The overall goal of this procedure is to allow whole organ and histological analysis of the mouse embryonic hindbrain during development. This method permits the study of developments on neurogenesis in the hindbrain of a mammalian model organism. The developing mouse hindbrain is an excellent model for studying the activity of neural progenesis due to its flat architecture which facilitates observation of neurogenesis patterns across the entire organ.
Begin this procedure with euthanizing a timed pregnant female mouse using an ethically approved procedure at the required gestational stage. Using sharp scissors, cut open the peritoneal cavity and carefully excise the uterus. Place the excised uterus containing the embryos into a 60 millimeter plastic dish containing 20 milliliters of ice cold PBS.
Perform all further dissection using a dissecting microscope. Using Watchmaker forceps number five, tear the uterine muscle wall to expose the embryos. Release each embryo by severing the umbilical cord and remove the yolk sac.
After transferring each embryo to a clean plastic dish with ice cold PBS, use Watchmaker forceps number 55 to sever the head. Optionally, retain a small piece of tissue for genomic DNA isolation and subsequent genotyping. Using Watchmaker forceps number 55, excise the tissue containing the hindbrain parallel to and immediately below the hindbrain neuro epithelium to separate it from facial and forebrain tissue.
Position the hindbrain and caudal head tissue, dorsal side up and identify the fourth ventricle which is covered by a thin tissue layer known as the roof plate. Carefully pierce the roof plate using Watchmaker forceps number 55 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. The hind brain should now be exposed in an open book preparation.
To prepare hindbrains for wholemount immuno labeling, use Watchmaker forceps number 55 to carefully tease away the remaining head of mesenchyme and any meninges attached to the peel side of the hindbrain. Remove the midbrain and spinal cord tissue to leave only the hindbrain tissue. To prepare hindbrains for vibratome and cryo sectioning, use Watchmaker forceps number 55 to remove the midbrain and spinal cord tissue.
Transfer the hindbrains from the plastic dish into round-bottom two milliliter tubes using a Pasteur pipette. After aspirating all the PBS, fix the hindbrains for two hours at four degrees Celsius with gentle agitation in freshly thawed 4%weight per volume formaldehyde dissolved in PBS. Finally, rinse the hindbrains three times with PBS.
If required, rehydrate hindbrains in serially decreasing delusions of methanol in PBS at room temperature for five minutes at each concentration. Then, transfer to the hindbrains to PBS. Permeabilize the hindbrains for 30 minutes at four degrees Celsius in PBS containing PBT with gentle agitation.
Then incubate the hindbrains for one hour at four degrees Celsius in PBT containing 10%heat inactivated goat serum. Next, incubate the hindbrains overnight as before in PBT containing 1%heat inactivated serum and primary antibodies. The next day, wash the hindbrains at four degrees Celsius with PBT for one hour each.
Following wash, incubate the hindbrains overnight at four degrees Celsius in PBT containing the appropriate fluorophore conjugated secondary antibodies targeted against the primary antibodies used. Keep the hindbrains in the dark from this point on to protect fluorophores from photobleaching. After washing as before with PBT, post-fix the hindbrains in 4%formaldehyde in PBS for 15 minutes at room temperature for long-term preservation of antibody binding.
Meanwhile, 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 the hindbrain. After briefly rinsing the hindbrain twice in PBS, transfer it into the pocket with a Pasteur pipette. Remove the excess liquid and add an appropriate anti-fade reagent to the pocket.
Then slowly cover the hindbrain 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 the slide at four degrees Celsius in the dark until image acquisition.
Embed hindbrains in molten agarose at 3%weight per volume prepared in distilled water. Cut transfers hindbrain sections to a thickness of 70 microns using a vibratome. Transfer each freshly cut section with a paintbrush into one well of a 24 well plate containing ice cold PBS.
After labeling the floating sections as detailed in the text protocol, wash them briefly in PBS. Carefully transfer floating sections to a glass microscope slide using a paintbrush. Mop up the excess PBS around the section using blotting paper or tissue.
Not the sections using 80%glycerol in PBS. And slowly cover them with a glass coverslip to avoid trapping air bubbles. Store the slide at four degrees Celsius in the dark until image acquisition.
Incubate the hindbrains in 30%weight per volume sucrose in PBS at four degrees Celsius to cryo protect hindbrains prior to freezing. Submerge the hindbrains in optical cutting temperature compound or OCT. Then, freeze them quickly by transferring the molds containing the hind rains to isopentane cooled to between 40 and 50 degrees Celsius on dry ice.
Next, cut the transfers hindbrain sections to a thickness of 10 microns using a cryostat. Transfer the hindbrain sections to electrostatically adhesive microscope slides. Incubate the slides at room temperature for 15 minutes to dry the sections to the slides.
Then wash the cryo sections with PBS to dissolve the OCT. Mark a hydrophobic barrier around cryo sections using a PAP pen. Image the samples using an Epi-fluorescent or confocal laser-scanning microscope equipped with lenses suitable for aqueous media mounted slides as well as optical filters suitable for the fluorophores used for immuno labeling.
To image whole hindbrain or a hindbrain section, use a lens for 10 times magnification. To visualize hindbrain areas for quantification use a 40 times magnification. And to visualize individual cells, use a lens with a 63 times magnification.
Shown here are representative results from wholemount immuno labeled hindbrains to observe dividing neural progenitor cells in the ventricular zone. These images were obtained by using the mitotic marker phospho histone H3 at consecutive stages of development. Transverse vibratome sections were labeled following a one-hour pulse of EdU to highlight hindbrain neural progenitor cells undergoing inter-kinetic nuclear migration as well as mitotic phospho-histone H3 positive cells at the ventricular surface.
The overall proportion of self-renewing neural progenitor cells can be determined by calculating the percentage of Ki67 BrdU double positive cells in yellow amongst all BrdU positive cells in red and yellow in transverse hindbrain cryo sections from embryos one day after BrdU administration. Hindbrain vibratome sections can also be immuno labeled with an antibody for the intermediate filament protein Nestin also known as RC-2 to visualize neural progenitor cell endfeet and protrusions. After watching this video, you should have a good understanding of how to analyze a flat mount preparation or transverse tissue sections of the hindbrain.
Once mastered, up to 10 hindbrains can be dissected in under one hour. While attempting this procedure, it is important to use ice cold PBS if first familiarizing yourself with hindbrain micro dissection as this facilitates separation from the meninges, in addition to ensuring tissue preservation. Following this procedure, other methods like flow cytometry can be performed on single cell suspensions of the hindbrain to separate neural progenitors from other cell types.
Establishing this technique has paved the way for researchers in the field of developmental neurobiology to explore mechanisms of mammalian neurogenesis by using the hindbrain as an alternative to the conventionally used spinal cord and forebrain models. This method can provide insight into mammalian hindbrain neurogenesis but can also be applied to other developing vertebrates given the degree of structural and molecular similarity between the species. This technique may aid the discovery of novel mechanisms controlling neurogenesis and help determine whether those identified elsewhere in the axis are recapitulated in this part of the vertebrate brain.
