Fine structural analysis of limb development in the wingless mutant chick embryo

Normal limb development is dependent on reciprocal interactions between the ectoderm and mesoderm (Zwilling, 1974). Saunders (1948) demonstrated the importance of the apical ectodermal ridge (AER) for continued distal development of the limb bud, and Zwilling & Hansborough (1956) later demonstrated that the mesoderm was necessary for continued maintenance of the AER.

Although considerable information has been obtained about the result of the interactions occurring during limb formation, little is known about the mechanisms responsible for these changes. Recent investigations have focused attention on the cellular and molecular aspects of these tissues. The presence of numerous gap junctions within the AER during the period when it exerts its influence on the mesoderm may be an important structural feature (Kelley & Fallon, 1976; Fallon & Kelley, 1977). Investigations of the cytoarchitecture of the developing mesoblast have suggested that cell-cell associations established by filopodial contacts and junctional specializations may be developmentally significant (Ede, Bellairs & Bancroft, 1974; Kelley & Fallon, 1978).

The wingless mutant chick provides a unique system for the study of limb development in which there is an abnormality in the developmental sequence. Wingless, a homozygous recessive trait, is characterized by the disappearance of the AER during the third day of development and the failure of distal wing structures to develop (Zwilling, 1949). Recombination grafts have shown that the mesoderm is defective and is unable to maintain the AER beyond the third day of development (Zwilling, 1956a). Experiments reported by Saunders (1972) with the wingless mutant suggest that the mutant tissues lack polarizing activity found in normal limb buds.

The present study was undertaken to determine if there are any fine structural modifications that might account for the failure of limb development in the wingless mutant. This study reports differences in gap junctions in the apical ectoderm and differences in mesodermal cell-cell association that may account for the failure of the mutant limb bud to undergo continued development.

White Leghorn eggs were purchased from the Pocosham Hatchery in Richmond, Virginia, and eggs from the wingless mutant line were obtained from Dr P. F. Goetinck at the University of Connecticut. Fertile eggs from both groups were incubated at 37 · 5 °C in a forced air incubator with controlled humidity. Incubation periods varied to produce a series of embryos ranging from stages 20 – 25 (Hamburger & Hamilton, 1951). At appropriate intervals, both normal and mutant eggs were opened and the embryos transferred to a sterile watch glass containing chick Ringer’s at 37 · 5 °C. Limb primordia were dissected free with iridectomy scissors and glass needles. The limb primordia were then transferred with a Spemann pipette to vials of fixative.

The morphological characteristics that identify the homozygous recessive wingless mutant cannot be seen until days of development (stage 21); therefore, in order to verify the phenotype of specimens younger than days, it was necessary to use a different harvesting procedure. To insure positive identification of homozygotes a window was cut in the egg shell and both fore and hind limb buds were excised from one side of the embryo and transferred to a vial of fixative with a Spemann pipette. The window was then covered with a circular 18 mm glass coverslip and the edges sealed with paraffin. The eggs were returned to the incubator until the embryos reached a stage when their phenotype could be positively identified (Zwilling, 1956b).

Specimens for transmission electron microscopy were fixed at 4 °C in 1·2% glutaraldehyde, 2% paraformaldehyde buffered with 0·1 M cacodylate (pH 7·4) for 30 to 60 min. Tissues were washed for 1 h in two changes 0·1 M cacodylate buffer (pH 7·4) and postfixed for 20 min in 2 % osmium tetroxide buffered with cacodylate at pH 7·4. After osmication some specimens were washed for 10 min in cold 0·05 M maleic acid buffer (pH 5·2) and stained en bloc for 30 min with 1 % uranyl acetate in 0·05 M maleic acid buffer at pH 5·2. Specimens that were not stained en bloc with uranyl acetate were stained with a 1 % aqueous solution of uranyl acetate after sectioning. Tissues were rinsed in two changes of maleic acid buffer or cacodylate buffer for 10 min dehydrated in a graded series of ethanol and embedded in Epon 812.

In order to demonstrate the presence of glycosaminoglycans (GAG), a second group of embryos was stained en bloc with ruthenium red (Luft, 1971). Limb buds were fixed for 1 h in a solution containing equal parts of 2·4% glutaraldehyde, 4% paraformaldehyde; 0·2 M cacodylate buffer, (pH 7·4) and ruthenium red (RR) stock solution containing 10 mg/ml in water. Specimens were washed in three changes of 0·15 M cacodylate buffer for 10 min and postfixed for 3 h at room temperature in a solution containing equal parts of 4% osmium tetroxide in distilled water, 0·2 M cacodylate (pH 7·4), and RR stock solution 10 mg/ml. Tissues were rinsed in buffer, dehydrated in a graded series of ethanol and embedded in Epon 812. Thin sections were cut, stained with Reynolds’ lead citrate (1963), and examined with a RCA EMU 3G or a Philips EM 201 electron microscope. Sections 0·5 μm thick, were cut and stained with toluidine blue, and examined by light microscopy.

Specimens processed for scanning electron microscopy were fixed in cold 1 ·2 % glutaraldehyde, 2 % paraformaldehyde in 0·1 M cacodylate buffer, (pH 7·4). After fixation, several limb buds were cut transversely along their anterior-posterior axis and postfixed for 20 min in 2 % osmium tetroxide. The specimens were dehydrated in a graded series of acetone, and critical-point dried with Freon 116. Specimens were mounted on brass stubs, coated with gold-palladium, and examined with a JEOL JSM-S1 scanning electron microscope.

After 3 days of development, histological differences between normal and wingless limb buds are apparent. The normal limb-bud ectoderm has a well developed AER while the AER of the wingless embryo is either lacking or reduced in height (Figs. 1 and 2).

The AER of the normal limb bud is composed of two cell layers - an outer peridermal layer and an inner basal cell layer. The basal cells are pseudostratified columnar. The apical cytoplasms of these cells contains well-developed Golgi complexes, numerous poly-ribosomes and a few profiles of rough endoplasmic reticulum (RER) (Fig. 3). The basal cells have numerous elongated mitochondria and many electron-dense vesicles approximately 160 nm in diameter located in their basal cytoplasm, (Fig. 4). Micropinocytotic vesicles are present in areas adjacent to the basal lamina, but they are most numerous along the lateral cell borders of the ectodermal cells (Fig. 5).

In the wingless embryo a region of compact ectodermal cells can be found in the area where the AER would normally develop (Fig. 2). In this region the ectoderm is composed of a thin peridermal cell layer and a basal cell layer. The basal cells differ from those found in the AER of normal embryos in that the cells are cubodial and the nuclei have an irregular shape. Residual bodies are found frequently in the basal cytoplasm and electron-dense vesicles are absent (Fig. 6).

The wingless embryo lacks a definitive subectodermal space and the mesodermal cells are compact and round (Fig 2). The mesodermal cells of the normal limb bud contain large round mitochondria, numerous polyribosomes and a few profiles of RER. The wingless mesenchymal cells lack the 200 nm electrondense vesicles occasionally found in normal mesenchyme (Fig. 7). Both normal and mutant mesodermal cells make contact with adjacent mesodermal cells by focal tight junctions, and occasionally well-developed gap junctions can be found (Figs. 8 and 9).

A comparison of normal and wingless limb buds reveals that the AER is well developed in the normal but is absent in the mutant. There is a prominent subectodermal space beneath the AER of the normal limb bud that is lacking in the mutant (Figs. 10 and 11). The AER of the normal limb bud reaches its maximum size at days. The periderm contains numerous degenerating profiles. The basal cells are columnar and contain electron-dense vesicles (Fig. 12). Cellular apposition is maintained between ectodermal cells by junctional complexes consisting of zonula occludens, zonula adherens, desmosomes, and gap junctions. In the perinuclear region of cells in the AER bundles of microtubules and microfilaments course perpendicular to the basal lamina (Fig. 14) and microfilaments run parallel to the basal lamina; these filaments are seen frequently in the basal cell filopodia (Fig. 15).

In contrast to the columnar basal cells found in the AER of the normal limb bud, the ectoderm of the wingless limb bud is composed of cuboidal basal cells with ovoid nuclei. Contacts between adjacent basal cells are not as extensive as those found in the normal AER, and gap junctions are not apparent. The apical ectoderm of the mutant is similar to that found in previous stages; it is reduced to a uniform height and electron-dense vesicles are not apparent. The basal cell surface has a scalloped appearance due to cytoplasmic protrusions (Fig. 13); and microtubules and microfilaments found in the normal AER are absent in the apical ectoderm of the mutant limb buds.

The limb-bud mesoderm of the normal embryo contains stellate cells with numerous filopodia approximately 0·2–0·3 μm in diameter and large extracellular spaces (Fig. 18). These cells are oriented at right angles to the overlying ectoderm. The subectodermal space of the normal contains slender mesodermal cell filopodia, which are in close association with the overlying ectoderm (Fig. 16). As in earlier stages, cell contacts within the mesoblast are made by focal tight junctions and gap junctions in both normal and mutant limb buds. The mesodermal cells of the mutant are round and have bulbous cytoplasmic extensions (Fig. 17); the amount of extracellular space is reduced, and a much greater expanse of the cell surface is in contact with adjacent mesodermal cells (Fig. 20). Mitotic figures were found frequently in both mutant and normal mesoderms. The mesodermal cells of the mutant are tightly packed against the basal lamina of the overlying ectoderm and contain little extracellular space (Figs. 19 and 20).

Expression of the mutation is variable in both wing and leg buds. In some cases thickened portions of ectoderm resemble that of stage-20 wingless ectoderm (Fig. 4). The cells are low columnar and contain numerous degenerating profiles in their basal cytoplasm. Ectodermal cell contacts are more extensive in these cases than those found in the ectoderm of the extreme mutants (Fig. 22). The extensive areas of ectodermal cellular apposition in the thickened ectoderm resembles the arrangement of the basal cells found in the AER of the normal limb bud. Although gap junctions are present in the thickened ectoderm, they are not as numerous as those found in the normal AER. Discontinuities of the basal lamina are found along portions of the thickened ectoderm. In regions where these discontinuities occur in the basal lamina, mesenchymal cell processes are in direct contact with the ectoderm (Figs. 22 and 23). In these intermediate examples, most cells of the mesoderm are round, but stellate cells with short filopodia can also be found (Fig. 21). There is more extracellular space between the mesodermal cells in these specimens than in the extreme mutants (Fig. 22).

The overlying ectoderm in the normal limb bud is separated from the mesoderm by a continuous basal lamina approxi-mately 70 nm thick. The basal lamina has an outer moderately electron-dense layer (lamina densa) that is composed of amorphous material. The lamina densa is separated from the ectoderm by a 30 nm electron-lucent space, the lamina lucida (Fig. 24). The basal lamina of the wingless limb-bud ectoderm in most cases resembles that of the normal, although the lamina lucida could no longer be distinguished in several specimens (Fig. 25).

Ruthenium red, a polycationic dye, imparts a density on cellular surfaces that is easily distinguished from the reaction obtained with lead citrate and uranyl acetate (Luft, 1971). The basal lamina of both normal and wingless mutant limb buds demonstrate a weak reaction with RR (Fig. 26); the basal lamina has a uniform distribution of 15–21 nm RR-positive particles intermeshed between fine filamentous material (Fig. 26). The material observed in the extracellular space consists of collagen fibres 40 nm in diameter, along with RR-positive particles 50–70 nm in diameter, which are interconnected by fine filamentous strands 7·5 nm in diameter. The fine filamentous strands also interconnect the RR-positive particles with the basal lamina and mesenchymal cells. RR-positive particles have a random distribution on the mesenchymal cell surface and they demonstrate a periodicity of binding on collagen (Fig. 27).

There are no apparent differences in the amount or distribution of extracellular materials in the normal and wingless limb buds, even though the extra-cellular space is reduced in the mutant mesoderm. There was a slight increase in the amount of collagen found in later stages (⁠ days), but it still consisted of small randomly oriented fibrils.

The results presented here are in agreement with Zwilling’s (1974) findings that continued elongation and distal development of the limb bud does not occur unless a morphologically well-defined AER is present. The AER of the normal limb bud demonstrates a high degree of specialization when compared to the dorsal and ventral ectoderm. The tall columnar cells of the ridge are in intimate contact with their neighbours and contain well-developed gap junctions that are not apparent in the dorsal and ventral ectoderm. The presence of gap junctions in the AER suggests that these cells are ‘electronically and metabolically’ coupled. This may be important for expression of inductive activity of the AER (Kelley & Fallon, 1976; Fallon & Kelley, 1977). Since the apical ectoderm of the wingless mutant contained few, if any gap junctions, the apparent lack of gap junctions in the mutant ectoderm may serve as a morphological indication of its waning inductive influence.

Numerous microfilaments and microtubules are found in the cells of the AER running perpendicularly to the basal surface, and well-developed microfilaments course transversely in the basal cytoplasm. It has been suggested that the arrangement of microfilaments in the AER may provide a ‘structural foundation’ for the ridge (Kelley & Fallon, 1976).

The apical region of the wingless limb bud is covered with an ectoderm that varies in appearance from a compact mass of ectodermal cells (no AER) to an abnormal AER that is reduced in height. The cells are cubodial, and there is a reduction in the number of organelles. While the electron-dense vesicles found in the normal limb bud are not present in the mutant numerous autophagosomes could be observed in the basal cytoplasm. Although autophagosomes occur in the normal limb-bud ectoderm, they are usually localized in the periderm and in the apical cytoplasm of the basal cells (Jurand, 1965; Ede et al. 1974). It would be tempting to correlate the disappearance of the electron-dense vesicles in the mutant ectoderm with a loss of inductive activity, but this would probably be ill-advised, since nothing is known about their molecular composition or function. These differences found in the ectoderm of the normal and mutant imply a change in the metabolic activities of the cells of the AER. The ectoderm of the mutant resembles that of the normal non-ridge ectoderm. Microtubules and microfilaments are less numerous and not as well organized as those found in the normal AER and gap junctions are only seen infrequently.

The present study shows that the limb-bud mesoderm cells of the normal embryo are stellate, while those of the severely affected mutants are round and have bulbous cytoplasmic expansions. Electron-dense vesicles are found in the normal limb mesenchyme but are not apparent in the mutant mesenchyme. This is the first study to report the occurrence of such vesicles, the function of which, however, is unknown.

Examination of the limb bud of the embryos of both genotypes reveals differences in their organization. The mesoderm of the normal limb bud contains large extracellular spaces, with the cells and their filopodia oriented toward the AER. The filopodia make contact with adjacent cells and the basal lamina of the overlying ectoderm. These cell processes may act as retractable probes that provide a means of cell-cell communication. The mutant mesoderm is composed of tightly packed cells that do not demonstrate the orientation toward the ectoderm found in the normal mesoderm. The filopodia are reduced in number and length. The differences in cell shape, organization, and orientation suggest that the gene defect may express itself by alterations in the cell membrane that could result in different cell behaviour and association. Contacts between both normal and mutant mesenchymal cells are maintained by focal tight junctions and gap junctions. Recent studies have shown that gap junctions are responsible for ionic coupling and the transfer of small molecules between cells (Cox, Krauss, Balis & Dancis, 1974;,Sheridan, 1974). The presence of gap junctions in the mesoderm could provide a means of cell communication and information transfer.

The normal limb bud has a well-developed subectodermal space that is traversed by numerous slender filopodia of the mesenchymal cells. This space is not apparent in the mutant. Studies of the talpid³ embryo, a polydactylous mutant, emphasized the importance of these differences. It was found that the subectodermal space in the talpid³ was wider and contained fewer cell processes than that of the normal (Ede et al. 1974). This feature may be important in order for interactions to take place between the ectoderm and mesoderm. The mesodermal cells of the talpid³ were also found to have a greater degree of adhesion (Ede & Flint, 1975). In the present study of the wingless mutant, the cell-to-cell contacts in the mesoderm are more extensive and the subectodermal space is reduced. The importance of the subectodermal space and the arrangement of the cells is unknown, but it could be possible that these play an important role in the establishment of positional information which Wolpert (1976) suggests is necessary for limb development.

Goetinck (1966) noted that the expression of a mutant phenotype is not solely due to the presence or absence of a mutant gene, but is dependent on the integrated action of the mutant genes with the whole genotype. Such variations are observed in the wingless embryo. Zwilling (1949) observed that the apical ridge did not disappear uniformly and small patches of thickened ectoderm were present. In the present study, it was found that the AER is not present in the mutant, although areas of thickened ectoderm can be found. In the regions where the ectoderm is thickened the mesoblast appears more like the normal and is composed of both stellate and round cells. It seems that the mesoderm exhibits an intermediate response in areas where the ectoderm is thickened. The cell-cell contacts are also not as extensive as those found in the mutant limb buds that did not have a thickened ectoderm. These findings are in agreement with the observations that the loss of the ridge can be correlated with the failure of further distal development (Saunders, 1948; Zwilling, 1949).

Previous reports by Jurand (1965) and Ede et al. (1974) state that the basal lamina of normal chick limb buds is discontinuous. Jurand (1965) found that disruptions occurred in the median aspect of the AER along the flat ‘blade like’ extensions of the basal cells. Ede et al. (1974) described interruptions of the basal lamina in the same region where it becomes continuous with extracellular material between adjacent cells. In the present study, examples were also found where the basal lamina of the normal ectoderm became continuous with extracellular material between adjacent cells; however, this is not interpreted to be a true discontinuation in the basal lamina. It is suggested that a true discontinuation in the basal lamina would be an area where the basal plasmalemma of the ectodermal cells is completely devoid of extracellular material.

In the region where the ectoderm of the mutant is thickened, the basal lamina is truly discontinuous and mesenchymal cell processes are in intimate contact with the ectodermal cell processes. In the mutant the lamina lucida was not apparent in several specimens. This suggests that the basal lamina of the wingless mutant may be defective. The ectodermal cells of the mutant have cytoplasmic extensions similar to those found when the basal lamina is removed by trypsin (Goel & Jurand, 1968). These findings support Zwilling’s hypothesis that the basal lamina may have a positive role in limb development by promoting cell migration, which may in turn be an important feature for continued distal development (Zwilling, 1961). There was no indication that a disruption of the basal lamina is necessary for continued limb development as suggested by Bell, Kaighn & Fessenden (1959).

Recent studies have focused attention on the importance of extracellular material and surface-associated material in embryonic development (Trelstad, 1973; Hay & Meier, 1974; Kelley, 1975; Lunt & Seegmiller, 1980; Stephens, Vasan & Lash, 1980). Ruthenium red was employed as an extracellular marker for glycosaminoglycans (Luft, 1971) in order to determine whether there was any difference in the composition and distribution of these substances in the basal lamina and extracellular matrix of normal and mutant limb buds. No differences are found in the RR staining of the basal laminae and extracellular matrix in normal and mutant limb buds. These findings do not exclude the possibility that the basal lamina may have a different chemical composition, since RR only reacts with polyanionic molecules with a high charge density and would not detect other differences (Luft, 1971).

One of the characteristic features of the wingless mutant is that the limb bud fails to elongate. It would be expected that failure of the limb bud to elongate may be due to an inhibition of mitotic activity or cell death. This does not seem to be the case in the wingless mutant during early development, because frequent mitotic figures could be found; furthermore there was no indication of an extensive area of cell death which has been reported in the sex-linked wingless mutant WS (Hinchliffe & Ede, 1973). A more tenable hypothesis for the failure of elongation would be that differences in cell-cell association (evidenced by the absence of slender filopodia found in the normal embryo) and cell surface alterations may ultimately reduce cell division.

Our results are in agreement with Zwilling’s (1974) conclusions that the mesoderm of the mutant is defective and that the loss of the AER results in cessation of distal development. However, the precise mechanisms of this interaction are still elusive. The gene defect has an effect on the shape and possibly the mobility of the mesodermal cells and also on the ability of the mutant to maintain a well-developed AER. The results indicate that normal limb development may depend on cell-to-cell interaction between the mesodermal cells and interactions between these cells with the basal lamina.

Further experimental studies of these differences between the mutant and normal limb development should provide a better understanding of embryonic induction.

Portions of this work were supported by Grant HD 09174 from the National Institute of Child Health and Human Development. The author wishes to thank Dr Paul F. Goetinck for his helpful suggestions and assistance in the preparation of the manuscript.