2 단계 접근-그리고 늦은-초기 조류 모델에서 기관 형성의 탐구: Thymus 및 부 갑 상선 동맥 Organogenesis 패러다임

The overall goal of this procedure is to provide a two-step approach to study the early and late stages of organogenesis by combining in vitro manipulation of embryonic tissues with in ovo grafting techniques using the avian model. In this method the embryonic territory comprising the prospective organ rudiment is isolated and grown in an organotypic system for 48 hours. By mimicking normal development this in vitro approach allows easy access to embryonic territories without the need for specific molecular markers and enables the experimental manipulation of early developmental stages in organ formation that rely on highly dynamic spatial modifications and complex tissue interactions.

Then cultured tissues are grafted onto the chorioallantoic membrane of a chicken embryo. The chorioallantoic membrane behaves as a vascular supplier of nutrients and allows gas exchanges to the grafted tissues enabling its development in ovo. This step is especially well suited to study late stages of organogenesis as fully formed organs can be achieved after 10 days of in ovo development.

In addition pharmacological agents can be administered along in vitro and in ovo development allowing the modulation of signaling pathways in a developmental stage specific manner. Ultimately the developing tissues can be collected at any time window to evaluate the progression of organogenesis and to be analyzed for gene expression and morphological features. By following organ formation outside the avian embryo this method can help elucidate key questions in developmental biology such as identifying the signals involved in distinct steps of organ formation.

This experimental procedure will be exemplified by studying the organogenesis of the thymus and parathyroid glands. In birds the epithelia of these organs derive from an endodermal common primordium of the third and the fourth pharyngeal pouches and emerge as discrete domains around embryonic day four in quail and four/five in the chicken. As development proceeds the organ's rudiments individualize and separate from the pharynx.

Later in development the thymic epithelium is colonized by hematopoietic progenitor cells. The method presented here starts with the isolation of the quail embryonic territory containing the presumptive territory of these organs, that is the pharyngeal region containing the third and fourth arches at embryonic day three. Fertilized quail eggs were incubated with the air chamber facing up for three days at 38 degrees centigrade in a humidified incubator.

To begin remove the quail eggs from the incubator. Perform egg manipulation procedures in sterile conditions using a horizontal laminar flow hood and sterilized instruments and materials. Prepare a large Pyrex bowl filled with cold PBS.

With curved scissors open the quail egg by tapping the shell and cutting a circular opening in the opposite side of the air chamber. Carefully pour down the embryo to the dish. Hold the embryo within thin forceps and with the help of the curved scissors make a cut into the vitelline membrane surrounding the yoke.

Cut around and externally to the circumference of the extra embryonic vessels in a continuous movement. Transfer the embryo to a small bowl with cold PBS. Then use a skimmer to transfer the embryo to a Pyrex Petri dish with black base containing cold PBS.

Under a stereo microscope hold the embryo to the bottom of the plate with thin insect pins. Place four pins forming a square shape in the extra embryonic region. With forceps remove the extra embryonic membranes of the cephalic region and put there a fifth pin.

Now the embryo is correctly positioned and the otic vesicle, the heart tube, and the first, second, and third pharyngeal arches are visible. Start to dissect the region of interest that is the third and the fourth pharyngeal arch region using Wecker eye scissors. Make a cut longitudinally and parallel to the embryo axis between the area of somites and neural tube and the pharyngeal arches.

Then remove the ventrally positioned heart tube by cutting it. Keep the scissors in the same position rotate the Petri dish to reposition the embryo according to the direction of the cut. To finish cut between the second and the third pharyngeal arches and below the fourth pharyngeal arch.

Aspirate the isolated tissues and transfer them to a glass dish filled with cold PBS using a sterile plastic pipette. Keep it on ice during the preparation of the in vitro assay. Fill one well from a six-well plate with 5 mL of culture medium and place a polycarbonate membrane insert on the well.

Under the stereo microscope transfer the explant by gentle sliding with the help of a spatula and forceps from the glass dish to the membrane surface. Alternatively explants can be cultured in floating membrane filters. For this procedure prepare a 35 millimeter Petri dish with 5 mL of culture medium.

Use forceps to float the membrane filter keeping a dry surface in contact with air. As before transfer the explant from the glass dish to the membrane surface by gentle sliding. The explants should be placed with the ventral side up and the dorsal side in contact with the membrane.

Add up to eight explants per membrane filter. Carefully place the six-well plate and the Petri dish containing the explants in a humidified incubator at 37 degrees centigrade with five percent carbon dioxide. After a 48 hour incubation period remove the plate and Petri dish from the incubator.

To collect the cultured explants from the six-well plate add PBS at room temperature to the membrane insert. Detach the cultured explants from the membrane by vigorous flushing using a sterile plastic pipette. With spatula and forceps transfer the cultured explants to a glass dish filled with PBS at room temperature.

Similarly collect the cultured explants from the floating membrane filter. In this case transfer the membrane filter with forceps to a new Petri dish filled with PBS at room temperature. Detach the explants from the membrane filter by vigorous pipetting.

Discharge the explant-free membrane filter after confirming that no explants remain attached to it. With a spatula and forceps carefully transfer the explants to 1 mL of total RNA isolation reagent to perform gene expression studies. Cultured explants can alternatively be grown in in ovo to compete organ formation.

First prepare the chorioallantoic membrane. For that remove from the incubator the chicken eggs with eight days of development. Eggs were incubated with the air chamber facing up in a humidified incubator at 38 degrees centigrade.

Cover the blunt end of the egg with clear plastic tape to prevent pieces of shell from falling into the air chamber. Tap the shell and cut a circular opening in the egg with curved scissors. The air chamber should become visible.

Slowly remove the white membrane of the air chamber with forceps. The chorioallantoic membrane is then visible and accessible for ectopic tissue transplantation. Do not to use PBS to hydrate to chorioallantoic membrane before or after transplantation since PBS promotes sliding and misplacement of the explants.

Start by performing small vascular lesions in the smaller vessels of the membrane. For this procedure use a microscalpel in a holder. In case of excess bleeding from the wound remove the blood by capillarity using the tip of a Pasteur pipette.

Transfer the explant to the wounded area of the membrane with a spatula and forceps. Cut a piece of a filter paper with a slightly higher size than the explant and put it on top of the explant. The filter paper helps tracking the explant location after its development in the chorioallantoic membrane.

If required the filter paper also allows daily drug delivery to the explant during in ovo development. Seal the egg window with clear plastic tape and identify it using a charcoal pencil. The plastic tape protects the embryo from dehydration during the incubation period.

Incubate the manipulated egg in a humidified incubator at 38 degrees centigrade. After 10 days of incubation collect the egg and carefully withdraw the plastic tape. Cut the chorioallantoic membrane around the filter region using curved scissors and transfer the explant with filter paper to a small Pyrex bowl filled with cold PBS.

With forceps transfer the explant to a 100 millimeter dish with black base filled with PBS. Gently remove the filter paper and the excess of membrane using Wecker eye scissors and forceps. With the skimmer transfer the grafts to a fixative solution and assess organ formation in paraffin sections by conventional histology immuno histochemistry.

Here is depicted the experimental design used to explore the early stages of thymus and parathyroid formation. As an example the expression of genes known to be involved in the development of the pharyngeal arch region was evaluated during the normal development by quantitative real-time PCR. Transcripts of the three genes Tbx1, Six1, and Bmp4 were detected in freshly isolated tissues and after 48 hours of culture.

To study the roles of Notch and Hedgehog signaling pathways pharmacological inhibitors were added to the culture medium during in vitro development. Expression levels of the three genes were significantly reduced in the third and the fourth pharyngeal arch region grown for 48 hours in the presence of Notch inhibitor when compared to controlled conditions. Conversely only Bmp4 transcripts were significantly reduced in cultured tissues when Hedgehog signaling was blocked.

To study the late stages of thymus and parathyroid gland organogenesis the cultured tissues were grafted onto chorioallantoic membrane and allowed to double up for a further ten days. Organ morphological analysis was performed by conventional histology and immunohistochemistry. Some representative results are the following:Grafts showed a fully formed chimeric thymus with QCPN positive, quail-derived thymic epithelium cells and lymphoid cells of chicken donor origin.

Additionally thymic and paratyphoid epithelia showed normal morphological features after cytokeratin detection. Thymic epithelium displayed a reticular architecture while parathyroid parenchymal cells were globular shaped arranged in clusters and encircled by numerous capillaries. After watching this video you should have a good understanding of how to use the two-step approach of in vitro and in ovo development using the avian model to investigate the role of the signaling pathways involved in the distinct steps of organ formation.