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. Author manuscript; available in PMC: 2015 Dec 20.
Published in final edited form as: FEBS Lett. 2014 Nov 20;588(24):4815–4824. doi: 10.1016/j.febslet.2014.11.016

Figure 7. Wnt1S224T mimics wild-type Wnt1 neuron specification activity in the developing spinal cord.

Figure 7

HH stage 11-14 chick embryos were electroporated with pCIG, pCIG.WNT1, pCIG.WNT1 S224A, pCIG.WNT1 S224C, or pCIG.WNT1 SS24T and harvested at HH stage 20-22. Transverse sections from embryos electroporated with pCIG, pCIG.WNT1 wild-type, pCIG.WNT1 S224A, pCIG.WNT1 S224C, and pCIG.WNT1 SS24T were stained with Islet1 to mark dI3 interneuron (dorsal) and motor neurons (ventral) and DAPI to mark cell nuclei. After collecting images via confocal microscopy, each image was cut into left and right halves using NIH Image J. A cell count program was run on all populations (DAPI, Islet1) on each half. A ratio was generated comparing the number of Di3 interneurons or motor neurons on the experimental (electroporated) side cell to that on the control (non-electroporated) side. (A, D, G, J, M) Merged images of electroporated spinal cords visualized with GFP (green), anti-Islet1 immunostaining (red) and DAPI (blue) to mark electroporated cells, neuron subtypes (DI3 is dorsal, MNs are ventral), and cell nuclei, respectively. (B, E, H, K, N) Expression of GFP in spinal cords electroporated with pCIG, pCIG.WNT1, pCIG.WNT1 S224A, pCIG.WNT1 S224C or pCIG.WNT1 S224T. (C, F, I, L, O) Expression of Islet1 marker in spinal cords electroporated with pCIG, pCIG.WNT1, pCIG.WNT1 S224A, pCIG.WNT1 S224C, or pCIG.WNT1 S224T. (P) The total number of neuron subtypes was normalized back to the total number of cells and reported as fraction of Islet1+ cells (DI3s dorsally and MNs ventrally) on the experimental and control sides of the spinal cord. Values reported on the Y axis are the fraction of DI3 or MNs on the experimental side divided by that on the control side. N=number of embryos analyzed while and n=number of sections: pCIG alone (N=3, n=35); WNT1 (N=7, n=71); WNT1 S224A (N=8, n=68); WNT1 S224C (N=4 n=55); and WNT1 S224T (N=3, n=68). Error bars represent +/− standard error. A Student’s t-test was used to assess statistical significance. Abbreviations are as follows: ISL1=Islet1; DI3=dorsal interneuron 3; and MN=motor neurons.