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Of Limb Morphogenesis in a Model System

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Of Limb Morphogenesis in a Model System DEVELOPMENTAL BIOLOGY 28, 113-122 (1972) Analysis of Limb Morphogenesis in a Model System ROBERT H. SINGER’. 2 Department of Biology, Brandeis University, Waltham, Massachusetts Accepted January 27, 1972 A method for analysis of chicken limb morphogenesis was devised. This method consisted of grafting a limb ectodermal jacket containing dissociated and pelleted mesenchymal cellular com- ponents to the host somites. Different cellular components stuffed into the ectoderm could be mixed in varied ratios. After 7 days the grafts were analyzed for outgrowth. Stage 19 mesoblast cells alone when treated as above gave limblike outgrowths with good digits, toes, and claws in all cases. However, mesoblasts from the proximal half of older limbs (stages 24, 25 and chondro- cytes) gave no outgrowths, and those from stage 23 gave outgrowths in 9% of the cases. In mixtures of 5% stage 19 cells with 95% chondrocytes, consistent morphogenesis (i.e., in 65% of grafts) oc- curred. The amount of morphogenesis (size of graft and perfection of digits) was directly propor- tional to the amount of stage 19 cells. However, these cells mixed with proximal cells of stages 23, 24, or 25 required higher proportions for equivalent morphogenesis. To obtain morphogenesis equivalent to the 5% mixture with chondrocytes, 10% stage 19 cells were needed when mixed with proximal stage 23, 25% with proximal stage 24 and 7% with proximal stage 25. Mixtures of stage 19 cells added to nonlimb (flank, stage- 19) mesoderm, formed large tumorous mounds of tissue with no limblike features. INTRODUCTION ectoderm ridge serves as a specific induc- The limb bud presents a good model of tive structure initiating and directing out- a developing system. The relationship be- growth and digit formation from the early tween the mesoblast and its ectodermal bud. The mesoderm, upon which the ridge cover, and the histogenesis and cytodif- exerts its effect, in turn supports the in- ferentiation of cartilage allows an investi- tegrity of the ridge by producing a “main- gation of several levels of organization in tenance factor” (see review by Zwilling, a self-contained and easily manipulated 1961). More recently, experimental inter- body part. Beginning with the pioneering est has enlightened cellular events perti- work of Saunders (1948) and Zwilling nent to limb morphogenesis including: (1949) the reciprocal interaction between the onset of histogenesis (Searls, 1965; limb bud ectoderm and mesoderm was Medoff, 1967); the stability of differenti- uncovered and identified. The relation- ated cellular phenotypes in culture (Coon, ship served as a model for the analysis of 1966; Cahn and Cahn, 1966; Nameroff the nature and the role of different tissues and Holtzer, 1967); the cellular programs in construction of the limb. The apical being followed during morphogenesis (Fallon and Saunders, 1968); and the time- ‘This research was completed under the direc- dependent decrease in the capability of tion of Professor Zwilling in partial fulfillment of the cells to change their morphogenetic pro- requirements for the degree of Doctor of Philosophy. The years I spent with Edgar Zwilling bring to mem- grams (Crosby, 1967; Cairns and Saunders, ory his kindness, integrity, and inspiration, and I 1954; Zwilling, 1955; Zwilling, 1968; dedicate my contributions in this work to his mem- Searls and Janners, 1969, 1971; Finch and ory. Supported by Grants Tl-HD-22 and HD-03465 Zwilling, 1971; Singer, 1972). from the National Institute of Child Health and Hu- The technique of independently manipu- man Development. ‘Present address: Department of Biology, 16. lating ectoderm and mesoderm in the 839, Massachusetts Institute of Technology, Cam- study of limb morphogenesis has allowed bridge, Massachusetts 02139. an elucidation of the developmental capa- 113 Copyright 0 1978 hy Academic Press, Inc. 114 DEVELOPMENTAL BIOLOGY VOLUME 28, 1972 bilities of combinations of nonlimb meso- form individual tissues (Crosby, 1967; derm with limb ectoderm as well as reveal- Zwilling, 1968; Singer, 1972). ing the patterns inherent in the limb The present work is a quantitative analy- mesoderm (Zwilling, 1955, 1956a, b; sis of the regulative capacities of meso- Saunders et al., 1959). More recently blast cells using the manipulative grafting Zwilling (1964) introduced a technique of procedures reviewed above. Young meso- studying the organizational and histo- blast cells were “diluted” with cells no genetic capacities of limb mesoblast cells longer capable of morphogenetic modula- which involved removal of the mesoblast, tion (no longer having “limbness” traits). dissociating the cells, then pelleting and The results show that such quantitative reimplanting them in an epidermal jacket, alterations elicit striking changes in and finally transplanting the stuffed ecto- morphogenetic expressions. derm to an ectopic flank site. Crosby (1967) used this method to measure the MATERIALS AND METHODS response to the apical ridge of mesoblast The method whereby cells are dissoci- cells in early stages before limb bud out- ated and stuffed into an ectodermal jacket growth (stage 13 of Hamburger and Hamil- is extensively described by Finch and ton, 1951). Her results affirmed the Zwilling (1971). About 50-60 leg buds, earlier conclusion of Zwilling (1955) and stage 19, were excised from White Leg- Saunders et al. (1957) that only limb meso- horn embryos and the mesoblast was derm is capable of growth and prolifera- denuded of its ectodermal covering with a tion when associated with the ridge and is 30-min treatment in a mixture of 2% tryp- the only mesoderm capable of supporting sin (Nutritional Biochemicals Corp.) (or inducing regeneration of) the ridge by and 1% pancreatin (Nutritional Biochemi- producing a maintenance factor (Amprino cals Corp.). All manipulations were done and Camosso, 1958; Saunders et al., 1957; in calcium- and magnesium-free Tyrode’s Searls and Zwilling, 1964). Moreover, solution (CMF). The freed mesoblast was Finch and Zwilling (1971) showed that then dissociated into a cell suspension by these properties are not expressed by further treatment with 1 ml of a 1% crys- mesoderm cells later than stage 24 or by talline trypsin solution (Worthington distal mesoblast tissue later than stage 26. Biochemical Corp.) for 20 min (38” C) . The The transient limb properties (“limbness”), mixture was poured into a full test tube which are typified by the response of (8 ml) of horse serum-Tyrode’s solution limb cells to the ridge resulting in growth (1: 1) and the cells were allowed to settle and morphogenesis of a limb, has been to the bottom. All but the bottom milliliter termed by Zwilling (1968) the “morpho- of horse serum solution was removed, and genetic phase” of limb development. The the cellular conglomerate was further dis- time when the limb cells acquire the po- rupted with a Vortex mixer. In the dissoci- tential for forming a limb precedes the ation of mesoblast cells from later stages, time when they become committed to a the cells from the dense chondrogenic cytodifferentiative path, for example as regions which failed to suspend even measured by the first appearance of en- after increased trypsinization (up to an zyme activity for synthesis of sulfated hour) were removed by filtration through mucopolysaccharides and other end prod- a 20-mesh Nitex cloth (Tobler, Ernst ucts (Searls, 1965; Medoff, 1967). Finally, and Traber; Elmsford, New York). the technique of dissociation and reim- For the preparation of chondrocytes plantation was also used to analyze the from stage 30 limbs (7 days), the legs capacity of mesoblast cells to sort out and were minced and then incubated with SINGER Analysis of Limb Morphogenesis 115 crystalline trypsin for 45 min. The tissue became firm. The pellet was removed clusters were dropped through a column of with a sterile spatula and placed in a dish CMF and further disrupted in the remain- containing horse serum solution. The pellet ing 1 ml after removing the supernatant. was subdivided into fragments each of After disruption, 4 ml of CMF was added which was stuffed into an ectodermal sac and mixed and the remaining chunks were which was just removed from stage 20 leg allowed to settle. The supernatant was buds. The stuffed ectoblasts were then discarded; the tissue pieces were then incubated in horse serum solution for 45 incubated in a depression dish in CMF min at 38°C. Eggs windowed at 4 days for 30 min, and the disruption procedures of incubation were used for grafting. An were repeated. The remaining tissue was excision of epidermis about one somite mainly cartilage rods. The rods were in- square was made over the somites of a cubated in the crystalline trypsin solution stage 20 animal just dorsal to the wing for 1 hr at 38”C, and then dropped through bud. The stuffed ectoblast was grafted to a test tube of horse serum solution; the the excised area with the free mesodermal supernatant was removed, and the re- area in contact with the graft site. The mainder was disrupted by Vortex agita- window was then sealed with Scotch tion. The cellular suspension was then brand tape and the egg was returned to passed through Nitex cloth to remove the incubator. After 2 days the grafts were undissociated rods. In this way it was observed and hosts which rejected grafts possible to obtain 6 million chondrocytes were discarded. Grafts were observed at from 12 limb buds. Viability was greater the seventh day and then fixed for histo- than 85% as measured by eosin exclusion. logical analysis. For the preparation of flank mesen- thyme cells, stage 19 embryos were re- RESULTS moved and resected in the region between Mixtures of Stage I9 Mesoblast Cells the limbs. The resected fragments were and Stage 30 cartilage Cells immersed in trypsin and pancreatin solu- tion for 20 min, the ectoderm teased away All the reconstituted limbs showed some and the flank mesoderm freed from the outgrowth by 7 days after grafting.
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