Chapter 9 Genetic Aspects of Skin and Limb Development*

CHAPTER 9

GENETIC ASPECTS OF

SKIN AND LIMB DEVELOPMENT* P . F . Goetinck DEPARTMENT OF ANIMAL GENETICS, STOFIRS AGRICULTURAL EXPERIMENT STATION, UNIVERSITY OF CONNECTICUT, STORRS, CONNECTICUT

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Differentiation of the Embryonic Chick Skin . . . . . . . . . . A. Description of Normal Feather and Scale Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Role of Dennis and Epidermis in Feather and Scale Differentiation .......................... C. Dermal-Epidermal Relationships in Mutants ...... D. Protein and RNA Synthesis during Feather Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Differentiation of the Embryonic Limb . . . . . . . . . . . . . . A. Description of the Early Stages of Limb Development in the Chick Embryo .................... B. The Role of the Ectoderin in Limb Outgrowth . . . C. Mesoderm-Ectoderm Interactions in Normal and Mutant Limb Development .................... IV. Summary and Concluding Remarks ................. References ......................................

253 254 254 255 258 261 263 263 264 270 277 281

1. Introduction

The differentiation of a number of embryonic structures is dependent on an interaction between the epithelial and mesenchymal components Scientific contribution No. 171 of the Agricultural Experiment Station, University of Connecticut. Contribution No. 129 of the Institute of Cellular Biology.

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of the particular system. Such interactions have been described in the development of the kidney (Grobstein, 1955), salivary gland (Grobstein, 1953), thymus ( Auerbach, 1960), pancreas ( Golosow and Grobstein, 1962), skin and feathers (Sengel, 1957; Saunders, 1958; Wessells, 1962; Rawles, 1963), and limbs (Zwilling, 1961; Amprino, 1963, 1965). The availability of a number of mutations which affect skin and limb development in the chick embryo makes these two systems particularly suitable for investigations of the genetic control of developmental processes. The present review will be limited to the effects of mutations on epitheliomesenchymal interactions in skin and limb development. The studies on mutant long-bone development in vitro (Wolff and Kieny, 1957, 1963; Kieny, 1962; Kieny and Abbott, 1962; Elmer and Pierro, 1964) will not be covered. II. Differentiation of the Embryonic Chick Skin

A. DESCRIPTION OF NORMALFEATHER AND SCALE DEVELOPMENT The purpose of the brief accounts in this and the following section is to provide the background for describing the genetic studies. Embryologically the skin of birds arises from two layers, the mesoderm and the ectoderm, and gives rise to a variety of regionally specific integumentary derivatives such as feathers, scales, beak, spurs, combs, wattles, and glands. The feathers are arranged in tracts which are separated by featherless areas. They first appear in the chick embryo anteriorly on the back between the sixth and seventh days of incubation. By the eleventh day all feather tracts are completed. Scales are first visible on the anterior part of the foot at about 11 days of incubation and at 12 days on the posterior part of the limb. Histologically, one of the first indications of feather and scale formation is seen as a condensation of the loosely arranged mesodermal cells into localized groupings immediately under the ectoderm (Fig. 1 ) . At the same time, or slightly before these dermal condensations become evident, the ectodermal cells associated with them elongate and slant inward at their distal end. Wessells (1965) has recently studied DNA synthesis in relation to feather germ formation. Cell density measurements and quantitative pulse-labeling experiments with tritiated thymidine in carefully staged skin suggest that the dermal condensations arise as a result of differential mitotic activity in the mesoderm. Before this stage, cells exhibiting mitotic activity and uptake of tritiated thymidine are

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distributed at random throughout the mesoderm. Once the dermal condensations have formed, their cells show no uptake of tritiated thymidine and no mitotic activity for about 20-30 hours. After this period of replicative inactivity, DNA synthesis starts again as outgrowth of the feather germ begins. During the formation of the feather one also observes differences in the distribution of several macromolecules. Thus, alkaline phosphatase appears in each dermal condensation of feathers and scales soon after this structure is formed, and at the same time the RNA concen-

FIG. 1. Transverse sections through middorsal skin of embryos ranging from 6% ( 1 ) to 8 days ( 4 ) of development. x 300.

tration increases rapidly in the basal cytoplasm of the epidermis overlying the dermal papilla (Hamilton, 1965; Thomson, 1964). Similarly, sulfated mucopolysaccharides are distributed uniformly throughout the dermis before germ formation and later become concentrated in distinct parts of the dermal condensations and the outgrowing feather (Sengel et al., 1962).

B. THEROLE OF DERMISAND EPIDERMIS IN FEATHER AND SCALE DIFFERENTIATION Interactions between the mesoderm and the epidennis have been shown to take place in skin differentiation by separating the embryonic skin into these component parts and recombining them with dermis or

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epidermis from other areas. The separation of the embryonic skin into its mesodermal and epidermal components can be achieved by incubating the skin in a dilute trypsin solution, After the appropriate recombinations, the composite skins are cultured either in vitro or on the chorioallantoic membrane of a chick embryo (Sengel, 1957, 1958; Rawles, 1963; Wessells, 1962, 1965). From these studies it is clear that the outcome of interactions between the dermis and epidermis differs depending on the age and site of origin of the particular components. For example, the epidermis in the tarsometatarsal region of the leg develops scales in response to interaction with the dermis of this area; it can be shown that tarsometatarsal dermis acquires the ability to induce scales only in later development, for, when tarsometatarsal dermis, derived from embryos after the thirteenth day of incubation, is combined with back epidermis from 5-82pday embryos, scales develop; however, when tarsometatarsal dermis from earlier embryos is used, it induces feather formation in back epidermis. On the other hand, the mesoderm of the spur primordium (located distally on the posterior part of the foot) already has a spur-inducing capacity in the 9-day embryo, since when combined with 5-8Q-day dorsal epidermis it causes it to form a spur. The dermis of the middorsum of =+-day embryos induces feathers when combined with tarsometatarsal epidermis from 9-12-day embryos. Beak dermis is strongly inductive, for it is capable of inducing beak formation in the middorsal epidermis of 8-8J-day embryos. On the basis of these results Rawles (1963) has classified the various sources of mesoderm according to "strength" of inductive activity, tarsometatarsal dermis being the weakest inducer, followed in increasing strength by middorsal and beak and spur mesoderm. The responsiveness of dorsal epidermis to mesodermal induction changes around 8-84 days of development ( Rawles, 1963). When this epidermis is recombined with tarsometatarsal mesoderm from 13-15-day embryos, it no longer forms scales, as it does when younger, but continues to differentiate in the feather direction. Presumably it has become by that time induced by its own dermis and hence is no longer responsive to the weak stimulus of the tarsometatarsal dermis. This restriction in response does not pertain to all mesodermal stimuli, however; as mentioned above, the developmental course of this epidermis can still be altered at this stage by combination with beak mesoderm, resulting in the formation of a beak. Further evidence for developmental lability of the epidermis and for the significance of the mesoderm in epidermal differentiation has been

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presented by McLoughlin ( 1961a,b). Recombinations of epidermis from 5-day embryonic limb buds with mesenchyme from proventriculus, gizzard, and heart of the same age resulted in each case in the formation of epithelial structures representative of the type of mesenchyme with which the epidermis was in contact. The nature of the interactions between the dermis and the epidermis in skin differentiation is not known. However, some of the dermal functions are indicated by experiments in which isolated epidermis was cultured in uiit~o.Under such conditions, the nuclei in the epidermis of 5-day limb buds show few mitotic figures ( McLoughlin, 1961a). Similarly, nuclei in isolated and cultured shank epidermis of ll-day-old chick embryos do not divide, as evidenced by failure to incorporate tritiated thymidine, and the cells in the basal layer lose their characteristic columnar shape (Dodson, 1963; Wessells, 1963). It is from this normally mitotically activr basal cell layer that the outermost cells are derived which synthesize keratin and become cornified. Both thymidine incorporation and the histodifferentiation of the epidermis can be restored by recombining it with dermis. Attempts at characterizing the conditions in the dermis responsible for maintaining the epidermis as an organized structure have met with some success. Dodson (1963) reported that dermis (either killed by repeated freezing and thawing or dissociated into single cells by trypsin) or collagen gels could sustain the orientation of the basal cells in epidermis cultured on an embryo extract-plasma clot. On the other hand, epidermis did not survive very long when supplied with dermis that was killed by freezing and thawing and subsequently trypsinized, or with heat-killed dermis. These results were confirmed by Wessells ( 1964), who used similar culture conditions. However, when a chemically defined medium was used, frozen-thawed dermis and tropocollagen failed to duplicate the effects obtained in the cultures on plasmaembryo extract medium. This led Wessells (1964) to investigate the effects of supplementing the defined medium with chicken plasma, chick embryo extract, and other large molecular substances. Of these only the embryo extract or a particulate fraction therefrom provided an effective supplement to the defined medium for maintaining basal cell orientation in the isolated epidermis cultured on a substratum of tropocollagen, frozen-thawed dermis, or millipore filter. The macromolecular fraction has been partially characterized and found to be nondializable, heat labile, and sensitive to proteolytic enzymes ( Wessells, 1964). Outgrowth of feather germs usually does not take place in culture

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when embryonic skin is explanted before 6-6i days of incubation (Sengel, 1958; Bell, 1964). After this stage normal feather development will take place in explants in culture. It may be significant that this stage corresponds in time to the formation of the dermal condensations. In fact, Wessells ( 1965 ) has shown by reciprocally exchanging epidermis and dermis from skin of predermal and postdermal condensation stages that only one of the two layers of the composite skin must have reached the dermal condensation stage in order for feather formation to take place in vitro. The percentage of feathers that develop in skin explants from embryos before the sixth day of incubation can be greatly increased by supplementing the defined medium with various proteins. Serum albumin is particularly suitable for this purpose (Bell, 1964). Sengel (1958) reported that early skin, which normally would not form feathers in vitro, will do so when cultured in association with neural tissue or when the medium is supplemented with a fraction of chicken brain. This active fraction was dialyzable, heat stable, and resistant to acid and alkaline hydrolysis (Sengel, 1964). Similarly, feathers failed to develop on the back of operated embryos from which the spinal cord had been removed very early in development. Therefore, the suggestion arises that neural tissue may play some role in feather differentiation and that its action may take place before the onset of the mesodermal-epidermal interactions described above. The nature of these neural-tissue or brain extract effects and their possible relationship to the action of the serum proteins mentioned above is not known. C. DERMAL-EPIDERMAL RELATIONSHIPSIN MUTANTS

Studies with normal embryonic skin have revealed that differentiation of the epidermis can be altered by recombining it with mesenchyme with different inductive capacities. That both induction and the response to it are under genetic control was shown by the analysis of a mutant in which ectodermal differentiation is absent and another in which the normal developmental pathway is altered. Birds which are homozygous for an autosomal recessive gene, “scaleless,” lack feathers in all but a few specific areas of the body. They also lack scales, footpads, and spurs ( Abbott and Asmundson, 1957). Reciprocal exchanges between leg-bud components of 39 -day scaleless and normal embryos resulted in expression of the scaleless phenotype only in composite limbs made up of scaleless epidermis and normal mesodermal

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constituents (Fig. 2). Leg buds composed of scaleless mesoderm and normal epidermis gave rise to normally scaled limbs (Goetinck and Abbott, 1960, 1963). These results were confirmed in in uitm experiments and extended to interchanges involving epidermis and dermis from areas which normally form feathers ( Sengel and Abbott, 1962, 1963). Thus the

FIG. 2 . Composite limbs which developed as flank-grafts. Left: limb composed of scaleless ectoderni and normal mesoderm. Although this limb is covered with some feathers, no scales are evident. Right: limb composed of scaleless mesoderm and normal ectoderni. x 2.8. (From Goetinck and Abbott, 1963. )

scaleless mutation affects only the epidermis in the interacting system in such a way that it is not competent to respond to the dermal induction. The scaleless mesoderm from both the shank and back areas reacts completely normally. Recently I have investigated the dermal-epidermal interactions in the limb buds of embryo of the Brahma breed by means of exchanges and recombinations of limb components ( Goetinck, 1966). This particular breed of chickens is characterized by having one to three rows of feathers

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along the fourth tarsometatarsus and on the fourth digit, leg areas which in other breeds are covered entirely by scales (Fig. 3 ) . This condition, known as ptilopody, is inherited as an autosomal dominant trait. In the embryo, feather germs appear on these leg areas first on the tenth day of development, scales on the eleventh day on the anterior part of the foot and on the twelfth day posteriorly. For the recombination experiments

FIG.3. Legs of 12-day-old embryos. Left: normal. Right: ptilopody. Note feathers along the fourth tarso-metatarsus in the mutant. x 3.5.

white leghorn embryos were used as a source of components of normally scaled limbs. Recombinations were made between 3-34-day-old hind limb bud parts of Brahma and of white leghorn embryos and the composite limb buds were grafted into the flanks of embryos and allowed to develop. Normal anteroposterior and dorsoventral relationships of the two components were maintained in recombining them into composite limb buds. It was first found that limb buds composed of Brahma mesoderm and of white leghorn epidermis gave rise to limbs with feathers along the fourth tarsometatarsus and on digit IV. However, when the reciprocal recombination-Brahma epidermis with white leghorn mesoderm-was

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analyzed, it too resulted in the formation of limbs with feathers distributed as in the intact Brahma. Therefore, unlike the scaleless mutation which affects only the epidermis, the ptilopody genotype affects both the limb mesoderm and limb epidermis, endowing both with feather-forming capacity in response to the white leghorn counterpart. In order to establish whether in the Brahma mutant the epidermis or the mesoderm determines the characteristic topographical localization of the feathers along the fourth tarsometatarsus, Brahma limb epidermis was recombined with a Brahma limb mesoderm with its anteroposterior axis reversed. The composite limbs had feathers along the fourth tarsometatarsus and on digit IV and the resulting feather pattern clearly demonstrated that it is the mesoderm which determines the localization of the feathers. These results are analogous to those presented by Zwilling (1959) from his studies on chicken-duck limb chimeras produced by combination of limb components. In this case duck-like interdigital webbing developed in limbs composed of chicken mesoderm and duck epidermis as well as in those made up of duck mesoderm and chicken epidermis. Zwilling suggested that a reinforcing mechanism for the development of webbing in the duck limb may have evolved through natural selection. It is possible that a similar reinforcing mechanism may have been brought about by artificial selection for ptilopody. The Brahma is a fancy breed of poultry selected continuously for specific breed characteristics; feathered shanks is one of the “desirable” characteristics in the Brahma breed. D. PROTEINAND RNA SYNTHESIS DURING FEATHER DEVELOPMENT The concept of differential gene activation as a fundamental aspect of histodifferentiation was formulated by Morgan (1934). We have since learned that genes determine the sequence of amino acids in proteins and that this step is mediated through RNA. Since different tissues of the same genotype contain a different array of proteins which appear at various times during development, the presence or absence of any protein in a cell is a reflection of the activated or repressed state of specific genes. With respect to embryonic skin we have already mentioned that alkaline phosphatase appears in the dermal condensation immediately after this structure is formed. Hamilton and his associates (see Hamilton, 1965, for a review) have stressed the importance of this enzyme for the continued growth and differentiation of the feather. The addition to the culture medium of compounds which inhibit alkaline phosphatase has

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been shown to arrest feather development. Furthermore, the addition of the enzyme to culture medium will stimulate feather outgrowth and increase the RNA in the basal parts of the cells in the epidermis. Hamilton has reported that this RNA is located in the mitochondria and that respiratory and mitochondria1 inhibitors will inhibit feather formation with a concomitant reduction in RNA content in the basal parts of the epidermaI cells. Various analogs of naturally occurring bases of RNA have also been shown to inhibit feather formation, and the results of their action may be noticed either in the epidermis or in the mesoderm. Other methods for the investigation of biochemical events which take place at the time of induction and during feather development have been used by Bell and his associates. Using double diffusion tests in agar, they detected three new antigens in 6-day-old skin ( Ben-Or and Bell, 1965). Of these three, one is skin-specific and two are stage-specific in that their presence could be established in other tissues by absorbing the antiserum with extracts from other organs. Since the time at which these antigens are first detected corresponds to the developmental stage at which formation of the dermal condensations is observed, it would be of interest to determine their localization, and particularly that of the skin-specific antigen, in the developing feather germ. Two additional stage-specific antigens become apparent at about 11 to 12 days of incubation, and another skin-specific antigen is found in 13-day embryos. None of these antigens has been identified; presumably they are new proteins which are being synthesized at the time of detection and their appearance therefore reflects the genetic activity of these tissues at a specific time of development. However, the requirement for messenger RNA as an intermediate between DNA and a protein to be synthesized does not permit one to use the detection of new proteins as a criterion for establishing the exact time of gene activation. Indeed, control mechanisms at the translation level have been postulated in the developing feather. The optical density profile of polysomes from embryonic skin and feathers in a sucrose density gradient remains unchanged between 5+ and 13 days of incubation, and it is different from that of other chick tissues examined in that it shows a sharp polysome peak which consists of four ribosomes and which has a sedimentation constant of 158 S (Bell, 1964; Scott and Bell, 1964). In skin from 9-13-day-old embryos this 158 S polysome population is characterized by and different from others in the skin in its resistance to ribonuclease. Of particular significance is the fact that no protein synthesis can be associated with the 158 S fraction up to 14 days. Beginning with

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14 days, however, the 158 S material becomes sensitive to ribonuclease and it is now activated to make protein for the first time. Electron micrographs of these inactive ribonuclease-sensitive polysomes show them to be arranged in tightly packed symmetrical squares, in contrast to the 158 S polysomes of 14-day feathers, which are strung out in a chain (Bell et al., 1965). At 14 days the polysome profile also changes and polysomes consisting of five and six ribosomes become evident in addition to the four-ribosome polysomes. It has been suggested that the inactive 158 S polysomes, whose messenger RNA is somehow protected against ribonuclease, are precursors of the five- and six-ribosome polysomes and that the appearance of the latter may indicate either that there is random attachment of ribosomes during translation of a single species of messenger RNA or, alternatively, that several species of messenger RNA may be found in the inactive 158 S material (Humphreys et aZ., 1964). It seems therefore that as early as 9 days of incubation messenger RNA is being synthesized in the embryonic skin which is being translated into protein (or proteins) only at 14 days. It should be stressed that a number of events take place at this stage of development; as mentioned earlier a new skin-specific antigen becomes apparent and X-ray diffraction studies of embryonic skin first show the adult pattern of p-keratin at about 14 days of incubation (Bell and Thathachari, 1963). Although it is very possible that all events which take place around 13 and 14 days are causally related, this very important question remains to be established. 111. Differentiation of the Embryonic Limb

A. DESCRIPTION OF THE EARLYSTAGESOF LIMB DEVELOPMENT IN THE CHICKEMBRYO The two main components of the embryonic limb are an internal mass of mesodermal cells, the mesoblast, which progressively gives rise to the skeleton, the muscles, and the dermis; and the epidermal jacket from which develops the integument of the limb with its differentiations (feathers, scales, spurs, etc.). The first indication of a limb bud is the persistent thickening of a longitudinal fold of the body wall lateral to the somites. As the mesoderm thickens at the limb sites, the overlying epidermis changes distally from a cuboidal to a columnar type of epithelium. At stage 18 (stages of Hamburger and Hamilton, 1951) the epithelium covering the distal tip of the limb bud forms a nipple-like crest or ridge of columnar cells, the apical epidermal ridge ( AER). Viewed in a

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cross section the cells of the AER are radially oriented toward a small sinus which lies at the immediate base of the AER (Fig. 4). Between stages 19 and 20 the limb becomes asymmetrical owing to a more rapid development of the postaxial side. The dorsal side of the bud is more convex than the ventral side, and the AER is located more ventrally to the midline. From stages 21 to 22, the asymmetries become even more pronounced and the limb bud develops most rapidly postaxially. Asso-

FIG.4. Cross section of a limb bud from a normal embryo (stage 19) showing the apical epidermal ridge. x 200.

ciated with the postaxial growth is a much thicker AER. At stage 24 the outline of the foot-paddle becomes asymmetrical, being more developed posteriorly than anteriorly. By stage 27 the digits may be distinguished and the major components of the leg are clearly recognizable. The AER, though somewhat flattened, is still present, whereas the rest of the epidermis remains cuboidal. B. THE ROLEOF

THE

ECTODERM IN LIMBOUTGROWTH

The importance of the ectoderm and more specifically the AER in the development of the limb of the chick embryo was first shown by Saunders

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(1948), who found that after the surgical removal of the AER the limb developed with its distal parts missing. The younger the embryos at the time of AER removal the greater was the deficiency of the terminal parts. Using similar surgical methods for the removal of the AER, Amprino and Camosso (1955a,b) confirmed these results. Of particular interest are the studies on wing development in a wingless mutation in the chicken ( Zwilling, 1919). In embryos homozygous for the recessive “wingless” gene wing buds develop through the third day of incubation, at which time their further development ceases and no distal parts are formed. Histological examinations showed degeneration of the AER of the wings. The suggested requirement for AER in the outgrowth and development of the distal limb parts received support from further studies. Zwilling (1955) removed the AER from extirpated limb buds by incubating them in a solution of the chelating compound disodium ethylenediaminetetraacetate (EDTA), He obtained no distal outgrowth following grafting of the denuded mesoblasts into the flank of embryos. Distal outgrowth is obtained from EDTA-denuded mesoblasts only if they are recovered with an AER (Zwilling, 1955; Gasseling and Saunders, 1961) or if the mesoblasts are deliberately (Bell et al., 1962) or accidentally contaminated with ectoderm. Goetinck and Abbott (1963) investigated the possibility of incomplete removal of ectoderm from limb buds of normally scaled stock after EDTA incubation by transplanting the mesoblasts into the flank of scaleless hosts. Since the development of the phenotype of scaleless ectoderm is autonomous even when it is in contact with normal mesoderm, this combination of phenotypically different grafts and hosts made it possible to ascertain the source of ectoderm in case distal structures developed from the presumably denuded mesoblasts. The results from these studies were clear. Of the grafts which could be recovered, 92y0 developed no distal structures. These grafts were covered with host (scaleless) ectoderm. The other 8% developed distal structures, but in each case the terminal parts of the grafts were covered with scales (Fig. 5). The use of scaleless ectoderm as a marker therefore clearly showed that the outgrowth in the exceptional cases was associated with contamination of the mesoblasts with donor ectoderm. HampC (1956, 1959) studied the development of limb buds from which the prospective area (mesoblast and AER) of the tarsometatarsals was excised. The most distal structure which developed in such limbs was the tibia; however, when an AER was placed back on the cut surface of the stump, limbs complete with metatarsals and phalanges developed. It ap-

FIG.5 ( a ) A limb which developed from an EDTA-denuded mesoblast. The transplant stock. The host embryo is scaleless. Incomplete removal of the epidermis from the transplanted on the terminal part of the limb. The arrow points to the line of demarcation of scaled and view of the grafted limb at the line of demarcation of scaled and scaleless tissues (arrow). 1963.)

was derived from normally scaled mesoblast is indicated by the scales scaleless tissues. x 3. ( b ) Enlarged x 20. (From Goetinck and Abbott,

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peared as if the AER induced the formation of the distal structures from more proximal parts of the mesoblast. Additional evidence for an inductive role of the AER epidermis came from the laboratory of Saunders (Saunders et al., 1955, 1957, 1959b). Blocks of mesoblast were transplanted from the prospective thigh region (i.e., proximal part of the leg bud) to various regions of the wing bud. Only when grafted in contact with the AER of the wing bud did this prospective thigh tissue give rise to distal leg structures, indicating that the AER influenced the development of the grafts. Using focused ultrasound to remove the AER, Bell et al. (1959) in an early report stated that 19% of the denuded mesoblasts developed distal structures following grafting to the flank or in the coelomic cavity of 3day host embryos. In a more recent publication (Bell et al., 1962) only 2% of the limb buds denuded by ultrasonication gave rise to distal structures, presumably owing to a more effective removal of the AER. However, another possibility had previously been raised; Bell et ul., (1959) made the observation that not only the epidermis but in some cases also the epidermal basement membrane breaks down as a result of ultrasonication and suggested that the absence of this structure may be a prerequisite for distal outgrowth of mesoblasts denuded of the AER. While this explanation may hold for mesoblasts deprived of basement membrane by ultrasonication, it is not more generally applicable. When the basement membrane of EDTA-denuded limb buds is removed with collagenase, the denuded mesoblasts do not develop distal structures when grown as flank grafts (Goetinck and Abbott, 1963). However, when such collagenase-treated mesoblasts are recovered with limb epidermis normal Iimbs develop. A different point of view toward the role of AER has been advanced by Amprino and Camosso (1955a,b). They too found that no distal outgrowth takes place after the surgical excision of the AER. They found, however, that distal outgrowth can take place if, in addition to the AER, a few cell layers of the underlying mesoderm are also removed. These authors proposed that the amount of distal outgrowth obtained is inversely related to the amount of cell death caused in the distal mesoderm. Although they do not view the epidermis as an entirely passive component of the limb, they do not ascribe an inductive role to the AER (Amprino, 1965). Amprino and Camosso (195%) have done carbon marking experiments which indicate that the epidermis grows distally more rapidly relative to the mesoderm and they have suggested that the AER

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may simply be an area where the ectodermal cells pile up, and not necessarily a site of specific induction. More recently, Searls and Zwilling (1964) found that a new AER may regenerate from residual epidermis; they cautioned that a variety of previous experiments in which distal limb parts developed following removal of the AER and presumably in

FIG. 6. Right leg of a 9G-day polydactylous embryo. The duplication involves only digit I (arrow). x 8.

its absence, may have to be reconsidered in the light of this finding. For a complete analysis of the two points of view on the role of the AER in limb development the reader is referred to the reviews by Zwilling (1961) and by Amprino ( 1965). While in the absence of AER distal limb parts fail to develop, grafting of an AER on each lateral surface of an EDTA-denuded mesoblast results in the formation of distal limb parts in two different planes (Zwilling, 1956b). A correspondence between AER and distal limb development

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is abundantly suggested by descriptive studies in four mutants which affect the normal morphology of the limb. Thus, embryos heterozygous

or homozygous for “dominant polydactyly” (Fig. 6 ) or its allele “duplicate” have a more extensive AER in the preaxial part of the limb, i.e., the site of supernumerary digit formation (Zwilling and Hansborough, 1956). Embryos homozygous for two autosomal recessive genes of different origin, “talpid“’ (Abbott et al., 1960) and “talpid”” (Ede and Kelly,

FIG.7. Left leg of dactylous foot. x 10.

it

B%-cIay talpid embryo. Seven digits are evident in this syn-

1964), have seven to eight syndactylous digits per foot. Of these, none can be identified as normal digits (Fig. 7 ) . During development the talpid limb buds have a very extensive AER covering the greatly enlarged distal area of the entire limb paddle. The area covered by the AER in talpid’ leg buds increases significantly over that of normal embryos and by stage 25 is 50% more extensive than in normal siblings ( Goetinck and Abbott, 1964). As mentioned earlier, embryos homozygous for two recessive wingless mutations of different origin have been examined (Fig. 8); in these the AER flattens out on the third day of development with a concomitant cessation of distal limb outgrowth ( Zwilling, 1949, 1956c) . The autosomal recessive trait, “eudiplopodia”

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(Rosenblatt et al., 1959), is characterized by having the supernumerary digits situated in a second plane above the normal toe complement, which is always present (Fig. 9 ) . The outgrowth of these digits is preceded by the formation of a secondary AER (Fig. 10) on the dorsal surface of the limb bud (Goetinck, 1964). The int,eractions between mesoblast and epidermis in these mutants will be the subject of the following section.

FIG. 8. Cross section through a 4-day wingless limb bud. In contrast to normal limb buds (see Fig. 4) the apical epidermal ridge is lacking in the mutant. x 300.

Finally, it might be pointed out that the morphological and functional differences between the AER and the rest of the limb epidermis are paralleled by sharply delineated metabolic activities in the AER, as revealed in histochemical studies. Thus, Hinrichsen (1956) reported a high concentration of ribonucleic acid in the AER of the mouse embryo, and has interpreted this to be indicative of active protein synthesis in this structure. Furthermore McAlpine (1956) and Milaire (1956, 1963, 1965) have demonstrated a high level of alkaline phosphatase in the AER in several mammalian species as well as in the chick embryo. C. MESODERM-E~ODERM INTERACTIONS IN NORMAL AND MUTANT LIMB DEVELOPMENT

In the preceding section results from several laboratories have been presented which have led to a wide, although not unanimous, acceptance of the inductive role of the AER. Some of the most cogent evidence bear-

FIG.9 ( a ) Left leg of a 9%-day eudiplopod embryo. The duplications of the digits are in two planes. of a 9%-day normal embryo. x 10. (From Goetinck, 1964.)

x

10. ( b ) Left leg

FIG. 10. Cross sections of eudiplopod hind limb buds, ( a ) Left limb, stage 24; the arrow points to the second epidermal ridge in the dorsal ectoderm. x 200. ( b ) Right limb, stage 27; the dorsal outgrowth seen in this section would have formed the supernumerary digits. The normal toe complement would have formed from the ventral outgrowth. Both areas of outgrowth are covered by an apical epidermal ridge. x 120. (From Goetinck, 1964.)

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ing on this problem comes from tissue recombination experiments using normal and mutant limb components. The first investigations on the interaction between mesoblast and limb epidermis in mutants were carried out by Zwilling ( 1 9 5 6 ~ and ) by Zwilling and Hansborough (1956). From these studies they advanced a hypothesis which postulated that the function of the AER, on which proximodistal outgrowth of the limb is dependent, is not autonomous but that it, in turn, depends for its continued activity on a factor in the mesoblast; they referred to the latter as the apical ectoderm maintenance factor ( AEMF). The hypothesis was based on an analysis of the results obtained in reciprocal recombinations between mutant and normal limb-bud components. Limbs combined from dominant polydactylous and normal components gave rise to the polydactylous phenotype only when the mesoblast was of polydactylous origin. The normal epidermis in this combination developed a more extensive AER in the preaxial area in addition to that found normally in the postaxial region. From these results it was proposed that the excessive preaxial AER developed in response to stimulation of the epidermis in this area by a mesodermal factor, the AEMF; it was further suggested that in the normal mesoblast AEMF activity is distributed only postaxially, i.e., in the direction of the normal AER, but that in the polydactylous mutation its distribution is extended also into the preaxial region, resulting in excessive AER formation. Alternatively, it may be that the cells in preaxial part of the mesoblast of polydactylous, but not of normal embryos, are capable of synthesizing the hypothetical AEMF. While the end result of these two possibilities would be the same, the mechanisms at the cellular level would be completely different. As we shall see later on, under certain experimental conditions the preaxial portion of the normal mesoblast may acquire and stabilize the AEMF (Saunders and Gasseling, 1963). The asymmetrical distribution of the AEMF activity is further supported by experiments in which the thicker portions of several AERs are placed in tandem along the distal edge of an EDTA-denuded mesoblast. In such experiments no supernumerary digits were obtained; rather, the ectoderm conformed to the normal asymmetrical pattern imposed by the mesoderm (Zwilling, 1956b). A situation anaIogous to that described for dominant polydactyly has been seen in the talpid' mutant (Goetinck and Abbott, 1964). In recombinations between limb-bud components of this mutant and of normal

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embryos, development of the talpid phenotype is associated with the talpid mesoblast, regardless of the genotype of the limb epidermis with which it is covered. In these two mutants, therefore, the genotype of the mesoblast determines the formation of the polydactylous limbs, and when normal epidermis is recombined with the mutant mesoblast a more extensive AER develops in the recombinant. On the other hand, the epidermis of these two mutants combined with normal mesoblast conforms to the normal pattern, although if left on the mutant mesoblasts the epidermis would have formed extra AER preaxially in the dominant pdydactylous limb and an extended AER over the whole limb paddle in talpid2 embryos. To account for the abnormal development of the limb in talpid2 it was proposed that the distribution of AEMF activity was affected in these embryos (Goetinck and Abbott, 1964); however, the situation in this mutant is probably different from and much more complex than in dominant polydactyly. As already mentioned, the entire talpid embryo is extremely abnormal; no digits can be recognized as normal, whereas in the dominant polydactylous limbs only the preaxial part of the foot is affected. It is therefore conceivable that an abnormal distribution of the hypothesized AEMF in talpid? embryos is a secondary aspect of a much more general developmental abnormality. That the postulated AEMF activity can also be lost as the result of mutations has been concluded from mesoblast-epidermis recombinations of limb-bud parts of “wingless” mutants (Zwilling, 1 9 5 6 ~ )Limbs . composed of mesoblasts of wingless embryos and of normal epidermis grow distally only somewhat more than intact limbs of wingless embryos. In combination with the mutant mesoblast the normal AER was not maintained in an active state; it later regressed and correspondingly limb outgrowth stopped. The combination of wingless epidermis and normal mesoblast also did not result in distal outgrowth, contrary to expectations that the postulated AEMF of the normal mesoblast might stimulate an AER in the wingless epidermis. Zwilling ( 1 9 5 6 ~ )proposed that either the epidermis at the stage tested had been irreversibIy affected by its association with the mutant mesoblast or that the wingless mutation affects both components of the developing limb bud. Recombinations with wingless epidermis from younger embryos could clarify this point. On the basis of the original work it was thought that only the epidermis of the distal edge of the limb bud, i.e., the normal AER, was capable of participation in distal limb outgrowth. Several cases are now known in which epidermis from other sites enables the formation of distal limb

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elements. The first was reported by Kieny (1960), who showed that the mesoblast provides the initial stimulus for the subsequent interactions between the two limb components in the chick, as it does in amphibians (Tschumi, 1957). Presumptive limb mesoderm from very young embryos (stages 15 to 18) was grafted under the flank epidermis of early embryos (stages 12 to 14) in an area which normally forms no limbs; it stimulated the formation of an ectopic AER and subsequently a complete limb developed. Recently Searls and Zwilling (1964) reported that terminal parts can develop when fragmented limb mesoblast is placed in the epidermal pouch of a tail bud. Again distal outgrowth was associated with the presence of an AER. The third case of exceptional limb outgrowth is found in the eudiplopodia mutant (Goetinck, 1964). In this mutant a supernumerary AER develops on the dorsal epidermis of the leg buds. In normal limbs this dorsal epidermis never develops an AER even when brought experimentally in contact with the distal tip of a normal mesoblast (Zwilling, 1956a). When the eudiplopod epidermis is combined with a normal mesoblast it forms a secondary AER and under its influence extra digits develop, but the combination of mutant mesoblast with normal epidermis results in a completely normal limb. It seems, therefore, that in this mutant in addition to the norma1 AER some of the dorsal leg epidermis becomes responsive at a specific stage of development to the stimulus for AER formation, resulting in the appearance of an additional AER and subsequent duplications of the distal leg parts. This mutant therefore lends strong support to the view that the AER acts as a distal limb outgrowth inductor. How this inductive interaction takes place in the eudiplopod limb or for that matter in normal development is not known. However, one might consider the dorsal ectoderm, which in normal limb buds as a rule does not form an AER, as being repressed for this event, without specifying at which of the many possible activity levels this repression could take place. Is it at the genomic level, or may we consider the basement membrane as a barrier to the interactions between the epidermis and the mesoblast? In the latter case, we would postulate a breakdown of the basement membrane in the dorsal surface of the eudiplopod limb bud at the site of AER formation. Such changes in the basement membrane were proposed by Balinsky (1956), who found in Amblystoma that the disappearance of the epidermal basement membrane precedes the development of supernumerary limbs.

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Amprino and Camosso (1958a,c, 1959; Amprino, 1965) and Saunders (Saunders et al., 1958, 1959a; Saunders and Gasseling, 1959, 1963; Gasseling and Saunders, 1960a,b) reported that duplicate hand parts developed when the apical zone ( AER with the mesoblast adjacent to it) of a 3-4-day wing is severed and replaced in reversed anteroposterior orientation. Amprino and Camosso have interpreted these results in terms of differences in the developmental states of the different regions of the mesoblast as indicating that the development of the less advanced distal parts is governed by the already more developed proximal tissues. Saunders, on the other hand, interprets the duplication of the hand parts in terms of the asymmetrical distribution of the AEMF. The reversal of the anteroposterior axis of the apical zone would result in two centers of AEMF activity: one preaxially in the reoriented distal part and one postaxially in the stump. Both centers of activity would have their effect through the AER, which in turn would induce the formation of duplicate parts. Indeed a more extensive AER becomes evident in the preaxial part of the reversed apical zone. Saunders and Gasseling (1963) have also shown that direct cell contact is not necessary for the transmission of AEMF activity, for duplicate hand parts will form when a strip of a millipore filter is inserted between the postaxial part of the stump and the preaxial part of the limb in a mediolateral direction but not, as judged from the spatial restrictions of the AEMF activity in the normal limb, in an anteroposterior direction. With respect to transmission of the postulated AEMF activity, normal preaxial mesoblast resembles wingless mesoblast in the following sense: When the latter is covered with a normal AER and grafted to the dorsal surface of a normal wing bud, the mutant mesoblast will elongate and maintain the AER as long as the graft is situated distally (Zwilling, cited in Saunders and Gasseling, 1963). However, as the graft shifts more proximally, owing to the elongation of the host wing, elongation of the mutant mesoderm ceases; this was attributed to the absence of AEMF activity in the more proximal areas of the older limb buds (Saunders et al., 1959a; Saunders and Gasseling, 1963). Whereas both wingless and normal preaxial mesoderm can transmit AEMF activity, the necessity of a continuous supply of this activity for the outgrowth of wingless mesoderm makes this mesoderm different from the preaxial mesoderm of the normal limb. AEMF activity can become stabilized in the latter in less than 12 hours, as shown from the duplications obtained when the normal anteroposterior position of the reversed apical zone is reestablished after

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10-12 hours (Saunders and Gasseling, 1963). The ability to transmit AEMF activity, however, is a unique property of limb mesoderm, whether from normal preaxial limb regions or from wingless wing buds, in that a normal AER quickly degenerates in association with nonlimb mesoderm even when the latter is backed by normal wing mesoblast ( Zwilling, 1964 ) . IV. Summary and Concluding Remarks

Skin and limb development depend on interactions between the epithelial and mesenchymal components which make up these structures. These two developmental systems are unique in that several mutations are known which upset the normal interactions and thus cause these structures to deviate from their normal developmental pathways. The mutations, their description, and the results of recombination experiments between mutant and normal tissues are summarized in Table I. The analyses of the mutants to date have shown that in some-ptilopody, interdigital webbing, and possibly wingless-both the mesoderm and the ectoderm can be affected through mutation; in others-scaleless, dominant polydactyly, talpid", and eudiplopodia-the effects of the mutations are observed only in one of the two components. Furthermore, the effects of the mutations can be observed through either a loss or a gain of properties which lead to abnormal development. While one would like to describe the action of these mutations in terms of specific macromolecules, the information presently available limits us to the detection of their effects at the tissue level. With the rapid advances in biochemistry it may be expected that we will soon be able to describe these developmental pathways in molecular terms and that the use of mutants will continue to be useful in the pursuit of these endeavors. It must be stressed, however, that the manifestation of a certain mutant phenotype is not determined by the mere presence or absence of a mutant gene or a pair of mutant genes but depends on an integrated action of the particular genes with the whole genotype of the developing individual. This is demonstrated by the variation observed in the expression of the phenotype in mutant individuals. For example, scaleless birds show a small amount of feathers in the later-appearing feather tracts, and the eudiplopod phenotype may range in its expression from a single duplicated toenail to a complete extra foot located on top of the normal toe complement. Furthermore, a gene or pair of mutant genes may express itself in one way in one genetic background and differently in another.

SUMMARY OF RECOMBINATIONS BETWEEN

TABLE I NORMAL AND MUTANT COMPONENTS OF EMBRYONIC SKINAND LIMB Source of

Description

Mesoderm

Ectoderm

Phenotype of composite organ

Scaleless

Autosomal recessive trait; scaleless birds lack scales, feathers, footpads, and spurs

Normal Scaleless

Scaleless Normal

Scaleless Normal

Ptilopody

Autosomal dominant trait; one to three rows of feathers along the 4th tarsometatarus and on the 4th digit replace the scales normally found

Normal Ptilopody

Ptilopody Normal

Ptilopody Ptilopody

Interdigital webbing characteristic of ducks

Duck Chick

Chick Duck

Webbed feet Webbedfeet

Autosomal dominant trait; duplications of the preaxial parts of the limbs; extension of the normal AER preaxially

Normal Polydactyly

Polydactyly Normal

Normal Polydactyly

Wingless

Autosomal recessive trait; wings and often the legs cease to develop; AER dcgencrates

Normal Wingless

Wingless Normal

Wingless Wingless

Talpid2

Autosomal recessive trait; of the 7 or 8 syndactylous digits per foot, none can be recognized as normal; the enlarged distal end of the limbs is covered with an extensive AER

Normal Ta1pid2

Talpidz Normal

Normal Talpidp

Phenotype

to

3

Skin

Webbing Limb Dominant polydactyly and its allele duplicate

td ?1 0

0 2

TABLE 1 (Continued) Source of Phenotype Eudiplopodia

Description Autosomal recessive trait; the development of the supernumerary digits, which are located in a second plane above the normal toe complement, is preceded by the formation of a secondary AER in the dorsal ectodenn

Mesoderm

Ectoderm

Normal Eudiplopodia

Eudiplopodia Normal

z.

v,

Phenotype of composite organ Eudiplopodia Normal

;4

9 i Z 4

8

km Y

280

P. F. COETINCK

Landauer ( 1948) has reported differences in the phenotypic expression of dominant polydactyly by selection. Similarly, Taylor et al. (1959) observed, after outcrossing carriers of the recessive lethal gene diplopodia (Taylor and Gunns, 1947) to an unrelated stock, that the offspring no longer segregated in a normal 3:l phenotypic ratio. Through selection they were successful in establishing three lines which differed significantly in their segregation ratios. One line was restored to the 3:l ratio characteristic of the original stock, and this level could not be exceeded by further selection. The line selected for low incidence of diplopodia gave rise to individuals producing only 2% of diplopod phenotypes in their offspring. The third line, intermediate in penetrance, fluctuated between 17 and 8% of mutant embryos in their offspring. Furthermore, as penetrance was reduced in the low-incidence line the expressivity of affected embryos was attenuated ( Abbott, 1959). From all considerations it seems that what is observed in the low-incidence line is a complete masking of the diplopodia phenotype in individuals homozygous for the once lethal gene. Selection has also been successful in increasing the number of feathers in homozygous scaleless chickens, but the selection for high feather number has had no effect on scale formation (Abbott, 1965). Several examples are known in which mutations present in a latent form in certain breeds have been discovered by outcrossing these breeds to stocks with different genetic histories (Landauer, 1965). These observations have been explained in terms of Waddington’s (1957) concept which formulates that the uniformity in the phenotypic expression of individuals in a population is the result of a buffering or canalization by natural selection of the many interacting developmental pathways, in spite of the genetic variability between the individuals of that population. The masking of mutants by selection is then visualized as restoring the canalization of the original or other related developmental pathways, the unmasking of mutants by outcrossing as a breaking up of the “epigenetic armor” (Landauer, 1965), which most certainly had been brought about by artificial selection in the specific breeds examined. While observations of this kind will by themselves surely not lead to an understanding of the primary gene events which control normal development, they have been presented here to emphasize the complexity of this control and to stress the importance of establishing and identifying the overall genotype in embryological and biochemical analyses in which specific mutants are used for studying the genetic control of developmental processes.

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