44 +/- 2.3 cmH2O (n = 23) with 2.5% agarose gels to 6713 +/- 960 cmH2O (n = 13) with 15% (P < 0.001). 5. Deoxycholate (2 mM) produced a decrease in fluid and Na+ absorption and reduced the suction pressure and power exerted by the colon.

Effects of freezing a segment of peripheral nerve on subsequent protein release and axonal regeneration in the frog.

Previous studies in frogs have shown that axons from the proximal stump of a cut nerve will grow toward the distal stump, possibly in response to diffusible trophic factors produced by cells of the nerve sheath. In the present experiments, the synthesis and release of proteins in vitro, from proximal and distal stumps of frog sciatic nerves, were studied 1, 4, and 14 days after nerve section in vivo. Using two-dimensional gel electrophoresis to separate released proteins, a marked increase in the synthesis of two lipoproteins of 37 and 67 kDa was seen, initially in both proximal and distal stumps, but by 14 days these proteins were produced exclusively by the distal stump. To see if the production of these proteins was correlated with subsequent reinnervation of the distal stump, isolated nerve segments were removed from the frogs and either replaced immediately or frozen (to kill sheath cells) and replaced. After 2 weeks, the pattern of newly synthesized proteins released by both the frozen and nonfrozen nerve segments was similar although freezing severely impaired the reinnervation of the nerve segment. These results suggest that although the 37- and 67-kDa lipoproteins may have a role in nerve regeneration, their presence per se is not sufficient to support the reinnervation of a distal stump of a cut peripheral nerve and that additional factors may therefore be required.

Concentration polarization of fluorescent dyes in rat descending colonic crypts: evidence of crypt fluid absorption.

1. Using confocal microscopy, the rate of fluid absorption into isolated perifused descending rat colonic crypt lumens is estimated from the concentration polarization and distribution of fluorescein sulphonate (FS) and fluorescein isothiocyanate-dextran (FITC-dextran; molecular weight, 10,000) within the crypt lumens and pericryptal fluid. 2. The probe dyes enter the crypt via the luminal opening, are concentrated in the lumen, then escape into the pericryptal space via the paracellular spaces spanning the crypt wall. 3. FITC-dextran is maximally accumulated at a luminal depth of 60 microns to 5 times the concentration at the crypt opening (p < 0.001) and penetrates 150-200 microns along the lumen. FS is maximally accumulated within crypt lumen close to the opening. At crypt luminal depths 10-60 microns from the opening FS is accumulated by a factor of 1.5-2.0 above that found in HgCl2-treated tissue (p < 0.001). 4. Dye enters the crypt lumen slowly from the basal side, but from this side does not accumulate above the bathing solution concentration. 5. HgCl2 (20 microM) or theophylline (10 mM) completely inhibit concentrative accumulation of FITC-dextran and FS within the crypts and pericryptal space (p < 0.001). 6. Computer simulation of the dye uptake indicates that the rate of water flow into the crypt luminal opening is 1 x 10(-3) cm s-1 which is equivalent to 15 microliters (cm mucosa)-2 h-1. Approximately 75% of the fluid entering the crypt is abosrbed across the proximal 50 microns of crypt wall as a consequence of the large osmotic pressure gradient between the pericryptal and crypt luminal solutions. A pericryptal diffusion barrier with lower permeability than that across the crypt wall is required to simulate dye accumulation in the pericryptal space. Differences between FITC-dextran and FS accumulation are explained by the lower diffusion coefficient within the crypt lumen, and lower crypt wall permeability of FITC-dextran.

Comparison of cattle and sheep colonic permeabilities to horseradish peroxidase and hamster scrapie prion protein in vitro.

BACKGROUND: Paracellular permeability to solutes across the descending colon is much higher in cattle than sheep. This is a possible route for transmission of infective materials, such as scrapie prion. AIMS: To compare the permeabilities of labelled scrapie prion protein and other macromolecules in bovine and ovine descending colons in vitro. METHODS: Using fresh slaughterhouse material, transepithelial fluxes of macromolecules across colonic mucosae mounted in Ussing chambers were measured by monitoring transport of either enzyme activity or radioactivity. RESULTS: The comparative bovine to ovine permeability ratio of the probes increased with molecular weight: from 3.1 (0.13) for PEG400 to 10.67 (0.20) (p<0.001) for PEG4000; and from 1.64 (0.17) for microperoxidase to 7.03 (0.20) (p<0.001) for horseradish peroxidase (HRP). The permeability of (125)I-labelled inactivated Syrian hamster scrapie prion protein (ShaPrP(sc)) was 7.02 (0.33)-fold higher in bovine than ovine colon (p<0.0025). In each species, the probe permeabilities decreased according to the formula: P = P(o). exp(-K.ra). The "ideal" permeabilities, P(o) are similar, however, K((ovine)) = 2.46 (0.20) cm/h/nm exceeds K((bovine)) = 0.85 (0.15) cm/h/nm (p<0.001) indicating that bovine colon has a higher proportion of wide pores than ovine. Image analysis confirmed that HRP permeated through the bovine mucosal layer via a pericryptal paracellular route much more rapidly than in sheep. CONCLUSIONS: These data may imply that scrapie prion is transmitted in vivo more easily across the low resistance bovine colonic barrier than in other species.

Regional differences in rat large intestinal crypt function in relation to dehydrating capacity in vivo.

1. Rat descending colon absorbed fluid against a large hydraulic resistance, imposed by 10 % agarose (w/v) gel plugs inserted in the lumen, by raising the tonicity of the absorbate from the gel to 880 +/- 54 mosmol kg-1; the tonicity of the absorbate from 2.5 % gels was 352 +/- 38 mosmol kg-1. The hypertonic absorbate generated an osmotic pressure which created a fluid tension in the crypt lumen. This was monitored as a suction tension in colonic luminal gels of 45.3 +/- 3 cmH2O with 2.5 % gels and 725 +/- 145 cmH2O with 10 % gels. The caecum was unable to absorb fluid against a significant hydraulic resistance. 2. Fluorescein isothiocyanate-labelled dextran (FITC dextran; molecular mass 10000 Da) accumulated within descending colonic crypt lumens by concentration polarization. Maximal accumulation at a depth of 20-40 micrometer below the mucosal surface was 5.68 +/- 0.2-fold above control levels. Caecal crypts accumulated dextran to a maximum of 1.8 +/- 0.17-fold above control levels. 3. The relationship between crypt luminal tension and suction tension of the distal colon was also demonstrated using paraffin, which occluded the crypt lumens with microscopic droplets and completely inhibited fluid absorption from high resistance luminal gels. 4. Reduction in dietary Na+ intake raised plasma aldosterone and the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. The caecum did not respond in this way to varied Na+ intake.

Dynamic left/right regionalisation of endogenous myosin light chain 3F transcripts in the developing mouse heart.

It has recently emerged that transcriptional differences exist between left and right cardiac chambers. An example is provided by transgenic mice with an nlacZ reporter gene under transcriptional control of the fast skeletal muscle alkali myosin light chain (MLC) 3 promoter and 3' enhancer, which express beta-galactosidase in a left ventricular-right atrial dominant pattern in the developing and adult heart. Here, we demonstrate that endogenous MLC3F transcripts are also left/right regionalised in the mouse heart during embryonic development. Regionalisation is observed as early as embryonic day (E) 8.5, and by E10.5 MLC3F transcripts are present predominantly in the future left ventricle and right atrium, and to a lesser extent in the left atrium. Subsequently, MLC3F transcripts are down-regulated in the left ventricle, and by E12.5 expression is restricted to both atria and left-ventricular trabeculae. No MLC3F protein can be detected in the adult or embryonic mouse heart, suggesting that post-transcriptional regulation prevents this fast myosin isoform contributing to myocardial contraction. Left ventricular-right atrial dominant MLC3F transgenes therefore reflect transitory left/right regionalisation of the endogenous gene, unlike other reported cases of transgene regionalisation. MLC3F transgenes, however, maintain an embryonic-like distribution throughout development suggesting that myocardial gene expression is controlled by distinct temporal, as well as spatial, regulatory modules.

Embryonic and fetal myogenic programs act through separate enhancers at the MLC1F/3F locus.

Embryonic and fetal stages of skeletal muscle development are characterized by the differential expression of a number of muscle-specific genes. These include the products of independent promoters at the fast myosin light chain 1F/3F locus. In the mouse embryo MLC1F transcripts accumulate in embryonic skeletal muscle from E9, 4-5 days before high-level accumulation of MLC3F transcripts. A 3' enhancer can activate MLC1F and MLC3F promoters in differentiated muscle cells in vitro and in transgenic mice; both promoters, however, are activated at the time of MLC1F transcript accumulation. We now demonstrate the presence of a second muscle-specific enhancer at this locus, located in the intron separating the MLC1F and MLC3F promoters. Transgenic mice containing the intronic, but lacking the 3' enhancer, express high levels of an nlacZ reporter gene from the MLC3F promoter in adult fast skeletal muscle fibers. In contrast to the 3' enhancer, the intronic element is inactive both in embryonic muscle cells in vivo and in embryonic myocyte cultures. The intronic enhancer is activated at the onset of fetal development in both primary and secondary muscle fibers, at the time of endogenous MLC3F transcript accumulation. Late-activated MLC3F transgenes thus provide a novel in toto marker of fetal myogenesis. These results suggest that temporal regulation of transcription at the MLC1F/3F locus is controlled by separate enhancers which are differentially activated during embryonic and fetal development.

Transplanted primary neonatal myoblasts can give rise to functional satellite cells as identified using the Myf5nlacZl+ mouse.

Myoblast transplantation is a potential therapeutic approach for the genetic modification of host skeletal muscle tissue. To be considered an effective, long-lived method of delivery, however, it is essential that at least a proportion of the transplanted cells also retain their proliferative potential. We sought to investigate whether transplanted neonatal myoblasts can contribute to the satellite cell compartment of adult skeletal muscle by using the Myf5nlacZ/+ mouse. The Myf5nlacZ/+ mouse has nlacZ targeted to the Myf5 locus resulting in beta-galactosidase activity in quiescent satellite cells. Following transplantation, beta-galactosidase-labelled nuclei were detected in host muscles, showing that donor cells had been incorporated. Significantly, beta-galactosidase-positive, and therefore donor-derived, satellite cells were detected. When placed in culture, beta-galactosidase marked myogenic cells emanated from the parent fibre. These observations demonstrate that cell transplantation not only results in the incorporation of donor nuclei into the host muscle syncytia, but also that the donor cells can become functional satellite cells. The Myf5nlacZ/+ mouse therefore provides a novel and specific marker for determining the contribution of transplanted cells to the satellite cell pool.

Suppression of atrial myosin gene expression occurs independently in the left and right ventricles of the developing mouse heart.

Many cardiac genes are broadly expressed in the early heart and become restricted to the atria or ventricles as development proceeds. Additional transcriptional differences between left and right compartments of the embryonic heart have been described recently, in particular for a number of transgenes containing cardiac regulatory elements. We now demonstrate that three myosin genes which become transcriptionally restricted to the atria between embryonic day (E) 12.5 and birth, alpha-myosin heavy chain (MHC), myosin light chain (MLC) 1A and MLC2A, are coordinately downregulated in the compact myocardium of the left ventricle before that of the right ventricle. alpha-MHC protein also accumulates in the right, but not left, compact ventricular myocardium during this period, suggesting that this transient regionalization contributes to fktal heart function. dHAND and eHAND, basic helix-loop-helix transcription factors known to be expressed in the right and left ventricles respectively at E10. 5, remain regionalized between E12.5 and E14.5. Downregulation of alpha-MHC, MLC1A, and MLC2A in iv/iv embryos, which have defective left/right patterning, initiates in the morphological left (systemic) ventricle regardless of its anatomical position on the right or left hand side of the heart. This points to the importance of left/right ventricular differences in sarcomeric gene expression patterns during fktal cardiogenesis and indicates that these differences originate in the embryo in response to anterior-posterior patterning of the heart tube rather than as a result of cardiac looping. Dev Dyn 2000;217:75-85.