Myocardial deletion of Smad4 using a novel alpha skeletal muscle actin Cre recombinase transgenic mouse causes misalignment of the cardiac outflow tract

S MAD4 acts as the converging point f or T GF β and BMP signaling in heart development. Here, we investigated the role of S MAD4 in heart development usi ng a novel α skeletal muscle actin Cre recombinase (MuCre) transgenic mouse strain. Lineage tracing using MuCre/ROSA26LacZ reporter mice indicated strong Cre-recombinase expression in developing and adult heart and skeletal muscles. In heart development, significant MuCre expression was noted at E11.5 in the atrial, ventricular, outflow tract and atrioventricular canal myocardium, but not in the endocardial cushions. MuCre-driven conditional deletion of Smad4 in mice caused double outlet right ventricle (DORV), ventricular septal defect (VSD), impaired trabeculation and thinning of ventricular myocardium, and mid-gestational embryonic lethality. In conclusion, MuCre mice effectively delete genes in both heart and skeletal muscles, thus enabling the discovery that myocardial Smad4 deletion causes misalignment of the outflow tract and DORV

OCRL1 engages with the F-BAR protein pacsin 2 to promote biogenesis of membrane-trafficking intermediates

Mutation of the inositol 5-phosphatase OCRL1 causes Lowe syndrome and Dent-2 disease. Loss of OCRL1 function perturbs several cellular processes, including membrane traffic, but the underlying mechanisms remain poorly defined. Here we show that OCRL1 is part of the membrane-trafficking machinery operating at the trans-Golgi network (TGN)/endosome interface. OCRL1 interacts via IPIP27A with the F-BAR protein pacsin 2. OCRL1 and IPIP27A localize to mannose 6-phosphate receptor (MPR)–containing trafficking intermediates, and loss of either protein leads to defective MPR carrier biogenesis at the TGN and endosomes. OCRL1 5-phosphatase activity, which is membrane curvature sensitive, is stimulated by IPIP27A-mediated engagement of OCRL1 with pacsin 2 and promotes scission of MPR-containing carriers. Our data indicate a role for OCRL1, via IPIP27A, in regulating the formation of pacsin 2–dependent trafficking intermediates and reveal a mechanism for coupling PtdIns(4,5)P2 hydrolysis with carrier biogenesis on endomembranes

Genetic Deletion of Laminin Isoforms β2 and γ3 Induces a Reduction in Kir4.1 and Aquaporin-4 Expression and Function in the Retina

Glial cells such as retinal Müller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Müller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Müller cells.The aim of the present study was to test the function of the β2 and γ3 containing laminins in murine Müller cells. We used knockout mice with genetic deletion of both β2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Müller cells from laminin β2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice.These findings demonstrate a strong impact of laminin β2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Müller cells

Modulation in voluntary neural drive in relation to muscle soreness

The aim of this study was to investigate whether (1) spinal modulation would change after non-exhausting eccentric exercise of the plantar flexor muscles that produced muscle soreness and (2) central modulation of the motor command would be linked to the development of muscle soreness. Ten healthy subjects volunteered to perform a single bout of backward downhill walking exercise (duration 30 min, velocity 1 ms−1, negative grade −25%, load 12% of body weight). Neuromuscular test sessions [H-reflex, M-wave, maximal voluntary torque (MVT)] were performed before, immediately after, as well as 1–3 days after the exercise bout. Immediately after exercise there was a −15% decrease in MVT of the plantar flexors partly attributable to an alteration in contractile properties (−23% in electrically evoked mechanical twitch). However, MVT failed to recover before the third day whereas the contractile properties had significantly recovered within the first day. This delayed recovery of MVT was likely related to a decrement in voluntary muscle drive. The decrease in voluntary activation occurred in the absence of any variation in spinal modulation estimated from the H-reflex. Our findings suggest the development of a supraspinal modulation perhaps linked to the presence of muscle soreness

Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival

Microanatomy of Adult Zebrafish Extraocular Muscles

Binocular vision requires intricate control of eye movement to align overlapping visual fields for fusion in the visual cortex, and each eye is controlled by 6 extraocular muscles (EOMs). Disorders of EOMs are an important cause of symptomatic vision loss. Importantly, EOMs represent specialized skeletal muscles with distinct gene expression profile and susceptibility to neuromuscular disorders. We aim to investigate and describe the anatomy of adult zebrafish extraocular muscles (EOMs) to enable comparison with human EOM anatomy and facilitate the use of zebrafish as a model for EOM research. Using differential interference contrast (DIC), epifluorescence microscopy, and precise sectioning techniques, we evaluate the anatomy of zebrafish EOM origin, muscle course, and insertion on the eye. Immunofluorescence is used to identify components of tendons, basement membrane and neuromuscular junctions (NMJs), and to analyze myofiber characteristics. We find that adult zebrafish EOM insertions on the globe parallel the organization of human EOMs, including the close proximity of specific EOM insertions to one another. However, analysis of EOM origins reveals important differences between human and zebrafish, such as the common rostral origin of both oblique muscles and the caudal origin of the lateral rectus muscles. Thrombospondin 4 marks the EOM tendons in regions that are highly innervated, and laminin marks the basement membrane, enabling evaluation of myofiber size and distribution. The NMJs appear to include both en plaque and en grappe synapses, while NMJ density is much higher in EOMs than in somatic muscles. In conclusion, zebrafish and human EOM anatomy are generally homologous, supporting the use of zebrafish for studying EOM biology. However, anatomic differences exist, revealing divergent evolutionary pressures

Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development

Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin β1 and β2 chains in various developing fetal organs is already available, a systematic localization of the laminin β1 and β2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin β1 and β2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin β1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin β2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development

Over-expression of adenosine deaminase in mouse podocytes does not reverse puromycin aminonucleoside resistance

<p>Abstract</p> <p>Background</p> <p>Edema in nephrotic syndrome results from renal retention of sodium and alteration of the permeability properties of capillaries. Nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats reproduces the biological and clinical signs of the human disease, and has been widely used to identify the cellular mechanisms of sodium retention. Unfortunately, mice do not develop nephrotic syndrome in response to PAN, and we still lack a good mouse model of the disease in which the genetic tools necessary for further characterizing the pathophysiological pathway could be used. Mouse resistance to PAN has been attributed to a defect in glomerular adenosine deaminase (ADA), which metabolizes PAN. We therefore attempted to develop a mouse line sensitive to PAN through induction of normal adenosine metabolism in their podocytes.</p> <p>Methods</p> <p>A mouse line expressing functional ADA under the control of the podocyte-specific podocin promoter was generated by transgenesis. The effect of PAN on urinary excretion of sodium and proteins was compared in rats and in mice over-expressing ADA and in littermates.</p> <p>Results</p> <p>We confirmed that expression of ADA mRNAs was much lower in wild type mouse than in rat glomerulus. Transgenic mice expressed ADA specifically in the glomerulus, and their ADA activity was of the same order of magnitude as in rats. Nonetheless, ADA transgenic mice remained insensitive to PAN treatment in terms of both proteinuria and sodium retention.</p> <p>Conclusions</p> <p>Along with previous results, this study shows that adenosine deaminase is necessary but not sufficient to confer PAN sensitivity to podocytes. ADA transgenic mice could be used as a background strain for further transgenesis.</p

Lung development in laminin γ2 deficiency: abnormal tracheal hemidesmosomes with normal branching morphogenesis and epithelial differentiation

BACKGROUND: Laminin γ2 (Lamc2), one of the polypeptides in laminin-332 (laminin-5), is prominent in the basement membrane of alveolar walls and airways of developing and adult lung. Laminins are important for lung morphogenesis and based on its localization, a function for laminin γ2 in lung development has been hypothesized. Targeted deletion of the laminin γ2 gene in mice results in skin blistering and neonatal death at 3–5 days after birth due to failure to thrive. METHODS: Examination of lung development in Lamc2-/- mice through 1–2 days postnatal was accomplished by morphometric analysis, lung bud culture, electron microscopy, immunohistochemical and immunofluorescence staining. RESULTS: Compared to littermate controls, Lamc2-/- lungs were similar in morphology during embryonic life. At post-natal day 1–2, distal saccules were mildly dilated by chord length measurements. Epithelial differentiation as evaluated by immunohistochemical staining for markers of ciliated cells, Clara cells, alveolar type I cells and alveolar type II cells did not reveal a difference between Lamc2-/- and littermate control lungs. Likewise, vascular development, smooth muscle cell differentiation, and elastic fiber formation looked similar, as did airway basement membrane ultrastructure. Branching morphogenesis by lung bud culture was similar in Lamc2-/- and littermate control lungs. Since laminin-332 is important for hemidesmosome formation, we examined the structure of tracheal hemidesmosomes by transmission electron microscopy. Compared to littermate controls, Lamc2-/- tracheal hemidesmosomes were less organized and lacked the increased electron density associated with the basement membrane abutting the hemidesmosome. CONCLUSION: These findings indicate that laminin γ2 and laminin-332, despite their prominence in the lung, have a minimal role in lung development through the saccular stage

Increasing Protein at the Expense of Carbohydrate in the Diet Down-Regulates Glucose Utilization as Glucose Sparing Effect in Rats

High protein (HP) diet could serve as a good strategy against obesity, provoking the changes in energy metabolic pathways. However, those modifications differ during a dietary adaptation. To better understand the mechanisms involved in effect of high protein diet (HP) on limiting adiposity in rats we studied in parallel the gene expression of enzymes involved in protein and energy metabolism and the profiles of nutrients oxidation. Eighty male Wistar rats were fed a normal protein diet (NP, 14% of protein) for one week, then either maintained on NP diet or assigned to a HP diet (50% of protein) for 1, 3, 6 and 14 days. mRNA levels of genes involved in carbohydrate and lipid metabolism were measured in liver, adipose tissues, kidney and muscles by real time PCR. Energy expenditure (EE) and substrate oxidation were measured by indirect calorimetry. Liver glycogen and plasma glucose and hormones were assayed. In liver, HP feeding 1) decreased mRNA encoding glycolysis enzymes (GK, L-PK) and lipogenesis enzymes(ACC, FAS), 2) increased mRNA encoding gluconeogenesis enzymes (PEPCK), 3) first lowered, then restored mRNA encoding glycogen synthesis enzyme (GS), 4) did not change mRNA encoding β-oxidation enzymes (CPT1, ACOX1, βHAD). Few changes were seen in other organs. In parallel, indirect calorimetry confirmed that following HP feeding, glucose oxidation was reduced and fat oxidation was stable, except during the 1st day of adaptation where lipid oxidation was increased. Finally, this study showed that plasma insulin was lowered and hepatic glucose uptake was decreased. Taken together, these results demonstrate that following HP feeding, CHO utilization was increased above the increase in carbohydrate intake while lipogenesis was decreased thus giving a potential explanation for the fat lowering effect of HP diets