Cryostat

Male WT mice between 12 and 23 wk of age were perfused with PBS followed by cold 4% PFA between 1300 and 1900 hours. Brains, thoracic spinal cords, and eyes (with attached optic nerve) were dissected and processed as previously described. Coronal and sagittal brain frozen sections of 20 µm thickness were cut using a Leica cryostat. Nonconsecutive coronal sections between 1 mm and −2 mm relative to bregma were collected, and nonconsecutive sagittal sections of one hemisphere per brain were collected. Spinal cord (transverse and longitudinal) and eye sections of 12-µm thickness were cut using a Leica cryostat. After sections were mounted on microscope slides, they were stained immunohistochemically for various antigens and imaged as described above. Qualitative assessment of CCN3 expression was performed using at least five animals.

Embryo heads were dissected on ice-cold PBS and placed in 4% paraformaldehyde/0.1 M phosphate buffer overnight at 4 °C, followed overnight by 15% and subsequent 30% sucrose/PBS at 4 °C. Fixed heads were then embedded in cryoprotectant Tissue-Tek O.C.T (Sakura), snap frozen in liquid nitrogen, and stored at −20 °C before sectioning. The 20-µm-thick mounted sections were obtained for histological analysis using a Cryostat (Leica). Both embryonic mouse and organoid frozen tissue were sectioned using a Cryostat (Leica), and 20-µm-thick sections were obtained for histological analysis. Samples were washed and blocked in PBS with 3% donkey serum (Millipore) and 0.25% Triton-X for 30 min prior to antibody incubation. Slides with primary antibody were kept at 4 °C for 24 to 48 h. Tissue was further washed and blocked again for 15 min followed by secondary antibody staining at room temperature for 1.5 h. For BrdU stains, mouse embryonic tissues were treated with 1 N HCl at 4 °C for 10 min, 2 N HCl at 37 °C for 30 min, and 0.1 M borate buffer (pH 8.8) for 10 min, followed by extensive washes with PBS. DAPI or Hoechst was used to counterstain cell nuclei. Tissues were coverslipped with ImmuMount (Thermo Fisher) and kept at 4 °C until imaged. Antibodies and dilutions used are outlined in SI Appendix, Table S2.

Snap-frozen hFCX (middle frontal gyrus at the level of mammillary body) and hTH (at the level of the subthalamic nucleus, red nucleus, and hippocampus) were sectioned on a cryostat (Leica) to collect 100 μm–thick sections for total RNA and nuclei isolation. Similarly, snap-frozen mouse brains were sectioned on a cryostat (Leica) and mFCX and mTH at the level of anterior hippocampus were micro-dissected. Total RNAs were extracted using the RNAeasy Mini kit (Qiagen) according to manufacturer’s instructions. RNA integrity was analyzed on an Bioanalyzer using RNA 6000 Nano kit (Agilent). Only samples with an RNA integrity number (RIN) of at least 6.5 were used to perform snRNA-Seq.