Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

This method can help answer key questions about the significance of gene expression dynamics in cell proliferation and differentiation, particularly the dynamics of Notch signaling molecules in neural stem cells. The main advantage of this technique is that we can monitor gene expression both in vitro and in vivo with a very high precision by live imaging. Demonstrating the procedure will be Hiromi Shimojo, the assistant professor from my laboratory who developed this new technology.

For Dll1 F luciferase reporter introduction into NPC, confirm a lack of response to pedal reflex in an embryonic day 12.5 to 14.5 pregnant mouse before making a two-to three-centimeter incision through the abdomen, along the midline. Place PBS-soaked gauze around the incision, and use ring-shaped forceps to carefully extract the right uterine horn. After counting the embryos, use a micro capillary to inject one to two microliters of mixed Dll1 F luciferase reporter DNA into the ventricle of the telencephalon of each embryo.

A successful injection will result in the observance of blue dye over the surface of the uterus. For electroporation, wet the uterus and the electrode with PBS, and gently grasp the head of the first embryo. Set the positively charged electrode to the side of the hemisphere in which the DNA was injected, and provide five 30-to 50-volt, 50-millisecond pulses, with a one-second pause between pulses, checking that bubbles are generated from the negatively charged electrode.

When all of the embryos have been injected, return the left uterine horn to the abdomen, and use a 17-millimeter needle to close the incision with a 4-0 silk suture, placing warm PBS into the abdominal cavity before completing the closure. To set up a dissociated NPC culture, first place the uterus from an embryonic day 12.5 to 14.5 pregnant Dll1 ubiquitinated luciferase transgenic mouse into a 10-centimeter Petri dish containing 25 milliliters of ice-cold PBS, and use micro scissors and fine forceps to remove the embryos from the uterus. After decapitating the embryos, place the heads into a new 10-centimeter Petri dish containing ice-cold DMEM/F12, and remove the epidermis and cartilage surrounding each brain.

Transfer the brains into a 35-millimeter dish under a dissecting microscope containing three milliliters of ice-cold N2B27 medium. Remove the right and left telencephalon from diencephalon. Use fine forceps to remove the meninges covering the surface of telencephalon and to dissect the dorsolateral part of cortex from the telencephalon.

When all of the neural tissues of interest have been isolated, use a P1000 pipette to transfer the samples into individual 1.5-milliliter tubes, and use a P200 pipette to aspirate any extra medium from each tube. Add 100 microliters of papain solution per cortex pair, and place the samples at 24 degrees Celsius for 15 minutes. At the end of the incubation, gently pipette the samples 10 times with a new P1000 pipette, and return the tubes to 24 degrees Celsius for an additional 15 minutes.

At the end of the incubation, gently pipette the samples again before collecting the digested tissues by centrifugation. Aspirate the supernatants, and gently resuspend each pellet with one milliliter of DMEM/F12 medium. Centrifuge the samples three more times as just demonstrated, gently resuspending each pellet with a fresh milliliter of medium after each centrifugation.

After the last centrifugation, resuspend the pellets in 500 microliters of N2B27 medium supplemented with one-millimolar luciferin, and seed one times 10 to the six cells from each sample onto individual poly-L-lysine-coated glass bottom dishes. Then, place the plates in the cell culture incubator for one hour. Once the cells have adhered to the dish, add two milliliters of fresh N2B27 medium plus one-millimolar luciferin to each plate.

To visualize luciferase reporter expression in NPC dissociation cultures, select the oil immersion objective, and place the sample dish onto the microscope stage. View the field manually to determine the best position for viewing the cells, and focus on the cells of interest. Click Live to acquire the test image, and use the Multi-Dimensional Acquisition program to run the time-lapse acquisition by two-dimensional luminescence and bright-field acquisitions for 24 hours.

To prepare developing cortex slice cultures, one day after reporter injection, harvest the embryonic day 13.5 to 14.5 embryo brains as demonstrated, and place the dish containing the brains onto the stage of a fluorescence stereoscopic microscope. Confirm that each cortex expresses the injected fluorescent reporter under the appropriate excitation light, and transfer the brains to a silicone rubber cutting board filled with 30 milliliters of DMEM/F12 medium, bubbled with 100%oxygen gas. Use a micro surgical knife and fine forceps to cut the border between the medial and lateral portion of the dorsal telencephalon, and separate the tissue into two hemispheres.

Then, use the knife to cut the cortex like stripes to obtain cortical slices, transferring the slices in medium from the cutting board into a new 35-millimeter Petri dish as they are acquired. When all of the slices have been collected, place the cut surface of each slice onto a culture insert set into a glass bottom dish containing enriched medium, adjusting the orientation of each slice with fine forceps as necessary. Then, remove any excess medium with a pipette, and place the dish into a multi-gas incubator set to 40%oxygen, 5%carbon dioxide, and 37 degrees Celsius for 30 minutes.

At the end of the incubation, add 300 microliters of enriched medium supplemented with one-millimolar luciferin to the culture dish. To visualize the luciferase reporter expression within the slice cultures, select the 40x objective, and place the sample dish onto the microscope stage. Acquire a test image of the fluorescence, and set the position and focus plane to the region of interest under the illumination of the appropriate excitation light.

Then, run the time-lapse acquisition by three-dimensional luminescence, fluorescence, and bright-field acquisitions for 24 hours. To monitor the promoter activity of the oscillating gene Dll1, ubiquitinated luciferase, a destabilized luciferase reporter with a half-life of about 10 minutes, can be used. Like a fluorescent reporter, the luciferase reporter can be used to monitor the expression dynamics of a protein by being fused to the gene coding sequence of interest.

To visualize reporter expression at the single-cell level in tissue cultures, the reporter gene can be transiently transfected into NPCs via in utero electroporation as demonstrated. In addition, microscope-based imaging enables the acquisition of bright-field, fluorescence, and chemiluminescence images. The reporter of Dll1 promoter activity exhibits an oscillatory expression in NPCs derived from the telencephalon of Dll1 ubiquitinated F luciferase reporter mice, with the destabilized luciferase reporter indicating a sharp up and down regulation of the expression of promoter activity.

In adjacent enhanced green fluorescent protein-positive cells carrying Hes1 reporter and mCherry-positive cells expressing Dll1 protein reporter that made contact with each other during observation, Hes1 reporter expression appears to start about 60 minutes after contact, suggesting that the time delay for the transmission of Notch signaling between the adjacent cells is about one hour. Furthermore, during signal transmission, Dll1 protein expression exhibits a dynamic translocation. When acquiring the bioluminescence imaging, keep the microscope room completely dark, as extraneous light can prevent an optimal acquisition.

Recent developments in optogenetics allows us to control gene expression with light. With this technique, we can simultaneously introduce various pattern of gene expression and monitor luciferase reporter responses.