遺伝子誘導の運命のマッピングへの実践的アプローチ：マークとトラックの細胞へのビジュアルガイドインビボでの

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. And advance in this concept is genetic inducible fate mapping or GIFM linking gene expression sulfate and cell behaviors in vivo to create fate maps based on genetic lineage. Hi, my name's Deborah Ellisor from Mark Services Laboratory and the department of Molecular Biology, cell biology and biochemistry at Brown University.

Today my colleagues Ashley Brown, Liz Norman Neen Hagen, Steve Brown, and I will demonstrate a procedure for genetic inducible fate mapping. We use this procedure in the lab to study early brain development. We can also apply this technique to gene inactivation studies and animal models of human disease.

So let's get started In X cre, E-R-M-G-F-P embryos. MGFP LAE is a reporter allele present in all cells represented by gray ovals X cre, er, where X represents gene regulatory elements. Controlling C ER expression is spatially restricted to gene X'S expression domain shown in blue and CRE ER protein is sequestered in the cytoplasm by HSP 90.

In the absence of Tamoxifen, the reporter will be off. Tamoxifen administration results in fate mapped cells in domain X because CER is released from HSP 90 translocates to the nucleus and removes the stop cassette from the reporter allele. Permanent and heritable recombination ensures that cells constituently express MG FP LA Z.The combination of reporter allele CER and tamoxifen results in marking when a cell population is initially marked in a primordial brain region of the embryo based on gene expression, these fate mapped cells are tracked even after the initial gene expression used to drive CER is extinguished.

In this way. The final distribution and terminal fate of a genetically defined cell lineage can be identified in the adult. This procedure begins with the preparation of a 20 milligram per milliliter stalk solution of tamoxifen dissolve 200 milligrams of the tamoxifen in 10 milliliters of pre-war corn oil in a glass inhalation vial.

Then incubate the solution at 37 degrees Celsius for two hours on a Tator vortex. The solution intermittently protect the prepared Tamoxifen stock solution from light by wrapping the vial with foil and stored at four degrees Celsius. The stock can be used for up to one month.

For fate mapping experiments, establish a breeding pair consisting of a wild type Swiss Webster female and a male carrying both a gene specific CreER allele and a reporter allele. For the purpose of this demonstration, we will use a win to one CreER MG FP male. Check the Swiss Webster female each morning for the appearance of a vaginal plug.

Designate the morning of the day of vaginal plug as seen as 0.5 days post cotus and calculate the date of Tamoxifen administration. Based on this starting point in this experiment, Tamoxifen will be administered on embryonic day 8.5. Using a one milliliter syringe with an animal feeding needle, draw up 200 microliters of the Tamoxifen stalk solution.

Firmly restrain the timed pregnant Swiss Webster female by grasping the nap of the neck and back to immobilize the head and turn the mouse over so the ventral side is facing up. Hold the tail between your free fingers to keep the body in a straight line. Next, place the feeding needle into the corner of the mouth and gently guide the needle parallel to the roof of the mouth until the tip of the feeding needle is approximately at the position of the eye.

Gently tilt the head back to gain access to the esophagus. Guide the needle past the epiglottis and down the esophagus toward the stomach. Be careful not to enter the trachea.

Once the feeding needle is in the stomach, administer the tamoxifen and remove the needle. Then return the female to her home cage until the date of dissection. On the date of dissection, E 12.5 embryos from the timed pregnant female are dissected free and then assessed for GFP marking.

In a GFP positive embryo, we observe that wind one derived neurons contribute to the midbrain, coddle hind brain and spinal cord. The MG FP reporter labels axonal projections, which can also be seen transfer embryos that are GFP positive by home mount to a Petri dish containing PPS and photograph them using picture frame After the embryos have been photographed, use forceps to pinch off a small piece of tail from each embryo and place each piece in a PCR tube. The tissues will be genotyped using PCR for both alleles to confirm results seen via whole mount analysis.

The following procedures enable us to analyze how the marked cells derived from embryonic regions populate the adult brain prior to starting the craniotomy. Confirmed by a toe pinch that the mouse is fully innu. The ized with nembutal perform incisions to gain access to the heart through the rib cage.

Next place a butterfly needle into the apex or left ventricle of the heart and secure with the C clamp. Create a fluid egress site in the right atrium with scissors, then perfuse with 10 to 15 milliliters of ice cold saline until fluid runs clear. Followed by 20 to 25 milliliters of 4%paraform aldehyde.

When the intracardiac profusion is complete, remove the head with scissors by cutting through the spinal column just above the shoulders. Run a scalpel along the dorsal midline of the head, rostral de cottle to cut through the scalp and expose the S skull. Using the scalpel, scrape away any excess tissue or muscle from along the side and posterior of the cranium.

With forceps, puncture the skull at the midline, just rostral to the olfactory bulbs and create a small hole to accommodate the tips of fine scissors. Insert the fine scissors into this hole and to make two bilateral incisions from the midline following the length of the olfactory bulbs. This cut will break the skull at the intersection of the nasal bone and frontal bone and provide good access for the scissors.

Cut along the sagittal sutures in the skull, making sure to keep the scissor tips angled away from the brain to avoid damaging the underlying tissue. Next, gently grasp the skull with forceps and peel away the bone along the medial incision to expose the brain. The skull may chip off in small pieces or break away in larger sections.

Continue using the forceps to remove all of the frontal parietal, inter parietal and occipital bones. Free the para oculi located along the lateral edges of the brain at the level of the cerebellum by gently pinching the spherical bones on each side. Use scissors to carefully cut the dorsal part of the bone that surrounds the brainstem.

Insert one side of the scissors just underneath the dorsal edge of the bone, beginning from where the spinal cord was cut and cutting towards the cerebellum. To free the brainstem and cerebellum. Remove the remaining bone from around the brainstem with the forceps.

Gently pull away the bones, turn the head dorsal side down and use the forceps to sever the cranial nerves and release the brain from the skull. Place the brain in a Petri dish of ice. Cold PPS Assess adult brain for GFP marking, using a fluorescent dissecting scope and photograph using picture frame.

In this example, the midbrain is marked by GFP in addition to its use for lineage analysis during embryogenesis and in the adult GIFM can be combined with other applications commonly used in developmental biology such as micro dissection and tissue explant experiments. For example, the ventral mesencephalon is the progenitor zone for developing dopamine neurons which express the gene wind one. Thus isolating the ventral mesencephalon by micro dissection allows for an in vitro setting to be established to further investigate its development.

This represents just one example of using GIFM to isolate a progenitor zone of interest defined by genetic history. To begin this procedure, transfer the previously identified GFP positive embryos to ice cold sterile PBS in a cell culture dish using fine scissors. Remove the head portion of an embryo by cutting transversely coddled to rommy.

One next, remove the rostral portion of the head with a coronal cut through the cephalon. This will expose a tube-like structure in which the fourth ventricle through the mesocephalic vesicle forms a conduit between dorsal and ventral tissues. Carefully insert the scissor tips into the fourth ventricle and snip along the dorsal midline coddled to rostral fully opening the tube, creating an open book preparation.

The ventral mesencephalon will now be exposed medially. While the two dorsal halves of the mesencephalon will reside laterally, it may be necessary to remove any remaining non neural tissue underneath the ventral mesencephalon. In order for the explan to lie flat, the explan should now resemble a butterfly in which the dorsal mesencephalon represents the wings and thrombo mere one the tail.

To further isolate the ventral mesencephalon for fax analysis or cell culture experiments, remove the lateral aspects of the tissue. Now we'll show some representative results of GIFM. In this experiment, WINT one expressing cells marked to E 8.5 are visualized to determine how wint one direct cells contribute to neural structures across development.

For example, wint one C EER MG FP embryos at E 12.5, exhibit GFP fluorescence primarily in the midbrain posterior hind brain and spinal cord at high magnification fine neuronal projections are seen innervating. The midbrain body wall limb and cranial facial region marked cells can also be visualized and analyzed at the cellular level by immunohistochemistry. As shown on this one micrometer thick optical section of an E 12.5 embryo marked by Tamoxifen administration at E 8.5 nuclear beta galacto antibody labeling in red and GFP antibody labeling in green indicates fate mapped cells shown in the ventral midbrain.

Here we combined cells are double positive because of the nature of the conditional MGFP reporter in the adult brain. The density of mature tissue may obscure GFP fluorescence emanating from internal brain structures. Therefore, whole mount fluorescence microscopy reveals only faint GFP fluorescence in the superior CUI of the adult assessing the win.

One lineage marked at E 8.5 on adult sections with low magnification microscopy reveals that the WIN one lineage gives rise to mid-brain structures, including the superior and inferior cui. In this higher magnification image taken from the ventral midbrain in the vicinity of dopaminergic neurons, nuclear beta cal positive cells and a rich GFP positive axonal plexus can be seen in contrast marking at E 9.5 results in substantial GFP labeling that is readily observed in the inferior colus of the midbrain by whole mount fluorescence. The WINT one lineage marked at E 9.5 on adult sections is concentrated in the inferior kaulu as seen with low magnification microscopy.

In this higher magnification image taken from the ventral midbrain in the of dopaminergic neurons, nuclear beta gel positive cells and a rich GFP positive axonal plexus can be seen. Thus while win one derived neurons marked to E 8.5 versus E 9.5 are progressively restricted from contributing to dorsal midbrain. The WIN one lineage persists in contributing to ventral midbrain.

We've just shown you the procedure for genetic inducible fate mapping to mark and track cell lineages in vivo. This allows us to mark cells based on their genetic lineage and track them over time even after gene expression has been extinguished. With tamoxifen administered at eight and a half, we've shown that the wit one domain contributes to the developing midbrain in addition to the entire midbrain in the adult.

In contrast, administering tamoxifen at a nine and a half when the wit one domain becomes restricted, results in more restricted contribution to the developing midbrain during embryogenesis, which persists into the adult. When doing this procedure, it's important to consider that the system marks cells, mosaically, and therefore not every cell in a particular structure may be labeled. This is likely due to the CRE er line or the conditional reporter allele that's being used.

Importantly, although cell marking is mosaic, the pattern and distribution of mar cells is highly reproducible. We have found that tamoxifen administration by oral gavage is advantageous compared to delivery by Interperitoneal injection because it minimizes inflammation in the interperitoneal space as well as mechanical damage to the embryos. So that's it.

Thanks for watching and good luck with your experiments.