Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling.

In the clinic, most cases of congenital heart valve defects are thought to arise through errors that occur after the endothelial-mesenchymal transition (EndoMT) stage of valve development. Although mechanical forces caused by heartbeat are essential modulators of cardiovascular development, their role in these later developmental events is poorly understood. To address this question, we used the zebrafish superior atrioventricular valve (AV) as a model. We found that cellularized cushions of the superior atrioventricular canal (AVC) morph into valve leaflets via mesenchymal-endothelial transition (MEndoT) and tissue sheet delamination. Defects in delamination result in thickened, hyperplastic valves, and reduced heart function. Mechanical, chemical, and genetic perturbation of cardiac forces showed that mechanical stimuli are important regulators of valve delamination. Mechanistically, we show that forces modulate Nfatc activity to control delamination. Together, our results establish the cellular and molecular signature of cardiac valve delamination in vivo and demonstrate the continuous regulatory role of mechanical forces and blood flow during valve formation.

BIN1 modulation in vivo rescues dynamin-related myopathy.

The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2RW/+ and Dnm2RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2RW/+ mice and rescued the perinatal lethality and survival of Dnm2RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations.

MTM1 overexpression prevents and reverts BIN1-related centronuclear myopathy.

Centronuclear and myotubular myopathies (CNM) are rare and severe genetic diseases associated with muscle weakness and atrophy as well as intracellular disorganization of myofibres. The main mutated proteins control lipid and membrane dynamics and are the lipid phosphatase myotubularin (MTM1), and the membrane remodelling proteins amphiphysin 2 (BIN1) and dynamin 2 (DNM2). There is no available therapy. Here, to validate a novel therapeutic strategy for BIN1- and DNM2-CNM, we evaluated adeno-associated virus-mediated MTM1 (AAV-MTM1 ) overexpression in relevant mouse models. Early systemic MTM1 overexpression prevented the development of the CNM pathology in Bin1mck-/- mice, while late intramuscular MTM1 expression partially reverted the established phenotypes after only 4 weeks of treatment. However, AAV-MTM1 injection did not change the DNM2-CNM mouse phenotypes. We investigated the mechanism of the rescue of the myopathy in BIN1-CNM and found that the lipid phosphatase activity of MTM1 was essential for the rescue of muscle atrophy and myofibre hypotrophy but dispensable for the rescue of myofibre disorganization including organelle mis-position and T-tubule defects. Furthermore, the improvement of T-tubule organization correlated with normalization of key regulators of T-tubule morphogenesis, dysferlin and caveolin. Overall, these data support the inclusion of BIN1-CNM patients in an AAV-MTM1 clinical trial.

Mice with muscle-specific deletion of Bin1 recapitulate centronuclear myopathy and acute downregulation of dynamin 2 improves their phenotypes.

Mutations in the BIN1 (Bridging Interactor 1) gene, encoding the membrane remodeling protein amphiphysin 2, cause centronuclear myopathy (CNM) associated with severe muscle weakness and myofiber disorganization and hypotrophy. There is no available therapy, and the validation of therapeutic proof of concept is impaired by the lack of a faithful and easy-to-handle mammalian model. Here, we generated and characterized the Bin1mck-/- mouse through Bin1 knockout in skeletal muscle. Bin1mck-/- mice were viable, unlike the constitutive Bin1 knockout, and displayed decreased muscle force and most histological hallmarks of CNM, including myofiber hypotrophy and intracellular disorganization. Notably, Bin1mck-/- myofibers presented strong defects in mitochondria and T-tubule networks associated with deficient calcium homeostasis and excitation-contraction coupling at the triads, potentially representing the main pathomechanisms. Systemic injection of antisense oligonucleotides (ASOs) targeting Dnm2 (Dynamin 2), which codes for dynamin 2, a BIN1 binding partner regulating membrane fission and mutated in other forms of CNM, improved muscle force and normalized the histological Bin1mck-/- phenotypes within 5 weeks. Overall, we generated a faithful mammalian model for CNM linked to BIN1 defects and validated Dnm2 ASOs as a first translatable approach to efficiently treat BIN1-CNM.

SCA7 Mouse Cerebellar Pathology Reveals Preferential Downregulation of Key Purkinje Cell-Identity Genes and Shared Disease Signature with SCA1 and SCA2.

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease mainly characterized by motor incoordination because of progressive cerebellar degeneration. SCA7 is caused by polyglutamine expansion in ATXN7, a subunit of the transcriptional coactivator SAGA, which harbors histone modification activities. Polyglutamine expansions in specific proteins are also responsible for SCA1-SCA3, SCA6, and SCA17; however, the converging and diverging pathomechanisms remain poorly understood. Using a new SCA7 knock-in mouse, SCA7140Q/5Q, we analyzed gene expression in the cerebellum and assigned gene deregulation to specific cell types using published datasets. Gene deregulation affects all cerebellar cell types, although at variable degree, and correlates with alterations of SAGA-dependent epigenetic marks. Purkinje cells (PCs) are by far the most affected neurons and show reduced expression of 83 cell-type identity genes, including these critical for their spontaneous firing activity and synaptic functions. PC gene downregulation precedes morphologic alterations, pacemaker dysfunction, and motor incoordination. Strikingly, most PC genes downregulated in SCA7 have also decreased expression in SCA1 and SCA2 mice, revealing converging pathomechanisms and a common disease signature involving cGMP-PKG and phosphatidylinositol signaling pathways and LTD. Our study thus points out molecular targets for therapeutic development, which may prove beneficial for several SCAs. Furthermore, we show that SCA7140Q/5Q males and females exhibit the major disease features observed in patients, including cerebellar damage, cerebral atrophy, peripheral nerves pathology, and photoreceptor dystrophy, which account for progressive impairment of behavior, motor, and visual functions. SCA7140Q/5Q mice represent an accurate model for the investigation of different aspects of SCA7 pathogenesis.SIGNIFICANCE STATEMENT Spinocerebellar ataxia 7 (SCA7) is one of the several forms of inherited SCAs characterized by cerebellar degeneration because of polyglutamine expansion in specific proteins. The ATXN7 involved in SCA7 is a subunit of SAGA transcriptional coactivator complex. To understand the pathomechanisms of SCA7, we determined the cell type-specific gene deregulation in SCA7 mouse cerebellum. We found that the Purkinje cells are the most affected cerebellar cell type and show downregulation of a large subset of neuronal identity genes, critical for their spontaneous firing and synaptic functions. Strikingly, the same Purkinje cell genes are downregulated in mouse models of two other SCAs. Thus, our work reveals a disease signature shared among several SCAs and uncovers potential molecular targets for their treatment.

Polyglutamine-expanded ATXN7 alters a specific epigenetic signature underlying photoreceptor identity gene expression in SCA7 mouse retinopathy.

BACKGROUND: Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder that primarily affects the cerebellum and retina. SCA7 is caused by a polyglutamine expansion in the ATXN7 protein, a subunit of the transcriptional coactivator SAGA that acetylates histone H3 to deposit narrow H3K9ac mark at DNA regulatory elements of active genes. Defective histone acetylation has been presented as a possible cause for gene deregulation in SCA7 mouse models. However, the topography of acetylation defects at the whole genome level and its relationship to changes in gene expression remain to be determined. METHODS: We performed deep RNA-sequencing and chromatin immunoprecipitation coupled to high-throughput sequencing to examine the genome-wide correlation between gene deregulation and alteration of the active transcription marks, e.g. SAGA-related H3K9ac, CBP-related H3K27ac and RNA polymerase II (RNAPII), in a SCA7 mouse retinopathy model. RESULTS: Our analyses revealed that active transcription marks are reduced at most gene promoters in SCA7 retina, while a limited number of genes show changes in expression. We found that SCA7 retinopathy is caused by preferential downregulation of hundreds of highly expressed genes that define morphological and physiological identities of mature photoreceptors. We further uncovered that these photoreceptor genes harbor unusually broad H3K9ac profiles spanning the entire gene bodies and have a low RNAPII pausing. This broad H3K9ac signature co-occurs with other features that delineate superenhancers, including broad H3K27ac, binding sites for photoreceptor specific transcription factors and expression of enhancer-related non-coding RNAs (eRNAs). In SCA7 retina, downregulated photoreceptor genes show decreased H3K9 and H3K27 acetylation and eRNA expression as well as increased RNAPII pausing, suggesting that superenhancer-related features are altered. CONCLUSIONS: Our study thus provides evidence that distinctive epigenetic configurations underlying high expression of cell-type specific genes are preferentially impaired in SCA7, resulting in a defect in the maintenance of identity features of mature photoreceptors. Our results also suggest that continuous SAGA-driven acetylation plays a role in preserving post-mitotic neuronal identity.

Loss of zebrafish Ataxin-7, a SAGA subunit responsible for SCA7 retinopathy, causes ocular coloboma and malformation of photoreceptors.

Polyglutamine (polyQ) expansion in Ataxin-7 (ATXN7) results in spinocerebellar ataxia type 7 (SCA7) and causes visual impairment. SCA7 photoreceptors progressively lose their outer segments (OSs), a structure essential for their visual function. ATXN7 is a subunit of the transcriptional coactivator Spt-Ada-Gcn5 Acetyltransferase complex, implicated in the development of the visual system in flies. To determine the function of ATXN7 in the vertebrate eye, we have inactivated ATXN7 in zebrafish. While ATXN7 depletion in flies led to gross retinal degeneration, in zebrafish, it primarily results in ocular coloboma, a structural malformation responsible for pediatric visual impairment in humans. ATXN7 inactivation leads to elevated Hedgehog signaling in the forebrain, causing an alteration of proximo-distal patterning of the optic vesicle during early eye development and coloboma. At later developmental stages, malformations of photoreceptors due to incomplete formation of their OSs are observed and correlate with altered expression of crx, a key transcription factor involved in the formation of photoreceptor OS. Therefore, we propose that a primary toxic effect of polyQ expansion is the alteration of ATXN7 function in the daily renewal of OS in SCA7. Together, our data indicate that ATXN7 plays an essential role in vertebrate eye morphogenesis and photoreceptor differentiation, and its loss of function may contribute to the development of human coloboma.

STIM1 over-activation generates a multi-systemic phenotype affecting the skeletal muscle, spleen, eye, skin, bones and immune system in mice.

Strict regulation of Ca2+ homeostasis is essential for normal cellular physiology. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling basal Ca2+ levels and intracellular Ca2+ store refilling, and abnormal SOCE severely impacts on human health. Overactive SOCE results in excessive extracellular Ca2+ entry due to dominant STIM1 or ORAI1 mutations and has been associated with tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK). Both disorders are spectra of the same disease and involve muscle weakness, myalgia and cramps, and additional multi-systemic signs including miosis, bleeding diathesis, hyposplenism, dyslexia, short stature and ichthyosis. To elucidate the physiological consequences of STIM1 over-activation, we generated a murine model harboring the most common TAM/STRMK mutation and characterized the phenotype at the histological, ultrastructural, metabolic, physiological and functional level. In accordance with the clinical picture of TAM/STRMK, the Stim1R304W/+ mice manifested muscle weakness, thrombocytopenia, skin and eye anomalies and spleen dysfunction, as well as additional features not yet observed in patients such as abnormal bone architecture and immune system dysregulation. The murine muscles exhibited contraction and relaxation defects as well as dystrophic features, and functional investigations unraveled increased Ca2+ influx in myotubes. In conclusion, we provide insight into the pathophysiological effect of the STIM1 R304W mutation in different cells, tissues and organs and thereby significantly contribute to a deeper understanding of the pathomechanisms underlying TAM/STRMK and other human disorders involving aberrant Ca2+ homeostasis and affecting muscle, bones, platelets or the immune system.

Tunable functionalization of nano-emulsions using amphiphilic polymers.

Nano-emulsions are defined as stable oil droplets sizing below 300 nm. Their singular particularity lies in the loading capabilities of their oily core, much higher than other kinds of carrier. On the other hand, functionalizing the dynamic oil/water interface, to date, has remained a challenge. To ensure the best anchoring of the reactive functions onto the surface of the droplets, we have designed specific amphiphilic polymers (APs) based on poly(maleic anhydride-alt-1-octadecene), stabilizing the nano-emulsions instead of surfactants. Aliphatic C18 chains of the APs are anchored in the droplet core, while the hydrophilic parts of the APs are poly(ethylene glycol) (PEG) chains. In addition, PEG chains are terminated with reactive (i) azide functions in order to prove the concept of the droplet decoration with clickable rhodamine (Rh-DBCO, specifically synthesized for this study), or (ii) biotin functions to verify the potential droplet functionalization with fluorescent streptavidin (streptavidin-AF-488). This study describes AP synthesis, physico-chemical characterization of the functional droplets (electron microscopy), and finally fluorescence labeling and droplet decoration. To conclude, these APs constitute an interesting solution for the stable functionalization of nano-emulsion droplets, paving a new way for the applications of nano-emulsions in targeting drug delivery.

In vivo phenotypic and molecular characterization of retinal degeneration in mouse models of three ciliopathies.

Cilia are highly conserved and ubiquitously expressed organelles. Ciliary defects of genetic origins lead to ciliopathies, in which retinal degeneration (RD) is one cardinal clinical feature. In order to efficiently find and design new therapeutic strategies the underlying mechanism of retinal degeneration of three murine model was compared. The rodent models correspond to three emblematic ciliopathies, namely: Bardet-Biedl Syndrome (BBS), Alström Syndrome (ALMS) and CEP290-mediated Leber Congenital Amaurosis (LCA). Scotopic rodent electroretinography (ERG) was used to test the retinal function of mice, Transmitted Electron microscopy (T.E.M) was performed to assess retinal structural defects and real-time PCR for targeted genes was used to monitor the expression levels of the major apoptotic Caspase-related pathways in retinal extracts to identify pathological pathways driving the RD in order to identify potential therapeutic targets. We found that BBS and CEP290-mediated LCA mouse models exhibit perinatal retinal degeneration associated with rhodopsin mislocalization in the photoreceptor and the induction of an Endoplasmic Reticulum (ER) stress. On the other hand, the tested ALMS mouse model, displayed a slower degeneration phenotype, with no Rhodopsin mislocalization nor ER-stress activity. Our data points out that behind the general phenotype of vision loss associated with these ciliopathies, the mechanisms and kinetics of disease progression are different.

Toward the Formulation of Stable Micro and Nano Double Emulsions through a Silica Coating on Internal Water Droplets.

Delivery systems able to coencapsulate both hydrophilic and hydrophobic species are of great interest in both fundamental research and industrial applications. Water-in-oil-in-water (w1/O/W2) emulsions are interesting systems for this purpose, but they suffer from limited stability. In this study, we propose an innovative approach to stabilize double emulsions by the synthesis of a silica membrane at the water/oil interface of the primary emulsion (i.e., inner w1/O emulsion). This approach allows the formulation of stable double emulsions through a two-step process, enabling high encapsulation efficiencies of model hydrophilic dyes encapsulated in the internal droplets. This approach also decreases the scale of the double droplets up to the nanoscale, which is not possible without silica stabilization. Different formulation and processing parameters were explored in order to optimize the methodology. Physicochemical characterization was performed by dynamic light scattering, encapsulation efficiency measurements, release profiles, and optical and transmission electron microscopies.

Correction: Pickering nano-emulsions stabilized by solid lipid nanoparticles as a temperature sensitive drug delivery system.

Correction for 'Pickering nano-emulsions stabilized by solid lipid nanoparticles as a temperature sensitive drug delivery system' by Sidy Mouhamed Dieng et al., Soft Matter, 2019, DOI: 10.1039/c9sm01283d.

Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice.

Myotubular myopathy, or X-linked centronuclear myopathy, is a severe muscle disorder representing a significant burden for patients and their families. It is clinically characterized by neonatal and severe muscle weakness and atrophy. Mutations in the myotubularin (MTM1) gene cause myotubular myopathy, and no specific curative treatment is available. We previously found that dynamin 2 (DNM2) is upregulated in both Mtm1 knockout and patient muscle samples, whereas its reduction through antisense oligonucleotides rescues the clinical and histopathological features of this myopathy in mice. Here, we propose a novel approach targeting Dnm2 mRNA. We screened and validated in vitro and in vivo several short hairpin RNA (shRNA) sequences that efficiently target Dnm2 mRNA. A single intramuscular injection of AAV-shDnm2 resulted in long-term reduction of DNM2 protein level and restored muscle force, mass, histology, and myofiber ultrastructure and prevented molecular defects linked to the disease. Our results demonstrate a robust DNM2 knockdown and provide an alternative strategy based on reduction of DNM2 to treat myotubular myopathy.

Myelinosomes act as natural secretory organelles in Sertoli cells to prevent accumulation of aggregate-prone mutant Huntingtin and CFTR.

Inappropriate deposition of insoluble aggregates of proteins with abnormal structures is a hallmark of affected organs in protein aggregation disease. Very rare, affected organs avoid aggregation naturally. This concerns atrophic testis in Huntington disease (HD). We aimed to understand how HD testis avoids aggregation. Using HD model R6/1 mice, we demonstrate that affected testis contain rare organelles myelinosomes. Myelinosomes secreted from testis somatic TM4 Sertoli cells provide the release of aggregate-prone mutant, but not normal Huntingtin (Htt) exon1. Myelinosomes also support the release of other aggregate-prone mutant protein responsible for cystic fibrosis (CF), F508delCFTR. The traffic and discharge of myelinosomes is facilitated by multivesicular bodies (MVB)s. Inhibition of MVB excretion induced reversible retention of both misfolded proteins inside TM4 Sertoli cells. We propose that myelinosome-mediated elimination of mutant proteins is an unusual secretory process allowing Sertoli cells getting rid of misfolded proteins to avoid aggregation and to maintain cell proteostasis.

Microfluidic-Assisted Production of Size-Controlled Superparamagnetic Iron Oxide Nanoparticles-Loaded Poly(methyl methacrylate) Nanohybrids.

In this paper, superparamagnetic iron oxide nanoparticles (SPIONs, around 6 nm) encapsulated in poly(methyl methacrylate) nanoparticles (PMMA NPs) with controlled sizes ranging from 100 to 200 nm have been successfully produced. The hybrid polymeric NPs were prepared following two different methods: (1) nanoprecipitation and (2) nanoemulsification-evaporation. These two methods were implemented in two different microprocesses based on the use of an impact jet micromixer and an elongational-flow microemulsifier. SPIONs-loaded PMMA NPs synthesized by the two methods presented completely different physicochemical properties. The polymeric NPs prepared with the micromixer-assisted nanoprecipitation method showed a heterogeneous dispersion of SPIONs inside the polymer matrix, an encapsulation efficiency close to 100 wt %, and an irregular shape. In contrast, the polymeric NPs prepared with the microfluidic-assisted nanoemulsification-evaporation method showed a homogeneous dispersion, an almost complete encapsulation, and a spherical shape. The properties of the polymeric NPs have been characterized by dynamic light scattering, thermogravimetric analysis, and transmission electron microscope. In vitro cytotoxicity assays were also performed on the nanohybrids and pure PMMA NPs.

Opposite phenotypes of muscle strength and locomotor function in mouse models of partial trisomy and monosomy 21 for the proximal Hspa13-App region.

The trisomy of human chromosome 21 (Hsa21), which causes Down syndrome (DS), is the most common viable human aneuploidy. In contrast to trisomy, the complete monosomy (M21) of Hsa21 is lethal, and only partial monosomy or mosaic monosomy of Hsa21 is seen. Both conditions lead to variable physiological abnormalities with constant intellectual disability, locomotor deficits, and altered muscle tone. To search for dosage-sensitive genes involved in DS and M21 phenotypes, we created two new mouse models: the Ts3Yah carrying a tandem duplication and the Ms3Yah carrying a deletion of the Hspa13-App interval syntenic with 21q11.2-q21.3. Here we report that the trisomy and the monosomy of this region alter locomotion, muscle strength, mass, and energetic balance. The expression profiling of skeletal muscles revealed global changes in the regulation of genes implicated in energetic metabolism, mitochondrial activity, and biogenesis. These genes are downregulated in Ts3Yah mice and upregulated in Ms3Yah mice. The shift in skeletal muscle metabolism correlates with a change in mitochondrial proliferation without an alteration in the respiratory function. However, the reactive oxygen species (ROS) production from mitochondrial complex I decreased in Ms3Yah mice, while the membrane permeability of Ts3Yah mitochondria slightly increased. Thus, we demonstrated how the Hspa13-App interval controls metabolic and mitochondrial phenotypes in muscles certainly as a consequence of change in dose of Gabpa, Nrip1, and Atp5j. Our results indicate that the copy number variation in the Hspa13-App region has a peripheral impact on locomotor activity by altering muscle function.

Conditional depletion of intellectual disability and Parkinsonism candidate gene ATP6AP2 in fly and mouse induces cognitive impairment and neurodegeneration.

ATP6AP2, an essential accessory component of the vacuolar H+ ATPase (V-ATPase), has been associated with intellectual disability (ID) and Parkinsonism. ATP6AP2 has been implicated in several signalling pathways; however, little is known regarding its role in the nervous system. To decipher its function in behaviour and cognition, we generated and characterized conditional knockdowns of ATP6AP2 in the nervous system of Drosophila and mouse models. In Drosophila, ATP6AP2 knockdown induced defective phototaxis and vacuolated photoreceptor neurons and pigment cells when depleted in eyes and altered short- and long-term memory when depleted in the mushroom body. In mouse, conditional Atp6ap2 deletion in glutamatergic neurons (Atp6ap2(Camk2aCre/0) mice) caused increased spontaneous locomotor activity and altered fear memory. Both Drosophila ATP6AP2 knockdown and Atp6ap2(Camk2aCre/0) mice presented with presynaptic transmission defects, and with an abnormal number and morphology of synapses. In addition, Atp6ap2(Camk2aCre/0) mice showed autophagy defects that led to axonal and neuronal degeneration in the cortex and hippocampus. Surprisingly, axon myelination was affected in our mutant mice, and axonal transport alterations were observed in Drosophila. In accordance with the identified phenotypes across species, genome-wide transcriptome profiling of Atp6ap2(Camk2aCre/0) mouse hippocampi revealed dysregulation of genes involved in myelination, action potential, membrane-bound vesicles and motor behaviour. In summary, ATP6AP2 disruption in mouse and fly leads to cognitive impairment and neurodegeneration, mimicking aspects of the neuropathology associated with ATP6AP2 mutations in humans. Our results identify ATP6AP2 as an essential gene for the nervous system.

The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development.

Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.

Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development.

We present an experimental method allowing the production of three-dimensional organ-like structures, namely microtissues (MTs), in vitro without the need for exogenous extracellular matrix (ECM) or growth factors. Submandibular salivary glands (embryonic day ED14), kidneys (ED13) and lungs (ED13) were harvested from mouse embryos and dissociated into single cells by enzyme treatment. Single cells were seeded into special hanging drop culture plates (InSphero) and cultured for up to 14 days to obtain MTs. This strategy permitted full control of the quantity of seeded cells. The development of the MTs into organs was followed histologically and immunohistochemically. Well-organized epithelial structures surrounded by a basal lamina were formed, as confirmed by transmission electron microscopy. Expression of E-cadherin, vimentin, fibronectin and α-SMA was compared in organs and corresponding MTs by real-time quantitative polymerase chain reaction. Branching morphogenesis was induced in MTs (as shown by histology and immunostaining for fibronectin and perlecan) and was conserved even after 14 days of culture. MTs continued their development and their epithelial structures were comparable with those of the physiological organ at postnatal day 2 (PN2). Expression of aquaporins was investigated to obtain better support for the functional differentiation of epithelial cells. Histogenesis proceeded and led to the start of organogenesis. This experimental model might improve our knowledge of epithelial-mesenchymal histogenesis and can be employed to study development or cellular organization during the embryonic formation of organs.