ABSTRACT
Distinct changes in epidermal cell shaping largely define the overall pattern of growth and form during generation of the ectodermal ridge and early stages of fin fold morphogenesis. The epidermal portion of the ridge and early fin fold are formed from a strip of epidermal cells that is only six to nine cells wide. There is apparently no increase in the number of these cells during initial formation of the ridge and its subsequent conversion into a fin fold which contains extracellular matrix fibres.
Epidermal cells adopt a wedge-shaped morphology during ridge production. Distinct changes in the shaping and contact relationships between basal portions of these cells generate intercellular spaces at several discrete loci within the ridge. These spaces become continuous with each other to form a subepidermal space. Hence, the subepidermal space is not produced by straightforward folding of an epidermal sheet.
Cells flanking the sides of the ridge start to flatten as it is converted into a fin fold. A continuous row of distinctive cells is positioned along the apex of the developing fold. The term ‘cleft cells’ is suggested for these apical cells. Each cleft cell retains a wedge-shaped form during fold formation and develops a basal cleft-shaped invagination. Invaginations are aligned in neighbouring cleft cells so that these cells cap the distal boundary of the subepidermal space where collagenous extracellular fibres called actinotrichia run anteroposteriorly along the length of the fin fold. This orientation is in direct contrast to the proximodistal orientation of actinotrichia within the remainder of the subepidermal space. During early stages of fold production a temporary set of previously unreported extracellular cross fibres spans the subepidermal space at right angles to actinotrichia. These configurations of extracellular fibres could be advantageous for maintaining the structural integrity of the early fin fold.
INTRODUCTION
The early stages of teleost fin fold morphogenesis involve the construction of a tissue with a relatively simple yet distinctive architecture. Peridermis and underlying epidermis form an elevated fin fold around a central subepidermal space (Geraudie, 1977). This space contains well-ordered arrays of extracellular fibres called actinotrichia (Bouvet, 1974).
The zebra fish Brachydanio rerio (Cyprinidae) has been used in this study of the initial stages of fin fold formation. This report indicates that epidermis rather than peridermis seems to play the major role in spatial organization of the fin fold. Furthermore, we have found that a limited number of epidermal cells are involved in the initial stages of fin fold formation; there are only six to nine cells/cross-sectional profile of a fold as the architecture outlined above is established. This system therefore provides a valuable opportunity for investigation of the impact of cell shape modulation and extracellular matrix organization during tissue shaping. In addition this system is of relevance to vertebrate limb morphogenesis in general. The caudal fin fold in Brachydanio develops from an apical ectodermal ridge-like structure as do all other teleost fins which have been examined (Bouvet, 1974; Geraudie & Françoise, 1973; Wood, 1982; Wood & Thorogood, 1984). Several aspects of structural organization that are reported in this account exhibit close similarities to those documented for the apical ectodermal ridges of other vertebrates (Saunders, 1948; Jurand, 1965; Ede, Bellairs & Bancroft, 1974; Goel & Mathur, 1977; Raynaud, Brabet & Adrian, 1979; Todt & Fallon, 1984). The important influence of the AER during limb development is well established (Saunders, 1948; Zwilling, 1955; Saunders, Gasseling & Cairns, 1959; Bell, Gasseling, Saunders & Zwilling, 1962; Stark & Searles, 1973; Ede, Hinchliffe & Balls, 1977).
Previous studies of teleost fin ultrastructure have mainly concentrated on morphogenetic stages which occur after the fin fold has been established (Geraudie & Françoise, 1973; Bouvet, 1974; Lanzing, 1976; Geraudie, 1977; Geraudie & Landis, 1982). This report deals mainly with specific changes in cell shaping and contact as well as extracellular matrix secretion and cytoskeletal deployment associated with the earliest stages of fin fold morphogenesis.
MATERIALS AND METHODS
Adult zebra fish (Brachydanio rerio) were mated in a Legg trap (Axelrod et al. 1962). Fertilized eggs were collected and incubated in fresh water at 27 °C. Stages in embryogenesis were distinguished using normal tables (Hisaoka & Battle, 1958). Tail buds were excised with fine tungsten needles prior to fixation for transmission electron microscopy. Two fixation procedures were used. Glutaraldehyde/osmium fixation was employed using a slight modification (50 mM-phosphate buffer at pH 7·6) of a protocol previously described by Tucker (1967). In addition, an osmium-ferricyanide fixation technique (Os, Fe-CN fixation) was used as described by McDonald (1984) except that immersion in tannic acid (1 %) was increased to 1 h. This protocol was used because it has been reported to improve the contrast and preservation of various cell components (McDonald, 1984). In developing fins extracellular matrix and certain membrane-associated materials exhibited increased contrast when fixed using the technique. Thin sections (50 nm) of tail buds embedded in Araldite were double stained with uranyl acetate and lead citrate for examination with a Philips EM 301.
RESULTS
The account that follows is based on examinations of dorsal portions of developing caudal fin folds. No marked changes in peridermal organization were detected during early fin fold morphogenesis. The main changes in epidermal cell organization and extracellular matrix deployment within the subepidermal space are summarized in Fig. 1.
Stage 19 (20−24 h postfertilization)
The tail bud is covered by two layers of ectodermal cells. A layer of cuboidal epidermal cells is overlain by a layer of partly flattened peridermal cells (Fig. 1A). Putative fin fold ectoderm does not exhibit any obvious differences in histological or fine structural organization that distinguish it from tail bud ectoderm which is not destined to contribute to fin fold construction.
Stage 20 (24−27h postfertilization)
Three distinct developmental phases can be distinguished.
i) Epidermal cells of the putative fin fold develop wedge-shaped cross-sectional profiles as they become ‘centered’ about an anteroposteriorly oriented axis on the mid-dorsal epidermal/mesodermal interface and group together to produce a local thickening of the ectoderm that forms an ectodermal ridge (Figs. 1B, 2). As a result of this change in cell shape the basal surfaces (those directed away from the peridermis) of these cells become reduced in area and lose contact with the underlying mesoderm (Figs. 1B, 2). Cross sections of the ridge include six to nine wedgeshaped epidermal cells (Fig. 2).
ii) Serial transverse sections reveal that cells which form a single row running along the entire length of the ectodermal ridge retain their wedge-shaped crosssectional profiles. These cells will be referred to as cleft cells. All other epidermal cells within a ridge become more or less rectangular in cross section and form the two closely apposed epidermal side walls of the ridge (Fig. 1C). As a consequence of this change in cell shaping the basal surfaces of epidermal cells in both side walls of a ridge increase in area. These surfaces are juxtaposed against the basal surfaces of other epidermal cells which contribute to the opposite side of a ridge.
iii) A subepidermal space develops between the apposed basal surfaces of epidermal cells flanking the sides of a ridge. Cell surfaces separate at several discrete loci to begin with (Figs. 1C, 3) and separation is often associated with the production of cell surface invaginations (Fig. 4). The extracellular spaces that are created by cell surface separation gradually become more extensive and unite with each other until an uninterrupted extracellular subepidermal space runs between epidermal cells flanking the sides of the ridge which has now become a recognizable fin fold.
Each extracellular space generated during the production of a subepidermal space is lined by a continuous basal lamina that is closely situated within 70 nm of the cell membranes of separating epidermal cell surfaces (Figs. 3,4). Dense bridges connect basal laminae to cell membranes (Fig. 4, arrows) and strands of electron-dense material run between basal laminae flanking either side of the developing subepidermal space (Fig. 4). Clusters of extracellular material which seem to represent a discontinuous basal lamina form at the epidermal-mesodermal interface adjacent to the subepidermal space (Fig. 5, small arrows). This material is replaced by a continuous basal lamina during stage 21.
Stage 21 (27−36 h postfertilization)
At the start of stage 21 the subepidermal space is a continuous extracellular cavity which extends from the base of the fin fold to the row of apical cleft cells (Fig. 5). Each cleft cell produces an invagination along the entire length of its narrow basal surface (Figs. 5, 6). The ends of adjacent cleft cells overlap and in some cases interdigitate (Fig. 7). The invaginations of adjacent cleft cells are well aligned with each other and run as a continuous subepidermal space along the entire length of a cleft cell row. Cleft cells retain their invaginations at least until the end of stage 25 (Fig. 1). However, cells flanking the subepidermal space do not retain the cleftlike invaginations which form during initiation of the subepidermal space (Stage 20, iii). In regions where cleft-shaped invaginations of cleft cells or other epidermal cells have recently been formed, the invaginated cell membranes are associated with a cytoplasmic layer of dense granular material (up to 0.5 μm thick) and small vesicles (Fig. 4).
The number of cross-sectional epidermal cell profiles included in a fin fold during early stage 21 is the same (six to nine) as those found in cross sections of ectodermal ridges at stage 20 prior to their conversion into a fin fold (compare Figs. 1B, C, D, 2, 5). During stage 21, however, the fin fold expands considerably; it increases in height from about 30 to 100 μm. As this increase occurs epidermal cells flanking the sides of the fin fold start to flatten and increase in number/cross section of a fin fold (compare Figs. 1, D & E).
At the start of stage 21, a set of previously undescribed extracellular cross fibres spans the subepidermal space (Fig. 1D). These fibres were readily apparent in the fin folds of tail buds fixed using the osmium-ferricyanide fixation procedure (Fig. 8). They were difficult to detect when the more conventional fixation procedure was used (see Materials and Methods). In addition, a considerable amount of less highly organized extracellular material with a dense granular appearance ramifies between the cross fibres and throughout most of the subepidermal space (Fig. 8). Where the cross fibres approach the surfaces of epidermal cells their ends penetrate the basal lamina and apparently terminate in direct contact with cell surface membranes (Fig. 9, arrows). Cross fibres are temporary components. They are numerous at the start of stage 21 but become increasingly sparse as actinotrichia start to polymerize within the subepidermal space. Well-defined cross fibres spanning the space were not detected at later stages in morphogenesis.
Actinotrichia start to assemble during stage 21 and increase in size during this and later stages. Throughout most of the fin fold actinotrichia form two distinct rows on either side of the subepidermal space adjacent to the basal surfaces of flanking epidermal cells. These extracellular fibres are proximodistally oriented in parallel arrays at right angles to the cross fibres (Fig. 1E, 9). Actinotrichial arrangement at the distal border of the supepidermal space is different. Cross sections of fin folds reveal cross-sectional profiles of actinotrichia within invaginations where cleft cells cap the subepidermal space (Fig. 6 arrows). This arrangement is in contrast to the longitudinal profiles obtained in more proximal regions of the subepidermal space (Fig. 6). Examination of freshly excised tail buds using differential interference microscopy, and vertical thin sections cut in the plane of a fin fold, show that the distal extremities of actinotrichia curve in a posterior direction as they enter the distal portions of the subepidermal space contained within cleft cell invaginations.
Networks of intermediate filament bundles assemble in epidermal cells flanking the sides of the subepidermal space during stage 21. These networks are highly localized. They are very closely juxtaposed against cell surface membranes facing the subepidermal space. Most of the bundles are positioned close to cell membrane regions directly beneath actinotrichia (Fig. 10 arrows).
Stages 22−25 (36−96 h postfertilization)
During stages 22−25 the fin fold continues to expand. It increases in height from about 100−220μm. Concurrently, epidermal cells flanking the sides of the fold become flattened compared with their more or less cuboidal shapes during the initial phases of stage 21 (compare Figs. 1E and F). These cells form a pavement epithelium flanking either side of the subepidermal space which increases considerably in width as well as height (Fig. 1F). The number of cross-sectional profiles of epidermal cells contributing to the side walls of a fold increases from about 16 at the end of stage 21 to about 40 during stages 24 and 25. Hence an increase in the number of epidermal cells and cell flattening both contribute to growth of the fin fold during stages 22−25. The increase in cell number might be due to epidermal cell proliferation within the fold and/or recruitment of adjacent epidermis into the fin fold.
As epidermal cells flatten an extensive system of intermediate filaments assembles into bundles which ramify throughout the cytoplasm of the fin fold epidermis (Fig. 11). A similar temporal relationship also occurs between intermediate filament assembly and peridermal cell flattening during earlier stages (20 and 21). In both epidermal and peridermal cells, intermediate filament bundles are associated with densely staining bodies (up to 0.25 μm in diameter) and with attachment desmosomes in regions where adjacent ectodermal cells make contact.
Surprisingly few microtubules were detected in epidermal cells during ectodermal ridge formation and the early stages of fin fold construction. Actinoid stress fibres are usually sufficiently robust to be preserved by the fixation procedures employed. However, such fibres were not detected.
DISCUSSION
The case for an apical ectodermal ridge
Similarities between early teleost fin folds and the apical ectodermal ridges of limb buds in higher vertebrates have been noted previously (see Introduction). For example, Wood (1982) describes killifish pectoral fin development as the modification of an apical ectodermal ridge to form a fin fold with a distinct subepidermal space. However, Geraudie (1978) uses the term ‘pseudoapical ectodermal ridge’ for the early fin fold in the trout pelvic fin on the grounds that it is ‘unlike the unfolded tetrapod apical ridge which is described as a thickening of the apical epidermis or a pseudostratified epithelium’. There have been no previous ultrastructural studies of thin sections of the very earliest stages of fin morphogenesis to unequivocably resolve whether fin construction starts with an ectodermal ridge or with a fold. This examination of the morphogenesis of a fin establishes that to begin with there is a ridge-shaped epidermal thickening (which lacks a subepidermal space) as distinct from an epidermal fold.
In this context it is worth noting that the apical ectodermal ridges of some other vertebrate limb buds display a feature which is spatially homologous with one found during transition from ridge to fin fold. In these buds a small cleft-shaped indentation runs along the mid-basal line of each apical ectodermal ridge. Such indentations have been variously described as ‘notches’ and ‘grooves’ in a number of systems (Saunders, 1948; Jurand, 1965; Ede et al. 1974; Goel & Mathur, 1977; Raynaud et al. 1979; Hurle & Fernandez-Teran, 1984; Todt & Fallon, 1984). It has been suggested that the notch in the chick wing bud apical ectodermal ridge may influence the positioning of distal wing elements (Todt & Fallon, 1984). An interesting comparison may be made between the suggested role of the notch and specification of the orderly arrangement of actinotrichia (which may help to define the position of bony fin rays (Geraudie & Landis, 1982)) in the subepidermal space of the fin fold.
Cell shape modulation
Fin fold morphogenesis provides a clear example of a situation where spatiotemporal integration of individual cell shaping plays a major role in establishing overall tissue shape. This is particularly clear so far as the epidermis is concerned because cell number does not increase during the establishment of the main spatial characteristics of a fin fold, and the developing fold is less than ten epidermal cells ‘wide’ during the period in question. Modulation of epidermal cell shaping reveals a considerable degree of spatial intricacy. For example, most epidermal cells exhibit a sequence of three distinct shape changes. All of these shape changes can be directly related to structural phases in the construction of a fin fold (Fig. 1).
Cleft cells
Cleft cells are geographically distinct from the other epidermal cells by virtue of their apical positions and can also be distinguished by their ‘morphological signature’.
Cleft cells are apparent in micrographs of developing trout fin buds (Bouvet, 1974, Figs 2, 3; Geraudie, 1977, Fig. 2). The texts of these reports do not draw attention to these cells, although Geraudie (1977) mentions ‘unstriated fibrils’ which presumably correspond to the cross-sectional profiles of actinotrichia in the cleft cell invaginations reported here.
There is evidence that cleft cells may be functionally, as well as morphologically, distinct from other epidermal cells within the fin fold. For example, actinotrichia are differently oriented where they lie in the cleft-shaped invaginations of cleft cells with respect to their orientation elsewhere in the subepidermal space.
Matrix organization in the subepidermal space
The dynamic and orderly layout of extracellular matrix fibres in the subepidermal space apparently provides a stable framework for support of the fin fold during early stages of morphogenesis. For example, at the start of stage 21, numerous cross fibres span the subepidermal space. These cross fibres presumably operate as trusses by acting in conjunction with the substantial basal lamina which flanks the sides of the subepidermal space. By the time that cross fibres depolymerize the actinotrichia have started to assemble and the cytoplasmic side walls of the subepidermal space are fortified by a juxtaposed meshwork of intermediate filaments, especially in regions subjacent to actinotrichia. These components presumably take over the mechanical role of the cross fibres. Elimination of cross fibres is probably necessary to leave a clear path for the invasion of the subepidermal space by migrating mesodermal cells which are apparently guided into the space by actinotrichia (Wood & Thorogood, 1984). The posterior curving and overlap of the distal extremities of actinotrichia in the clefts of cleft cells probably contributes to maintenance of the structural integrity of the actinotrichial arrays. Such overlap only occurs prior to formation of the basement lamella which may subsequently stabilize the arrangement of the closely associated actinotrichia.
Formation of the subepidermal space
The subepidermal space is not produced by a straightforward folding of the epidermis. It results from the separation of the closely apposed basal surfaces of epidermal cells. In this respect, the procedure is similar to the development of ducts in mouse mammary glands. These ducts result from the fusion of extracellular spaces which form at the apical surfaces of a solid mass of cells, apparently as a consequence of changes in cell adhesion and contact relationships (Hogg, Harrison & Tickle, 1983). It is unlike the formation of some other extracellular cavities such as the cavity inside most vertebrate neural tubes (see Karfunkel, 1974) and the lumen of the mammalian salivary gland (Spooner & Wessels, 1972) where epithelial sheets curve or fold to enclose pre-existing extracellular spaces. There is a similarity to neural tube production and salivary gland morphogenesis in terms of the involvement of wedge-shaped cells.
Fin fold morphogenesis
The most important feature to emerge from this investigation is evidence that several different procedures are substantially involved in fin fold generation. For example, there is a well-defined sequence of epidermal cell shape changes and surface contact relationships. These apparently require alteration in both cyto-skeletal organization and cell surface adhesion. The shaping sequence is complemented by intricate modulations in the layout of extracellular matrix fibres in the subepidermal space. This emphasizes the possibility that the extracellular dynamics of actinotrichial and cross-fibre deployment are important for stabilizing and promoting new aspects of epidermal architecture. The idiosyncrasies of cleft cell relationships with the subepidermal space and actinotrichial orientation may be an indication that this ‘special’ row of cells plays a key role in coordinating the pattern of intra- and extra-cellular events. The main challenge now, is to more accurately assess the impact of each of these procedures and components on fold morphogenesis and to elucidate how integration of their contributions is controlled. This is currently being pursued by analyses of perturbations of in vitro fin fold morphogenesis induced by a range of experimental interventions.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Medical Research Council. We would like to thank Mr J. B. Mackie and Mr D. L. J. Roche for their excellent technical assistance.