Dissection and Culture of Embryonic Spinal Commissural Neurons

Moore, Sam; Kennedy, Timothy E.

doi:10.1002/0471142301.ns0320s45

Cited by 7 publications

(4 citation statements)

References 26 publications

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“…All reagents used for neuronal and cell cultures were from Thermo Fisher Scientific (USA) unless otherwise specified. Explants and dissociated neurons of mouse embryonic dorsal spinal cord (DSC) were dissected and cultured following previously described methods (40,41). The culturing medium recipe is neurobasal medium supplemented with B27 (1×), Penicillin-Streptomycin (1×) and GlutaMAX-1 (1×).…”

Section: Methodsmentioning

confidence: 99%

The m6A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

Zhuang

Zhu

et al. 2019

Nucleic Acids Research

157

135

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N 6 -Methyladenosine (m 6 A) is a dynamic mRNA modification which regulates protein expression in various posttranscriptional levels. Functional studies of m 6 A in nervous system have focused on its writers and erasers so far, whether and how m 6 A readers mediate m 6 A functions through recognizing and binding their target mRNA remains poorly understood. Here, we find that the expression of axon guidance receptor Robo3.1 which plays important roles in midline crossing of spinal commissural axons is regulated precisely at translational level. The m 6 A reader YTHDF1 binds to and positively regulates translation of m 6 A-modified Robo3.1 mRNA. Either mutation of m 6 A sites in Robo3.1 mRNA or YTHDF1 knockdown or knockout leads to dramatic reduction of Robo3.1 protein without affecting Robo3.1 mRNA level. Specific ablation of Ythdf1 in spinal commissural neurons results in pre-crossing axon guidance defects. Our findings identify a mechanism that YTHDF1-mediated translation of m 6 A-modified Robo3.1 mRNA controls pre-crossing axon guidance in spinal cord.

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Section: Methodsmentioning

confidence: 99%

The m6A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

Zhuang

Zhu

et al. 2019

Nucleic Acids Research

157

135

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“…To address the temporal and spatial activation of signal transduction mechanisms activated by netrin-1 downstream of DCC, we utilized a CFP/YFP FRET biosensor for the Rho GTPase Cdc42 [ 16 , 17 ]. The cDNA encoding the reporter was transfected into E13 rat spinal commissural neurons (1 DIV) isolated and cultured as described [ 7 , 11 ]. When grown in dispersed cell culture, as described here, embryonic rat spinal commissural neurons respond to netrin-1 as a chemoattractant for up to at least 4 DIV [ 18 ], and rapidly respond to bath application of netrin-1 with increased numbers of filopodia and growth cone expansion, characteristic of membrane extension evoked by a chemoattractant [ 7 ].…”

Section: Resultsmentioning

confidence: 99%

“…Staged pregnant Sprague-Dawley rats were obtained from Charles River Laboratories Canada (St Constant, QC) and killed by carbon dioxide overdose followed by cervical dislocation. Embryos were obtained by Caesarian section and embryonic day 13 (E13; vaginal plug is E0) dorsal spinal cords isolated and dissociated as described [ 11 ]. Neurons were plated at 350,000 cells/well in 35 mm, 14 mm glass No.…”

Section: Methodsmentioning

confidence: 99%

FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones

et al. 2016

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Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

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“…We use tungsten wire with 0.008 inch diameter (#214071 from Leico Industries). Needles are electrolytically sharpened using a small plastic beaker of 1 N NaOH in which the needle and needle holder complete a circuit using a 6 V power supply (e.g., see Moore and Kennedy [2008]). We use a modified microscope lamp power supply (for 6 V lamps); the wires that would normally attach to the bulb are instead attached to alligator clips.…”

Section: Tungsten Needlesmentioning

confidence: 99%

Special Considerations for Making Explants and Transplants with Xenopus tropicalis

Fisher

Grainger

2019

Cold Spring Harb Protoc

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Although Xenopus laevis is an important model organism for embryological experimentation, the smaller, more genetically tractable, and faster developing Xenopus tropicalis provides advantages for using genetic approaches to understand developmental mechanisms. Explant cultures and transplants of X. tropicalis embryonic tissues present unique opportunities to examine embryonic tissue determination in a simplified setting. Here we demonstrate preparation of explants and transplants of preplacodal head ectoderm in order to illustrate these approaches; however, these methods apply broadly to tissues throughout the embryo. We focus on technical adjustments to accommodate the differences in size, tissue character, and rate of development between X. laevis and X. tropicalis. With only modest modifications, X. tropicalis embryos are quite amenable to the same kinds of experimental manipulations as X. laevis. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Bovine serum albumin (BSA), fraction V (10 mg/mL stock solution in H 2 O) Dilute to 20-100 µg/mL in 1× MBS for coating dishes and pipettes.

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Dissection and Culture of Embryonic Spinal Commissural Neurons

Cited by 7 publications

References 26 publications

The m6A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

The m6A reader YTHDF1 regulates axon guidance through translational control of Robo3.1 expression

FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones

Special Considerations for Making Explants and Transplants with Xenopus tropicalis

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