Xenbase Image ID: 1055

Fig. 2. The mode of molecular action of Shisa2 and 3. (A-B") ER localization of Shisa2 and 3. HEK293T cells transfected with Shisa2 (A) or Shisa3-HA (B) were stained for Shisa (green) and an ER marker calreticulin (red). Cells were transfected with 20 ng of Shisa2 or Shisa3-HA DNA in a 96-well plate. (C) Shisa2 and 3 cellautonomously suppressed the Wnt signal in the receptor cells. Ligand cells expressing Wnt3a (W) and receptorcells expressing Fz and Lrp6 together with TOPFLASH reporter (F/L) were prepared independently and mixed for stimulation. The non-cell-autonomous action of Wnt3a elevated the reporter activity (threefold; lane 2). When either Shisa2 or 3 was coexpressed with the Wnt ligand, reporter activity was not affected (lanes 5, 6); however, expression of Shisa2 or 3 in the receptor cells suppressed the reporter activity below the basal level (lanes 3, 4). Each experiment was carried out at least in triplicate, and error bars represent the standard deviation. (D,E) Shisa2 and 3 physically interact with Fz (D) and FGFR (E). Fz- and FGFR-tagged HA were coexpressed together with Shisa2 or -3-tagged Flag, and immunoprecipitated with anti-Flag mAb. Cells were transfected in the 12-well plate with DNAs: FGFR-HA, 300 ng; Fz8-HA, 200 ng; Shisa2-Flag, 500 ng; Shisa3-Flag, 500 ng. (F-I") Shisa2 and -3 retain the Fz and FGFR in the ER in COS cells. DsRedER marked ER. (F-F") Transfected with Fz8-GFP alone (surface expression of Fz, n=24, 100%). (G-G") Transfected with Fz8- GFP and shisa2 (ER retention of Fz, n=21, 100%). (H-H") Transfected with FGFR-GFP alone (surface expression of FGFR, n=20, 95%). (I-I") Transfected with FGFR-GFP and Shisa2 (ER retention of FGFR, n=22, 81%). Cells were transfected in 96-well glass chambers with DNAs: Fz8- GFP, 10 ng; FGFR-GFP, 20 ng; Shisa2, 180 ng; pDsRed-ER, 10 ng. (J-J") Whole-mount in situ hybridization of Otx2, dorsal view at stage 10.5. Shisa1MO (10 ng) was unilaterally injected into two right side blastomeres at the four-cell stage. Reduced Otx2 expression in the Shisa1MO injected side; n=28/40 (J') and was rescued by co-injection of Shisa2 RNA (10 pg per blastomere); n=32/42 (Jâ ³). (J) Uninjected embryo. Image published in: Nagano T et al. (2006) Copyright © 2006. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Gene Synonyms Species Stage(s) Tissue otx2.S otx-2, otx2-a, otx2-b, otxA, Xotx-2, Xotx2 X. laevis Throughout NF stage 10.5 involuted dorsal mesoderm endomesoderm anterior neuroectoderm Image source: Published Larger Image Printer Friendly View

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