Cas Adaptor Proteins Coordinate Sensory Axon Fasciculation.

Development of complex neural circuits like the peripheral somatosensory system requires intricate mechanisms to ensure axons make proper connections. While much is known about ligand-receptor pairs required for dorsal root ganglion (DRG) axon guidance, very little is known about the cytoplasmic effectors that mediate cellular responses triggered by these guidance cues. Here we show that members of the Cas family of cytoplasmic signaling adaptors are highly phosphorylated in central projections of the DRG as they enter the spinal cord. Furthermore, we provide genetic evidence that Cas proteins regulate fasciculation of DRG sensory projections. These data establish an evolutionarily conserved requirement for Cas adaptor proteins during peripheral nervous system axon pathfinding. They also provide insight into the interplay between axonal fasciculation and adhesion to the substrate

Neural crest stem cell maintenance by combinatorial Wnt and BMP signaling

Canonical Wnt signaling instructively promotes sensory neurogenesis in early neural crest stem cells (eNCSCs) (Lee, H.Y., M. Kléber, L. Hari, V. Brault, U. Suter, M.M. Taketo, R. Kemler, and L. Sommer. 2004. Science. 303:1020–1023). However, during normal development Wnt signaling induces a sensory fate only in a subpopulation of eNCSCs while other cells maintain their stem cell features, despite the presence of Wnt activity. Hence, factors counteracting Wnt signaling must exist. Here, we show that bone morphogenic protein (BMP) signaling antagonizes the sensory fate-inducing activity of Wnt/β-catenin. Intriguingly, Wnt and BMP act synergistically to suppress differentiation and to maintain NCSC marker expression and multipotency. Similar to NCSCs in vivo, NCSCs maintained in culture alter their responsiveness to instructive growth factors with time. Thus, stem cell development is regulated by combinatorial growth factor activities that interact with changing cell-intrinsic cues

The RacGAP β2-Chimaerin Selectively Mediates Axonal Pruning in the Hippocampus

SummaryAxon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) β2-Chimaerin (β2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds β2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. β2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/β2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling

A Late Role for bmp2b in the Morphogenesis of Semicircular Canal Ducts in the Zebrafish Inner Ear

BACKGROUND:The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear. METHODOLOGY/PRINCIPAL FINDINGS:We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/-)) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal. CONCLUSIONS/SIGNIFICANCE:Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species

Sculpting Neural Circuits by Axon and Dendrite Pruning

The assembly of functional neural circuits requires the combined action of progressive and regressive events. Regressive events encompass a variety of inhibitory developmental processes, including axon and dendrite pruning, which facilitate the removal of exuberant neuronal connections. Most axon pruning involves the removal of axons that had already made synaptic connections; thus, axon pruning is tightly associated with synapse elimination. In many instances, these developmental processes are regulated by the interplay between neurons and glial cells that act instructively during neural remodeling. Owing to the importance of axon and dendritic pruning, these remodeling events require precise spatial and temporal control, and this is achieved by a range of distinct molecular mechanisms. Disruption of these mechanisms results in abnormal pruning, which has been linked to brain dysfunction. Therefore, understanding the mechanisms of axon and dendritic pruning will be instrumental in advancing our knowledge of neural disease and mental disorders

Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh

The inner ear is partitioned along its dorsal/ventral axis into vestibular and auditory organs, respectively. Gene expression studies suggest that this subdivision occurs within the otic vesicle, the tissue from which all inner ear structures are derived. While the specification of ventral otic fates is dependent on Shh secreted from the notochord, the nature of the signal responsible for dorsal otic development has not been described. In this study, we demonstrate that Wnt signaling is active in dorsal regions of the otic vesicle, where it functions to regulate the expression of genes (Dlx5/6 and Gbx2) necessary for vestibular morphogenesis. We further show that the source of Wnt impacting on dorsal otic development emanates from the dorsal hindbrain, and identify Wnt1 and Wnt3a as the specific ligands required for this function. The restriction of Wnt target genes to the dorsal otocyst is also influenced by Shh. Thus, a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types

A luciferase fragment complementation assay to detect focal adhesion kinase (FAK) signaling events

Integrin Adhesion Complexes (IACs) serve as links between the cytoskeleton and extracellular environment, acting as mechanosensing and signaling hubs. As such, IACs participate in many aspects of cellular motility, tissue morphogenesis, anchorage-dependent growth and cell survival. Focal Adhesion Kinase (FAK) has emerged as a critical organizer of IAC signaling events due to its early recruitment and diverse substrates, and thus has become a genetic and therapeutic target. Here we present the design and characterization of simple, reversible, and scalable Bimolecular Complementation sensors to monitor FAK phosphorylation in living cells. These probes provide novel means to quantify IAC signaling, expanding on the currently available toolkit for interrogating FAK phosphorylation during diverse cellular processes

Specification of the mammalian cochlea is dependent on Sonic hedgehog

Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain unclear. This study identifies Sonic hedgehog (Shh) secreted by the notochord and/or floor plate as a primary regulator of auditory cell fates within the mouse inner ear. Whereas otic induction proceeds normally in Shh(−/−) embryos, morphogenesis of the inner ear is greatly perturbed by midgestation. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia failed to develop in the absence of Shh. The origin of the inner ear defects in Shh(−/−) embryos could be traced back to alterations in the expression of a number of genes involved in cell fate specification including Pax2, Otx1, Otx2, Tbx1, and Ngn1. We further show that several of these genes are targets of Shh signaling given their ectopic activation in transgenic mice that misexpress Shh in the inner ear. Taken together, our data support a model whereby auditory cell fates in the otic vesicle are established by the direct action of Shh