The overall goal of this experimental procedure is to map the spatio-temporal gene expression dynamics of the vertebrate segmentation clock using fixed tissue sampling. This method allows an unbiased assessment of oscillatory gene dynamics in the presomitic mesoderm, and this is crucial to understanding the molecular mechanisms governing vertebrate somatogenesis. The main advantage of this technique is that many samples can be generated and processed simultaneously, allowing the high three part analysis of gene expression dynamics in fixed tissue.
Demonstrating the procedure will be Dr.Charlotte Bailey, who's a former post-doc in my laboratory. After obtaining a mouse uterine horn according to the text protocol, under a stereo microscope, used curved scissors to cut the thick muscular membrane of the uterine horn and carefully extract each embryo. With curved scissors and fine forceps, dissect away the amniotic sac from each embryo.
Then using either a surgical needle or curved scissors, harvest the tail tissue from each embryo by cutting the embryo anterior to the rear limb buds. Balance the tail tissue ventral side down. Then generate pairs of PSM explants by dissecting the tail tissue into two halves along the midline, using a gentle rocking motion with the needle.
Ensure that the neural tube, notochord, and PSM tissue are equally divided between the two explants. Then pipette each contralateral PSM explant onto the underside of a 35mm plastic culture dish lid in a small volume of pre-warmed culture medium. Place the dish on the top of the lid and quickly invert it, so that the PSM tissue is suspended from the lid in a hanging drop of medium.
Then culture the PSM explants in a humidified chamber at 37 degrees Celsius for one to two hours. Transfer pairs of PSM explants to the individual wells of a 24 well tissue culture plate. Then add 4%paraformaldehyde in PBS to the samples and incubate the plate at room temperature for one hour, or at four degrees Celsius on a rocking platform overnight.
Following the incubation, use PBS to wash the sample wells at room temperature on a rocking platform. With a fine plastic Pasteur pipette, exchange the PBS solution on the samples three to four times. To carry out immunohistochemistry, add 2%Triton X-100 in PBS to one PSM explant from each embryonic pair and incubate the samples at room temperature on a rocking platform for one hour to wash them.
Use PBS to briefly rinse the samples. Then replace the PBS with blocking solution and incubate the samples at four degrees Celsius on a rocking platform overnight. Using working buffer, dilute the desired primary antibodies.
In this example, antibodies to Delta-like 1 and Notch1 are used at a dilution of 1:25. Add the antibodies to the explants and incubate the samples on a rocking platform at four degrees Celsius for three to five days. Following the incubation, use PBS to wash the samples two times for five to ten minutes each.
Then perform three washes in 0.3%Triton X-100 in PBS at room temperature on a rocking platform. After diluting secondary antibodies in working buffer, add 250-500 microliters of the antibody solution to each sample well, taking care not to use the last few microliters of solution, which may contain antibody aggregates. With tin foil, cover the plate and incubate the samples in secondary antibody solution in the dark at four degrees Celsius for three to five days.
After the incubation, use PBST to wash the samples twice for ten minutes each. Then use PBS to wash the tissue once at room temperature on a rocking platform for five minutes. Following the text protocol, dehydrate and rehydrate the remaining contralateral PSM explants through an ethanol PBST dilution series.
Add ten micrograms per milliliter of Proteinase K in 0.1%PBST to the explants, and incubate the tissue without agitation for five minutes. Then quickly remove the Proteinase K solution and use PBST to briefly rinse the samples. Add 4%formaldehyde and 0.1%glutaraldehyde in PBST to the PSM explants to postfix them and incubate the samples for 30 minutes.
Then, after using PBST to wash the samples twice for ten minutes each, add 50%hybridization mix to the tissue and incubate at 65 degrees Celsius without agitation for ten minutes. After incubating the samples and hybridization mix according to the text protocol, remove the solution and add 0.25 to 0.5 milliliters of pre-warmed hybridization mix containing a digoxigenin-labeled antisense RNA probe against a known segmentation clock component. Use sticky tape to seal the plate and incubate the samples at 65 degrees Celsius for two nights.
Following the incubation, use pre-warmed 50%hybridization mix in TBST to wash the samples for 15 minutes. Then use TBST to rinse the samples twice before incubating the tissue in TBST at room temperature on a rocking platform for 30 minutes. Next, pre-incubate the explants in blocking solution for a minimum of two hours.
Then replace the solution with fresh blocking solution, containing a 1:200 dilution of HRB conjugated anti-digoxigenin antibody. Incubate the samples at four degrees Celsius overnight. The following day, use TBST to rinse the samples three times at room temperature and transfer them to individual wells of a new 24 well tissue culture plate.
Then with TBST, wash the explants three times for one hour each. Use a tyramide signal amplification kit to visual the detected mRNA before preparing the samples for imaging. Prepare one charged adhesion glass slide for each explant pair by removing the adhesive liner from a 0.12 mm thick imaging spacer and sticking it onto a glass slide.
After positioning a pair or explants onto the slide according to the text protocol, allow the samples to adhere to the slide for 45-60 seconds, until the tissue begins to appear sticky and translucent without completely drying out. In the meantime, use forceps to remove the remaining adhesive liner from the spacer and add a large drop of dual function mounting medium and clearing solution to the samples in the center of the spacer. Apply a circular coverslip across the samples ensuring that the mounting medium is evenly distributed and that all the edges make contact with the spacer.
Then place the coverslip slide upside down on some low lint paper. Press down firmly to ensure that the coverslip fully adheres to the spacer and that any excess mounting medium is removed. Repeat this process until no more mounting medium blots the paper.
Clean and label the slide and store it in the dark until imaging. Then carry out imaging and analysis according to the text protocol. In this experiment, Delta-Like 1 and Notch1 protein expression are shown to oscillate out of synchrony by temporarily arranging embryos according to the nascent transcription of the Notch regulated segmentation clock gene intronic lunatic fringe detected in the one half of each explant pair.
The panels are arranged according to phase one, two, and three of the segmentation clock cycle. The extent of the expression domains for Delta-Like 1, Notch1, and intronic lunatic fringe along the anteroposterior axis of the PSM are demarcated by color coded bars. As shown here, a plot is generated to quantify Delta-Like 1, Notch1, and intronic lunatic fringe signal intensity in relation to the anteroposterior axis of the PSM and illustrates axial variation and signal intensity across the PSM throughout the clock cycle.
Kymographs visualize the spatial distribution of Delta-Like 1, Notch1, and intronic lunatic fringe across numerous PSMs. Each row of the kymograph represents the signal intensity of an individual PSM explant. Quantification of Delta-Like 1, Notch1, and intronic lunatic fringe signal intensity in relation to the anteroposterior axis of the PSM reveals clear oscillatory expression dynamics for these targets.
Finally, these kymographs show the spatial distribution of the cleaved and activated form of the Notch receptor NICD, Delta-Like 1, intronic lunatic fringe, and Notch1 across numerous PSMs. This technique has aided researchers in the field of vertebrate's mitogenesis to better understand segmentation gene oscillation dynamics in the chick and mouse presomitic mesoderm. Once mastered, this technique can be performed on large numbers of samples allowing the expression profiles of several mRNAs and proteins of interest to be analyzed simultaneously.
Following this procedure, additional quantification of the image data can provide an understanding of temporal delays in gene expression as well as the subcellular localization of both the RNA and protein signals of interest to the researcher. After watching this video, you should have a good understanding of how to map the spatial-temporal gene expression dynamics of the vertebrate segmentation clock using thick tissue sampling. This can be followed by automated image analysis as detailed in the text protocol.
While attempting this procedure, it's important to ensure equal distribution of PSM, notochord, and neural tube between PSM explants and to carefully optimize antibody dilutions before starting. Remember that working with formamide, paraformaldehyde, and glutaraldehyde can be extremely hazardous. Take care to wear personal protection equipment and work in a fume hood where applicable.
