NF-protocadherin and TAF1 regulate retinal axon initiation and elongation in vivo

NF-protocadherin (NFPC)-mediated cell–cell adhesion plays a critical role in vertebrate neural tube formation. NFPC is also expressed during the period of axon tract formation, but little is known about its function in axonogenesis. Here we have tested the role of NFPC and its cytosolic cofactor template-activating factor 1 (TAF1) in the emergence of the Xenopus retinotectal projection. NFPC is expressed in the developing retina and optic pathway and is abundant in growing retinal axons. Inhibition of NFPC function in developing retinal ganglion cells (RGCs) severely reduces axon initiation and elongation and suppresses dendrite genesis. Furthermore, an identical phenotype occurs when TAF1 function is blocked. These data provide evidence that NFPC regulates axon initiation and elongation and indicate a conserved role for TAF1, a transcriptional regulator, as a downstream cytosolic effector of NFPC in RGCs

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus

Background: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes

Mechanosensing is critical for axon growth in the developing brain.

During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signaling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell axons. In vivo atomic force microscopy revealed a noticeable pattern of stiffness gradients in the embryonic brain. Retinal ganglion cell axons grew toward softer tissue, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically and knocked down the mechanosensitive ion channel piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness, read out by mechanosensitive ion channels, is critically involved in instructing neuronal growth in vivo.This work was supported by the German National Academic Foundation (scholarship to D.E.K.), Wellcome Trust and Cambridge Trusts (scholarships to A.J.T.), Winston Churchill Foundation of the United States (scholarship to S.K.F.), Herchel Smith Foundation (Research Studentship to S.K.F.), CNPq 307333/2013-2 (L.d.F.C.), NAP-PRP-USP and FAPESP 11/50761-2 (L.d.F.C.), UK EPSRC BT grant (J.G.), Wellcome Trust WT085314 and the European Research Council 322817 grants (C.E.H.); an Alexander von Humboldt Foundation Feodor Lynen Fellowship (K.F.), UK BBSRC grant BB/M021394/1 (K.F.), the Human Frontier Science Program Young Investigator Grant RGY0074/2013 (K.F.), the UK Medical Research Council Career Development Award G1100312/1 (K.F.) and the Eunice Kennedy Shriver National Institute Of Child Health & Human Development of the National Institutes of Health under Award Number R21HD080585 (K.F.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/nn.439

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons.

Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.This work was supported by Wellcome Trust Grants (085314/Z/08/Z and 203249/Z/16/Z) to C.E.H. and (100329/Z/12/Z) to W.A.H., European Research Council Advanced Grant (322817) to C.E.H., Champalimaud Vision Award to C.E.H. and by the Netherlands Organization for Scientific Research (NWO Rubicon 019.161LW.033) to M.K. CFK acknowledges funding from the UK Engineering and Physical Sciences Research Council, EPSRC (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z) and the UK Medical Research Council, MRC (grants MR/K015850/1 and MR/K02292X/1) and Infinitus (China) Ltd

E3 Ligase Nedd4 Promotes Axon Branching by Downregulating PTEN

SummaryRegulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo

Signaling Mechanisms Underlying Slit2-Induced Collapse of Xenopus Retinal Growth Cones

SummarySlits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in -1

<p><b>Copyright information:</b></p><p>Taken from "Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in "</p><p>http://www.biomedcentral.com/1471-213X/7/107</p><p>BMC Developmental Biology 2007;7():107-107.</p><p>Published online 27 Sep 2007</p><p>PMCID:PMC2147031.</p><p></p>neurons including the axons (the ventricle and neuropil are outlined in white). The arrow indicates a bundle of axons travelling in the neuropil). b: Radial-glia like morphology of GAP-RFP transfected cells lining the ventricle. c-e: Co-expression of GAP-GFP (c) and acetylated-tubulin (d) in superficial layers (e- merge). f: Wholemount brain preparation from an electroporated embryo showing different axon tracts. The brain outline was drawn based on the corresponding bright field image. Di., diencephalon; OT, optic tectum; Tel., telencephalon; Epi., epiphysis. Scale bars: 100 μm in f; 50 μm in a; 10 μm in b-e

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in -5

<p><b>Copyright information:</b></p><p>Taken from "Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in "</p><p>http://www.biomedcentral.com/1471-213X/7/107</p><p>BMC Developmental Biology 2007;7():107-107.</p><p>Published online 27 Sep 2007</p><p>PMCID:PMC2147031.</p><p></p>tagged MO. Large numbers of cells can be loaded with MO in both the brain (a) and the eye (c). Microanatomy of both structures appears normal (b and d). e-f: Co-electroporation of pCS2GAP-GFP with lissamine-tagged special delivery MO. e: A higher magnification image of a co-electroporated brain. The MO signal was de-saturated in Photshop in order to facilitate observation of MO and membrane GFP co-expression (arrowhead). f: An image of eye-targeted co-electroporation illustrating the extent of co-electroporation and the sizes of MO and DNA electroporated regions. g: Frontal section of a MO/GFP co-electroporated embryo showing that GFP can be used to trace the axons of electroporated cells (arrowheads indicate axons at different points in their pathway). h and i: Examples of embryos electroporated with pCS2GFP in the presence (i) or absence (h) of anti-GFP MO. Morphology of the eye appeared normal in both conditions (left panel). The GFP signal was sharply reduced in the anti-GFP MO condition when analyzed 12 h after electroporation (central panels). A decrease in electroporation efficiency was not a confounding factor in this experiment as the Special Delivery lissamine-tagged MO control is efficiently loaded in both conditions (far right panel). j: Quantification of results presented in h and i (n indicates the number of embryos analyzed). Anti-GFP MO only affects expression of pCS2GFP but not of pEGFP (Clontech). k: Anti-GFP MO was co-electroporated with GFP and GAP-RFP. 48 h after electroporation, GFP and RFP fluorescence was quantified on sections and the ratio between the two calculated. (n refers to the numbers of sections quantified [3 embryos were analyzed for control and 6 for MO]). Statistical analysis: Mann-Whitney test; probabilities are indicated together with the S.E.M. l-m: Sections through an eye lipofected with GFP (green, l and m) and subsequently loaded with lissamine-tagged MOs (red) using electroporation (merge, m). n-q: Electroporated embryos can be a source of modified cells for studies. Explants and cells cultured from MO (n and o) or DNA (GFP) (p and q) electroporated embryos. Scale bars: 400 μm in h; 100 μm in a; 50 μm in d, f, and g; in 25 μm e and m; 20 μm in n; 10 μm in o