The overall goal of this procedure is to dissect an ex vivo culture, mouse, embryonic thyroid implants, or lobes to allow the study of morphogenetic and differentiation events occurring during thyroid development. This is accomplished by first removing the upper jaw and the lower part of the body of embryonic day. E 12.5 to E 14.5 mouse embryos to isolate the neck region of the embryos.
In the second step, the neural tube, lateral tissues and tongue are removed to expose the upper part of the trachea and esophagus. Next, the thyroid bud, regions, or lobes are further cleaned of any undesired tissues. In the final step, the thyroid implants are plated for culture on a coated microwell or on a filter.
Ultimately, time lapse imaging, immunos staining or gene expression analysis can be performed to study the epithelial morphogenesis three dimensional organization and follicles, vascularization of the implants or thyroid progenitor differentiation. This method can help answer key developmental and differentiation. Question about thyroid epithelial cell organization in 3D, lumen formation, cell differentiation, polarization, and growth.
Demonstrating the procedure will be Fidel Marcel, a graduate student, and Myla LaCorte, a postdoc both from my laboratory Under sterile condition in the laminar flow hood. Two hours before the isolation of the thyroid tissue dilute sterile Type one collagen to a 50 microgram per milliliter. Final concentration in 10 millimolar hydrogen chloride, and place the collagen on ice.
Then cover each of the wells of a plastic culture chamber with 200 microliters of the diluted collagen and incubate the chamber at room temperature After two hours, aspirate the collagen. Rinse the wells with M1 99 media and add a minimum volume of 200 microliters of culture medium to each. Well place the coated chambers in the incubator and then proceed with the thyroid dissection.
First, place the dissected uterus into a 10 centimeter plate containing HBSS. Next, use forceps to hold one extremity of the uterus and then open the uterus with small scissors to consecutively liberate each of the embryos. Use the scissors to separate the embryos from the placenta, and then transfer the embryos within their extra embryonic membranes into a new 10 centimeter plate containing fresh HBSS.
Next, continue the dissection under a stereo microscope for each embryo. First, remove the yolk sack and the amnion. Then holding tungsten needles in glass capillaries as a knife and fork.
Remove the top part of the head, including the upper jaw and the ears. Hold the embryo on its back. Then remove an extra piece of the neural tube and after remove the lower part of the embryo just under the anterior limbs and above the heart.
To orient the tissue, transfer the embryo slice containing the tongue its neck region into a new 10 centimeter plate containing fresh HBSS and remove the neural tube and tissues posterior to the esophagus and trachea. Now gently section the tissue slice along both sides of the tongue. Then locate the esophagus and trachea, dissect away the tongue, leaving the retinoid swelling and the tissue on both sides of the esophagus and trachea.
After removing the tongue, dissect away the esophagus and place the trachea ventral side up. Then dissect away the undesired tissues such as the thymus and all the tissues located laterally to the pharyngeal arch arteries. Then for the E 13.5 or E 14.5 embryos, visualize the thyroid lobes on each side of the trachea and further dissect the lobes from the trachea.
For E 12.5 embryos, the dissection can be performed as just demonstrated until after excising the tongue and esophagus. Then after locating the pharyngeal arch arteries, remove all the lateral tissues as for E 13.5 to 14.5 embryos. Finally, using a pre wetted filter tip on a P 200 micro pipette, transfer the isolated tissue into a 35 millimeter culture plate containing prewarm culture medium.
Before plating the E 13.5 to E 14.5 thyroid X explan. First, wash the tissue by successive transfer into three wells of a 24 well plate containing one milliliter of prewarm culture medium to plate the lobes onto the pre-coated chambers. First, aspirate the culture medium and use a micro pipette with filtered tips to carefully plate one thyroid lobe into each of the culture chambers.
Remove any excess medium and then place the culture chamber into a tissue culture incubator to allow the explants to attach to the matrix. Then after two hours, gently add 200 to 300 microliters of prewarm culture medium to each well and place the chambers back into the incubator to plate the E 12.5 trachea region or thyroid lobes on semi porous filters. First, fill the wells of a 24 well plate with hundred and 30 microliters of Prewarm culture.
Medium place filters onto the medium. Taking care to avoid trapping air bubbles below the culture filter. Then use a micro pipette with filtered tips to carefully transfer the thyroid explan or lobes, and a minimal volume of medium onto the center of each of the filters to a maximum of four x explan per filter.
When fl the X explan onto the filter, it's important to transfer them to the center of the filter with a minimal amount of medium to avoid medium to contact and create meniscus with the filter wall. Finally, remove any excess medium and use a tungsten needle to space out thes. Then place thes in the tissue culture incubator for up to seven days changing culture medium every other day under the laminar flow hood.
After one day in culture on a filter, the midline on laa is visible as an elongated tissue spanning the top length and forming two lobes on each side of the trachea. With culture, the lobes expand undergoing morphogenesis organization of the epithelial cells into macroscopically visible follicular structures. As demonstrated in these images, the organization and polarization of the e coherent, positive epithelial cells can be visualized by ezrin immuno labeling.
Over time, the ezrin positive intracellular structures fused with the pole of the cell that will become the apical pole. During this process, endothelial cells positive four pcam proliferate and organize around the developing follicles to form angio follicular units. To quantify the differentiation of the thro, cytes and C cells, gene expression can be assayed by R-T-Q-P-C-R of RNA, isolated from the cultured thyroid lobes.
While the expression of the thyroid transcription factor, NK X 2.1 does not change during the culture, the expression of the thy specific thyroglobulin and of C cell specific calcitonin dramatically increases indicating a functional differentiation. After watching this video, you should have a good understanding of how to dissect and culture thyroid explan. You are not ready to perform gain and loss of function experiment using wild type or NOCO embryos.
