Figure 1: GLAST::CreER^T2 marks α-tanycytes subpopulations in the adult hypothalamus.

(a) Left hand panel: coronal section (c.s.) through third ventricle (3V), immunolabelled with Vimentin. The Vimentin^+ive process distinguishes tanycytes (false-coloured in right-hand side of image) from ependymocytes. Central panel cartoon shows position and process projection of tanycyte subtypes: purple, α1; green, dα2; blue, vα2; red, β. Note β-tanycytes divide into medial β2 and lateral β1 subsets^13,14. Right-hand (rh) panel shows high-power magnifications of boxed regions. Ventrally, all ventricular cells appear to have a Vimentin^+ive process; medially, many ventricular cells have a Vimentin^+ive process; dorsally, the ependymocyte/α1 boundary is indistinct, with intermingling of tanycytes and ependymocytes. (b–d): Confocal images, showing c.s. through central hypothalamus, counter-stained with DAPI. (b) Double-labelling shows co-expression of Nestin/Vimentin in tanycytes. High-power views (rh panels) show co-expression in most cells. (c) Double-label analysis of Vimentin/GFAP reveals that dα2-tanycytes (boxed, green arrowhead) and some α1-tanycytes (purple arrowhead) but not vα2- (blue arrowheads) or β-tanycytes (red arrowhead) co-express Vimentin/GFAP. (d) Co-labelling with GFAP and Hu reveals long-process dα2-tanycytes (green arrowhead), short-process α1-tanycytes (purple arrowhead) and GFAP^−ive vα2-tanycytes (blue arrowhead). (e,f): Schematics, showing (e) GLAST::CreER^T2 mice²⁷ crossed to R26R mice. Recombination was induced with tamoxifen. Cre-recombinase expressed under GLAST promoter is translocated to nucleus where it floxes a neo cassette, leading to either lacZ expression and βgal production, or to GFP expression, under control of the ubiquitous ROSA26 promoter. (f) Mice were analysed acutely (5 or 10 days) or after longer-term chase periods (6 weeks, 9 months). (g–i): GLAST::CreER^T2 mice express the reporter, β-gal, in α-tanycytes. Inset in g shows pseuodocoloured regions as in (a). High-power views (taken from regions equivalent to those boxed in (g) show expression in α2-tanycytes (h) but not β-tanycytes (i). (j,k): Double or triple labelling of GFP, Nestin and DAPI (j) or GFP, GFAP and DAPI (k). Yellow arrowheads show GFP expression in dα2-tanycytes. Right-hand panels show high-power views of boxed regions. VMN, ventromedial nucleus; ARC, arcuate nucleus; ME, median eminence. Scale bars:100 μm (a–d,j,k); 20 μm (h,i, rh panels in j,k).