Depending on the latter, the donor embryo will be older or younger than the recipient see Le Douarin, 1982, and references therein. The neural tube is then removed using either a Spemann pipette or micropipette, taking care not to damage the underlying notochord. After Indian ink injection, if necessary, the vitelline membrane is torn open with a microscalpel at the site chosen for the graft. Focal Injections of DiI into Neural Folds Premigratory neural crest cells arise from the dorsal neural folds shortly after tube closure in the chick embryo. With the spoon beneath the embryo, grasp one corner of the vitelline membrane with fine-tipped forceps and gently peel the membrane with the attached embryo from the surface of the yolk Fig. Very fine heterochromatin dots and an occasional pale staining nucleolus are shown.
Lo sviluppo embrionale della quaglia giaponese. A rhodorsil base Rhbne-Poulenc is now preferred to paraffin. Reprinted with permission from Hallonet and Le Douarin 1993. Many of the growth factors acting on somatic or hematopoietic tissues also are expressed within the brain. The delimited tissue comprises the button itself, plus narrow strips of the neural plate bordering it. Figure 18 summarizes the major events in the development of the hemopoietic system, all of which were 51 2.
Tosney 93, 109 , Department of Biology, The University of Michigan, Ann Arbor, Michigan 48109 L. Quail—Chick Transplantations Sites of erythropoiesis and hemopoietic cell production 0 1 2 3 4 5 6 7 16 8 Yolk sac compartment 21 days Embryonic compartment Quail and chick Intraaortic clusters Quail Paraaortic foci Chick Thymus 1st receptive 2nd receptive period period Refractory Refractory Pre-receptive period period period 3rd receptive period Quail 0 5 11 12 16 17 18 19 20 21 days 12 13 14 Hatching 18 19 20 21 6 6 7 8 Chick Hatching Bursa of fabricius Quail 7 11 8 14 16 21 days 16 21 days Chick Bone marrow Quail 9 10 Chick Fig. Neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. Alternatively, if you have only cleaned some yolky endoderm, manipulate the node to the denuded site. Although no immune reaction against the graft takes place during embryogenesis when the immune system is building up, the transplant triggers its own rejection which occurs at various times after birth.
Quail and chick are closely related in taxonomy, although they diVer by their size at birth the quail weight is about 10 g and that of the chick is 30 g and by the duration of their incubation period 17 days for the quail and 21 days for the chick. The protocols emphasize microsurgery, histology, and cellular and molecular marking, which are not covered in the usual molecular biology methods manuals. In order to determine the fate of the mass of tissue situated under the median pit, we replaced this structure of the chick in ovo by its quail counterpart see Fig. In both staining methods, mouse neural crest cells can be distinguished from chick cells by the appearance of condensed heterochromatin in their nuclei arrows , whereas the heterochromatin in chick nuclei appears evenly distributed arrowheads. Resulting chimeras are incubated for the desired duration to analyze neural crest migration or diVerentiation. Cell labeling has revolutionized the long-held classical view of the ontogeny of the hemopoietic system by revealing one of its essential features: that stromal cells of hemopoietic organs do not yield hemopoietic cells. For controls, orthotopic grafts and sham operations can 66 Peter Y.
Alternative Culture Methods A number of less common embryo culture methods have been devised that may prove useful for some applications. Separate staging systems are available for duck Koecke, 1958 and quail Padgett and Ivey, 1960 but these can be adapted to the scheme used for chick Schneider and Helms, 2003; Yamashita and Sohal, 1987. Classically, neural tube transplantations have provided a wealth of information about migratory pathways and, in particular, neural crest derivatives in avian embryos see Chapter 2. Because the explant will often float to the top, use a blunt tunsgsten needle to gently poke the tissue into the liquid collagen and to position the explant in the desired orientation. Further applications still need to be devised, either to follow up initiated investigations or to explore new issues. The collagen mixture will turn from a bright yellow to a faint orange color. Make two lateral cuts between the neural tube and the ectoderm.
Prepare glass knifes by pulling thin glass rods to a sharp tip using an electrode puller. The fate map of the cephalic neural primordium at the presomitic to the 3-somite stage in the avian embryo. A novel method to bursectomize avian embryos and obtain quail-chick bursal chimeras. The protocol for coating dishes with most matrix molecules is similar. The graft, marked with a few particles of carbon black, is deposited near the grafting site. Usually, we introduce the needle in between both tissues at the caudal portion of the open neural plate, and slide the needle toward the anterior region of the embryo, separating them precisely at the border. A PetroV—Heuser bacterial cell counter is used to count the number of cells in the suspension, although other types of hemocytometers can be used.
. Subsequently, species-specific antibodies that recognize either quail or chick cells have been developed. Let the membrane rest on the bottom of the dish, inner surface containing the embryo facing upward. Culture of the Avian Embryo 15 Fig. Because the vitelline membrane and blastoderm adhere to the filter paper, the annulus can then be lifted from the yolk by means of forceps, with the embryo lying taut across the center. Introduction The formation of the embryo involves intricate cell movements, cell proliferation, and differentiation. A current and authoritative guide, Methods in Avian Embryology presents a combination of classical embryological techniques and modern molecular biological approaches to studying the developing avian embryo.
Take care while removing excess saline from the operated embryo so that the grafted node remains in place. Agar clots Spratt, 1947a,b; New, 1966 have superseded plasma as a culture substrate and many workers currently use them for culturing young gastrula and neurula stage embryos Mitrani and Shimoni, 1990; Eyal-Giladi et al. To avoid any possible contribution from the ablated tissue, it is removed from the egg by capillary action through a micropipette. Chick—chick chimeras have been constructed according to the same pattern and were able to hatch Petitte et al. Сваливаем все в один архив, заливаем на ifile.
Embryos can also be put in sterilized dishes without black wax, if desired. The origin of lymphoid stem cells studied in chick yolk sac-embryo chimaeras. This staining is performed on 5-pm paraffin sections. The following protocol, adapted from Cohen and Konigsberg 1975 , works well as a complete medium for neural crest cells. First, young embryos in New culture can easily be studied using either transmitted or incident light.
Operations on Primitive Streak Stage Avian Embryos reveal that stage 3c a short streak with a groove is only observed in a minority of embryos. These results therefore suggested that multipotent cells might be still present at later stages of melanocyte development. However, no individual organism can serve to answer all embryological questions. The neural crest forms transiently in the dorsal neural primordium to yield migratory cells that will invade nearly all tissues and later, differentiate into bones and cartilages, vascular smooth muscle cells, connective tissues, neurons and glial cells of the peripheral nervous system, endocrine cells, and melanocytes. The method, described next, which permits precise replacement of the recipient bursa by a foreign rudiment, is well tolerated so that animals hatch and survive until adulthood.