Supplementary Figure S1: Tanycytes are restricted to the central/posterior hypothalamus

Transcription

1 Supplementary Figure S1: Tanycytes are restricted to the central/posterior hypothalamus a: Expression of Vimentin, GFAP, Sox2 and Nestin in anterior, central and posterior hypothalamus. In the anterior hypothalamus, with the exception of a minor cell population adjacent to the PVN, intermediate filament labelling was never detected in cells with the long basal process characteristic of tanycytes, confirming previous morphologically classifications of these cells as ependymocytes 16,20 b: Double-label analysis of Sox2 and GFAP shows overlapping and intermingled cells in the ventricular zone in d 2- and 1-tanycyte-containing regions (green, purple arrowheads respectively) Scale bars:100 m. 1

2 Supplementary Figure S2: GLAST::CreER T2 activity in tanycytes, but not SVZ astrocytes Coronal sections taken through the central hypothalamus of 5-day recombined GLAST::CreER T2 mice. a,b show GFP-reporter (z/eg) mice; c,d show -gal reporter mice. a,b: Reporter expression around third ventricle, after recombination (a). No expression is detected in uninduced control mice (b). c,d: Single (c) or dual (d) channel view after GFAP-double-labelling. Reporter activity is detected in ventricular zone cells at the level of 2-tanycytes (pseudocoloured as in Figure 1a). Doublelabelling (d) shows co-expression of reporter and GFAP in d 2-tanycytes (region of green arrowhead). In the v 2-tanycyte region (blue arrowhead) SVZ astrocytes can be distinguished through morphology and expression of GFAP, but do not express active GLAST::CreER T2. Scale bars: 100 m (a-c); 20 m (c) 2

3 Supplementary Figure S3: GLAST::CreER T2 activity in parenchymal regions Coronal sections taken through the central hypothalamus of 5-day recombined GLAST::CreER T2 z/eg mice. (a) A small minority of reporter-positive cells in the parenchyma are GFAP+ (yellow arrow (1), graph; n=5, unpaired t-test was used to calculate p values, * indicates p<0.05, error bars represent SEM). (b-d) The majority of GLAST::CreER T2 activity in parenchymal cells is detected in cells with a particular morphology, but that do not express GFAP, NeuN, NG2 or Nestin (green arrows (3). Red arrows (2) show reporter-negative parenchymal cells that are GFAP +ive, NG2 +ive or NeuN +ive. Scale bars: 20 m 3

4 Supplementary Figure S4: Temporal increase in GLAST::CreER T2 -labelled astrocytes, neurons and progenitors a: Heat maps, showing GFAP +ive Reporter +ive (Rp+) cells in each field of view (FOV) at 5-10 days post-recombination, and after 6-week or 9-month chase. Right hand panel shows schematic, indicating saturating analyses of hypothalamic hemi-section. b: Left hand graph shows increase in proportion of GFAP +ive Rp +ive cells over time. Note this includes VZ dorsal 2-tanycytes and non-vz astrocytes. Right hand graph shows comparative increase in mean number of VZ and non-vz Rp +ive GFAP +ive cells. Both significantly increase over time. n=4-6 each. Unpaired t-test was used to calculate p values, * indicates p<0.05, error bars represent SEM. c: Heat maps showing location of NeuN +ive reporter cells immediately after recombination (no NeuN +ive cells) and after 6-week or 9-month chase. d: Graph shows increase in proportion of NeuN +ive Rp +ive cells over time. n=4 each. Unpaired t-test was used to calculate p values, * indicates p<0.05, error bars represent SEM. e,f,i: Coronal sections taken through the central hypothalamus of GLAST::CreER T2 mice (LacZ reporter) after acute recombination (e) or 6 week chase (f, i), double-labelled to detect doublecortin. After acute recombination, reporter activity is detected in 2 tanycytes and unkown parenchymal cells (red arrow 3 in e). Green arrow (2) shows reporter -ive doublecortin +ive cell. After 6 week chase (f,i), reporter activity is detected in doublecortin +ive parenchymal cells lying close to tanycyte processes (yellow arrow 1 in f). Right hand panels in f show high power single and dual channel views of boxed region. Green arrow 2 in (i) points to doublecortin +ive Rp -ive cell with astrocytic morphology. g: Heat maps, showing doublecortin +ive Reporter +ive (Rp+) cells in FOV at 5-10 days postrecombination, and after 6-week chase. h: Graph show increase in proportion of doublecortin +ive Rp +ive cells over time. n=4. Unpaired t-test was used to calculate p values, * indicates p<0.05, error bars represent SEM. Scale bars: 100 m (a,c,e,g); 10 m (f,i) 4

5 Supplementary Figure S5: FGF2 promotes -tanycyte proliferation Quantification of newborn cells by location (a, c) or after double-labelling with Vimentin or GFAP to distinguish tanycyte subsets (b, d) in acutely-culled (a, b) or 6-week chased (c, d) mice. n=3-6 each. Unpaired t-test was used to calculate p values, * indicates p<0.05, error bars represent SEM. 5

6 Supplementary Figure S6: Accurate subdissection of A-P hypothalamic regions Coronal sections through anterior (a) central (c) and posterior (e) hypothalamus. Immunohistochemical and in situ analyses reveal distinctive regional signatures. Subdissected tissues (b,d,f), analysed by immunohistochemistry or in situ hybridisation, express identical profiles, evidence to the accuracy of their dissection. Scale bars: 100 m 6

7 Supplementary Figure S7: Expression of Fgf10 and Fgf18 in tanycyte subsets a-c: Expression of Fgf-10, Fgf-18 and GFAP in serial adjacent coronal sections through central hypothalamus. d,e: High power views of the v 2 region showing Fgf-10 and Fgf-18 expression in tanycytes, adjacent to the ventricle (green dots). Black dotted line indicates SVZ Scale bars: 100 m (a-c); 12 m (d,e) 7

8 Supplementary Figure S8: Hypothalamic neurospheres express Fgf10 and Fgf18 Lane 1 shows RNA ladder (Invitrogen); lanes 2-5 show RNA products amplified from neurospheres; lanes 6-8 show RNA products amplified from whole adult hypothalami. 8