Development 101, 501-515 (1987) 501 Printed in Great Britain © The Company of Biologists Limited 1987 Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation WILLIAM L. TODT* and JOHN F. FALLONt Department of Anatomy, The University of Wisconsin, 1300 University Avenue, Madison, Wisconsin 53706, USA * Present address: Department of Physiology and Biophysics, University of California, Irvine, CA 92717. USA t Author for reprints Summary The ability of the anterior apical ectodermal ridge to by necrosis in the distal, anterior mesoderm at 48 h promote outgrowth in the chick wing bud when postoperatively and subsequent anterior truncation. disconnected from posterior apical ridge was exam- Clearly, healthy posterior limb bud mesoderm is ined by rotating the posterior portion of the stage- needed for anterior limb bud survival and develop- 19/20 to stage-21 wing bud around its anteroposterior ment. We propose that anterior truncation is the axis. This permitted contact between the anterior and direct result of anterior mesodermal cell death and posterior mesoderm, without removing wing bud that this may not be related to positional specification tissue. In a small but significant number of cases of anterior cells. In our view, cell death of anterior (10/54), anterior structures (digit 2) formed spatially mesoderm, after posterior mesoderm removal, should isolated from posterior structures (digits 3 and 4). not be used as evidence for a role in position specifi- Thus, continuity with posterior ridge is not a pre- cation by the polarizing zone during the limb bud requisite for anterior-ridge function in the wing bud. stages of development. We suggest that the posterior Nevertheless, posterior-ridge removal does result in mesoderm that maintains the anterior mesoderm need anterior limb truncation. To investigate events leading not be restricted to the mapped polarizing zone, but is to anterior truncation, we examined cell death pat- more extensively distributed in the limb bud. terns in the wing bud following posterior-ridge re- moval. We observed an abnormal area of necrosis Key words: limb development, apical ectodermal ridge, along the posterior border of the wing bud at 6-12 h wing bud, cell death, polarizing zone, chick, pattern following posterior-ridge removal. This was followed formation. Introduction & Fallon, 1982) and the specification of distal limb elements ceases, resulting in a stage-dependent trunc- The avian limb bud consists of a mesodermal core ation of the formed limb (Saunders, 1948; Summer- covered with simple cuboidal epithelium on the bell, 1974; Rowe & Fallon, 1982). It is clear that dorsal and ventral surfaces and capped apically by a normal development of the limb depends on com- pseudostratified columnar ectodermal ridge. Inter- munication between the mesoderm and ectoderm. actions necessary for normal limb formation occur Communication is also required along the antero- between the mesoderm and apical epithelium. In- posterior axis of the limb. Posterior tissue develops itially, the limb mesoderm induces formation of the normally if the anterior tissue is removed. However, ridge (Kieny, 1960, 1968; Saunders & Reuss, 1974) anterior development is truncated if the posterior and subsequently maintains it as well (Zwilling & tissue is removed (Warren, 1934; Hinchliffe & Gum- Hansborough, 1956; Fallon, Boutin & Carrington, pel-Pinot, 1981). Insertion of a barrier between the 1986). Removal of the apical-ectodermal ridge results anterior and posterior tissue is also followed by in necrosis in the subjacent mesoderm (Rowe, Cairns anterior truncation (Warren, 1934; Summerbell, 502 W. L. Todt and J. F. Fallon 1979; Kaprio, 1981). In addition to anterior trunc- experimentally isolated from posterior ridge provided ation, cell death in the anterior mesoderm is observed that limb tissue is not removed and contact between following these operations (see Hinchliffe & Grif- the anterior and posterior mesoderm is reestablished. fiths, 1984). These data demonstrate that normal We also report specific cell death patterns in the anterior development is dependent on posterior tis- mesoderm following posterior-ridge removal. These sue. results permit insights into the ectodermal-mesoder- A series of experiments beginning with Saunders & mal and mesodermal-mesodermal interactions oc- Gasseling (1968) demonstrates that posterior border curring during limb development. mesoderm can bring about polarized duplications when a small piece is grafted into an anterior limb bud Materials and methods site. This has been interpreted to mean that posterior border mesoderm establishes a gradient of a morpho- Fertile White Leghorn eggs were incubated at 37-38°C for gen that prescribes the anteroposterior axis during 3 days, candled, windowed, sealed with Scotch magic tape normal limb development (see Tickle, Summerbell & and reincubated until they reached the desired stage Wolpert, 1975). Thus, in this view, normal limb (Hamburger & Hamilton, 1951). To operate on the em- development is dependent on active production of the bryo, a small hole was made in the membranes overlying morphogen and continuous maintenance of the gradi- the wing bud and two drops of sterile Tyrode's solution ent until all limb structures are specified. The pro- were added. The wing bud apex was stained lightly with posed morphogen has not been identified. However, Nile blue A and a camera-lucida drawing of the wing bud and adjacent somites was made (final magnification, X290). provocative results have been achieved with retinoic Following each operation described below, two drops acid (Tickle, Alberts, Wolpert & Lee, 1982; Summer- of penicillin-streptomycin (500 units ml"1; 500mcgml~'; bell, 1983; Eichele, Tickle & Alberts, 1985; Thaller & GIBCO) were added and eggs were resealed. We observed Eichele, 1987) in that it can mimic the action of better development of the anterior tissue in rotation exper- grafted posterior border mesoderm and cause polar- iments if the embryos were held at room temperature for a ized duplications. short period. Therefore, in all types of experiments, some Summerbell (1979) and Hinchliffe and co-workers embryos were left at room temperature (25°C) for 1 to 1-5 h (Hinchliffe & Gumpel-Pinot, 1981; Hinchliffe & Grif- after the operation while others were reincubated immedi- fiths, 1984; Wilson & Hinchliffe, 1987) argue that ately. Only in the case of 180° posterior-bud rotation did anterior defects seen after posterior-bud removal or room temperature affect the results (see below). Embryos were checked several times in the 2 days following the barrier insertion are due to isolation of the anterior operation and prepared for cell death studies or allowed to mesoderm from the polarizing influence of the pos- develop until the skeletal patterns could be analysed. terior mesoderm. However, their work does not rule out the possibility that separation of the anterior Experimental manipulations (Fig. 1) ridge from the posterior ridge is responsible for Partial bud removal anterior truncation. If the anterior ridge became To remove part of the wing bud, a cut was made through nonfunctional when separated from the posterior the wing bud, parallel to the proximodistal axis, with a pair ridge, the results obtained would mimic the surgical of sharpened forceps. The location of the cut was marked removal of the anterior ridge, i.e. cell death would on the camera-lucida drawing. A cut was then made along occur in the distal, anterior mesoderm and truncation the base of either the anterior (Fig. 1A) or posterior of digital skeletal structures would result. As men- (Fig. IB) portion of the bud. The excised tissue was tioned above, cell death and truncation are seen removed from the egg. following barrier insertion and posterior-bud removal Partial ridge removal (Hinchliffe & Gumpel-Pinot, 1981; Hinchliffe & Grif- In order to remove a portion of the apical ectodermal ridge, fiths, 1984). Rowe & Fallon (1981) demonstrated that the region of ectoderm to be removed was stained lightly posterior-ridge removal, leaving the anterior apical with Nile blue A. Two cuts were made through the ridge and the posterior mesoderm in situ, results in ectodermal ridge using a sharpened tungsten needle. One truncated anterior development. To understand why cut was near the boundary between digits 2 and 3 (see such different manipulations (posterior-bud removal, below and Fig. 2). The second cut was made within 150/im barrier insertion and posterior-ridge removal) all of the anterior (Fig. 1C) or posterior (Fig. ID) junction of result in anterior truncation, two questions must be the limb bud and body wall. The locations of the cuts were marked on the camera-lucida drawing. Then the dorsal answered. (1) Is anterior apical ridge inactivated ectoderm proximal to the apical ridge was cut along the when separated from posterior ridge? (2) What cell extent of the ridge to be removed. The apical ridge was death patterns accompany posterior-ridge removal? teased off the apex of the bud. In the last step for ridge We examined the ability of anterior ridge to sup- removal, the ventral ectoderm proximal to the ridge was cut port normal anterior limb development and found and the apical ectoderm, including the apical ridge, was that anterior ridge can support development when removed from the egg. Posterior ridge and anterior wing bud patterning 503 Posterior-bud rotation and the posterior part of the bud was rotated 180° around To rotate the posterior portion of the wing bud, a cut was the pin. A second pin was inserted through the rotated made parallel to the proximodistal axis and the location was tissue and into the body wall, fixing the tissue in the rotated marked on the camera-lucida drawing. A platinum pin position (Fig. IF). (0025mm diameter, Goodfellow Metals, Cambridge) was inserted parallel to the anteroposterior axis (Fig. IE). A cut Cell death studies was made along the base of the posterior portion of the bud Regions of cell death were examined in ovo for some embryos at each stage (19/20 through 21) and for each type of operation.