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Effect of removal of the apical ectodermal ridge on the rate of cell division in the subridge mesenchyme of the embryonic chick wing
DEVELOPMENTAL
BIOLOGY
24,
Effect of Removal
465-476
(1971)
of the Apical
Ectodermal
Ridge on
the Rate of Cell Division in the Subridge Mesenchyme of the Embryonic Chick Wing’ ’ ’ MARTHA
Y. JANNERS AND ROBERT L. SEARLS
Department of Biology, Temple University, Philadelphia,
Pennsylvania 19122
Accepted October 27, 1970 INTRODUCTION
The early limb bud of the embryonic chick (3-5 days of embryonic development) consists of mesoderm with an ectodermal covering. A thickening of the ectoderm at the apex of the limb (the apical ectodermal ridge) has been demonstrated to be essential for normal limb development: when the apical ectodermal ridge is removed, short limbs develop that are deficient in distal elements (Saunders, 1948; Hampe, 1959; Barasa, 1960); and when a second ridge is grafted to a limb mesoblast, a second outgrowth occurs that gives rise to distal elements (Zwilling, 1956; Saunders and Gasseling, 1968). That outgrowth occurs only in the presence of a ridge, and apparently in response to a ridge, suggests that one function of the apical ectodermal ridge is to maintain a particular growth pattern in the limb mesoblast. However, no clear evidence for a region of high mitotic activity in proximity to the apical ectodermal ridge has been found in normal limbs (Saunders, 1948; Camossoet al., 1960; Cairns, 1966; Janners and Searls, 1970). In an attempt to discover the effect of the apical ectodermal ridge on the pattern of cell division in the mesoderm subjacent to the ridge, we have compared the labeling index (the percentage of cells labeled within 30 minutes after an injection of tritiated thymidine) in wings that have had the apical ridge removed to the labeling index in wings with intact apical ridges. In these experiments we also measured the width (distance from body wall to apex of the wing) of I Supported in part by National Science Foundation grant GB-4846. The material in this paper is taken from a thesis submitted by Martha Y. Janners in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of Virginia, Charlottesville, Virginia. zCommunication concerning this paper should be with Robert L. Searls, Biology Department, Temple University, Philadelphia, Pennsylvania 19122. 465 Copvrkht
0 1971 by Academic
Press. Inc.
466
JANNERS
AND
SEARLS
the operated wings at various times after the removal of the apical ectodermal ridge in order to determine the effect of ridge removal on the rate of outgrowth of the operated wings. It has been found that ridge removal has no effect on the labeling index in any region of the wing at any time within 36 hours after the operation. Despite the observed constancy of the labeling index, a lag in wing outgrowth that lasted for about 12 hours was observed immediately after the operation. These results are discussed as they relate to the role of the apical ectodermal ridge in the normal development of the embryonic chick wing. MATERIALS
AND
METHODS
Fertilized White Cornish Cross (Brown Cornish male x Arbor Acres White Rock female) eggs were incubated at 37”. After 2 days of incubation, a window was cut in the shell of each egg (Zwilling, 1959) and the eggs were returned to the 37” incubator. Surgical removal of the apical ectodermal ridge was performed on the right wing buds of stage 19 (Hamburger and Hamilton, 1951) and stage 20 embryos according to the procedure outlined by Saunders (1948). Following the operation, the window was sealed with Scotch tape and the egg was returned to the 37O incubator. The embryos were kept out of the 37” incubator for as short a time as possible so that cooling of the embryos would not influence the rate of ceil division or the rate of wing outgrowth. At various times after the removal of the apical ridge, each embryo received 10 &i of tritiated thymidine (0.10 ml; New England Nuclear Corp.; greater than 10 Ci/mmole; diluted with Tyrode’s solution to 100 &i/ml) by injecting directly into the yolk through a hole drilled in the large end of the egg. One-half hour after the injection of thymidine, the embryos were removed from the yolk, and the wings were fixed in glutaraldehyde (2.5% glutaraldehyde, 0.1 M disodium phosphate, 0.2 M sucrose). The operated wing and the contralateral control wing were embedded in paraffin, and serial 5 ~1 sections were cut in cross section in cranial-caudal sequence. The paraffin was removed with xylene, the sections were hydrated through a graded series of alcohols to water and were dipped in Kodak NTB-3 photographic emulsion while still wet. They were allowed to drain and dry and were stored over Drierite at 4O. After 3-4 weeks, the emulsion was developed in Kodak D-19 (Caro and Tubergen, 1962), and the sections were stained with hematein by a double bath technique (Sea&, 1967).
EFFECT
OF
REMOVAL
OF
THE
APICAL
ECTODERMAL
RIDGE
467
The percentage of cells that had taken up tritiated thymidine was determined under oil at X 500 using a Wild binocular microscope equipped with a drawing tube. A square field was drawn on a piece of paper and viewed through the drawing tube. The field outlined an area on the slide approximately 0.1 mm on a side. This field usually contained between 150 and 250 nuclei. The number of labeled and unlabeled nuclei within the standard field was recorded. A nucleus was considered to be labeled if it had over it 3 or more silver grains more than would be expected from background. Four fields were counted in each wing: (1) the region immediately beneath the apical ectodermal ridge, (2) the dorsal proximal region, (3) the central proximal region, and (4) the ventral proximal region (Fig. 1). These regions were counted in every tenth section of the craniocaudal sequence through each wing. In order to reduce statistical scatter, the data from three sequential counted sections (e.g., the lOth, the ZOth, and the 30th) were averaged. This procedure gave average values for the percentage of cells in DNA synthesis in overlapping 150 ,J units in the wing for each region. RESULTS
Table 1 summarizes the experiments that were done. The apical ectodermal ridge was removed at stages 19 and 20, and the operated wing and the contralateral control wing were fixed at various times after the operation. The embryos received 10 &i of tritiated thymidine 30 minutes before the wings were fixed. After preparation of the wings as autoradiographs, the sections were examined to discover
STAGE
22+
FIG. 1. Position of the fields counted. SR the subridge region; mal region; D, the dorsal proximal region; V, the ventral proximal
C, the central region.
proxi-
468
JANNERS
TIME
OF FIXATION=
Within 10 hours, stage 22 or younger Stage Stage Total
19 20
o Time
SEARLS
TABLE 1 AND STAGE OF EMBRYO
10-20 hours , stage 22 to 24
10 9 19 of fixation
AND
after
removal
More than 30 hours, older than stage 26
20-30 hours, stage 24 to 26
7 6 13
is the time
AT FIXATION
4 II 15
5 3 8
Total
26 29 55
of the ridge.
sf”/;~‘l’~~“~ -I
JO-
*
F Z
0
A0
n A
i E a
0
CONTROL EXPERIMENTAL
20
t---CRANIO-CAUDALW 5~
SECTIONS
FIG. 2. The labeling index through a wing bud from which the apical ridge had been removed 4 hours previously during stage 19. The squares represent the values for the subridge area; the triangles values for the dorsal and ventral proximal areas; and the circles the values for the central proximal area.
whether the ridge had been completely removed and the extent of the damage to the underlying mesoderm. The success of the operation was recorded, and the percentage of the mesodermal cells that became labeled in each of the four regions was determined. Figure 2 shows the result obtained when the apical ectodermal ridge of the right wing bud was removed at stage 20, and the experimental wing and its contralateral control were fixed 4 hours later. The apical ectodermal ridge had been completely removed in this case, and no surgical damage to the underlying mesoderm could be detected. At the time of fixation, the ectoderm had not yet healed over the mesoblast and necrotic cells were present in the subridge mesoderm. In such cases, the exposed mesoderm was contracted (i.e., the limb was shorter than expected for the time of wing development
EFFECT
OF
REMOVAL
OF THE
APICAL
ECTODERMAL
RIDGE
469
and more nuclei than normal were present within the standard field in the subridge region). The percentage of labeled nuclei in each region of the wing was similar in the experimental wing (closed symbols) to the percentage in the control wing (open symbols); the variation in percentage of labeled nuclei being no greater than the variation previously observed between normal wings from embryos from the same stage (Janners and Searls, 1970). Figure 3 shows the result obtained when the ridge was removed at stage 20, and the experimental wing and its contralateral control wing were fixed 20.5 hours after the operation. The apical ectoderma1 ridge had been completely removed in this case; and if any damage had been done surgically to the underlying mesoderm, that damage could no longer be detected, By the time of fixation, the ectoderm had healed over the mesoblast to form a smooth continuous ectodermal covering. The absence of the apical ectodermal ridge had no effect on the percentage of mesodermal cells incorporating tritiated thymidine either at the apex of the wing or at the base of the wing. The region of low labeling index in the chondrogenic core of the wing (the central proximal region, midway between the cranial and caudal extremes of the wing) was of the same shape and width in the experimental wing as in the control wing. Fifty-five experimental wings were compared with their contra-
4Or
5
IO-
0
. o
l
.
.
0
0
0
b
l
0 A0
CONTROL
s n
E a
A 0
EXPERIMENTAL
0
f---
CRANIO-CAUDALd 5~ SECTIONS
FIG. 3. The labeling index through a wing bud from which the apical ridge had been removed 20.5 hours earlier during stage 19. The squares represent the values for the subridge region, the triangles values for the dorsal and ventral proximal regions, and the circles the values for the central proximal region.
470
JANNERS
AND
SEARLS
lateral control unoperated wings. Some of these showed only partial removal of the apical ectodermal ridge, and some showed surgical damage to the underlying mesoderm. In most of the cases where ridge removal was complete, only a few sections midway between the cranial and caudal extremes of the wing were counted. In cases where ridge removal was only partial, sections were counted both where the ridge had been removed and where some of the ridge remained. A complete determination of the labeling index in the four regions in every tenth section in the craniocaudal sequence of the two wings was completed for 10 pairs of wings. In no case did we find a difference in labeling index in any region of the wing greater than the difference found previously between normal wings from different embryos of the same stage (Janners and Searls, 1970). It is known that short incomplete limbs develop following removal of the apical ectodermal ridge (Saunders, 1948; Hampe, 1959; Barasa, 1960). Yet, removal of the apical ridge had no obvious effect on the percentage of cells incorporating tritiated thymidine, and therefore, presumably, no effect on the rate of cellular proliferation in the mesoderm. In order to determine the effect of removal of the apical ridge on the rate of limb outgrowth, the apical ridge was removed from 8 embryos when the width of the wing (distance from the body wall to the apex of the wing) was between 0.32 and 0.48 mm (stage 19 to stage 20). The width of the wing was measured again at the time of fixation and, if the wing developed for sufficient time before it was fixed, once or twice between the time of the operation and the time when it was fixed. The limbs were prepared as autoradiographs, the sections were examined to ensure that the ridge had been removed completely and that little or no surgical damage had been done to the apical mesoderm, and the labeling index in the four regions of the operated wing was compared with the labeling index in the four regions of the contralateral control wing. The rate of increase in width of the wing following removal of the apical ridge was compared with the rate of increase in width of the wing during normal development. The preparation of the standard curve for the rate of increase in the width of the wing during normal development has been described previously (Janners and Searls, 1970). The width of the wing at the time of the operation (0.32-0.48 mm) was placed at the proper time on the normal growth curve (16-20 hours of wing development), and subsequent measurements of wing width were plotted at the appropriate times thereafter. The operated
EFFECT
OF
REMOVAL
OF
THE
TIME
APICAL
IN
ECTODERMAL
RIDGE
471
HOURS
FIG. 4. Rate of increase of the width of the wing following ridge removal. is the standard curve for rate of increase of the width of the wing (Janners 1970). The points show the increase in the width of the operated wings.
The curve and Searls,
wings were found to cease outgrowth from the time of the operation, and sometimes to decrease slightly in width (Fig. 4). Operated wings did not resume outgrowth until about 12 hours after the operation. When they started to increase in width, the rate of increase in width in operated wings closely paralleled the rate of increase in width of normal wings. During the time when there was little increase in the width of the experimental wings (12 hours postoperatively), the labeling index was the same in the experimental wings as in the contralateral control wings. DISCUSSION
If the apical ectodermal ridge is removed at an early stage in limb development (stage 19 or stage 20), the limb that develops is approximately half the size of a normal limb, has proximal elements that are normal in size and morphology, but has no distal elements (Saunders, 1948; Hampe, 1959, Barasa, 1960). This result mimics the mutant condition Wingless in which the thickening of the ectoderm
472
JANNERS
AND
SEARLS
at the apex of the wing degenerates leaving a continuous smooth covering over the limb mesoblast, and limb outgrowth fails to occur (Zwilling, 1949). If a limb mesoblast is supplied with a second apical ectodermal ridge, a second outgrowth appears that gives rise to distal elements (Zwilling, 1956; Saunders and Gasseling, 1968). This result mimics the mutant condition Eudiplopodia in which a second ectodermal ridge arises on the dorsal surface of the limb and a duplication of distal elements occurs (Goetinck, 1964). If the ridge is wider than normal, the channel of mesodermal outgrowth is wider, and polydactylous limbs develop (Zwilling and Hansborough, 1956; Saunders and Gasseling, 1963; Abbott et al., 1960; Goetinck and Abbott, 1964). The distal morphology of the mature limb can be predicted from the size, shape, and location of the apical ectodermal ridge during early limb development. The role of the apical ectodermal ridge in limb outgrowth and limb morphogenesis has been subject to considerable discussion. The scheme of limb development espoused by Saunders and by Zwilling has been described by Zwilling (1968) as follows. “The apical ectodermal ridge, initially induced by mesoderm of the early limb field, evokes the outgrowth of the underlying mesoderm.” In contrast, Amprino and his associates have suggested that “all developmental factors reside in the mesoderm of the limb district and govern its outgrowth, the gradual individuation of the territories, and the determination and differentiation of the latter . . .” (Amprino, 1965). It seems to be generally agreed that the presence of the ridge is essential for limb development. However, Saunders and Zwilling have both stated that the ridge induces outgrowth of the limb. Amprino has suggested that “the arrest of limb development [following removal of the apical ridge] may depend on other factors-namely, the disruption of the growth pattern of the mesoderm following alterations of the local homeostasis which occur when the mesoderm is exposed abruptly to abnormal environmental conditions” (Amprino, 1965). We have found that surgical removal of the apical ectodermal ridge has no effect on the percentage of cells that become labeled during a short exposure to tritiated thymidine (the percentage of the cells in the DNA synthetic phase of the division cycle, S). It does not seem to us possible that the rate of cellular proliferation could decrease without producing a change in the percentage of cells in the S phase of the division cycle. If the length of the division cycle were
EFFECT
OF
REMOVAL
OF THE
APICAL
ECTODERMAL
RIDGE
473
to become longer without change in the labeling index, then the length of the S phase must become longer in proportion to the increase in length of the other phases of the cycle. If some of the cells were to cease dividing without change in the labeling index, then the length of the S phase must get longer in proportion to the number of cells that cease dividing while the length of the other phases of the division cycle does not change. However, it has been proposed that the length of the S phase of the division cycle is that portion of the cycle least likely to change in length (Cameron and Greulich, 1963; Cameron, 1964; Defendi and Manson, 1963). The length of the S phase of the division cycle in the chick has been measured to be between 4 and 6 hours from 3 days of embryonic development to 35 days post-hatching (Fujita, 1962: 4-6 hours; Cameron, 1964: 5-6 hours; Okazaki and Holtzer, 1965: 5 hours; Marchok and Herrmann, 1967: 5.85 hours; Mitriou et al., 1968: 4-5.5 hours; Janners and Searls, 1970: 5.6 hours). It seems to us unlikely that the S phase of the division cycle would change in length following removal of the apical ectodermal ridge. We conclude, therefore, that surgical removal of the apical ridge has no effect on the rate of cellular proliferation in the wing bud mesoderm. Immediately after removal of the apical ectodermal ridge, there was a time period of about 12 hours during which the wing bud did not increase in width (the distance from the body wall to the apex of the wing). After this period, the experimental wings increased in width at about the same rate as the normal wings. It has been suggested (Amprino, 1965) that the slowing of limb outgrowth following ridge removal is the result of surgical trauma to the mesoderm. A wave of necrosis in the subridge mesoderm after surgical removal of the ridge has been observed in the present experiments and has been extensively studied by Cairns (in preparation, we wish to thank Dr. John Cairns for the opportunity to examine a preliminary manuscript). Loss of mesodermal cells may explain how limb outgrowth can cease while the rate of cellular proliferation remains constant. If cells were lost as a result of the operation but the remaining cells continued to divide at a constant rate, a time interval would be required for sufficient new cells to be added to restore the original width of the wing and before outgrowth relative to the width of the wing at the time of the operation could become evident. Loss of cells may explain the slowing of wing outgrowth following surgical removal of the apical ectodermal ridge, but it does not ex-
474
JANNERS
AND
SEARLS
plain the resulting change in wing morphology. Limbs that develop following ridge removal are not only short, but they are also lacking in distal limb elements. (a) These distal deficiencies are probably not due to destruction of part of a mosaic system; it has been demonstrated that normal distal elements can form from proximal limb mesoderm (Zwilling, 1956, 1964; Saunders et al., 1957, 1959; Cairns, 1965; Hampe, 1959). (b) The distal deficiencies cannot be explained as the result of insufficient cells to support normal distal development. Barasa (1964) has demonstrated that up to 90% of the wing mesoderm of a stage 19 embryo may be surgically removed without preventing normal development of the operated wing. Barasa’s experiment has been repeated in this laboratory; it has been found that if 50% or more of the wing mesoderm was removed from the wing of a stage 19 embryo without damage to the apical ectodermal ridge, the wing undergoes a lag in outgrowth very similar to the lag observed after removal of the apical ridge (Searls, unpublished observations). However, a normal wing may develop after removal of mesoderm, a wing with distal deficiencies after removal of the ridge. The apical ectodermal ridge evidently has some effect on the differentiation of the cells immediately beneath the ridge. If the apical ectodermal ridge is removed from the wing at stage 19 or stage 20, the normal differentiative changes occur in the proximal regions of the wing during stage 22. The cells in the central proximal region of the wing become smaller and more rounded and incorporate increased amounts of ““S-sulfate into mucopolysaccharide whereas the cells in the dorsal and ventral proximal regions have increased amounts of cytoplasm and incorporate decreased amounts of YSsulfate into mucopolysaccharide. However, after ridge removal the cells in the subridge region, which normally do not change in cell morphology or in rate of mucopolysaccharide synthesis until stage 24, change during stage 22 so that they become similar to the cells in the dorsal and ventral proximal regions (Searls, 1965). It is not clear to us how an effect of the ridge on the differentiation of subridge mesenchyme cells could be responsible for distal development of the limb. More important, it is not clear to us how an effect of the ridge on the differentiation of subridge mesenchyme cells could explain why a secondary outgrowth appears after the grafting of an extra ridge. The results of the experiments reported here suggest that the ridge is not an outgrowth inducer in the sense that it induces or maintains cell division patterns or increased rates in the mesoblast. The ridge may be an outgrowth inducer in the sense that it permits
EFFECT
OF
REMOVAL
OF
THE
APICAL
ECTODERMAL
RIDGE
475
growth in the proper direction and thereby shapes the growing mesoblast. It cannot be denied that the apical ectodermal ridge is essential for normal limb development, but the precise role of the ridge remains obscure. SUMMARY
The apical ectodermal ridge of the embryonic chick limb has been demonstrated to be essential for normal limb outgrowth and development. However, the rate of cell division in the mesenchyme close to the ridge has been found to be similar to the rate of cell division in other regions of the limb. In the present experiments, the rate of cell division in the mesenchyme close to the ridge and in three other regions of the limb in normal limbs has been compared with the rate of cell division in the same four regions after removal of the ridge. It has been found that removal of the apical ectodermal ridge has no effect on the rate of cell division in any region of the limb. REFERENCES ABBOTT, U. K., TAYLOR, L. W., and ABPLANALP, H. (1960). Studies with Talpid2, an embryonic lethal of the fowl. J. Hered. 51, 195-202. AMPRINO, R. (1965). Aspects of limb morphogenesis in the chicken. In “Organogenesis” (R. L. DeHaan and H. Ursprung, eds.), pp. 255-281. Holt, New York. BARASA, A. (1960). Consequenza dell’ablazione della cresta ectodermica apicale sullo sviluppo dell’abbozzo dell’ala nell’embrione di ~0110. Riu. Biol. 52, 257-292. BAKASA, A. (1964). On the regulative capacity of the chick embryo limb bud. Experientia 20, 443. CAIRNS, J. M. (1965). Development of grafts from mouse embryos to the wing bud of the chick embryo. Deuelop. Biol. 12, 36-52. CAIRNS, J. M. (1966). Cell generation times and growth of the chick wing bud. Amer. Zool. 6, 328. CAMERON, I. L. (1964). Is the duration of DNA synthesis in somatic cells of mammals and birds a constant? J. Cell Biol. 20, 185. CAMERON, I. L., and GREULICH, R. C. (1963). Evidence for an essentially constant duration of DNA synthesis in renewing epithelia of the adult mouse. J. Cell Biol. 18, 31-40. CAMOSSO, M. V., JACOBELLI, V., and PAPPALETTERA, N. (1960). Ricerche descrittive e sperimentale sull’organogenesi dell’abbozzo dell’ala dell’embrione di polio. Riu. Biol. 52, 323-357. CARO, L. G., and TUBERGEN, R. P. (1962). High resolution autoradiography I. Methods. J. Cell Biol. 15, 173-188. DEFENDI and MANSON, L. A. (1963). Analysis of life cycle in mammalian cells. Nature (London) 198, 359-360. FUJITA, S. (1962). Kinetics of cell proliferation. Exp. Cell Res. 28: 52-60. GOETINCK, P. F. (1964). Studies on avian limb morphogenesis. II. Experiments with the polydactylous mutant Eudiplopodia. Deuelop. Biol. 10, 71-91.
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GOETINCK, P. F. and AFIBOT~, U. K. (1964). Studies in limb morphogenesis. I. Experiments with the polydactylous mutant Tulpidz. J. Exp. 2001. 154, 7-19. HAMBURGER, V., and HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HAMPE, A. (1959). Contribution a l’etude du developpement et de la regulation des deficiences et des excedents dans la patte de l’embryon de poulet. Arch. Anut. Micros. Morphol. Exp. 48, 345-478. JANNERS, M. Y., and SEARLS, R. L. (1970). Changes in rate of cellular proliferation during differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing. Develop. Biol. 23, 136-165. MARCHOK, A. C., and HERRMANN, H. (1967). Studies of muscle development. I. Changes in cell proliferation. Deuelop. Biol. 15, 129-155. MITRIOU, P., LANG, W., and MAUER, W. (1968). Autoradiographische Bestimmung des Mardierungindex der S-Phase und der Generationszeit einiger Zellarten von 2-35 Tage alten Kiiken. 2. Zelljorsch. Mikrosk. Amt. 90, 68-80. OKAZAKI, K., and HOLTZER, H. (1965). An analysis of myogenesis in vitro using fluorescein-labeled antimyosin. J. Histochem. Cytochem. 13, 726-739. SAUNDERS, J. W., JR. (1948). The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 108, 363-403. SAUNDERS, J. W., JR., and GASSELING, M. T. (1963). Transfilter propagation of apical ectoderm maintenance factor in the chick embryo wing bud. Develop. Biol. 7, 64-78. SAUNDERS, J. W., JR., and GASSELING, M. T. (1968). Ectodermal-mesenchymal interactions in the origin of limb symmetry. In “Epithelial Mesenchymal Interactions (R. Billingham, ed.), pp. 78-97. Williams and Wilkins, Baltimore, Maryland. SAUNDERS, J. W., JR., CAIRNS, J. W., and GASSELING, M. T. (1957). The role of the apical ridge of ectoderm in the differentiation of the morphological structure and inductive specificity of limb parts in the chick. J. Morphol. 101, 57-88. SAUNDERS, J. W., JR., GASSELING, M. T., and CAIRNS, J. W. (1959). The differentiation of prospective thigh mesoderm grafted beneath the apical ectodermal ridge of the wing bud in the chick embryo. Deuelop. Biol. 1, 281-301. SEARLS, R. L. (1965). An autoradiographic study of the uptake of S-35 sulfate during the differentiation of limb bud cartilage. Develop. Biol. 11, 155-168. SEARLS, R. L. (1967). The role of cell migration in the development of the embryonic chick limb bud. J. Exp. Zool. 166, 39-50. ZWILLING, E. (1949). The role of epithelial components in the developmental origin of the “wingless” syndrome of chick embryos. J. Exp. Zool. 111, 175-187. ZWILLING, E. (1956). Interaction between limb bud ectoderm and mesoderm in the chick embryo, II. Experimental limb duplication. J. Exp. Zool. 132, 173-187. ZWILLING, E. (1959). A modified chorioallantoic grafting procedure. Transplant. Bull. 6, 115-116. ZWILLING, E. (1961). Limb morphogenesis. Aduan. Morphogenesis 1, 301-330. Aca-
demic Press, New York. ZWILLING,
Develop.
E. (1964). Development Biol. 9, 20-37. E. (1968). Morphogenetic
of fragmented
and dissociated
limb
bud mesoderm.
phases in development. Develop. Biol. Suppl. 2, 184-207. ZWILLING, E., and HANSBOROUGH, L. A. (1956). Interaction between limb bud ectoderm and mesoderm in the chick embryo. III. Experiments with polydactylous limbs. J. Exp. Zool. 132, 219-239.
ZWILLING,
BIOLOGY
24,
Effect of Removal
465-476
(1971)
of the Apical
Ectodermal
Ridge on
the Rate of Cell Division in the Subridge Mesenchyme of the Embryonic Chick Wing’ ’ ’ MARTHA
Y. JANNERS AND ROBERT L. SEARLS
Department of Biology, Temple University, Philadelphia,
Pennsylvania 19122
Accepted October 27, 1970 INTRODUCTION
The early limb bud of the embryonic chick (3-5 days of embryonic development) consists of mesoderm with an ectodermal covering. A thickening of the ectoderm at the apex of the limb (the apical ectodermal ridge) has been demonstrated to be essential for normal limb development: when the apical ectodermal ridge is removed, short limbs develop that are deficient in distal elements (Saunders, 1948; Hampe, 1959; Barasa, 1960); and when a second ridge is grafted to a limb mesoblast, a second outgrowth occurs that gives rise to distal elements (Zwilling, 1956; Saunders and Gasseling, 1968). That outgrowth occurs only in the presence of a ridge, and apparently in response to a ridge, suggests that one function of the apical ectodermal ridge is to maintain a particular growth pattern in the limb mesoblast. However, no clear evidence for a region of high mitotic activity in proximity to the apical ectodermal ridge has been found in normal limbs (Saunders, 1948; Camossoet al., 1960; Cairns, 1966; Janners and Searls, 1970). In an attempt to discover the effect of the apical ectodermal ridge on the pattern of cell division in the mesoderm subjacent to the ridge, we have compared the labeling index (the percentage of cells labeled within 30 minutes after an injection of tritiated thymidine) in wings that have had the apical ridge removed to the labeling index in wings with intact apical ridges. In these experiments we also measured the width (distance from body wall to apex of the wing) of I Supported in part by National Science Foundation grant GB-4846. The material in this paper is taken from a thesis submitted by Martha Y. Janners in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of Virginia, Charlottesville, Virginia. zCommunication concerning this paper should be with Robert L. Searls, Biology Department, Temple University, Philadelphia, Pennsylvania 19122. 465 Copvrkht
0 1971 by Academic
Press. Inc.
466
JANNERS
AND
SEARLS
the operated wings at various times after the removal of the apical ectodermal ridge in order to determine the effect of ridge removal on the rate of outgrowth of the operated wings. It has been found that ridge removal has no effect on the labeling index in any region of the wing at any time within 36 hours after the operation. Despite the observed constancy of the labeling index, a lag in wing outgrowth that lasted for about 12 hours was observed immediately after the operation. These results are discussed as they relate to the role of the apical ectodermal ridge in the normal development of the embryonic chick wing. MATERIALS
AND
METHODS
Fertilized White Cornish Cross (Brown Cornish male x Arbor Acres White Rock female) eggs were incubated at 37”. After 2 days of incubation, a window was cut in the shell of each egg (Zwilling, 1959) and the eggs were returned to the 37” incubator. Surgical removal of the apical ectodermal ridge was performed on the right wing buds of stage 19 (Hamburger and Hamilton, 1951) and stage 20 embryos according to the procedure outlined by Saunders (1948). Following the operation, the window was sealed with Scotch tape and the egg was returned to the 37O incubator. The embryos were kept out of the 37” incubator for as short a time as possible so that cooling of the embryos would not influence the rate of ceil division or the rate of wing outgrowth. At various times after the removal of the apical ridge, each embryo received 10 &i of tritiated thymidine (0.10 ml; New England Nuclear Corp.; greater than 10 Ci/mmole; diluted with Tyrode’s solution to 100 &i/ml) by injecting directly into the yolk through a hole drilled in the large end of the egg. One-half hour after the injection of thymidine, the embryos were removed from the yolk, and the wings were fixed in glutaraldehyde (2.5% glutaraldehyde, 0.1 M disodium phosphate, 0.2 M sucrose). The operated wing and the contralateral control wing were embedded in paraffin, and serial 5 ~1 sections were cut in cross section in cranial-caudal sequence. The paraffin was removed with xylene, the sections were hydrated through a graded series of alcohols to water and were dipped in Kodak NTB-3 photographic emulsion while still wet. They were allowed to drain and dry and were stored over Drierite at 4O. After 3-4 weeks, the emulsion was developed in Kodak D-19 (Caro and Tubergen, 1962), and the sections were stained with hematein by a double bath technique (Sea&, 1967).
EFFECT
OF
REMOVAL
OF
THE
APICAL
ECTODERMAL
RIDGE
467
The percentage of cells that had taken up tritiated thymidine was determined under oil at X 500 using a Wild binocular microscope equipped with a drawing tube. A square field was drawn on a piece of paper and viewed through the drawing tube. The field outlined an area on the slide approximately 0.1 mm on a side. This field usually contained between 150 and 250 nuclei. The number of labeled and unlabeled nuclei within the standard field was recorded. A nucleus was considered to be labeled if it had over it 3 or more silver grains more than would be expected from background. Four fields were counted in each wing: (1) the region immediately beneath the apical ectodermal ridge, (2) the dorsal proximal region, (3) the central proximal region, and (4) the ventral proximal region (Fig. 1). These regions were counted in every tenth section of the craniocaudal sequence through each wing. In order to reduce statistical scatter, the data from three sequential counted sections (e.g., the lOth, the ZOth, and the 30th) were averaged. This procedure gave average values for the percentage of cells in DNA synthesis in overlapping 150 ,J units in the wing for each region. RESULTS
Table 1 summarizes the experiments that were done. The apical ectodermal ridge was removed at stages 19 and 20, and the operated wing and the contralateral control wing were fixed at various times after the operation. The embryos received 10 &i of tritiated thymidine 30 minutes before the wings were fixed. After preparation of the wings as autoradiographs, the sections were examined to discover
STAGE
22+
FIG. 1. Position of the fields counted. SR the subridge region; mal region; D, the dorsal proximal region; V, the ventral proximal
C, the central region.
proxi-
468
JANNERS
TIME
OF FIXATION=
Within 10 hours, stage 22 or younger Stage Stage Total
19 20
o Time
SEARLS
TABLE 1 AND STAGE OF EMBRYO
10-20 hours , stage 22 to 24
10 9 19 of fixation
AND
after
removal
More than 30 hours, older than stage 26
20-30 hours, stage 24 to 26
7 6 13
is the time
AT FIXATION
4 II 15
5 3 8
Total
26 29 55
of the ridge.
sf”/;~‘l’~~“~ -I
JO-
*
F Z
0
A0
n A
i E a
0
CONTROL EXPERIMENTAL
20
t---CRANIO-CAUDALW 5~
SECTIONS
FIG. 2. The labeling index through a wing bud from which the apical ridge had been removed 4 hours previously during stage 19. The squares represent the values for the subridge area; the triangles values for the dorsal and ventral proximal areas; and the circles the values for the central proximal area.
whether the ridge had been completely removed and the extent of the damage to the underlying mesoderm. The success of the operation was recorded, and the percentage of the mesodermal cells that became labeled in each of the four regions was determined. Figure 2 shows the result obtained when the apical ectodermal ridge of the right wing bud was removed at stage 20, and the experimental wing and its contralateral control were fixed 4 hours later. The apical ectodermal ridge had been completely removed in this case, and no surgical damage to the underlying mesoderm could be detected. At the time of fixation, the ectoderm had not yet healed over the mesoblast and necrotic cells were present in the subridge mesoderm. In such cases, the exposed mesoderm was contracted (i.e., the limb was shorter than expected for the time of wing development
EFFECT
OF
REMOVAL
OF THE
APICAL
ECTODERMAL
RIDGE
469
and more nuclei than normal were present within the standard field in the subridge region). The percentage of labeled nuclei in each region of the wing was similar in the experimental wing (closed symbols) to the percentage in the control wing (open symbols); the variation in percentage of labeled nuclei being no greater than the variation previously observed between normal wings from embryos from the same stage (Janners and Searls, 1970). Figure 3 shows the result obtained when the ridge was removed at stage 20, and the experimental wing and its contralateral control wing were fixed 20.5 hours after the operation. The apical ectoderma1 ridge had been completely removed in this case; and if any damage had been done surgically to the underlying mesoderm, that damage could no longer be detected, By the time of fixation, the ectoderm had healed over the mesoblast to form a smooth continuous ectodermal covering. The absence of the apical ectodermal ridge had no effect on the percentage of mesodermal cells incorporating tritiated thymidine either at the apex of the wing or at the base of the wing. The region of low labeling index in the chondrogenic core of the wing (the central proximal region, midway between the cranial and caudal extremes of the wing) was of the same shape and width in the experimental wing as in the control wing. Fifty-five experimental wings were compared with their contra-
4Or
5
IO-
0
. o
l
.
.
0
0
0
b
l
0 A0
CONTROL
s n
E a
A 0
EXPERIMENTAL
0
f---
CRANIO-CAUDALd 5~ SECTIONS
FIG. 3. The labeling index through a wing bud from which the apical ridge had been removed 20.5 hours earlier during stage 19. The squares represent the values for the subridge region, the triangles values for the dorsal and ventral proximal regions, and the circles the values for the central proximal region.
470
JANNERS
AND
SEARLS
lateral control unoperated wings. Some of these showed only partial removal of the apical ectodermal ridge, and some showed surgical damage to the underlying mesoderm. In most of the cases where ridge removal was complete, only a few sections midway between the cranial and caudal extremes of the wing were counted. In cases where ridge removal was only partial, sections were counted both where the ridge had been removed and where some of the ridge remained. A complete determination of the labeling index in the four regions in every tenth section in the craniocaudal sequence of the two wings was completed for 10 pairs of wings. In no case did we find a difference in labeling index in any region of the wing greater than the difference found previously between normal wings from different embryos of the same stage (Janners and Searls, 1970). It is known that short incomplete limbs develop following removal of the apical ectodermal ridge (Saunders, 1948; Hampe, 1959; Barasa, 1960). Yet, removal of the apical ridge had no obvious effect on the percentage of cells incorporating tritiated thymidine, and therefore, presumably, no effect on the rate of cellular proliferation in the mesoderm. In order to determine the effect of removal of the apical ridge on the rate of limb outgrowth, the apical ridge was removed from 8 embryos when the width of the wing (distance from the body wall to the apex of the wing) was between 0.32 and 0.48 mm (stage 19 to stage 20). The width of the wing was measured again at the time of fixation and, if the wing developed for sufficient time before it was fixed, once or twice between the time of the operation and the time when it was fixed. The limbs were prepared as autoradiographs, the sections were examined to ensure that the ridge had been removed completely and that little or no surgical damage had been done to the apical mesoderm, and the labeling index in the four regions of the operated wing was compared with the labeling index in the four regions of the contralateral control wing. The rate of increase in width of the wing following removal of the apical ridge was compared with the rate of increase in width of the wing during normal development. The preparation of the standard curve for the rate of increase in the width of the wing during normal development has been described previously (Janners and Searls, 1970). The width of the wing at the time of the operation (0.32-0.48 mm) was placed at the proper time on the normal growth curve (16-20 hours of wing development), and subsequent measurements of wing width were plotted at the appropriate times thereafter. The operated
EFFECT
OF
REMOVAL
OF
THE
TIME
APICAL
IN
ECTODERMAL
RIDGE
471
HOURS
FIG. 4. Rate of increase of the width of the wing following ridge removal. is the standard curve for rate of increase of the width of the wing (Janners 1970). The points show the increase in the width of the operated wings.
The curve and Searls,
wings were found to cease outgrowth from the time of the operation, and sometimes to decrease slightly in width (Fig. 4). Operated wings did not resume outgrowth until about 12 hours after the operation. When they started to increase in width, the rate of increase in width in operated wings closely paralleled the rate of increase in width of normal wings. During the time when there was little increase in the width of the experimental wings (12 hours postoperatively), the labeling index was the same in the experimental wings as in the contralateral control wings. DISCUSSION
If the apical ectodermal ridge is removed at an early stage in limb development (stage 19 or stage 20), the limb that develops is approximately half the size of a normal limb, has proximal elements that are normal in size and morphology, but has no distal elements (Saunders, 1948; Hampe, 1959, Barasa, 1960). This result mimics the mutant condition Wingless in which the thickening of the ectoderm
472
JANNERS
AND
SEARLS
at the apex of the wing degenerates leaving a continuous smooth covering over the limb mesoblast, and limb outgrowth fails to occur (Zwilling, 1949). If a limb mesoblast is supplied with a second apical ectodermal ridge, a second outgrowth appears that gives rise to distal elements (Zwilling, 1956; Saunders and Gasseling, 1968). This result mimics the mutant condition Eudiplopodia in which a second ectodermal ridge arises on the dorsal surface of the limb and a duplication of distal elements occurs (Goetinck, 1964). If the ridge is wider than normal, the channel of mesodermal outgrowth is wider, and polydactylous limbs develop (Zwilling and Hansborough, 1956; Saunders and Gasseling, 1963; Abbott et al., 1960; Goetinck and Abbott, 1964). The distal morphology of the mature limb can be predicted from the size, shape, and location of the apical ectodermal ridge during early limb development. The role of the apical ectodermal ridge in limb outgrowth and limb morphogenesis has been subject to considerable discussion. The scheme of limb development espoused by Saunders and by Zwilling has been described by Zwilling (1968) as follows. “The apical ectodermal ridge, initially induced by mesoderm of the early limb field, evokes the outgrowth of the underlying mesoderm.” In contrast, Amprino and his associates have suggested that “all developmental factors reside in the mesoderm of the limb district and govern its outgrowth, the gradual individuation of the territories, and the determination and differentiation of the latter . . .” (Amprino, 1965). It seems to be generally agreed that the presence of the ridge is essential for limb development. However, Saunders and Zwilling have both stated that the ridge induces outgrowth of the limb. Amprino has suggested that “the arrest of limb development [following removal of the apical ridge] may depend on other factors-namely, the disruption of the growth pattern of the mesoderm following alterations of the local homeostasis which occur when the mesoderm is exposed abruptly to abnormal environmental conditions” (Amprino, 1965). We have found that surgical removal of the apical ectodermal ridge has no effect on the percentage of cells that become labeled during a short exposure to tritiated thymidine (the percentage of the cells in the DNA synthetic phase of the division cycle, S). It does not seem to us possible that the rate of cellular proliferation could decrease without producing a change in the percentage of cells in the S phase of the division cycle. If the length of the division cycle were
EFFECT
OF
REMOVAL
OF THE
APICAL
ECTODERMAL
RIDGE
473
to become longer without change in the labeling index, then the length of the S phase must become longer in proportion to the increase in length of the other phases of the cycle. If some of the cells were to cease dividing without change in the labeling index, then the length of the S phase must get longer in proportion to the number of cells that cease dividing while the length of the other phases of the division cycle does not change. However, it has been proposed that the length of the S phase of the division cycle is that portion of the cycle least likely to change in length (Cameron and Greulich, 1963; Cameron, 1964; Defendi and Manson, 1963). The length of the S phase of the division cycle in the chick has been measured to be between 4 and 6 hours from 3 days of embryonic development to 35 days post-hatching (Fujita, 1962: 4-6 hours; Cameron, 1964: 5-6 hours; Okazaki and Holtzer, 1965: 5 hours; Marchok and Herrmann, 1967: 5.85 hours; Mitriou et al., 1968: 4-5.5 hours; Janners and Searls, 1970: 5.6 hours). It seems to us unlikely that the S phase of the division cycle would change in length following removal of the apical ectodermal ridge. We conclude, therefore, that surgical removal of the apical ridge has no effect on the rate of cellular proliferation in the wing bud mesoderm. Immediately after removal of the apical ectodermal ridge, there was a time period of about 12 hours during which the wing bud did not increase in width (the distance from the body wall to the apex of the wing). After this period, the experimental wings increased in width at about the same rate as the normal wings. It has been suggested (Amprino, 1965) that the slowing of limb outgrowth following ridge removal is the result of surgical trauma to the mesoderm. A wave of necrosis in the subridge mesoderm after surgical removal of the ridge has been observed in the present experiments and has been extensively studied by Cairns (in preparation, we wish to thank Dr. John Cairns for the opportunity to examine a preliminary manuscript). Loss of mesodermal cells may explain how limb outgrowth can cease while the rate of cellular proliferation remains constant. If cells were lost as a result of the operation but the remaining cells continued to divide at a constant rate, a time interval would be required for sufficient new cells to be added to restore the original width of the wing and before outgrowth relative to the width of the wing at the time of the operation could become evident. Loss of cells may explain the slowing of wing outgrowth following surgical removal of the apical ectodermal ridge, but it does not ex-
474
JANNERS
AND
SEARLS
plain the resulting change in wing morphology. Limbs that develop following ridge removal are not only short, but they are also lacking in distal limb elements. (a) These distal deficiencies are probably not due to destruction of part of a mosaic system; it has been demonstrated that normal distal elements can form from proximal limb mesoderm (Zwilling, 1956, 1964; Saunders et al., 1957, 1959; Cairns, 1965; Hampe, 1959). (b) The distal deficiencies cannot be explained as the result of insufficient cells to support normal distal development. Barasa (1964) has demonstrated that up to 90% of the wing mesoderm of a stage 19 embryo may be surgically removed without preventing normal development of the operated wing. Barasa’s experiment has been repeated in this laboratory; it has been found that if 50% or more of the wing mesoderm was removed from the wing of a stage 19 embryo without damage to the apical ectodermal ridge, the wing undergoes a lag in outgrowth very similar to the lag observed after removal of the apical ridge (Searls, unpublished observations). However, a normal wing may develop after removal of mesoderm, a wing with distal deficiencies after removal of the ridge. The apical ectodermal ridge evidently has some effect on the differentiation of the cells immediately beneath the ridge. If the apical ectodermal ridge is removed from the wing at stage 19 or stage 20, the normal differentiative changes occur in the proximal regions of the wing during stage 22. The cells in the central proximal region of the wing become smaller and more rounded and incorporate increased amounts of ““S-sulfate into mucopolysaccharide whereas the cells in the dorsal and ventral proximal regions have increased amounts of cytoplasm and incorporate decreased amounts of YSsulfate into mucopolysaccharide. However, after ridge removal the cells in the subridge region, which normally do not change in cell morphology or in rate of mucopolysaccharide synthesis until stage 24, change during stage 22 so that they become similar to the cells in the dorsal and ventral proximal regions (Searls, 1965). It is not clear to us how an effect of the ridge on the differentiation of subridge mesenchyme cells could be responsible for distal development of the limb. More important, it is not clear to us how an effect of the ridge on the differentiation of subridge mesenchyme cells could explain why a secondary outgrowth appears after the grafting of an extra ridge. The results of the experiments reported here suggest that the ridge is not an outgrowth inducer in the sense that it induces or maintains cell division patterns or increased rates in the mesoblast. The ridge may be an outgrowth inducer in the sense that it permits
EFFECT
OF
REMOVAL
OF
THE
APICAL
ECTODERMAL
RIDGE
475
growth in the proper direction and thereby shapes the growing mesoblast. It cannot be denied that the apical ectodermal ridge is essential for normal limb development, but the precise role of the ridge remains obscure. SUMMARY
The apical ectodermal ridge of the embryonic chick limb has been demonstrated to be essential for normal limb outgrowth and development. However, the rate of cell division in the mesenchyme close to the ridge has been found to be similar to the rate of cell division in other regions of the limb. In the present experiments, the rate of cell division in the mesenchyme close to the ridge and in three other regions of the limb in normal limbs has been compared with the rate of cell division in the same four regions after removal of the ridge. It has been found that removal of the apical ectodermal ridge has no effect on the rate of cell division in any region of the limb. REFERENCES ABBOTT, U. K., TAYLOR, L. W., and ABPLANALP, H. (1960). Studies with Talpid2, an embryonic lethal of the fowl. J. Hered. 51, 195-202. AMPRINO, R. (1965). Aspects of limb morphogenesis in the chicken. In “Organogenesis” (R. L. DeHaan and H. Ursprung, eds.), pp. 255-281. Holt, New York. BARASA, A. (1960). Consequenza dell’ablazione della cresta ectodermica apicale sullo sviluppo dell’abbozzo dell’ala nell’embrione di ~0110. Riu. Biol. 52, 257-292. BAKASA, A. (1964). On the regulative capacity of the chick embryo limb bud. Experientia 20, 443. CAIRNS, J. M. (1965). Development of grafts from mouse embryos to the wing bud of the chick embryo. Deuelop. Biol. 12, 36-52. CAIRNS, J. M. (1966). Cell generation times and growth of the chick wing bud. Amer. Zool. 6, 328. CAMERON, I. L. (1964). Is the duration of DNA synthesis in somatic cells of mammals and birds a constant? J. Cell Biol. 20, 185. CAMERON, I. L., and GREULICH, R. C. (1963). Evidence for an essentially constant duration of DNA synthesis in renewing epithelia of the adult mouse. J. Cell Biol. 18, 31-40. CAMOSSO, M. V., JACOBELLI, V., and PAPPALETTERA, N. (1960). Ricerche descrittive e sperimentale sull’organogenesi dell’abbozzo dell’ala dell’embrione di polio. Riu. Biol. 52, 323-357. CARO, L. G., and TUBERGEN, R. P. (1962). High resolution autoradiography I. Methods. J. Cell Biol. 15, 173-188. DEFENDI and MANSON, L. A. (1963). Analysis of life cycle in mammalian cells. Nature (London) 198, 359-360. FUJITA, S. (1962). Kinetics of cell proliferation. Exp. Cell Res. 28: 52-60. GOETINCK, P. F. (1964). Studies on avian limb morphogenesis. II. Experiments with the polydactylous mutant Eudiplopodia. Deuelop. Biol. 10, 71-91.
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GOETINCK, P. F. and AFIBOT~, U. K. (1964). Studies in limb morphogenesis. I. Experiments with the polydactylous mutant Tulpidz. J. Exp. 2001. 154, 7-19. HAMBURGER, V., and HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HAMPE, A. (1959). Contribution a l’etude du developpement et de la regulation des deficiences et des excedents dans la patte de l’embryon de poulet. Arch. Anut. Micros. Morphol. Exp. 48, 345-478. JANNERS, M. Y., and SEARLS, R. L. (1970). Changes in rate of cellular proliferation during differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing. Develop. Biol. 23, 136-165. MARCHOK, A. C., and HERRMANN, H. (1967). Studies of muscle development. I. Changes in cell proliferation. Deuelop. Biol. 15, 129-155. MITRIOU, P., LANG, W., and MAUER, W. (1968). Autoradiographische Bestimmung des Mardierungindex der S-Phase und der Generationszeit einiger Zellarten von 2-35 Tage alten Kiiken. 2. Zelljorsch. Mikrosk. Amt. 90, 68-80. OKAZAKI, K., and HOLTZER, H. (1965). An analysis of myogenesis in vitro using fluorescein-labeled antimyosin. J. Histochem. Cytochem. 13, 726-739. SAUNDERS, J. W., JR. (1948). The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 108, 363-403. SAUNDERS, J. W., JR., and GASSELING, M. T. (1963). Transfilter propagation of apical ectoderm maintenance factor in the chick embryo wing bud. Develop. Biol. 7, 64-78. SAUNDERS, J. W., JR., and GASSELING, M. T. (1968). Ectodermal-mesenchymal interactions in the origin of limb symmetry. In “Epithelial Mesenchymal Interactions (R. Billingham, ed.), pp. 78-97. Williams and Wilkins, Baltimore, Maryland. SAUNDERS, J. W., JR., CAIRNS, J. W., and GASSELING, M. T. (1957). The role of the apical ridge of ectoderm in the differentiation of the morphological structure and inductive specificity of limb parts in the chick. J. Morphol. 101, 57-88. SAUNDERS, J. W., JR., GASSELING, M. T., and CAIRNS, J. W. (1959). The differentiation of prospective thigh mesoderm grafted beneath the apical ectodermal ridge of the wing bud in the chick embryo. Deuelop. Biol. 1, 281-301. SEARLS, R. L. (1965). An autoradiographic study of the uptake of S-35 sulfate during the differentiation of limb bud cartilage. Develop. Biol. 11, 155-168. SEARLS, R. L. (1967). The role of cell migration in the development of the embryonic chick limb bud. J. Exp. Zool. 166, 39-50. ZWILLING, E. (1949). The role of epithelial components in the developmental origin of the “wingless” syndrome of chick embryos. J. Exp. Zool. 111, 175-187. ZWILLING, E. (1956). Interaction between limb bud ectoderm and mesoderm in the chick embryo, II. Experimental limb duplication. J. Exp. Zool. 132, 173-187. ZWILLING, E. (1959). A modified chorioallantoic grafting procedure. Transplant. Bull. 6, 115-116. ZWILLING, E. (1961). Limb morphogenesis. Aduan. Morphogenesis 1, 301-330. Aca-
demic Press, New York. ZWILLING,
Develop.
E. (1964). Development Biol. 9, 20-37. E. (1968). Morphogenetic
of fragmented
and dissociated
limb
bud mesoderm.
phases in development. Develop. Biol. Suppl. 2, 184-207. ZWILLING, E., and HANSBOROUGH, L. A. (1956). Interaction between limb bud ectoderm and mesoderm in the chick embryo. III. Experiments with polydactylous limbs. J. Exp. Zool. 132, 219-239.
ZWILLING,