ISPD gene mutations are a common cause of congenital and limb-girdle muscular dystrophies

Dystroglycanopathies are a clinically and genetically diverse group of recessively inherited conditions ranging from the most severe of the congenital muscular dystrophies, Walker-Warburg syndrome, to mild forms of adult-onset limb-girdle muscular dystrophy. Their hallmark is a reduction in the functional glycosylation of α-dystroglycan, which can be detected in muscle biopsies. An important part of this glycosylation is a unique O-mannosylation, essential for the interaction of α-dystroglycan with extracellular matrix proteins such as laminin-α2. Mutations in eight genes coding for proteins in the glycosylation pathway are responsible for ∼50% of dystroglycanopathy cases. Despite multiple efforts using traditional positional cloning, the causative genes for unsolved dystroglycanopathy cases have escaped discovery for several years. In a recent collaborative study, we discovered that loss-of-function recessive mutations in a novel gene, called isoprenoid synthase domain containing (ISPD), are a relatively common cause of Walker-Warburg syndrome. In this article, we report the involvement of the ISPD gene in milder dystroglycanopathy phenotypes ranging from congenital muscular dystrophy to limb-girdle muscular dystrophy and identified allelic ISPD variants in nine cases belonging to seven families. In two ambulant cases, there was evidence of structural brain involvement, whereas in seven, the clinical manifestation was restricted to a dystrophic skeletal muscle phenotype. Although the function of ISPD in mammals is not yet known, mutations in this gene clearly lead to a reduction in the functional glycosylation of α-dystroglycan, which not only causes the severe Walker-Warburg syndrome but is also a common cause of the milder forms of dystroglycanopathy

Conditional Inactivation of N-WASP in the mouse: Analyses of N-WASP function

N-WASP is a potent activator of the Arp2/3 complex in vitro and harbours key regulatory functions for cellular actin assembly in response to signalling. To analyse N-WASP function, mice carrying a loxP flanked N-WASP allele were generated using gene targeting in embryonic stem cells and the Cre/loxP recombination system. N-WASPflox/flox mice allow the conditional inactivation of the N-WASP gene by Cre recombinase mediated deletion in a tissue specific and temporally controlled manner. The N-WASP gene was disrupted in the mouse germline, which led to embryonic lethality. To elucidate the role of N-WASP at the cellular level, embryonic fibroblasts from N-WASPflox/flox mice were immortalized and various N-WASP-defective cell lines were selected upon transient expression of Cre recombinase. These N-WASP-defective fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia by microinjection of constitutively active Cdc42, but failed to support the intracellular motility of Shigella flexneri. Reconstitution experiments using GFP-tagged N-WASP and various GFP-N-WASP mutants revealed a recruitment mechanism independent of the CRIB domain and Cdc42. In addition, enteropathogenic and enterohemorrhagic Escherichia coli were incapable of inducing the formation of actin pedestals in N-WASP-defective cells. In reconstitution experiments, N-WASP recruitment and EPEC pedestal formation was shown to be independent of small Rho family GTPases and the CRIB domain, but to be mediated by a recruitment domain in the amino-terminal half of N-WASP. These results prove the essential role of N-WASP for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N-WASP is dispensable for filopodia formation.N-WASP aktiviert den Aktinfilament-Nukleator Arp2/3 Komplex in Abhängigkeit von Signaltransduktionsvorgängen und spielt damit eine Schlüsselrolle in der Regulation des Aktinzellskeletts. Um die Funktion von N-WASP zu untersuchen, wurden durch gezielte Mutagenese mittels homologer Rekombination in embryonalen Stammzellen Mäuse hergestellt, die ein loxP flankiertes N-WASP Allel tragen und die damit eine gewebsspezifische und zeitlich kontrollierte Inaktivierung des N-WASP Gens in Abhängigkeit der Cre Rekombinase erlauben. Um die Rolle von N-WASP während der Embryonalentwicklung zu erforschen, wurde das N-WASP Gen in der Keimbahn der Maus inaktiviert, was zum Absterben der betroffenen Embryonen führte. Um die Funktion von N-WASP auf zellulärer Ebene zu analysieren, wurden mehrere N-WASP-defekte embryonale Fibroblastenzelllinien etabliert. Diese N-WASP-defekten Zellen zeigten keine auffälligen morphologischen Veränderungen, vor allem war die durch konstitutiv aktives Cdc42 induzierte Ausbildung von Filopodien nicht beeinträchtigt. Eine Analyse der durch pathogene Bakterien induzierten Reorganisationsvorgänge des Aktinzellskeletts zeigten, dass ohne N-WASP die intrazelluläre Fortbewegung von Shigella flexneri unterbunden war. Rekonstitutionen mit verschiedenen GFP-N-WASP Mutanten zeigten, dass N-WASP unabhängig von der CRIB Domäne an Shigellen rekrutiert wird und Rho GTPasen nicht zur Rekrutierung und Motilität beitragen. Weiterhin konnten enteropathogene und enterohämorrhagische Escherichia coli in N-WASP-defekten Zellen keine Aktinpodeste mehr induzieren. Es konnte gezeigt werden, dass N-WASP über eine im Aminoterminus liegende Rekrutierungsdomäne rekrutiert wird. Diese Resultate zeigen die essentielle Rolle des N-WASP Proteins in den von diesen Pathogenen induzierten Reorganisationsvorgängen des Aktinzellskeletts in vivo und beweist zudem zum ersten mal, dass, zumindest in diesen Fibroblasten, N-WASP für eine Ausbildung von Filopodien nicht notwendig ist

Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells

In mammalian cells, actin dynamics is tightly controlled through small GTPases of the Rho family, WASP/Scar proteins and the Arp2/3 complex. We employed Cre/loxP-mediated gene targeting to disrupt the ubiquitously expressed N-WASP in the mouse germline, which led to embryonic lethality. To elucidate the role of N-WASP at the cellular level, we immortalized embryonic fibroblasts and selected various N-WASP-defective cell lines. These fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia, but failed to support the motility of Shigella flexneri. In addition, enteropathogenic Escherichia coli were incapable of inducing the formation of actin pedestals in N-WASP-defective cells. Our results prove the essential role of this protein for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N-WASP is dispensible for filopodia formation

In vivo dynamics of axon pathfinding in the Drosophila CNS: A time-lapse study of an identified motoneuron

We developed a system for time-lapse observation of identified neurons in the central nervous system (CNS) of the Drosophila embryo. Using this system, we characterize the dynamics of filopodia and axon growth of the motorneuron RP2 as it navigates anteriorly through the CNS and then laterally along the intersegmental nerve (ISN) into the periphery. We find that both axonal extension and turning occur primarily through the process of filopodial dilation. In addition, we used the GAL4-UAS system to express the fusion protein Tau-GFP in a subset of neurons, allowing us to correlate RP2's patterns of growth with a subset of axons in its environment. In particular, we show that RP2's sharp lateral turn is coincident with the nascent ISN. (C) 1998 John Wiley & Sons, Inc

Cdc42/N-WASP signaling links actin dynamics to pancreatic β cell delamination and differentiation.

Delamination plays a pivotal role during normal development and cancer. Previous work has demonstrated that delamination and epithelial cell movement within the plane of an epithelium are associated with a change in cellular phenotype. However, how this positional change is linked to differentiation remains unknown. Using the developing mouse pancreas as a model system, we show that β cell delamination and differentiation are two independent events, which are controlled by Cdc42/N-WASP signaling. Specifically, we show that expression of constitutively active Cdc42 in β cells inhibits β cell delamination and differentiation. These processes are normally associated with junctional actin and cell-cell junction disassembly and the expression of fate-determining transcription factors, such as Isl1 and MafA. Mechanistically, we demonstrate that genetic ablation of N-WASP in β cells expressing constitutively active Cdc42 partially restores both delamination and β cell differentiation. These findings elucidate how junctional actin dynamics via Cdc42/N-WASP signaling cell-autonomously control not only epithelial delamination but also cell differentiation during mammalian organogenesis

N-WASP is a novel regulator of hair-follicle cycling that controls antiproliferative TGF beta pathways

N-WASP is a cytoplasmic molecule mediating Arp2/3 nucleated actin polymerization. Mice with a keratinocyte-specific deletion of the gene encoding N-WASP showed normal interfollicular epidermis, but delayed hair-follicle morphogenesis and abnormal hair-follicle cycling, associated with cyclic alopecia and prolonged catagen and telogen phases. The delayed anagen onset correlated with an increased expression of the cell-cycle inhibitor p21CIP, and increased activity of the TGF beta pathway, a known inducer of p21CIP expression. Primary N-WASP-null keratinocytes showed reduced growth compared with control cells and enhanced expression of the gene encoding the cell-cycle inhibitor p15INK4B, a TGF beta target gene. Inhibition of TGF beta signaling blocked overexpression of p15INK4B and restored proliferation of N-WASP-deficient keratinocytes in vitro. However, induction of N-WASP gene deletion in vitro did not result in obvious changes in TGF beta signaling or growth of keratinocytes, indicating that the in vivo environment is required for the phenotype development. These data identify the actin nucleation regulator N-WASP as a novel element of hair-cycle control that modulates the antiproliferative and pro-apoptotic TGF. pathway in keratinocytes in vivo and in vitro

N-WASP is a novel regulator of hair-follicle cycling that controls antiproliferative TGFβ pathways

N-WASP is a cytoplasmic molecule mediating Arp2/3 nucleated actin polymerization. Mice with a keratinocyte-specific deletion of the gene encoding N-WASP showed normal interfollicular epidermis, but delayed hair-follicle morphogenesis and abnormal hair-follicle cycling, associated with cyclic alopecia and prolonged catagen and telogen phases. The delayed anagen onset correlated with an increased expression of the cell-cycle inhibitor p21CIP, and increased activity of the TGF beta pathway, a known inducer of p21CIP expression. Primary N-WASP-null keratinocytes showed reduced growth compared with control cells and enhanced expression of the gene encoding the cell-cycle inhibitor p15INK4B, a TGF beta target gene. Inhibition of TGF beta signaling blocked overexpression of p15INK4B and restored proliferation of N-WASP-deficient keratinocytes in vitro. However, induction of N-WASP gene deletion in vitro did not result in obvious changes in TGF beta signaling or growth of keratinocytes, indicating that the in vivo environment is required for the phenotype development. These data identify the actin nucleation regulator N-WASP as a novel element of hair-cycle control that modulates the antiproliferative and pro-apoptotic TGF. pathway in keratinocytes in vivo and in vitro