The Grafts

3.2.1. Neural Tube Transplantations (Fig. 2)

Orthotopic transplantations of fragments of neural tube have allowed the construction of a neural crest fate map (3) and the detection of definite crest cell migration pathways (14). The rules of this operation are based on the fact that neural crest cells start migrating first in the cephalic region and then progressively from rostral to caudal when neural tube forms. The interspecific graft is performed at a level where crest cells are still inside the apex of the neural tube, i.e., in the neural folds in the cephalic area, at the level of the last formed somites in the cervical and thoracic regions, and at the level of the segmental plate in the lumbo-sacral region. The operation has to be made on the length of no more than five to six somites to take into account the rostro-caudal differential state of evolution of the neural crest. Donor and host embryos are strictly stage-matched (see Note 4).

1. Excision of the host neural tube: The selected neural tube fragment is excised from the host embryo by microsurgery in ovo. A longitudinal slit through the ectoderm and between the neural tube and the adjacent paraxial mesoderm, at the chosen level, is made bilaterally using a microscalpel. The neural tube is then gently separated from the neighboring mesoderm and cut out transversally, ros-trally, and caudally without damaging the underlying notochord and endoderm. The fragment of neural tube is then progressively severed from the notochord and finally sucked out using a calibrated glass micropipet (see Note 8).

2. Preparation of the graft: The transverse region of the stage-matched donor embryo comprising the equivalent fragment of neural tube plus surrounding tissues (ectoderm, endoderm, and mesoderm) is retrieved with iridectomy scissors and subjected in vitro to enzymatic digestion (pancreatin, Gibco, one-third in PBS or Tyrode) for 5-10 min on ice or at room temperature according to the stage of the embryo (see Note 7). Then tissues are dissociated using two smooth microscalpels

CHICK HOST QUAIL DONOR

CHICK HOST QUAIL DONOR

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Fig. 2. Scheme of the orthotopic quail/chick neural tube transplantation. A chick embryo in ovo (A1) is microsurgically deprived of its neural tube (A2) at the level of the last formed somites. The corresponding fragment of blastoderm of a quail at the same stage (B1) is enzymatically dissociated. The isolated quail neural tube (B2) is orthotopically grafted into the chick embryo (A3).

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Fig. 2. Scheme of the orthotopic quail/chick neural tube transplantation. A chick embryo in ovo (A1) is microsurgically deprived of its neural tube (A2) at the level of the last formed somites. The corresponding fragment of blastoderm of a quail at the same stage (B1) is enzymatically dissociated. The isolated quail neural tube (B2) is orthotopically grafted into the chick embryo (A3).

(see Note 11), and finally, the isolated neural tube fragment is rinsed with PBS or Tyrode supplemented with bovine serum to inhibit the action of the proteolytic enzymes. It is then ready to be grafted.

3. Grafting procedure: The donor neural tube is transferred to the host embryo using a calibrated glass micropipet and placed in the groove produced by the excision, in the normal rostro-caudal and dorso-ventral orientation (see Note 15).

Heterotopic graftings were instrumental to study whether the fate of the neural crest is specified when the operation is carried out (9). The graft is taken at a more rostral or more caudal level than the acceptor level. Depending on the latter, the donor embryo is older or younger than the recipient (see Note 16).

Partial dorso-ventral orthotopic graftings have also been made in order to localize possible early segregation of precursors in the neural tube (15).

Fig. 3. Scenario of the orthotopic graft of brain vesicles. (A) 12-somite chick embryo in ovo after injection of a solution of Indian ink under the blastoderm. Brain vesicles are well delineated. (B) Longitudinal incisions are made between the cephalic neural tube and the head mesenchyme to delimit the brain excision (arrows). (C) After a transversal section at the level of the mesencephalo-metencephalic constriction, the prosencephalon and the mes-encephalon are separated from the head mesoderm and endoderm. The notochord (N) is then visible. (D) The excised chick brain vesicles are discarded. (E) The equivalent quail brain vesicles (Q) are grafted into the chick host. Pro: prosencephalon; Mes: mesencepha-lon; Met: metencephalon; S12: somite 12. A bar = 0.05 mm; B, C, D, E bar = 0.05 mm.

Fig. 3. Scenario of the orthotopic graft of brain vesicles. (A) 12-somite chick embryo in ovo after injection of a solution of Indian ink under the blastoderm. Brain vesicles are well delineated. (B) Longitudinal incisions are made between the cephalic neural tube and the head mesenchyme to delimit the brain excision (arrows). (C) After a transversal section at the level of the mesencephalo-metencephalic constriction, the prosencephalon and the mes-encephalon are separated from the head mesoderm and endoderm. The notochord (N) is then visible. (D) The excised chick brain vesicles are discarded. (E) The equivalent quail brain vesicles (Q) are grafted into the chick host. Pro: prosencephalon; Mes: mesencepha-lon; Met: metencephalon; S12: somite 12. A bar = 0.05 mm; B, C, D, E bar = 0.05 mm.

3.2.2. Orthotopic Transplantations of Brain Vesicles (Fig. 3)

This operation has been devised to label defined regions of the neuroepithe-lium and thus to study cell migrations and morphogenetic movements during brain development (4,16,17). It was also applied to the transfer of a genetic behavioral or functional trait from donor to recipient in either xenogeneic or isogeneic combinations (8,18,19).

1. Excision of brain vesicles from donor and host embryos: Equivalent brain vesicles are excised microsurgically in the same way in stage-matched donors and recipients (see Note 17). The dorsal ectoderm is slit precisely at the limit between the neural tissue and the cephalic mesenchyme on each side of the selected part of the brain. The neural epithelium is loosened from the cephalic mesenchyme, then cut out transversally at the chosen rostral and caudal levels, and finally severed from the underlying notochord.

2. Exchange of brain vesicles: The transfer of brain vesicles from the quail to the chick and vice versa (or from a mutant to a normal chick embryo) is made using a calibrated glass micropipet. The piece of neural tissue is inserted into the groove made by the excision, with the normal rostro-caudal and dorso-ventral orientation, and then adjusted (see Note 18).

3. Modifications of the technique consist of orthotopic partial dorsal or dorsolateral grafts of brain vesicles (17,20-22). Heterotopic grafts have also been performed to study specific problems (20,23-25).

3.2.3. Neural Fold and Neural Plate Transplantations

Orthotopic and isochronic grafts have been made in order to map the early rostral or caudal neural primordium (5,26-29).

1. Excision of precise pieces of the neural fold or neural plate in the chick host: Very thin microscalpels (made up from insect pins or steel needles sharpened on an oil stone) are used to excise precise fragments of the folds and neural plate in 0- to 5-somite stage chick embryos in ovo. An ocular micrometer is used to measure the pieces of tissue to be removed.

2. Excision of equivalent pieces of tissue from the quail donor: The grafts are excised from stage-matched quail in vitro using the same method. They are not subjected to enzymatic treatment.

3. Graft: Pieces of quail tissue are grafted orthotopically into the chick host.

Heterotopic grafts have also been made to establish the degree of autonomy of precise territories of the rhombencephalon (30,31).

Simple excisions combined with adjacent orthotopic grafts have been performed in order to identify neural crest cells differentiating at the level of the excision (32).

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