James Cohen

King's College London | KCL · Centre for Developmental Neurobiology

Quantification of the number of ectopic motor neurons in chick embryos electroporated in the ventral neural tube with EGFP-shRNAs. Table providing a quantitative analysis of the occurrence of ectopic motor neurons after electroporation of chick embryos in the ventral neural tube with Npn-2, Plexin-A2, MICAL3 EGFP-shRNA or empty vector. Embryos were...

Targeted delivery of EGFP expression plasmids by selective electroporation towards either the ventral neural tube or the neural crest. Confocal micrographs of transverse vibratome sections (75 μm). (a) An example of a HH23 chick embryo electroporated in the ventral neural tube with the pCA-EGFPm5-mU6 plasmid, as detailed in the Materials and method...

Npn-2 B and Npn-2 E shRNA reduce expression of V5-tagged cNpn-2 in HEK cells. Vectors co-expressing EGFP and shRNA targeted at Npn-2 were co-transfected with pcDNA3.1-V5-cNpn-2 [19] at a ratio of 5:1. At 2 days post-transfection, cells were fixed and stained with V5 antibodies. The data show efficient knockdown of V5-tagged Npn-2 with Npn-2 B and N...

Confirmation of knockdown of Plexin mRNAs by ventral targeted shRNA in the chick spinal cord. Embryos were electroporated at E2 (HH stage 13–15) in the ventral neural tube with shRNA vectors targeting (a,b) Plexin-A1, (c,d) Plexin-A2 or (e,f) Plexin-A4. The effect of each shRNA on expression was assessed after 48–54 hours (HH 23–24) by in situ hybr...

Screen of shRNA vectors targeting Npn or Plexin-A receptors in the chick for the ability to induce ectopic motor neuron migration. The prevalence of ectopic motor neurons after ventral electroporation of shRNA vectors targeting plexin-A and neuropilin receptors in the chick was assessed. In a first round of analysis, three different vectors were te...

Table of shRNA target sequences and SFOLD scores (on a 0–20 scale). Note that the Npn-1B and Npn-2 B shRNAs we published previously have a relatively low SFOLD score, yet efficiently reduced expression of their targets [19].

Specificity of knockdown by Plexin-A2 shRNA. An embryo was electroporated at E2 (HH stage 13–15) in the ventral neural tube with an shRNA vector targeting Plexin-A2. The effect at HH stage 23 of shRNA electroporation (a) on expression of (b) Plexin-A1, (c) -A2 and (d) -A4 was assessed on adjacent transverse cryosections of hindlimb region spinal co...

Egr2/Krox-20 expressing BC cells persist in Npn-2 mutant mice. In situ hybridisation for Egr2 on transverse cryosections (30 μm) obtained from E11.5 mouse embryos at hindlimb level. The results show no obvious difference in Egr2 expression in BC cells located at the DREZ or MEP in (a) wild-type, (b) heterozygous or (c) Npn-2 null mice. Bar = 100 μm...

Background In developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement cam...

The drg11 gene is a member of the vertebrate aristaless-related gene family and encodes a paired homeodomain transcription factor. Its expression is largely restricted to PNS neurons subserving somatosensory functions and their CNS targets in rodents. The phenotype of drg11 null mice suggests that it is crucial for the proper development in the emb...

Boundary cap (BC) cells are neural crest derivatives that form clusters at the surface of the neural tube, at entry and exit points of peripheral nerve roots. Using various knock-in alleles of the mouse gene Egr2 (also known as Krox20), the expression of which, in trunk regions, is initially restricted to BC cells, we were able to trace BC cell pro...

The chick embryo is widely used for the study of vertebrate development, but a general, reliable loss-of-function strategy for the analysis of gene function is currently not available. By using small inhibitory hairpin RNA (siRNA) molecules generated by the mouse U6 promoter, we have applied an RNA interference approach to achieve quantitative knoc...

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap...