M11217

To enable determination of tetrode locations, after the last recording day we anaesthetised mice using an isoflurane chamber and pentobarbital (100–150 μl) and applied a 2-s ~20 µA current to burn the tissue at the tip of the electrodes. We then intracardially perfused phosphate-buffered saline (PBS, Gibco, 70011044, 10 times diluted with distilled water) for 2 min, then 4% paraformaldehyde (PFA, Sigma Aldrich, 30525-89-4) in 0.1 M phosphate buffer (PB, Sigma Aldrich, P7994) for 4 min at a 10 ml/min flow rate. We left the brains in 4% PFA in 0.1 M PB for 16 h, then transferred them to 30% sucrose (Sigma Aldrich, S0389) in PBS until they sank.
We cut 50 µm sagittal sections of the fixed brains using a freezing microtome. Sections were processed to label them with primary antibody rat anti-mCherry (Invitrogen M11217, 1:1000) followed by secondary antibody goat anti-rat Alexa 555 (Invitrogen A-21434, 1:1000) and stained with either NeuroTrace 640/660 (Invitrogen N21483, 1:500) or NeuroTrace 435/455 (Invitrogen N21479, 1:500) following procedures described previously^{43 (link)}. Images were taken on a Zeiss Axio Scan Z1 using a ×10 objective and visually inspected to determine the final position of the recording electrodes (see Supplementary Note 1).

To prepare the paraffin-embedded histology sections, samples were decalcified with 10% EDTA for 3 weeks at room temperature after image collection by micro-CT. Sections were stained with H&E for morphological analysis.
EdU-incorporated proliferative cells were detected using the Click-iT EdU Cell Proliferation Kit for Imaging Alexa Fluor 488 dye (Invitrogen) according to manufacturer instructions. Following the detection of EdU-labeled cells, molecular markers were co-labeled by immunohistochemistry, as previously described^{40 (link)}. The primary antibodies used were as follows: mCherry (1:2,000; M11217; Invitrogen), Gli1 (1:400; 78259; NOVUS, Littleton, CO, USA), Ring1b (1:100; 101273; Abcam, Cambridge, UK), Cbfa1/Runx2 (1:400; 035; MBL Life Sciences, Nagoya, Japan), Sp7/Osterix (1:800; 22552; Abcam), and PCNA (1:200; 2586; Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were as follows: goat anti-rabbit IgG antibody Alexa Fluor 555 (1:400; Invitrogen) and goat anti-mouse IgG antibody Alexa Fluor 555 (1:400; Invitrogen). For the detection of Gli1, Alexa Fluor 555 Tyramide SuperBoost Kit goat anti-rabbit IgG (Invitrogen) was used to amplify the immunoreactivity according to manufacturer instructions. The samples were enclosed with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA).