Deletion‐transplantation experiments on embryos of Fundulus heteroclitus. I. The posterior embryonic shield

Abstract

Experiments are described in which the differentiative capacity of the posterior embryonic shield of the teleost, Fundutus heteroclitus, was investigated by means of deletion-transplantation techniques.The posterior embryonic shield, removed from early gastrulae to immediate postgastrulae and grafted into the pericardial cavity of slightly older embryos, exhibits considerable growth and differentiates typical trunk and tail structures involving all germ layers.The donor embryos, in general, develop normal heads to pectoral fin regions. They lack all trunk structures except for gut and some gut derivatives. They frequently differentiate a very small "tail" with unpaired somites. The "tail" sometimes flattens and forms fin rays.Collateral experiments involving the deletion and grafting of various parts of the germ ring provide data in agreement with the conclusion that the posterior embryonic shield supplies the bulk of material for the formation of the trunk and tail in teleosts.The experiments are discussed from the standpoint of their elucidation of gastrulation, trunk and tail formation, embryonic regulation of defects, and endoderm formation in teleosts.A number of attempts have been made to map the teleost blastoderm. Oppenheimer ( ' 3 6 ) and Pasteels ('36) used vital stains; Brummett ('54) used carbon particles; and Ballard ('66, '68) used a combination of these techniques. There were discrepancies between the results and interpretation of the vital staining experiments of Oppenheimer and Pasteels on the one hand and the carbon marking experiments of Brummett on the other regarding the role of the cells of the 180" germ ring (that part of the germ ring 180" from the posterior embryonic axis) in the formation of the tail bud blastema. The vital staining experiments suggested that these cells form the bulk of the tail bud, while the carbon marking experiments indicated that they contribute little or nothing to its formation. The latter experiments suggested that the entire tail bud is formed by a convergence of cells less than 90" on either side of the posterior embryonic axis.Limitations of both the vital staining and the carbon marking techniques, however, preclude an unequivocal answer to the question of the origin of the tail bud blastema. It seemed advisable, therefore, to devise another experimental approach to this problem. It was thought that the use of in vitro cultures of parts of the embryonic shield and germ ring might provide the answers desired. When initial attempts to devise a suitable in vitro culture method for these tissues met with little success, however, an in vivo technique was tried with gratifying results. It is felt that the data obtained from the series of experiments which followed, contribute significantly to the further understanding of morphogenetic movements and differentiation in the teleost embryo. MATERIALS AND METHODSThese experiments were performed on living embryos of the common killifish, Fundulus heteroclitus, collected at Beaufort, North Carolina. The fish were...

OXYGEN CONSUMPTION IN ANTERIORVERSUSPOSTERIOR EMBRYONIC SHIELD OFFUNDULUS HETEROCLITUS

“…The data suggests, then, that the anterior shield is not significantly ahead of the posterior shield along the differentiative pathway. This interpretation is reasonable in the light of the results of grafting experiments (Brummett, 1968(Brummett, , 1969 which reveal similar time scales for graft differentiation whether the tissue is derived from the anterior or posterior shield.…”

“…Following fertilization, the eggs were kept at room temperature (approximately 24 C). When the eggs reached the desired stage of gastrulation [Oppenheimer (1937) stages 12, 14 and 16 which correspond to Armstrong and Child (1965) stages 15, 18, and 20], they were washed in 3% formalin-sea water to reduce the possibility of contamination, rinsed in filtered sea water, and dechorionated in sterile Tyrode solution (without bicarbonate) according to methods previously described (Brummett, 1968).…”

“…Operating in sterile Tyrode solution, portions of the embryonic shield were carefully excised with finely sharpened watchmaker's forceps (Brummett, 1968(Brummett, , 1969.…”

“…Deletion-grafting experiments (Brummett, 1968) demonstrate that the posterior shield tissue in Fundulus is not limited to chordamesoderm but consists, rather, of cells which are already determined to form all tissues appropriate to posterior trunk and tail. The regions of the frog gastrula measured by Sze (1953), then, are not directly comparable to the teleost tissues used in the experiments reported here.…”

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It has been demonstrated (Brummett, 1968(Brummett, , 1969 that the embryonic shield of Fnndnlns has the fate of its various organs and tissues determined early in gastrulation. When the anterior shield is excised and implanted into the pericardial chamber of an older embryo it differentiates brain and eyes; posterior shield, similarly handled, forms spinal cord, somitic muscle, gut, and other structures appropriate to trunk and tail.

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