Shh upregulates Lrrn2 in rhombomere 4. (A-A") Co-electroporation of Shh and GFP results in overexpression throughout the left side of the hindbrain as visualised by anti-GFP labelling, reflecting a similar pattern of Shh misexpression (data not shown). This results in an upregulation of Lrrn2 expression only in r4. (A) Lrrn2 expression, (A') GFP expression, (A") overlay. (B, B') The same embryo, following immunocytochemistry with anti-Isl1/2 antibody (green) to label motor neurons, showing the normal pattern of Isl1/2 antibody staining in motor neurons lying adjacent to the floor plate (yellow brackets); (B) photo taken with focal plane set ventrally, and (B') a more dorsal focal plane showing upregulation of Isl1/2 throughout the left side of the hindbrain, as one would predict (expanded yellow bracket). Dashed line indicates the plane of section of (C-C"'). (C-C"') Transverse sections through the hindbrain at the level of r4. (C) Overlay of Isl1/2 in green and GFP in red, indicating unilateral Shh overexpression and induction of Isl1/2 expression. Inset shows high power image. (C'-C"') The expansion of Lrrn2 expression can be seen to be confined to the mantle layer (C") and colocalises with upregulation of Islet1/2 (Isl1/2 alone (C'); overlay with Lrrn2 (C"'); inset shows high power view with Lrrn2 expression pseudocoloured in magenta to more easily visualise overlap with Isl1/2 staining). Scale bar: 100 μm; 50 μm (inset).