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

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

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

Brief Communications 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

E3 Ligase Nedd4 Promotes Axon Branching by Downregulating PTEN

Regulated 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

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in -6

<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>in the main channel of the electroporation chamber, while the electrode tips (0.5 mm wide) were positioned in the transverse channel. A diagram of the setup is presented as an insert with channel (outlines in red). b, c: Representative images of embryos electroporated in 1× MMR and 0.1× MBS. Bright field images (left panel) and GFP fluorescence (right panel) of living embryos 12 h after electroporation. No morphological abnormalities are observed. d: Histograms presenting the relative transfection efficiencies (blue) evaluated from observation of embryos as shown in c and d. The percentage of embryos showing macroscopic damage (red) was recorded for each condition. Different parameters are listed in the following order: Voltage, pulse duration, interpulse space and number of pulses. e, f: Electroporation resulted in a high percentage of transfected cells without affecting brain microanatomy. Nls-GFP signal (e) was observed in many nuclei (f) from the ventricle to the most superficial layer 48 h after electroporation. The transfected hemi-brain was outlined in white. Scale bars: 400 μm in b and c; 100 μm in e

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in -0

<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>in the main channel of the electroporation chamber, while the electrode tips (0.5 mm wide) were positioned in the transverse channel. A diagram of the setup is presented as an insert with channel (outlines in red). b, c: Representative images of embryos electroporated in 1× MMR and 0.1× MBS. Bright field images (left panel) and GFP fluorescence (right panel) of living embryos 12 h after electroporation. No morphological abnormalities are observed. d: Histograms presenting the relative transfection efficiencies (blue) evaluated from observation of embryos as shown in c and d. The percentage of embryos showing macroscopic damage (red) was recorded for each condition. Different parameters are listed in the following order: Voltage, pulse duration, interpulse space and number of pulses. e, f: Electroporation resulted in a high percentage of transfected cells without affecting brain microanatomy. Nls-GFP signal (e) was observed in many nuclei (f) from the ventricle to the most superficial layer 48 h after electroporation. The transfected hemi-brain was outlined in white. Scale bars: 400 μm in b and c; 100 μm in e

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in -4

<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>n. a: A dorsal view of an embryo doubly transfected. Retinal axons (red in b and c) navigate normally to the tectum, passing through a transfected region of the diencephalon (green in c) (dashed line indicates the OT boundary). Eye- and ventral-targeted electroporation can be combined (d). Frontal section showing axons from the transfected retina (red) that have crossed the transfected midline (GFP-transfected) and growing dorsally towards tectum (arrow). e-g: Electroporation can be performed on embryos lipofected in the eyes. e: High magnification of two GFP lipofected axons passing through a cluster of electroporated tectal cells. f and g: Frontal sections of an embryo lipofected in the eye and electroporated in the brain. Retinal axons in the dorsal brain (green: f, g) traversed the transfected cells (red: g). Outlines of brains in wholemounts (b, c, e) and sections (f, g) were drawn based on bright field images and DAPI counterstainings respectively. Epi., epiphysis; Di., diencephalon; OT, optic tectum; Tel, telencephalon. Scale bars: 400 μm in a; 100 μm in b-g

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in -2

<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>rcentages of nls-GFP positive cells 12 h after transfection at stage 26, 28 or 32 (n represents the number of sections analyzed from 3 embryos). Similar results were obtained at 48 h post electroporation (data not shown). b-d: Distribution of transfected cells depended on the stage of embryos electroporated. Distribution of nls-GFP transfected cells 48 h afterwards in embryos electroporated at stage 28 (b) and 32 (c). Note that the density of cells (DAPI) is lower laterally. d: Histograms showing decreases in the fraction of cells transfected in the superficial third of the brain when embryos were electroporated at stage 32 as compared to stage 28. e: A cluster of superficially located cells can be selectively transfected by injecting the DNA solution under the skin (the pia and epidermis are outlined in white). f-h: Time course of GFP expression in embryos electroporated at stage 29/30 (20 V/25 ms/1 s/8 x). The fractions as well as mean intensities of GFP positive cells were quantified (h) from sections (examples: f and g) (15 sections from 3 embryos were analyzed for the 6 h and 48 h time points and 39 sections from 3 embryos for the 24 h time-point). Differences between the time points were statistically significant using a Mann-Whitney test; probabilities are indicated together with the standard error (S.E.M). Outlines of the brains are presented (ventricle on the left). Scale bars: 100 μm in e; 50 μm in b, c, f and g