Assessment of laser-synthesized Si nanoparticle effects on myoblast motility, proliferation and differentiation: towards potential tissue engineering applications

International audienceDue to their biocompatibility and biodegradability and their unique structural and physicochemical properties, laser-synthesized silicon nanoparticles (Si-NPs) are one of the nanomaterials which have been most studied as potential theragnostic tools for non-invasive therapeutic modalities. However, their ability to modulate cell behavior and to promote proliferation and differentiation is still very little investigated or unknown. In this work, ultrapure ligand free Si-NPs of 50 ± 11.5 nm were prepared by femtosecond (fs) laser ablation in liquid. After showing the ability of Si-NPs to be internalized by murine C2C12 myoblasts, the cytotoxicity of the Si-NPs on these cells was evaluated at concentrations ranging from 14 to 224 μg mL−1. Based on these findings, three concentrations of 14, 28 and 56 μg mL−1 were thus considered to study the effect on myoblast differentiation, proliferation and motility at the molecular and phenotypical levels. It was demonstrated that up to 28 μg mL−1, the Si-NPs are able to promote the proliferation of myoblasts and their subsequent differentiation. Scratch tests were also performed revealing the positive Si-NP effect on cellular motility at 14 and 28 μg mL−1. Finally, gene expression analysis confirmed the ability of Si-NPs to promote proliferation, differentiation and motility of myoblasts even at very low concentration. This work opens up novel exciting prospects for Si-NPs made by the laser process as innovative tools for skeletal muscle tissue engineering in view of developing novel therapeutic protocols for regenerative medicine

HOX epimutations driven by maternal SMCHD1/LRIF1 haploinsufficiency trigger homeotic transformations in genetically wildtype offspring

The body plan of animals is laid out by an evolutionary-conserved HOX code which is colinearly transcribed after zygotic genome activation (ZGA). Here we report that SMCHD1, a chromatin-modifying enzyme needed for X-inactivation in mammals, is maternally required for timely HOX expression. Using zebrafish and mouse Smchd1 knockout animals, we demonstrate that Smchd1 haplo-insufficiency brings about precocious and ectopic HOX transcription during oogenesis and embryogenesis. Unexpectedly, wild-type offspring born to heterozygous knockout zebrafish smchd1 mothers exhibited patent vertebrate patterning defects. The loss of maternal Smchd1 was accompanied by HOX epi-mutations driven by aberrant DNA methylation. We further show that this regulation is mediated by Lrif1, a direct interacting partner of Smchd1, whose knockout in zebrafish phenocopies that of Smchd1. Rather than being a short-lived maternal effect, HOX mis-regulation is stably inherited through cell divisions and persists in cultured fibroblasts derived from FSHD2 patients haploinsufficient for SMCHD1. We conclude that maternal SMCHD1/LRIF1 sets up an epigenetic state in the HOX loci that can only be reset in the germline. Such an unusual inter-generational inheritance, whereby a phenotype can be one generation removed from its genotype, casts a new light on how unresolved Mendelian diseases may be interpreted

Complex 4q35 and 10q26 Rearrangements

International audienceBackground and Objectives After clinical evaluation, the molecular diagnosis of type 1 facioscapulohumeral dystrophy (FSHD1) relies in most laboratories on the detection of a shortened D4Z4 array at the 4q35 locus by Southern blotting. In many instances, this molecular diagnosis remains inconclusive and requires additional experiments to determine the number of D4Z4 units or identify somatic mosaicism, 4q-10q translocations, and proximal p13E-11 deletions. These limitations highlight the need for alternative methodologies, illustrated by the recent emergence of novel technologies such as molecular combing (MC), single molecule optical mapping (SMOM), or Oxford Nanopore-based long-read sequencing providing a more comprehensive analysis of 4q and 10q loci. Over the last decade, MC revealed a further increasing complexity in the organization of the 4q and 10q distal regions in patients with FSHD with cis -duplication of D4Z4 arrays in approximately 1%–2% of cases. Methods By using MC, we investigated in our center 2,363 cases for molecular diagnosis of FSHD. We also evaluated whether previously reported cis -duplications might be identified by SMOM using the Bionano EnFocus FSHD 1.0 algorithm. Results In our cohort of 2,363 samples, we identified 147 individuals carrying an atypical organization of the 4q35 or 10q26 loci. Mosaicism is the most frequent category followed by cis -duplications of the D4Z4 array. We report here chromosomal abnormalities of the 4q35 or 10q26 loci in 54 patients clinically described as FSHD, which are not present in the healthy population. In one-third of the 54 patients, these rearrangements are the only genetic defect suggesting that they might be causative of the disease. By analyzing DNA samples from 3 patients carrying a complex rearrangement of the 4q35 region, we further showed that the SMOM direct assembly of the 4q and 10q alleles failed to reveal these abnormalities and lead to negative results for FSHD molecular diagnosis. Discussion This work further highlights the complexity of the 4q and 10q subtelomeric regions and the need of in-depth analyses in a significant number of cases. This work also highlights the complexity of the 4q35 region and interpretation issues with consequences on the molecular diagnosis of patients or genetic counseling

miR-376a-3p and miR-376b-3p overexpression in Hutchinson-Gilford progeria fibroblasts inhibits cell proliferation and induces premature senescence

International audienc

HRAS germline mutations impair LKB1/AMPK signaling and mitochondrial homeostasis in Costello syndrome models

Germline mutations that activate genes in the canonical RAS/MAPK signaling pathway are responsible for rare human developmental disorders known as RASopathies. Here, we analyzed the molecular determinants of Costello syndrome (CS) using a mouse model expressing HRAS p.G12S, patient skin fibroblasts, hiPSC-derived human cardiomyocytes, a HRAS p.G12V zebrafish model, and human fibroblasts expressing lentiviral constructs carrying HRAS p.G12S or HRAS p.G12A mutations. The findings revealed alteration of mitochondrial proteostasis and defective oxidative phosphorylation in the heart and skeletal muscle of CS mice that were also found in the cell models of the disease. The underpinning mechanisms involved the inhibition of the AMPK signaling pathway by mutant forms of HRAS, leading to alteration of mitochondrial proteostasis and bioenergetics. Pharmacological activation of mitochondrial bioenergetics and quality control restored organelle function in HRAS p.G12A and p.G12S cell models, reduced left ventricle hypertrophy in CS mice, and diminished the occurrence of developmental defects in the CS zebrafish model. Collectively, these findings highlight the importance of mitochondrial proteostasis and bioenergetics in the pathophysiology of RASopathies and suggest that patients with CS may benefit from treatment with mitochondrial modulators

<i>Fat1</i> expression at late stages of muscle differentiation.

<p>(<b>A</b>) <i>Fat1</i> expression was visualized in E13.5 embryos or in neonate (P0) muscle by β-galactosidase staining or by in situ hybridization with a <i>Fat1</i> 3′UTR RNA probe. (<b>B</b>–<b>D</b>) Immunolocalization of FAT1 (anti-FAT1-ICD from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003550#pgen.1003550-Hou1" target="_blank">[35]</a>, green) was performed in E12.5 mouse embryo (<b>C</b>), and on adult (<b>B, D</b>) muscle fibers on longitudinal muscle cryosections from wild type (<b>B</b>, <b>C_1–3, D_1,4</b>), from <i>Fat1^ΔTM/ΔTM</i> embryos (<b>C_4–6</b>), and from <i>Fat1^LacZ/LacZ</i> (<b>D_2–3, D_5–6)</b> mice, combined with either antibodies against alpha-actinin (red, <b>B₅</b>), DHPR (Cacna1s) (red, <b>B_2,3</b>), or RyR (red, <b>B₄</b>), or with Phalloidin (red, <b>C, D</b>). In <b>D</b>, Green channel images (FAT1) were first captured with either identical exposure time between wild type and mutants (<b>D_1,4</b> and <b>D_2,5</b>, 421 ms), or with longer exposure time (<b>D_3,6</b>, 2222 ms). This indicates that the epitope detected by the anti-FAT1-ICD antibody (from ref <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003550#pgen.1003550-Hou1" target="_blank">[35]</a>) is present in reduced but detectable amounts in <i>Fat1^LacZ/LacZ</i> muscles. This observation was made when <i>Fat1^LacZ/LacZ</i> mice (n = 2 at P0; and n = 3 at adult stages) displayed severe muscle defects at the stage of dissection, indicating that levels of FAT1 protein inversely correlate with phenotype severity. Scale bars: (<b>B</b>–<b>D</b>) 4 µm, (<b>C</b>) 6 µm.</p

Presymptomatic adult <i>Fat1</i> mutant mice show selective defects in scapular muscles.

<p>(<b>A</b>) Adult <i>Fat1^LacZ/LacZ</i> mice show visible scapular winging (orange arrow) at stages prior to detectable weight loss (defined as presymptomatic). Pictures (extracted from movies) show a posture in which the mice challenge their shoulder girdle muscles by extending their head as far rostral as possible. At 7 weeks, wasting of the rhomboid muscles can already be detected in presymptomatic <i>Fat1^LacZ/LacZ</i> mice as they move on a cage grid. Note the large gap (orange arrow) between scapulas (where rhomboids normally maintain scapulas attached to the dorsal spine), not visible in the corresponding position in the wild type littermate. (<b>B</b>) At advanced symptomatic stages (30% weight loss, anesthetized mice), there is marked curvature of the spine in the upper back and shoulder area, also visible through X-ray <i>post-mortem</i> imaging. (<b>C</b>) Kaplan-Meier plot showing survival of wild type, <i>Fat1^LacZ/+</i>, and <i>Fat1^LacZ/LacZ</i> mice. Most <i>Fat1^LacZ/LacZ</i> mice die between 2 and 4 months, with a median survival of 3 months, while a small group survives beyond 6 months. (<b>D</b>) Masses of dissected muscles of <i>Fat1^LacZ/LacZ</i> mice at presymptomatic disease stage (0% weight loss, n = 3) relative to age-matched controls (n = 6; average wild type weight defined as 100%). (<b>E</b>) Motor performance defects in presymptomatic adult <i>Fat1^LacZ/LacZ</i> mice. Rotarod analysis shows that the latency to fall off from the rod was significantly shorter in presymptomatic adult <i>Fat1^LacZ/LacZ</i>. In this set of experiments, additional <i>Fat1^LacZ/LacZ</i> mice that were symptomatic at the stage when training started had died by the time the test was performed and are therefore not included in the graph. (<b>F</b>) Scapular muscle dissection in adult wild type and <i>Fat1^LacZ/LacZ</i> mice reveals a pronounced reduction in volume and thickness of the <i>rhomboid superficialis</i> (Rh. Sup.) and <i>rhomboid profundus</i> (Rb. P.). This likely underlies the scapular winging phenotype. In the top pictures, the <i>trapezius cervicalis</i> (Trap) has been removed on the right side of each mouse to uncover the other scapular muscles (<i>rhomboids</i>: Rho; <i>levator scapula</i>: LS). Yellow dotted lines indicate the extent of the scapula, red and orange dotted lines that of the two <i>rhomboid</i> muscles. The intermediate magnification highlights the respective shapes of the <i>rhomboid superficialis</i> (orange dotted line) and <i>rhomboid profundus</i> (purple dotted line). (<b>G</b>) Phalloidin staining of flat-mounted <i>rhomboid superficialis</i> muscles of wild type and <i>Fat1^LacZ/LacZ</i> mice at presymptomatic (middle panel) or advanced disease (20% weight loss; bottom panel) stages shows that early defects of myofiber orientation precede reduction of myofibre diameter. Scale bars: (<b>F</b>) 2 mm; (<b>G</b>) 300 µm.</p

Selective changes in <i>Fat1</i> mutant mice recapitulate the clinical picture of FSHD.

<p>(<b>A</b>) Schematic representation of the human 4q35.2 region, including 5 Mb upstream of the FSHD-associated <i>D4Z4</i> repeat array. (<b>B–C</b>) Retinal defects and exudative vasculopathy in adult <i>Fat1^LacZ/LacZ</i> retinas. <i>Fat1^LacZ/LacZ</i> eyes have an opaque appearance, in contrast to wild type eyes (<b>B</b>; yellow arrow). Removal of the cornea reveals absence of opening of the pigmented retina (aniridia), which therefore covers the lens and prevents light from entering the eye. (<b>C</b>) Retinal vasculature visualized using isolectinB4 (GS-IB4) staining of flat-mounted adult retinas from wild type and <i>Fat1^LacZ/LacZ</i> mice. The retina of <i>Fat1^LacZ/LacZ</i> mice displayed zones in which the normal net of secondary and tertiary vessels was replaced by disorganized vasculature, revealing numerous intra-retinal microvascular abnormalities, including IB4-binding microaneurysms (orange arrows). Insert: Example of severe retinal detachment (red arrows) observed in <i>Fat1^LacZ/LacZ</i> eyes, visible even through the lens prior to its removal during dissection. (<b>D</b>) The shape of the inner ear was visualized at E12.5 in WT and <i>Fat1^ΔTM/ΔTM</i> embryos owing to the MLC3F-2E transgene, which is expressed in the developing inner ear in addition to differentiating muscles. Micrographs show an area of the face around the ear. This area shows: left: the masserter muscles (unaffected), bottom: a stream of muscle cells migrating subcutaneously from the second brachial arch (future subcutaneous muscles of the face, which migration path is visibly affected); and top right: the inner ear structure with the endolymphatic duct (ed), a long tube oriented dorsally, finishing with an enlarged area called the endolymphatic sac (es). Both the ed and es are reduced in half <i>Fat1^ΔTM/ΔTM</i> inner ears examined (frequently asymmetric). (<b>E</b>) Quantification of the inner ear shape defect was performed by measuring the area occupied by the endolymphatic duct (ed) and endolymphatic sac (es), as illustrated with the red dotted lines in (<b>D</b>). Each value for a given genotype were plotted on a vertical line, to illustrate the scale of variability of mutant phenotypes. Scale bars: (<b>B,C₃</b>) 0,5 mm; (<b>C_1–2</b>) 200 µm; (<b>C_4–5</b>) 80 µm, (<b>C₆</b>) 30 µm.</p

FAT1 protein and RNA levels are mis-regulated in human foetal FSHD tissues.

<p>(<b>A</b>) Immunolocalization of FAT1 (Rb-1465 anti FAT1-ICD, green) and DHPR (Cacna1s, magenta) in longitudinal sections from human quadriceps biopsies from a control (top) or and FSHD (F1, bottom) foetus with 1.5 D4Z4 repeats. (<b>B</b>) qPCR analysis of <i>FAT1</i> mRNA levels in quadriceps (3 left graphs) and deltoid muscles (middle graph) and in brain (right graph), comparing respectively with age-matched control foetuses (blue bars), a 26 weeks old FSHD1 foetus (F1) harbouring 1.5 D4Z4 repeats in the 4q35 region (dark red bars), a 16 weeks old FSHD1 foetus harbouring 4.3 D4Z4 repeats at 4q35 region (F2), and twin FSHD1 foetuses aged 28 weeks, with 7 D4Z4 repeats. (<b>C</b>) Analysis of the regulatory status of the promoter region by Chromatin immunoprecipitation. The respective level of the following histone marks: H3K27me3 (silenced chromatin; <b>C-left</b>), and H3K4m3 (promoter active; <b>C-right</b>), in muscle extracts from four age matched controls (ct1 to 4) or four FSHD1 foetuses (F1 to F4) are shown. Relative quantities were normalized with the level of histone marks at the promoter of the <i>GUSB</i> gene as internal control, and expressed as % of control 1 (ct1). Scale bars: (<b>A</b>) 50 µm.</p

The transmembrane domain of FAT1 is required to polarize muscle migration.

<p>(<b>A</b>) Schemes representing the main protein product expected from a wild type, a <i>Fat1^LacZ</i>, and a <i>Fat1^ΔTM</i> locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. (<b>B</b>) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 <i>Fat1^LacZ/LacZ</i>, wild type, and <i>Fat1^ΔTM/ΔTM</i> embryos using indicated antibodies, which targeted epitopes are positioned in (<b>A</b>). (<b>C</b>) Whole mount LacZ staining of E12.5 <i>Fat1^LacZ/LacZ</i> mutant embryo. (<b>D</b>) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and <i>Fat1^ΔTM/ΔTM</i> embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 <i>Fat1^ΔTM/ΔTM</i> embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). (<b>E</b>) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003550#pgen.1003550.s004" target="_blank">Figure S4A</a>). The profile of <i>Fat1</i> RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. <i>Fat1^ΔTM/ΔTM</i> embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in <i>Fat1^LacZ/LacZ</i> embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.</p