Visualization of miRNAs expressed at different levels in the mouse brain. (a) Low magnification images of nervous system specific miR–124 (orange) shows broad expression [is it possible to say that expression is restricted to neurons from these low mag pictures, Previous studies show miR-124 is restricted to the nervous system (ref #3) and based on dual labeling of miR-124 by ISH and labeling neurons with a specific antibody via immunostain, we show miR-124 is mostly present in neurons (Supplementary Fig. 3)]. (b–c) High magnification images of miR–124 demonstrate ubiquitous expression in the neurons of the cerebellum (top, orange), (b) cerebral cortex and hippocampus (c). (d) Higher magnification images show that miR–124 signals are not present in all cells, marked with arrows. Bottom panels show with 4’, 6–diamino–2–phenylindole dihydrochloride (DAPI) stain. (e) Fluorescence images of mouse brain sections probed for highly expressed miR–9 (, red) localized in Purkinje cells of the cerebellum. miR-410 (f) and miR–370 (g), have intermediate expression. miRNAs differing by 3 nucleotides, miR–26b (h) and miR–26a (i) are differentially expressed. Panels on the right show DAPI stain (e-i). Scale bars, 500 µm (f–i).

As noted in the Checklist I forwarded to you, we require 6 bandwidth around a cropped gel which would make fig. 1 very large – it would take space that is – in my view – better devoted to fig. 2 and fig. 3. The whole text and figures have to fit on 3 print pages We made the appropriate changes.

Formaldehyde + EDC–fixed sections show miRNA localized in the dendrites of neurons. (a) Low magnification fluorescent images demonstrate a robust and broad distribution of miR–370 expression (white) with intense staining in the hippocampus (inset). (b,c) High magnification images show a single neuron in the CA1 region of the hippocampus for which miR–370 is localized both in the cell body and the dendrites (arrows) (c,d), represent inset (a). miR–370 localization extends 50 µm from the cell body (b), and 30 µM in other neurons (c,d). (e) The mature miRNA miR–9 expressed in dendrites of Purkinje cells in the cerebellum, yet the miR–9* (f) was absent in dendrites. (g) Pyramidal cells in the hippocampus (arrows) show staining of miR–124 in hippocampus dendrites and in neurons located in the pons.