The Rac-GAP alpha2-Chimaerin Signals via CRMP2 and Stathmins in the Development of the Ocular Motor System.

A precise sequence of axon guidance events is required for the development of the ocular motor system. Three cranial nerves grow toward, and connect with, six extraocular muscles in a stereotyped pattern, to control eye movements. The signaling protein alpha2-chimaerin (α2-CHN) plays a pivotal role in the formation of the ocular motor system; mutations in CHN1, encoding α2-CHN, cause the human eye movement disorder Duane Retraction Syndrome (DRS). Our research has demonstrated that the manipulation of α2-chn signaling in the zebrafish embryo leads to ocular motor axon wiring defects, although the signaling cascades regulated by α2-chn remain poorly understood. Here, we demonstrate that several cytoskeletal regulatory proteins-collapsin response mediator protein 2 (CRMP2; encoded by the gene dpysl2), stathmin1, and stathmin 2-bind to α2-CHN. dpysl2, stathmin1, and especially stathmin2 are expressed by ocular motor neurons. We find that the manipulation of dpysl2 and of stathmins in zebrafish larvae leads to defects in both the axon wiring of the ocular motor system and the optokinetic reflex, impairing horizontal eye movements. Knockdowns of these molecules in zebrafish larvae of either sex caused axon guidance phenotypes that included defasciculation and ectopic branching; in some cases, these phenotypes were reminiscent of DRS. chn1 knock-down phenotypes were rescued by the overexpression of CRMP2 and STMN1, suggesting that these proteins act in the same signaling pathway. These findings suggest that CRMP2 and stathmins signal downstream of α2-CHN to orchestrate ocular motor axon guidance and to control eye movements.SIGNIFICANCE STATEMENT The precise control of eye movements is crucial for the life of vertebrate animals, including humans. In humans, this control depends on the arrangement of nerve wiring of the ocular motor system, composed of three nerves and six muscles, a system that is conserved across vertebrate phyla. Mutations in the protein alpha2-chimaerin have previously been shown to cause eye movement disorders (squint) and axon wiring defects in humans. Our recent work has unraveled how alpha2-chimaerin coordinates axon guidance of the ocular motor system in animal models. In this article, we demonstrate key roles for the proteins CRMP2 and stathmin 1/2 in the signaling pathway orchestrated by alpha2-chimaerin, potentially giving insight into the etiology of eye movement disorders in humans.

In Vivo and In Vitro Knockdown Approaches in the Avian Embryo as a Means to Study Semaphorin Signaling.

A combination of both in vivo and in vitro techniques is invaluable for studying semaphorin signaling in the avian central nervous system. Here we describe how both types of approaches can be used to compliment each other in order to unravel the role that semaphorins play during embryonic development and elucidate the functional consequences of semaphorin knockdown using RNA interference vectors. We describe and discuss specifically the use of in ovo electroporation and primary oculomotor neuron culture to identify the role of semaphorins in oculomotor neuron migration and assess functional consequences of semaphorin disruption in this system.

α2-Chimaerin regulates a key axon guidance transition during development of the oculomotor projection.

The ocular motor system consists of three nerves which innervate six muscles to control eye movements. In humans, defective development of this system leads to eye movement disorders, such as Duane Retraction Syndrome, which can result from mutations in the α2-chimaerin signaling molecule. We have used the zebrafish to model the role of α2-chimaerin during development of the ocular motor system. We first mapped ocular motor spatiotemporal development, which occurs between 24 and 72 h postfertilization (hpf), with the oculomotor nerve following an invariant sequence of growth and branching to its muscle targets. We identified 52 hpf as a key axon guidance "transition," when oculomotor axons reach the orbit and select their muscle targets. Live imaging and quantitation showed that, at 52 hpf, axons undergo a switch in behavior, with striking changes in the dynamics of filopodia. We tested the role of α2-chimaerin in this guidance process and found that axons expressing gain-of-function α2-chimaerin isoforms failed to undergo the 52 hpf transition in filopodial dynamics, leading to axon stalling. α2-chimaerin loss of function led to ecotopic and misguided branching and hypoplasia of oculomotor axons; embryos had defective eye movements as measured by the optokinetic reflex. Manipulation of chimaerin signaling in oculomotor neurons in vitro led to changes in microtubule stability. These findings demonstrate that a correct level of α2-chimaerin signaling is required for key oculomotor axon guidance decisions, and provide a zebrafish model for Duane Retraction Syndrome.

Spongious hypertrophic cardiomyopathy in patients with mutations in the four-and-a-half LIM domain 1 gene.

BACKGROUND: X-linked myopathy with postural muscle atrophy is a novel X-linked myopathy caused by mutations in the four-and-a-half LIM domain 1 gene (FHL1). Cardiac involvement was suspected in initial publications. We now systematically analyzed the association of the FHL1 genotype with the cardiac phenotype to establish a potential cardiac involvement in the disease. METHODS AND RESULTS: Seventeen male patients and 23 female mutation carriers were compared with healthy controls. Every patient underwent a comprehensive clinical and cardiovascular workup. ECG abnormalities occurred frequently in affected males and were less frequent in heterozygous females. Both male and female mutation carriers had increased myocardial mass (affected males=115.1±25.3 g/m(2); heterozygous females=95.1±19.6 g/m(2); controls=89.0±15.6 g/m(2) and 72.6±12.6 g/m(2); respectively) with increased wall thickness (typically midventricular and apical segments) mainly in affected males. Longitudinal systolic function was reduced in affected males (radial systolic strain: affected males=24.6±11.8%; male controls=43.2±14.8%; P=0.002). Diastolic dysfunction occurred in both affected males and heterozygous females. Cardiac MRI revealed a morphological hallmark of X-linked myopathy with postural muscle atrophy; a characteristic spongious structure and replacement fibrosis indicated by late enhancement could be detected in most affected males. X-linked myopathy with postural muscle atrophy was associated with reduced exercise capacity in affected males but not in heterozygous female mutation carriers. CONCLUSIONS: X-linked myopathy with postural muscle atrophy patients consistently showed electrical, functional, and characteristic morphological cardiac abnormalities that translate into reduced exercise capacity. Reduced systolic and diastolic function is associated with a novel type of spongious hypertrophic cardiomyopathy. An unexpected finding was that some cardiac abnormalities were also present in heterozygous female mutation carriers.

Four and a half LIM protein 1C (FHL1C): a binding partner for voltage-gated potassium channel K(v1.5).

Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G(0)/G(1) phase. Furthermore, low expression of K(v1.5), a voltage-gated potassium channel known to alter myoblast proliferation during the G(1) phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between K(v1.5) and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and K(v1.5) within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of K(v1.5) with FHL1C in Xenopus laevis oocytes markedly reduced K(+) currents when compared to oocytes expressing K(v1.5) only. We here present the first evidence on a biological relevance of FHL1C.