The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

All animal work was carried out in accordance with UK Home Office and local ethical guidelines.

1. Summary of Steps and Timing

1. Perform timed matings of adult mice from a strain suitable to answer the biological question under investigation to obtain embryonic day (e) 9.5-e13.5 pregnancies; requires 12 – 15 days.
2. Optionally, prepare 5-bromo-2'-deoxyuridine (BrdU)/5-ethynyl-2'-deoxyuridine (EdU) solution and perform injection (Protocol section 2); requires ~ 1 h on the day before or on the day of embryo isolation, depending on the desired length of EdU/BrdU labeling.
3. Perform embryo isolation and hindbrain dissection (Protocol section 3); requires ~ 10 min/embryo.
4. Perform wholemount immunofluorescence labeling (Protocol section 4); requires 3 days.
5. Section using a vibratome and perform floating section immunofluorescence labeling (Protocol section 4): requires 2 days.
6. Section using a cryostat and perform immunofluorescence labeling of cryosections (Protocol section 5); requires 2 days.

2. Inject Pregnant Female Mouse with BrdU or EdU (Optional)

1. Dissolve BrdU or EdU in sterile phosphate buffered saline (PBS) to concentrations of 10 mg/mL and 1 mg/mL, respectively.
   Caution: BrdU and EdU are toxic; wear appropriate protection.
2. Weigh the pregnant mouse and calculate the volume of BrdU or EdU solution that should be administered to reach 100 mg/kg BrdU or 5 mg/kg EdU.
3. Inject BrdU or EdU solution through the intraperitoneal route either 1 h or 1 day before collecting the embryos, depending on the required length of labeling.
   NOTE: Labeling for 1 h visualizes hindbrain cells in S-phase. Labeling for 1 day visualizes the progeny of hindbrain NPCs.

3. Dissection of Hindbrains from e9.5 – e13.5 Mouse Embryos

1. Euthanize a timed-pregnant female mouse using an ethically approved procedure at the required gestational stage (e.g., cervical dislocation). Using sharp scissors, cut open the peritoneal cavity and carefully excise the uterus. Place the excised uterus containing the embryos into a 60 mm plastic dish containing 20 mL ice-cold PBS.
   NOTE: Aseptic technique is not required.
2. Perform all further dissection using a dissecting microscope. Using watchmaker forceps number 5, tear the uterine muscle wall to expose the embryos, release each embryo by severing the umbilical cord and remove the yolk sac.
3. Using a sterile Pasteur pipette with a wide-bore opening, transfer each embryo into a clean plastic dish with ice-cold PBS.
4. Using watchmaker forceps number 55, sever the head (Figure 1E).
5. Optionally, retain a small piece of tissue (e.g., ¼ of a yolk sac or approximately 2 mm tail tip) for genomic DNA isolation and subsequent genotyping.
6. Using watchmaker forceps number 55, excise the tissue containing the hindbrain parallel to and immediately below the hindbrain neuroepithelium to separate it from facial and forebrain tissue (Figure 1F).
7. Position the hindbrain and caudal head tissue dorsal side up and identify the 4^th ventricle, which is covered by a thin tissue layer (roof plate). Carefully pierce the roof plate using watchmaker forceps number 55 (Figure 1G) and peel away excess tissue with forceps, moving rostrally along the midline over the midbrain, and then caudally over the posterior hindbrain and spinal cord (Figure 1G); the hindbrain should now be exposed in an open book preparation (Figure 1H).
8. Prepare hindbrains for wholemount immunolabeling (Option 1).
   1. Using watchmaker forceps number 55, carefully tease away remaining head mesenchyme and any meninges attached to the pial side of the hindbrain using forceps (Figure 1I). Remove the midbrain and spinal cord tissue (Figure 1J) to leave only the hindbrain tissue (Figure 1K).
      NOTE: This step of the procedure should not be followed at e9.5 or e10.5, because attempting to do so would likely damage the hindbrain neuroepithelium; moreover, removing the meninges is not essential because the hindbrain neuroepithelium is sufficiently thin at this stage to allow efficient penetration of antibodies for immunolabeling. This step should, however, be followed from e11.5 onwards, when the meningeal tissue consolidates, to enhance antibody penetration into the hindbrain neuroepithelium. Dissection is easiest when performed in ice-cold PBS (use clean PBS for each hindbrain).
9. Prepare hindbrains for vibratome and cryosectioning (Option 2)
   1. Using watchmaker forceps number 55, remove the midbrain and spinal cord tissue (Figure 1J).
      NOTE: It is not necessary to remove mesenchyme and meninges of hindbrains intended for sectioning (as shown in Figure 1I).
10. Transfer hindbrains from the plastic dish to round-bottomed 2 mL tubes using a Pasteur pipette, aspirate all PBS, and fix for 2 h at 4°C with gentle-agitation in freshly thawed 4% w/v formaldehyde dissolved in PBS.
    Caution: Formaldehyde is toxic; wear appropriate protection.
11. Rinse hindbrains 3x times with PBS. Store at 4 °C in PBS if immunolabeling will begin within 2-3 days or replace PBS with 50% methanol/50% PBS for 5 min before transferring to 100% methanol for longer storage at -20^°C.

4. Wholemount Immunofluorescence

1. If required, rehydrate hindbrains in serially decreasing dilutions of methanol in PBS (e.g., 75% methanol, 50% methanol, 25% methanol) at room temperature (RT) for 5 min each and then transfer to PBS.
   NOTE: A graded series of methanol is required to gently rehydrate hindbrains and ensure proper tissue preservation.
2. Permeabilize hindbrains for 30 min at 4 °C in PBS containing 0.1% Triton X-100 (PBT) with gentle agitation.
3. Incubate hindbrains for 1 h at 4 °C in PBT containing 10% heat-inactivated goat serum with gentle agitation.
   NOTE: Use serum from the host species that the secondary antibodies were raised in. For primary antibodies raised in goat, incubate in serum-free protein block, for example 5% bovine serum albumin in PBS or a suitable commercial alternative (see Table of Materials). This will reduce non-specific staining frequently observed when using primary antibodies raised in goat.
4. Incubate hindbrains overnight at 4 °C in PBT containing 1% heat-inactivated serum and primary antibodies with gentle agitation (e.g., rabbit anti-phospho histone H3 [pHH3] diluted 1:400).
   NOTE: For primary antibodies raised in goat, use PBT without serum.
5. Wash the hindbrains at 4 °C 5x with PBT for 1 h each.
6. Incubate hindbrains overnight at 4 °C in PBT containing appropriate fluorophore-conjugated secondary antibodies (e.g., goat anti-rabbit Alexa Fluor 488) at 1:200 in PBT targeted against the primary antibodies used. Keep hindbrains in the dark from here on to protect fluorophores from photobleaching.
   NOTE: For primary antibodies raised in goat, use anti-goat Fab fragments of secondary antibodies to reduce non-specific staining.
7. Wash the hindbrains at 4 °C 5x with PBT for 1 h each.
8. Postfix the hindbrains in 4% formaldehyde in PBS for 15 min at RT for long term preservation of antibody binding. Briefly rinse twice in PBS.
9. Cover a glass microscope slide with two layers of black electrical tape and excise a small square from the layered tape to create a pocket large enough to hold one hindbrain.
10. Transfer each hindbrain into a pocket with a Pasteur pipette, remove excess liquid and add an appropriate antifade reagent to the pocket before covering it slowly with a glass coverslip to avoid trapping air bubbles under the coverslip. Seal the coverslip and affix it to the slide with a thin layer of nail polish. Store slide at 4 °C in the dark until image acquisition (Protocol section 7).

5. Vibratome Sectioning and Floating Section Immunofluorescence

1. If required, rehydrate hindbrains from methanol as described in step 4.1.
2. Embed hindbrains in molten 3% w/v agarose prepared in distilled water.
   NOTE: Allow molten agarose to cool to approximately 55°C briefly before embedding to prevent heat damage to hindbrain.
3. Cut transverse hindbrain sections to a thickness of 70 µm using a vibratome. Transfer each freshly cut section with a paintbrush into one well of a 24-well plate containing ice-cold PBS.
4. Label the floating sections as described in steps 4.2-4.7, but modify the steps as follows: wash the floating sections at 4 °C 3x with PBT for 15 min each, decrease incubation time for secondary antibodies to 2 h, and incubate in secondary antibody at RT.
5. Optionally, after labeling with antibodies for other epitopes as described in step 5.4, detect EdU⁺ nuclei using an EdU labeling kit according to the manufacturer's instructions. Incubate the floating sections in the reaction cocktail for 30 min at 37 °C in the dark. Wash hindbrain sections at 4 °C 3x with PBT for 15 min each.
6. Optionally, after labeling for other epitopes as described in step 5.4, detect BrdU⁺ nuclei as follows.
   1. Incubate floating sections in 2 N hydrochloric acid at 37 °C for 30 min in the dark.
   2. Incubate floating sections in 0.1 M sodium borate buffer pH 8.5 twice for 5 min each at RT in the dark to neutralize the hydrochloric acid.
   3. Wash floating sections 3x briefly in PBS at 4 °C.
   4. Perform immunofluorescence labeling for BrdU as described in step 5.4.
7. Incubate the floating sections for 2 min at RT in 10 µg/mL 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS to counterstain cell nuclei.
8. Postfix the floating sections in 4% formaldehyde for 15 min at RT.
9. Wash floating sections briefly in PBS. Carefully transfer floating sections to a glass microscope slide using a paintbrush. Mop up excess PBS around the section using blotting paper or tissue.
10. Mount sections using 80% glycerol in PBS and cover slowly with a glass coverslip to avoid trapping air bubbles. Store slide at 4 °C in the dark until image acquisition (step 7).

6. Cryostat Sectioning and Cryosection Immunofluorescence Labeling

1. If required, rehydrate hindbrains from methanol as described in step 4.1.
2. Incubate hindbrains in 30% w/v sucrose in PBS to cryoprotect hindbrains prior to freezing.
   NOTE: Hindbrains are ready for freezing when they have sunk to the bottom of the tube, which typically takes ≤ 2 h for e9.5-10.5 hindbrains and 3-6 h for e11.5-13.5 hindbrains.
3. Submerge hindbrains in optical cutting temperature compound (OCT) and freeze quickly by transferring molds containing hindbrains in OCT to isopentane cooled to between -40 °C and -50 °C on dry ice.
   NOTE: Frozen, embedded hindbrains can be stored at -20 °C short term and -80°C long term until sectioning.
4. Cut transverse hindbrain sections to a thickness of 10 µm using a cryostat and transfer to electrostatically adhesive microscope slides.
   NOTE: Hindbrain cryosections can be stored at -20 °C short term and -80 °C long term until labeling.
5. Incubate slides at RT for 15 min to dry sections to the slides. Wash cryosections with PBS to dissolve OCT and mark a hydrophobic barrier around cryosections using a PAP pen. Repeat steps 5.4-5.8.
6. Repeat step 5.10 using a polyvinyl alcohol-based mounting medium in place of glycerol.

7. Image Acquisition

1. Image samples using an epifluorescent or confocal laser-scanning microscope equipped with lenses suitable for aqueous media-mounted slides and optical filters suitable for the fluorophores used for immunolabeling.
2. To image the whole hindbrain or a hindbrain section, use a lens for 10x magnification (e.g., Figure 2B); to visualize hindbrains areas for quantification, use a 40X magnification (e.g., Figure 2D) and to visualize individual cells, use a lens with a 63x magnification (e.g., Figure 2C).

8. Alternative Methods

1. Following step 3.8, homogenize unfixed hindbrains to produce a single cell suspension for flow cytometry applications¹⁵.
2. Following step 3.8, homogenize unfixed hindbrains to extract RNA from single cell suspensions for RT-qPCR (e.g., ¹⁶).
   NOTE: Ensure all reagents and equipment are kept sterile and RNase-free throughout to prevent degradation of RNA.
3. Following step 3.8, homogenize unfixed hindbrains to isolate NPCs and propagate them in vitro as neurospheres for analysis of hindbrain NPC behavior¹⁷.
   NOTE: Ensure all reagents and equipment are kept sterile to prevent bacterial/fungal contamination of neurosphere cultures.