J. Embryol. exp. Morph. 80, 21-41 (1984) 21 Printed in Great Britain © The Company of Biologists Limited 1984 Development of the apical ectodermal ridge in the chick wing bud By WILLIAM L. TODT AND JOHN F. FALLON1 Department of Anatomy, The University of Wisconsin, Madison, Wisconsin 53706, U.S.A. SUMMARY Histological examination of the stage-18 to stage-23 chick wing bud apex revealed the following. Initially, the wing bud was covered by a cuboidal to columnar epithelium with an overlying periderm. Thickening of the apical ectoderm was not obvious until late stage 18 (36 pairs of somites), after the appearance of the wing bud. At late stage 18, cells of the inner layer of ectoderm had elongated slightly along an axis perpendicular to the epithelial-mesenchymal interface. Well-defined apical ectodermal ridge morphology, i.e., pseudostratified columnar epithelium with an overlying periderm, was not apparent until stage 20. Subsequently the ridge lengthened along the anteroposterior perimeter of the wing bud. We demonstrated histologically that the apical ectodermal ridge of the wing bud was asymmetric with respect to the anteroposterior axis, in that there was more ridge associated with posterior mesoderm. Other observations include the spatial and temporal location of a groove in the base of the thickest part of the ridge. The groove can be correlated with the specification of distal wing elements. The groove was first seen at stage 20 and became more prominent through stage 23. An anteroposterior progression of ectodermal cell death was also observed. This began at late stage 18 and continued through each of the stages examined. INTRODUCTION The development of the limb bud of the chick embryo is dependent upon a series of interactions between the limb bud mesoderm and overlying ectoderm. The mesoderm induces the formation of a pseudostratified columnar epithelium at the apex of the bud (Kieny, 1968; Saunders & Reuss, 1974) which is called the apical ectodermal ridge (Saunders, 1948). The ridge is distinct from the simple cuboidal epithelium of the dorsal and ventral limb bud surfaces (Ede, Bellairs & Bancroft, 1974; Fallon & Kelley, 1977). Removal of the ridge results in limbs with predictable terminal deficiencies. The longer the mesoderm is associated with the ridge, the more distally complete the resultant limb will be (Saunders, 1948; Summerbell, 1974; Rowe & Fallon, 1982). Thus, the ridge may be viewed as an inducer of normal avian limb bud outgrowth (Saunders, 1977). In a previous systematic study of the chick wing bud, Jurand (1965) presented representative sections of the wing bud apex from stages 16 to 31. Sections were selected and interpreted from serially sectioned material embedded in paraffin. 1 For reprint requests. 22 W. L. TODT AND J. F. FALLON This work constitutes the most informative description of ridge development to date. However, there are limitations inherent in the use of paraffin embedded material. These include the inability to control the orientation of the tissue when it is sectioned and difficulty in interpreting the position of the sections relative to the tissue as a whole. To facilitate further morphological and experimental studies on the development and function of the apical ectodermal ridge, we have done a systematic study of ridge development using electron microscopic fixation procedures and plastic embedment. The results of this study are the subject of this report. MATERIALS AND METHODS Fixation and embedment Standard techniques were used to obtain stage-18 to -23 White Leghorn embryos (Hamburger & Hamilton, 1951). Embryos were fixed in a solution of formaldehyde (2 %), glutaraldehyde (2-5 %), and picric acid (0-02 %), buffered with 0-075 M-phosphate buffer, pH7-3 (Fallon & Kelley, 1977). They were then lightly postfixed in osmium tetroxide to facilitate counting of the somites and visualization of the tissue. Wing buds and adjacent tissues were embedded flat in epon. The tissue was placed with the dorsal wing bud surface just beneath the surface of the block to allow clear visualization of the tissue. Both wing buds from at least one embryo at each stage were examined. The data from right and left wing buds from the same embryo were in agreement. Sectioning Camera-lucida drawings were made of embedded tissue (e.g., Fig. IB), paying special attention to the outline of the wing bud, the adjacent somites (stages 18 to 20) and the anterior and posterior j unctions of the wing bud and body wall (stages 20 to 23). To obtain accurate measurements of ectodermal thickness, it was necess- ary that the sections be cut in the dorsoventral plane perpendicular to the apical epithelium (cross sections). At each location, three ad j acent sections were cut, 1 to 1-5 jum thick. To insure that cross sections were in fact cut, the block was then realigned with the camera-lucida drawing and the position (location and orienta- tion) of the face of the block was recorded on the drawing. In addition to recording the position of the sections, it was possible to predict the change in orientation of the block needed to remain perpendicular to the apical epithelium of the bud for cutting the next group of cross sections. By repeating the procedure of cutting and marking, cross sections from 20 to 40 different locations along the apex of each bud were obtained. Sections were stained with azure II-methylene blue (0-5 %). Determination of location of sections For stages 18 to 20, the location of the cross sections could be expressed with reference to the adjacent somites. At later stages, when reference to the somites Apical ridge development in the chick wing bud 23 was difficult, the location was expressed relative to the distance along the antero- posterior perimeter of the wing bud. The anteroposterior perimeter was defined as the outline of the wing bud from the anterior to posterior junction of the bud and body wall, as seen by viewing the dorsal surface of the wing bud. Thus, 0 % and 100 % of the anteroposterior perimeter represented the anterior and posterior junctions of the wing bud and body wall, respectively (see Figs 4A and 4B). Stage 20 was a transition between the two methods as values relative to both the perimeter and the somites could be assigned to each location (see Fig. 4A). As wing outgrowth continues, the wing level somites increase in size and the anteroposterior perimeter increases in length (compare Figs 4B and 8B). Therefore, it is stressed that direct comparison of graphs from different stages may be deceptive. It should be noted also that 50 % of the anteroposterior perimeter did not necessarily correspond to the theoretical anteroposterior mid- line (axial line; see Figs 4A, 6A and 8A). Determination of ectodermal thickness A filar micrometer was used to measure ectodermal thickness for the three sections at each location. These measurements were made along a line which passed through the thickest portion of the apical ectoderm (including periderm) and was normal to the line tangential to the base of the ectoderm. The ectoder- mal thickness for each location was calculated by averaging these measurements. Because of the possibility that the plane of section was not always exactly perpen- dicular to the apical epithelium, the average thickness was corrected for this potential problem. A correction factor was calculated taking into account, 1) the rate of change in ectodermal thickness in a given region, as determined from the raw data, and 2) the orientation of the plane of section with respect to the line perpendicular to the outline of the wing bud at that location, as measured on the camera-lucida drawing. To keep the magnitude of these corrections in perspective, we point out that only 1-8 % (15 of 834) of the locations examined had a correction greater than 2-0 %. Only 2 of these 15 corrections were larger than 5 %, viz., 6-6 % and 7-3 %. It is equally important to note that measurements from adjacent sections at the same location could show variation of 2 %, probably due to the unevenness of the outer peridermal surface (cf. Camosso & Roncali, 1968). Thus, while the magnitude of the correction did not significantly alter the data presented in this report, it was utilized for consistency. RESULTS Data for some of the epithelial characteristics described in this section are summarized in Table 1. 24 W. L. TODT AND J. F. FALLON Table 1. Summary of wing bud apical epithelial characteristics Stage No. Thickest Limits of ectodermal Contour of (Pairs of somites) examined ectoderm Gum) cell death ectodermal base 18 6 20 no cell death smooth (32-33) 18+ 5 24-30 somites 15-17 smooth (36) 19 5 30-35 mid somite 16 - smooth (38) mid somite 18 20 7 38-44 somites 16-18 slight notch (41-42) (0% to 60% of perimeter) 21 5 37-45 20% to 60% of notch perimeter 23 5 33-38 20% to 83% of notch perimeter Stage 18 (32—33 pairs of somites, Table 1 and Fig. 1) The wing bud ectoderm at this early stage consisted of irregular cuboidal to columnar cells with an overlying simple squamous periderm. Five wing buds were examined at the level of somite 15 (Figs 1A, B), and in three of these a thickening was seen at this level. The thickening was localized ventrally and was not considered a ridge, as no prominence was seen in the outline of the outer ectodermal surface (Figs 1A, B, C). The apical ectoderm around the level of the Fig. 1. Stage 18 (32-33 pairs of somites). (A) The relationship between ectodermal thickness and location (relative to somite level) for six wing buds. Solid dots are data from the wing bud pictured in (B). Letters along the abscissa correspond to locations in (B) and to micrographs (C) through (H). It is crucial to point out that the graph does not represent the outline of the wing bud, but the change in ectodermal thickness along the anteroposterior extent of the wing bud. This cautionary note also applies to Figs 2A, 3A, 4A, 6A and 8A. (B) Camera-lucida drawing of the wing bud indicated by solid dots in (A). Somites are numbered at the left and the curve indicated by the arrows is the outline of the wing bud. Lines intersecting the wing bud outline show the position of sections examined for that wing bud. Heavy lines have letters corresponding to those along the abscissa in (A) and to micrographs (C) through (H). Scale bars (A and B) represent 250 fim. (C) through (H) Micrographs of cross sections of the wing bud at positions in- dicated by the corresponding letters in (B). The micrographs are oriented with distal mesoderm covered by apical ectoderm. Dorsal is to the left, ventral to the right. Note the anterior thickening (C, arrows). Anterior ectoderm is thicker than posterior ectoderm (compare D and G). No ectodermal cell death is seen. Scale bar (C) represents 20/xm. Apical ridge development in the chick wing bud 25 junction of somites 17 and 18 was 17 to 20/im thick. Intercellular spaces were generally smaller than those in adjacent dorsal and ventral ectoderm, and sug- gestions of nuclear stratification were apparent (e.g., Figs ID to IF). Posteriorly, there was a decrease in apical ectodermal thickness so that at the level of the junction of somites 20 and 21, it was 9 to 15 /im thick (Figs 1A, H). Ectodermal cell death was not observed at this stage. Late stage 18 (36 pairs of somites, Table 1 and Fig. 2) At the level of somite 15 (Figs 2A, B), a thickening of ectoderm was seen on the ventral surface of the wing bud (Figs 2A, B, C), near the lateral body fold (Hamburger & Hamilton, 1951). Around the level of somite 18, the apical epithelium was thickened to at least 24/im. From examination of cross sections 40 H 15 E 250 jim 3 30 D 75 20 -F -G 20 10- H D E H 250 fi 15 | 16 17 I 18 | 19 20 I 21 I Anterior Somite level Posterior Fig. 1 26 W. L. TODT AND J. F. FALLON A 40 -i Fig. 2. Late stage 18 (36 pairs of somites). (A) The relationship between ectodermal thickness and location (relative to somite level) for five wing buds. Solid dots are data from wing bud pictured in (B). Letters along the abscissa correspond to locations in (B) and to micrographs (C) through (H). (B) Camera-lucida drawing of the wing bud indicated by solid dots in (A). Somites are numbered at the left and the curve indicated by the arrows is the outline of the wing bud. Lines intersecting the wing bud outline show the position of sections examined for that wing bud. Heavy lines have letters corresponding to those along the abscissa in (A) and to micrographs (C) through (H). Scale bars (A and B) represent 250//m. (C) through (H) Micrographs of cross sections of the wing bud at positions in- dicated by the corresponding letters in (B). Micrographs are oriented with distal mesoderm covered by apical ectoderm. Dorsal is to the left, ventral to the right. Note the anterior thickening (C) very close to the lateral body fold (arrow). Cell death is seen in some anterior apical ectoderm (D, arrowheads). Dorsoventral asymmetry is seen clearly in (F). Scale bar (C) represents 20fim. Apical ridge development in the chick wing bud 27 A 50- 40- 250jun ° °° "*> o» " * O 30- • 0 ° O ° ° 0 • 0 20- o°o o o° o o° 00 0 • UJ 10- C D E F G H 16 17 1 18 1 19 1 20 Fig. 3. Stage 19 (38 pairs of somites). (A) The relationship between ectodermal thickness and location (relative to somite level) for five wing buds. Solid dots are data from wing bud pictured in (B). Letters along the abscissa correspond to locations in (B) and to micrographs (C) through (H). (B) Camera-lucida drawing of the wing bud indicated by solid dots in (A). Somites are numbered at the left and the curve indicated by the arrows is the outline of the wing bud. Lines intersecting the wing bud outline show the position of sections examined for that wing bud. Heavy lines have letters corresponding to those along the abscissa in (A) and to micrographs (C) through (H). Scale bars (A and B) represent 250 ^m. (C) through (H) Micrographs of cross-sections of the wing bud at positions in- dicated by the corresponding letters in (B). Micrographs are oriented with distal mesoderm covered by apical ectoderm. Dorsal is to the left, ventral to the right. Note the anterior ectodermal thickening (C) very close to the lateral body fold (arrow) and the ectodermal cell death (E, arrowheads). Obvious dorsoventral asymmetry is seen, in (F). Scale bar (C) represents 20/im. 28 W. L. TODT AND J. F. FALLON 50" 16 17 18 19 20 ? 40- 250 finn 8 °c 1/3 •o o <U 0 0 ck30- o o tihi o *° o al derm20- 8O a t> W a «p o o 10- ABC D E F GH IJ KLM N O PQ R i "If —T 0 20 40 60 80 100 R 250jon (Anterior) Anteroposterior perimeter {%) (Posterior) Fig. 4. Stage 20 (41-42 pairs of somites). (A) The relationship between ectodermal thickness and location (relative to the anteroposterior perimeter) for seven wing buds. Approximate somite levels are indicated across the top. Solid dots are data from wing bud pictured in (B). Letters along the abscissa correspond to locations in (B) and to the micrographs in Fig. 5. To determine the position of the axial line, indicated by the X (47-5 % ± 0-9 S.D.), a line representing the base of the wing bud was drawn by connecting the anterior and posterior junctions of the bud and the body wall (see Fig. 4B). The axial line was drawn perpendicular to the base of the bud, midway between the anterior and posterior junctions of the bud and the body wall. (B) Camera-lucida drawing of the wing bud indicated by the solid dots in (A). The curve indicated by the arrows is the outline of the wing bud; the anterior and pos- terior boundaries of the wing bud are indicated by the arrowheads. Lines intersecting the wing bud outline mark the position of the sections examined for that wing bud. Heavy lines have letters corresponding to those along the abscissa in (A) and to the micrographs in Fig. 5. Scale bars (A and B) represent 250//m. Fig. 5. Stage 20 (41-42 pairs of somites). Micrographs of cross sections of the wing bud at the locations indicated by the corresponding letters in Fig. 4B. Distal mesoderm is covered with apical ectoderm. Dorsal is to the left, ventral to the right. Micrographs are lettered in sequence, (A) being the most anterior (cranial). In addition, the micrographs are paired. For example, (A) and (R) are approximately the same distance from the axial line, as are (B) and (Q), etc. Note the apical ectodermal cell death (A through F, see arrowheads) and dorsoventral asymmetry (e.g., I through K). Compare the subectodermal region in (A) and (R). In addition, a slight notch is evident in the base of the ectoderm of some sections (see arrow, L). Anteroposterior asymmetry of the entire ridge with respect to the axial line is apparent when comparing (A) through (D) with (O) through (R). Scale bar (A) represents 20/im. Apical ridge development in the chick wing bud 29 A B Fig. 5 EMB80 30 W. L. TODT AND J. F. FALLON between these two regions, it was seen that the thickenings were continuous. In the region opposite somite 18, the ectoderm comprised elongated cells which were often radially arranged (Fig. 2E). In most of the cross sections (43 of 56) in this region, an obvious dorsoventral asymmetry was seen in that the transition from apical to ventral ectoderm was more pronounced than the transition from apical to dorsal ectoderm. In addition, the thickest ectoderm was located slightly ventral with respect to the apex of the mesodermal core as seen in cross sections (Fig. 2F; cf., Zwilling, 1961). The intercellular spaces were smaller than those seen at the previous stage. Apical ectoderm at the level of the junction of somites 20 and 21 was similar to adjacent dorsal and ventral ectoderm (12 to 18|Um, Fig. 2H). The occurrence of an average of two necrotic loci in each cross section of the apical ectoderm at a given location was considered evidence of cell death. Ectodermal cell death was evident in all wing buds at this stage and was limited to anterior ectoderm opposite somites 15 through 17 (Fig. 2D). The precise distribution within this region varied from bud to bud. However, it should be noted that cell death in the apical ectoderm was not seen posterior to the level of the middle third of somite 17. Stage 19 (38 pairs of somites, Table 1 and Fig. 3) In three wing buds examined at the level of somite 16 (Figs 3A, B), a thicken- ing was seen in the ventral ectoderm near the lateral body fold (Figs 3A, B, C). As seen in the previous stage, the thickening was continuous with the thickening of the apical epithelium of more posterior wing bud levels (Figs 3C, D, E). The apical ectoderm at the level of the caudal 2/3 of somite 18 and the cranial 2/3 of somite 19 was consistently thicker than 27 /mi. The architecture was similar to that of the thickest regions of late stage-18 wing bud ectoderm, but there were fewer and smaller intercellular spaces (compare Figs 2F and 3F). It should be noted that the thickest ridge was in the posterior half of the wing bud around the level of the junction of somites 18 and 19. Posteriorly, the apical ectoderm decreased in thickness to about 15/im and was similar to adjacent dorsal and ventral ectoderm (Fig. 3H). The base of the ectoderm was smooth at all antero- posterior levels. Cell death in the ectoderm was seen in only two of the wing buds examined (arrowheads, Fig. 3E). Ectodermal cell death was not seen posterior to the level of the middle of somite 18 in any stage-19 wing bud examined. Stage 20 (41-42 pairs of somites, Table 1 and Figs 4 and 5) Anteriorly, at approximately the level of somite 16 (from 0 % to about 15 % of the anteroposterior perimeter, Figs 4A, B; 5A, B), the apical ectoderm was comprised of cuboidal to columnar cells with an overlying periderm and a smoothly contoured base. Suggestions of nuclear stratification were evident in some parts of the ectoderm. There were large intercellular spaces and the