The origin and movement of the limb-bud epithelium and mesenchyme in the chick embryo as determined by radioautographic mapping

By G L E N N 1 0 Author's address: Department of Pediatrics, The Johns Hopkins Hospital , Baltimore, Maryland 21205 , U.S.A 1 From the Department of Pediatrics, The Johns Hopkins Hospital of the limb-bud epithelium and mesenchyme in SUMMARY The origin of the limb-bud cells was determined by tracing the movements of [3H]thymidinelabelled grafts excised from late medium-streak to 5-somite stage chick embryos and transplanted to the epiblast, streak, and endoderm-mesoderm of similarly staged recipient embryos. Although exact definition of the prelimb areas was not possible because of the small number of grafts placed at each developmental stage, the study showed in general that at the late medium-streak stage the future limb-bud epithelium is in the epiblast (dorsal) layer near the lateral margin of the area pellucida. It moves medially toward the embryonic axis, just lateral to the premesoderm cells which will be invaginated at the primitive streak. With regression of the streak, the limb-bud epithelium moves relatively anteriorly into a position dorsal to the limb-bud mesoderm, beginning at least as early as the early head-fold stage. - The limb-buds of the chick embryo begin to form at about the 30-somite stage, when the somatic mesoderm along the right and left flanks begins to accumulate into mounds of mesenchyme, covered by a layer of epidermal ectoderm. previous investigators (Wolff, 1936; Rudnick, 1945; Chaube, 1959) have identified areas of limb-bud-forming tissue in the right and left flanks of embryos prior to the 30-somite stage, the position of this tissue prior to the head-fold stage has not been investigated. Using radioautographic analysis the present investigation traces the movement of [3H]thymidine-labelled transplants from their original positions in the epiblast, streak and endoderm-mesoderm layer of recipient embryos into the limbbuds of these embryos. Although the small number of grafts placed prevents exact definition of the prelimb areas, these regions are more precisely defined than they were previously, and the mesoderm and ectoderm layers are treated separately. MATERIALS AND METHODS The methods of preparation of recipient and [3H]thymidine-labelled donor embryos and of transplantation and radioautographic analysis of the grafts were identical to those described in previous publications (Rosenquist, 1966, 19706) and the description will not be repeated here. The late medium-streak to head-process stages of both donor and host embryos have been described previously (Rosenquist, 1970a) and are shown diagrammatically in Fig. 1A-C. Early head-fold (EHF) stage embryos had an elongated head process and a depression at the site of the future head fold, which had not as yet formed a pocket (Fig. 1D). Host embryos fixed prior to the 7-somite stage were normal when examined with the naked eye or microscopically. In those host embryos which survived to the 26-somite to early limb-bud stage the heart was beating, but extra-embryonic circulation had ceased and the development of the embryos was retarded compared to that of similar embryos incubated in ovo. The number of pairs of somites could not always be determined accurately (Fig. 2B, E). In the older host embryos which had not as yet developed limb-buds, the anterior and posterior limb regions, and their dorsal and ventral portions, were designated in cross-sections on the basis of their distance from the anterior intestinal portal, primitive streak, midline axis and nephrotome in comparison with embryos which had already developed limb-buds. This stage is referred to as the 26-30-somite stage in the Table and text, and is equivalent to stage 1 (Hamburger, 1938) and stage 15 (Hamburger & Hamilton, 1951). Host embryos which had developed limb-buds (stages 2-3 of Hamburger, 1938; stages 16-17 of Hamburger & Hamilton, 1951) were said to be at the early limb-bud (ELB) stage. In these embryos the portion of the ectoderm and somatic mesoderm lateral to the crest of the ectodermal cone is referred to in the text and Table as the future ventral portion of the limb, while the portion of the ectoderm and somatic mesoderm medial to the ectodermal cone is called the future dorsal portion of the limb. Although a part of each of the labelled transplants lay in the limb-budforming region of its host embryo, the number of embryos investigated was relatively small, and each transplant contained cells other than those destined for the limb-buds (as indicated in Table 1). Therefore the positions of the transplants at each stage in Fig. 1 suggest the location of the limb-bud cells at that stage, but do not define it precisely. The mapping of the limb-bud regions is based upon the following assumptions: (1) that since previous studies have established the general position of the premesoderm and pre-ectoderm cells in the epiblast layer of the chick blastoderm without mapping every part of that layer at each stage (Rosenquist, 1966), a small number of transplants carefully placed can demonstrate the position of more specific portions of the mesoderm and ectoderm, such as the limb-buds. (2) That similar graft positions in different embryos of the same stage are homologous even if the embryos were incubated for different lengths of time, and that the migration pathways followed by more than one accurately placed graft can be combined to follow movements of a group of cells through several overlapping stages of development. (3) That maps of presumptive organ-forming regions of the embryo are valid even if structures in the recipient embryos other than the organ to be mapped contain labelled cells. Throughout the text and figures, an asterisk (*) after the embryo number indicates that the position shown is that of the graft after its migration in the host embryo. At the late medium-streak stage the cells which would form the epithelium of the limb-buds were in the epiblast layer at the lateral edge of the area pellucida, lateral to the primitive streak; in some cases they may have been on or outside the boundary between the area pellucida and the area opaca (embryos 1-5, Table 1, Fig. 1A). At the early head-fold stage, the cells which would form the epithelium of the anterior limb-bud were in the epiblast layer near the anterior half of the streak, about halfway between the streak and the lateral margin of the area pellucida (embryos 6, 7, Table 1, Fig. ID). By the 4-7-somite stage, the transplant in embryo 6* had migrated anteriorly and laterally to a position between the anterior end of the streak and the somite region (Table 1, Fig. 1E). If embryo 6 had been allowed to develop to the limbbud stage, labelled cells would have migrated into the anterior limb-bud, as did those in embryo 7* (Fig. I D , G). Therefore, the transplants in embryos l*-4*, which had reached positions similar to that of the graft in embryo 6* (Fig. 1E), also were considered to be destined for the anterior limb-bud. The transplant in embryo (...truncated)