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. 2020 Jan 9;21(3):e48512. doi: 10.15252/embr.201948512

Figure EV2. Related to Fig 1. Peripheral nerve growth and motor neuron cultures in microfluidic chambers.

Figure EV2

  1. WT and crmp2 −/− E11.5 embryos stained with antibody against neurofilaments. All peripheral nerves are present in crmp2 −/−. III—oculomotor, IV—trochlear, V—trigeminal, VII—facial, VIII—vestibulocochlear, IX—glossopharyngeal, X—vagal, and XI—accessory nerve. Op—ophthalmic branch, Mx—maxillar branch, and Md—mandibular branch of the trigeminal nerve.
  2. Whole‐mount immunolabeling of embryos at E12.5 (WT n = 4, crmp2 −/− n = 5, 2 litters), note the increased branching of the Op branch in crmp2 −/−. Scale bars: 500 μm.
  3. Quantification of the number of the Op branches (WT 67 ± 4.5, crmp2 −/− 86 ± 10.3, P < 0.05), mean ± SD are shown, *P < 0.05, t‐test.
  4. Increased levels of CRMP4 and CRMP1 in crmp2 −/− brain lysates.
  5. Adjacent coronal slices of E12.5 embryonic head sections stained with anti‐neurofilaments and anti‐CRMP4 antibodies to confirm the presence of CRMP4 in peripheral nerves. Arrows indicate the same nerves in different sections. Scale bars: 200 μm.
  6. Adjacent coronal sections of E12.5 embryonic heads stained with anti‐neurofilaments and anti‐CRMP2/2A antibodies to confirm the presence of CRMP2 isoforms in peripheral nerves. Arrows indicate the same nerves in different sections. Scale bars: 100 μm.
  7. Growth cone collapse assay revealed decreased reactivity to Sema3A in DRGs isolated from crmp2 −/− embryos. DRGs (n ≥ 3 explants per genotype and condition) were stimulated with different Sema3A concentrations for 30 min, fixed, and stained for actin (green) and β3‐tubulin (red). Scale bars: 100 μm, mean ± SD, ***P < 0.001, 2‐way ANOVA with Bonferroni's multiple comparison test.
  8. Experimental design of motor neuron cultures in microfluidic chambers using spinal cord explants. Dashed squares in the second row show areas that were scanned and analyzed.