Pathways of cytodifferentiation during the metamorphosis of the epibranchial cartilage in the salamander Eurycea bislineata

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Pathways of Cytodifferentiation during the Metamorphosis of the Epibranchial Cartilage in the Salamander Eurycea bislineata PERE Museum

of Comparative

Received

ALBERCH Zoology,

December

AND

Harvard

EMILY

University,

47, 1985; accepted

in revised

A. GALE Cambridge, firm

Massachusetts March

02138

11, 1986

The metamorphosis of the epibranchial-a skeletal element of the hyobranchial apparatus-is characterized by the degeneration of the larval cartilaginous element coupled with the formation of an adult cartilage in the same position. By integrating histological evidence from serial sections, in toto clearing and staining, TEM, SEM, and [‘Hlthymidine autoradiography, we found that larval chondrocytes die and do not participate in the formation of the adult cartilage. Meanwhile, the adult element forms from a small group of cells located in the mid-ventral region of the larval perichondrium. At the onset of metamorphosis, this group of perichondral cells begins to proliferate and they assume a mesenchymal morphology to subsequently undergo chondrogenesis. Although we only document a specific case, we believe that this process of apportionment of prospective larval and adult cell development is characteristic of highly specialized metamorphic systems in all groups. In general, differentiation is an irreversible process and larval cells once part of a differentiated tissue cannot dedifferentiate and participate in the formation of a new adult structure. To circumvent this constraint during the evolution of divergent larval and adult morphologies, organisms have evolved compartmentalized metamorphic systems. IT 1986 Academic Press, Inc. INTRODUCTION

The amphibian life cycle can be viewed as a two-step developmental process. The first stage occurs in the egg in which a larva is generated. The larva itself is a stable state; it maintains its shape and overall structural organization for an indefinitely long period of time, often years, and, in the case of neotenic salamanders, such as the axolotl, it is retained forever. Metamorphosis, triggered by a complex set of global cues (e.g., Dodd and Dodd, 1976; and Gilbert and Frieden, 1981), results in a second-order developmental process in which new genes are expressed and new morphogenetic processes unfold to give rise to the adult morphology. Our research addresses the general issue of the relationships between these two developmental processes in amphibians. If embryonic development is accepted to be a process characterized by a progressive restriction of the fate of any given population of cells, we can inquire how the organism succeeds in forming a fully differentiated larva while still being able to retain the potential to undergo a second developmental process in which an, often, completely different adult morphology is generated. In holometabolous insects, this problem is solved by the organism becoming physically subdivided in populations of larval and adult cells. Adult cells remain sequestered in an undifferentiated state in the imaginal disks while larval cells undergo morphogenesis and differentiation. At metamorphosis most larval structures degenerate and the imaginal discs are activated to form 233

the adult elements. The holometabolous insects, probably representing the most evolved complex life cycle, have circumvented the problem of larval differentiation by evolving parallel developmental systems. An equivalent physical apportionment of prospective larval and adult cells has not been reported in amphibians. Distinct pockets of undifferentiated cells have never been described in amphibian larva, to our knowledge. In this paper, we examine the issue of the fate of larval cells and of the developmental origin of the cells that generate the adult tissues during amphibian metamorphosis. The results that we report here deal with the remodeling of the epibranchial cartilages in the hyobranchial skeleton of the lungless salamander Eurycea bislineata. This urodele exhibits a complex life cycle characterized by an aquatic larval stage and an adult terrestrial period. At metamorphosis major structural changes occur related to this change in habitat (see Wilder (1925) for a comprehensive review). The hyobranchial apparatus is one of the musculo-skeletal systems that is most profoundly affected. In the larva, the hyobranchial skeleton is composed of a medial plate and four arches: the ceratohyal and three epibranchials (Fig. la). There is some controversy regarding the homology of the epibranchials; some authors, e.g., Duellman and Trueb (1985, p. 300), refer to them as ceratobranchials. Here, we follow the nomenclature used by Wilder (1925) and Lombard and Wake (1976) among others. The hyobranchial cartilages are used as support for the external gills, ventilation during aquatic branchial respiration, and suction 001%1606/86 Copyright All rights

$3.00

8 1986 by Academic Press, Inc. of reproduction in any form reserved.

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FIG. 1. Diagram illustrating the morphology and developmental fates at metamorphosis of the larval (A) and adult (B) hyobranchial apparatus. Stippling in (A) indicates that the elements disappear during metamorphosis; cross hatching indicates the derivatives of the central basal plate, while vertical hatching indicates remodeling of the ceratohyals. Heavy stippling illustrates the elements appearing de wvo in the adult. Bbi, basibranchial 1; Bp, branchial plate; Cb,, ceratobranchial 1; Cb2, ceratobranchial2; Ch, ceratohyal; el, larval epibranchial 1, e2, larval epibranchial 2; e3, larval epibranchial 3; E, adult epibranchial; L, lingual cartilage; R, radials; U, urohyal.

feeding (Lombard and Wake, 1976 and 1977). During metamorphosis, major morphogenetic changes, involving degeneration of elements, differentiation of new ones and remodeling of the ones being retained, are observed (see Fig. 1 for a summary of these metamorphic changes). The resultant adult hyobranchial skeleton (Fig. 1B) is quite different in overall morphology reflecting its new function as the skeleton of a sophisticated tongue projection apparatus (see Wake (1982) for a review of the functional changes in the larval vs adult hyobranchial skeleton). Smith (1920) performed a detailed histological analysis of the metamorphosis of the hyobranchial apparatus in Eurycea bislineata. She discovered the surprising fact that the adult epibranchial cartilage is not a remodeled larval first epibranchial in spite of both elements being located in the same position (Fig. 1). By examining a series of specimens at various stages of metamorphosis, Smith (1920) showed that during metamorphosis the first larval epibranchial is reabsorbed and a new element undergoes chondrogenesis. In Fig. 2, we show the simultaneous presence of a budding adult epibranchial and a degenerating larval first epibranchial at midmetamorphosis. Our research has focused on this intriguing developmental system-the replacement of the first epibranchial cartilage during metamorphosis (Fig. 2). In particular, we are interested in the fate of the larval chondrocytes as well as the developmental origin of the mesenchymal cells that participate in the chondrogenesis of the adult element. Two possible pathways of cellular differentiation are involved in this developmental process. These two alternative hypotheses are illustrated

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in Fig. 3. In the first, the larval chondrocytes undergo dedifferentiation at the onset of metamorphosis, return to a mesenchymal morphology and participate in the chondrogenesis of the adult cartilage (Fig. 3A). Alternatively, the larval cells, locked into a terminal differentiated state, are unable to participate in the formation of the adult element and undergo cell death. Meanwhile, the adult chondrocytes form from mesenchymal cells originating from a population of cells that have retained their morphogenetic potentiality throughout the larval period (Fig. 3B). In the first hypothesis (Fig. 3A), the process of differentiation is reversible, i.e., differentiated cells can go back and return to an embryonic undifferentiated state and participate in a second order morphogenetic process. The second hypothesis (Fig. 3B) implies that differentiation is irreversible and that differentiated cells have two options at metamorphosis: to remain in the same state allowing the tissue to undergo only minor remodeling or to die. In this case, new morphogenetic processes at metamorphosis are carried out by cells that have retained an undifferentiated state. To be able to carry out this latter process it is necessary during early embryonic development to compartmentalize the system into prospective larval vs prospective adult cells. These two populations of cells will respond differently to global cues in spite of the fact that they both will end up forming the same kind of differentiated tissue (Alberch, 1986). Previous experimental and histological studies have shown that larval chondrocytes respond to rising levels of thyroid hormones by undergoing autophagocytosis (Alberch et al., 1985). The concentrations of thyroxine or thyronine required to trigger this response fall within the physiological levels, measured using radioimmunoassay, in naturally metamorphosing Eurycea (Albereh et al., 1986). We continue this research here by reporting results addressing the origin of the mesenchymal cells that form the adult epibranchial cartilage. MATERIALS

AND

METHODS

The adults and larvae of Eurycea bislineata used in this study were collected in Massachusetts and New Hampshire. The larvae were maintained in bowls containing five to seven animals per liter of 50% Holtfreter’s solution with 0.1 g/liter Mg SO4 7HZ0 added. They were kept in a temperature controlled room at 15-18°C (12L/12D light cycle) and fed brine shrimp or pieces of earthworms three times per week. Adults were kept in the same room in plastic boxes with moist paper towels and fed Drosophila twice a week. The developmental stage of a metamorphosing animal was determined using the criteria outlined in Alberch et al (1986). The cellular and morphological examination

FIG. 2. The replacement of the larval lirst epibranchial (er) by the adult element (E) in Eurycrc~ Inslineata. (A) Cross section through posterior region of the head in a larval animal. Note the three larval epibranchials (e,, e,, and ea). Bar equal to 100 em, (8) In a metamorphosing specimen, the adult epibranchial (E), characteristically surrounded by muscle bundles, can be seen next to the degenerating larval element (el). Bar equal to 100 Wm. (C) Similar stage as seen in (B) in a ventral view of a specimen cleared and stained for cartilage matrix preparation The adult element (E) can be seen next to the degenerating condition, indicated by weak staining of the matrix in larval element (el). Larval epibranchials e2 and e3 will completely degenerate at a later stage. Bar equal to 200 pm. (D) Semithin (1 pm) plastic section of an adult (E) and larval (e,) first epibranchial at a similar stage to those seen in (B) and (C). Note the chondrogencsis of the adult element and the vacuolization of the larval chondrocytes. Bar equal to 100 pm.

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FIG. 3. Two hypothetical pathways of cellular differentiation during the metamorphosis of the epibranchial cartilage in urodeles. (A) This pathway involves dedifferentiation and redifferentiation, thus requiring the developmental process to be reversible; (B) this pathway outlines two parallel pathways involving heterochronic differentiation but not reversibility of differentiated states.

of the hyobranchial apparatus was carried out using the following techniques. (1) One hundred and four specimens (17 larvae, 23 prometamorphic, 20 early metamorphic, 18 midmetamorphic, 10 late metamorphic, and 16 adults) were fixed in formalin, embedded in paraplast and serially sectioned at lo-pm slices. The sections were stained with modified trichromate Heidenhain’s Azan technique (Baldauf, 1958). (2) One hundred and six specimens (45 larvae, 26 metamorphosing, and 35 adults) were cleared with trypsin and KOH and stained in toto with Alcian blue 8GX (for cartilage matrix) and alizarin red S (for calcium deposits) (modified after Wassersug, 1976). (3) Skeletal elements were removed from animals anesthesized in a 1:lOOO MS222 solution and fixed with 2.5% glutaraldehyde and 1% osmium tetroxide in cacodylate buffer at a pH of 7.2. A total of 103 epibranchials extracted from animals at various metamorphic stages were embedded in Epon 812. Ultrathin sections and lpm sections were obtained with a glass knife on a Sorvall MTBB ultra microtome. Thin sections were examined and photographed on a Zeiss 9-S2 transmission electron microscope. (4) Elements removed for eight animals at various stages of metamorphosis were fixed in the same manner as the specimens for TEM. They were dehydrated, sputter coated with gold palladium, and viewed and photographed using an AMRAY 1000 scanning electron microscope. For a more detailed description of the techniques used see Alberch et al. (1985). Twenty-eight animals (‘7 larvae, 14 prometamorphic, 3 early metamorphic, 2 midmetamorphic, 2 late metamorphic) were used for autoradiography. The animals were anesthesized with 1:lOOO MS222 injected with 0.05 Ci of [H3]thymidine (New England Nuclear) in 0.05 ml of sterile water, then returned to Holtfreter’s solution. Two specimens received this treatment only once; 26 animals received this treatment six times at 12-hr intervals. All specimens were preserved 4 hr after the last injection. After being fixed in 10% buffered formalin and embedded in paraplast, they were sectioned for auto-

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radiography. Control sections and labeled autoradiographs were obtained by alternatively cutting ten sections at 10 /*rn and then ten sections at 6 pm. The lo-pm sections were stained using modified trichromate Heidenhain’s Azan technique (Baldauf, 1956). The 6-pm sections were dipped in undiluted Illford emulsion K-5d for 1 set, dried in a high humidity 25°C room, and stored in plastic opaque slide boxes at 4°C for 2 weeks (Bogoroch, 1972). The slides were developed in midrodol-X and fixed in Kodak rapid fixer with hardener. The slides were post-stained with Toluidine Blue 0, Giemsa, or Nuclear Fast Red using methods described by Humason (1979). In analyzing the autoradiography sections, cell counts of labeled and unlabeled cells were done in specific areas of the head. These data were used to determine the percentage of labeled cells in a specific area at a given stage. RESULTS

We have compiled a detailed description of the changes in gross morphology, cell ultrastructure, and patterns of cell proliferation that accompany the metamorphosis of the epibranchial cartilages in Eurgcea tn’slineata. This was accomplished by integrating histological evidence from paraplast lo-pm sections, in toto clearing and staining, semithin and ultrathin (TEM) Epon sections, SEM, and [3H]thymidine autoradiography. We report our observations according to metamorphic stage. Our stages are based on the classification presented in Alberch et al. (1986), in which four metamorphic stages are defined in E. bislineata on the basis of a combination of external and osteological features. Larva. Prior to metamorphosis, there are three epibranchials in the larva (Figs. 1A and 2A). The first epibranchial, when viewed with SEM, presents a smooth perichondral surface with the cell-cell boundaries faintly outlined (Figs. 4A and B). Autoradiography shows sparse cell labeling throughout the head. Only the outer epithelium, which is regularly shed by the animal, shows heavy labeling. A number of chondrocytes in the epibranchials may be labeled but there are no concentrations of labeling indicating localized areas of rapid cell proliferation (Fig. 5). Ultrastructurally, the chondrocytes have a typical morphology (e.g., see Sheldon, 1983). They show large, approximately rounded, nuclei and relatively large amounts of cytoplasm completely filling the lacunar space. The cytoplasm has few vacuoles, a well developed rough endoplasmic reticulum and numerous mitochondria (Fig. 6A). Prometamcn-phosis. The earliest signs of metamorphosis are indicated by the onset of proliferation in a group of perichondral cells in the ventro-lateral side of the first epibranchial (Fig. 7). This area of perichondral cell proliferation is highly localized and its position ex-

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./I ‘/ ,

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FIG. 5. Representative autoradiographic labeling in a larval animal prior to metamorphosis. The two photographs are of the same section. The right one was exposed to enhance the contrast which emphasizes the darkly labeled cells that have undergone mitosis in the 7%hr period prior to fixation. el, first epibranchial; g, second epibranchial. Note the sparse labeling that includes one larval chondroeyte and contrast it with the localized heavy labeling in Figs. ‘7 and 8. Bar equal to 500 pm.

tremely consistent from specimen to specimen. It marks the point where the adult epibranchial will bud off the larval element. This initial stage of localized perichondral cell proliferation is followed by a morphogenetic transformation of the actively dividing perichondral cells, shown in Fig. 7, into mesenchymal cells. This transformation is illus-

trated in Figs. 4C and D, in which the presence of perichondral cells emerging from the connective tissue layer can be observed. This is not an artifact of preparation group of cells emerging since the presence of a localized from the perichondral layer and beginning to take a mesenchymal morphology is consistently observed in SEM preparations of specimens at this developmental

FIG. 6. TEM comparison of chondrocytes from the first larval epibranchial prior to (A) and during metamorphosis (B). This view depicts the characteristics of the larval chondrocyte prior to metamorphosis, completely filling its lacunae with a cytoplasm rich in organelles, including mitochondria and an extensive rough endoplasmic reticulum (arrow). In contrast, larval chondrocytes during metamorphosis (B) show clear signs of degeneration including deformed nuclei, highly vacuolated cytoplasm, reduction in the number of mitochondria, reduction in the extent of the rough endoplasmic reticulum and extensive presence of lysosomes (not evident at this low magnification). Bars equal to 3 ,cm.

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perichondrium but it is much less dense than in the ventral side. The dorsal perichondrium is one cell thick, while three to four layers of cells are observed in the ventral region (Fig. 10A). At the budding point, all the perichondral cells, 75% of the cells undergoing chondrogenesis to form the adult epibranchial and many of the cells of the surrounding tissue are autoradiographically labeled (Fig. 10B). Posteriorly, at the point where the adult element is physically separated from the larval cartilage (see Fig. 9), the larval perichondral labeling gradually decreases in density to about 65% of the cells having divided. High-percentage labeling is observed in

A FIG. ‘7. Autoradiographically (el) and second (ee) epibranchials the presence of a group of cells, larval first epibranchial (arrow), eration in the 72 hr previous observed in the perichondrium chondrocytes. Bar equal to 100

labeled section through the larval first at the onset of metamorphosis. Note localized in the ventral region of the that have undergone mitotic prolifto fixation. No equivalent labeling is of the second epibranchial or in the pm.

stage. It also agrees with the evidence obtained from thin plastic sections. These cells-which, we propose, are the ones that will later form the adult elementstill retain extensive areas of cell-cell contact. It looks as if they are being “pushed up” as a layer by the actively proliferating cells underneath. This perception is supported by the fact that later on the perichondrium becomes locally thickened to three to four layers of actively dividing cells (Fig. 8). The cells in the outer layers assume a more mesenchymal morphology. This initial region of perichondral cell proliferation eventually expands dorsally to include most of the ventral half of the midregion of the first epibranchial. The active proliferation of the ventral perichondrium drastically contrasts with the unlabeled larval chondrocytes or the very scarcely labeled perichondria of epibranchials two and three. Early metamcn-ph.osis. At this stage the mesenchymal cells originating from the ventral region of the perichondrium have condensed and begun to undergo chondrogenesis. The adult cartilage has become a discrete structure budding off the larval element (Fig. 9). This stage is characterized by the highest amount of labeling observed in the epibranchial region during metamorphosis (Fig. 10). Just anterior to the point where the adult epibranchial is budding off, in the region immediately posterior to the epibranchial-ceratobranchial articulation, 90% of all the ventral perichondral cells and 80% of the surrounding mesenchyme have divided in a 3-day time span. There is some labeling in the dorsal

.

FIG. 8. (A) Autoradiographically labeled section of larval first (er) and second (ez) epibranchials at late prometamorphosis. (B) Close-up of the boxed region in (A); note the heavy labeling in the ventral region of the first epibranchial. There are three to four layers of labeled cells in the perichondrial region as well as mesenchyme cells located in the surrounding tissue. Note that none of the larval chondrocytes has undergone cell division during the 72-hr period prior to fixation. Bars equal to 100 pm.

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FIG. 10. Autoradiography at various levels, corresponding to the ones indicated in Fig. 9, in an early metamorphic animal. (A) A vertical plane anterior to the point of budding (level A in Fig. 9); note extensive proliferation of the ventral half of the perichondral region of e,. (B) The point of budding of the adult epibranchial (E), level B in Fig. 9; note extensive labeling in the ventral perichondral region, and in the adult chondrocytes and chondroblasts. (C) A plane posterior to the point of budding (level C in Fig. 9); the adult epibranchial is separated from the larval (el), reduced perichondral labeling; note the extensive labeling of adult chondrocytes and chondroblasts; the outer mesenchymal cells around E will probably differentiate into muscle. (D) A plane immediately posterior to the tip of the developing adult (E) (level D in Fig. 9). Notice the sudden reduction in the amount of labeling. Note the lack of labeled larval chondrocytes in any of these sections. All sections at the same magnification. Bar equal to 100 Km.

the adult chondroblasts and chondrocytes throughout the length of the elongating element (Fig. 1OC). The labeling decreases abruptly posterior to the tip of the developing adult element (Fig. 10D). No signs of cell division in the larval chondrocytes were observed. Chondrogenesis of the adult epibranchial follows the typical pattern, with cells forming well defined Anikin cell arrangements (Gould et al., 1974; Ede, 1983) consisting of rounded chondroblasts concentrically surrounded by elongated mesenchymal cells (Fig. 2D). Midmetamorphosis. The first obvious signs of degeneration of the larval epibranchials are observed at this stage, although increasing vacuolization of the larval

chondrocytes was present since prometamorphosis. Ultrastructural evidence provided by TEM indicates that the morphology of the larval chondrocytes has radically changed from the one observed in these same cells prior to the onset of metamorphosis (Fig. 6). All larval chondrocytes are characterized by numerous signs of undergoing autophagocytosis including a highly vacuolated cytoplasm (Fig. 6B), numerous lysosomes, absence of an organized rough endoplasmic reticulum, and swollen mitochondria. As expected from cells in the process of dying no autoradiographic labeling was observed in larval chondrocytes. The lacunae of these larval chondrocytes become enlarged.

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In the adult epibranchial, we now observe signs of anterior elongation in addition to its posterior growth. It, thus, forms a rod of cartilage that runs parallel to the larval element and they are only joined at the initial budding point. Figure 11 illustrates an autoradiographically labeled specimen at the stage at which the larval and adult elements have broken off their connection. Notice that the fission has occurred through the larval cartilage; a piece of larval matrix has remained attached to the adult element. The adult matrix can be clearly distinguished from decaying matrix by cell morphology, heavy cell proliferation, and distinct metachromatic staining. As the adult epibranchial becomes an independent unit, most of its growth appears to be by internal chondrocyte proliferation rather than by the apposition of mesenchymal cells. This is evidenced by the reduction in autoradiographic labeling in the neighboring cells, while the adult chondrocytes are heavily labeled (Fig. 12). Late metamorphosis. The adult epibranchial continues its development, while the larval element is reabsorbed. At this stage most of the enlarged lacunae in the larval matrix contain only cellular debris rather than cells. There are no signs of invasion of the cartilage matrix by blood vessels. Most of the final resorption seems to be mediated by phagocytic monocytes that surround the amorphous pieces of cartilage matrix-the remains of the larval element. The adult element continues its elongation by growth of its apical tip. This is evidenced by the high density of cell proliferation at the distal tip

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FIG. 12. Autoradiographically labeled section of a late-metamorphic animal at the level of the growing tip of the adult epibranchial, which at this stage has elongated beyond the tip of the larval first epibranehial. This section intends to illustrate that the elongation of E occurs by rapid proliferation of the chondrocytes (most of them show labeling) rather than by distal apposition of mesenchymal cells. M, subarcualis rectus muscle. Bar equal to 500 pm.

relative to that of the more proximal region. Only about 25% of the chondrocytes are labeled in the anterior region, while about 50% of the cells have divided at the posterior tip. Posterior to that point labeling drops to the low levels seen throughout the head. DISCUSSION

Larval Chondrocytes Undergo Autophagocytosis in the Morphogenesis and Do Not Participate of the Adult Cartilage

FIG. 11. Autoradiographically labeled section of a late midmetamorphic animal at the level where the adult element (E) breaks off from the larval (ei). The arrow indicates a piece of degenerating matrix that has remained attached to the adult, heavily labeled, element. The labeled cells on the surface of ei may be phagocytic monocytes. Bar equal to 100 pm.

As experimentally shown by Alberch et al. (1985), larval chondrocytes in the epibranchials of Eurycea bislineata undergo rapid ultrastructural modifications after only 24 hr of exposure to physiological levels of thyroxine and/or thyronine. The most obvious morphological changes entail the formation of large vacuoles, an increase in the number of lysosomes, swollen mitochondria, disappearance of the rough endoplasmic reticulum, and the deformation of the nucleus (Fig. 6B). These are all signs of cellular autophagocytosis (e.g., Hinchliffe, 1981). After a few days unequivocal evidence of cell death is observed in the larval chondrocytes of both naturally, and experimentally induced, metamorphosing animals. The cytoplasm has been completely reabsorbed and the nucleus is pycnotic and deformed (in the most advanced cases, only cellular debris is observed in the lacunae). Probably the decaying larval chondrocytes secrete en-

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zymes that digest the surrounding cartilage matrix, since the lacunae appear abnormally enlarged in metamorphosing larval epibranchials (e.g., Fig. 2D). Additional work is required to elucidate the biochemistry and cell dynamics that accompany the complete degeneration and removal of the larval cartilages. Our current evidence suggests that the initial stages of cartilage degeneration result from the autophagocytosis of the larval chondrocytes but that, after the death of the chondrocytes, the complete resorption of the matrix involves phagocytic monocytes that may be the cells observed on the surface of the degenerating larval element (Fig. 11). The hypothesis of the death of the larval chondrocytes is further supported by the autoradiographic evidence that no cell division occurs in the larval chondrocytes after the onset of metamorphosis. Cells That Participate in the Chondrogenesis of the Adult Epibranchial Originate from a Localized Region in the Larval Perichmdrium

Mesenchymal

The data reported indicate that at the onset of metamorphosis a relatively small group of perichondral cells located in the mid-ventral region of the larval first epibranchial become “activated” (Fig. 7). By this we mean that the cells began a very rapid rate of cell proliferation coupled with a transformation from perichondral to mesenchymal cells (Figs. 7-10). These mesenchymal cells undergo normal chondrogenesis to form the adult epibranchial (Fig. 10). We are currently studying the details of this perichondral to mesenchymal transformation using monoclonal antibodies to cell surface molecules. The pattern of growth of the adult epibranchial is diagrammatically illustrated in Fig. 13. After the initial condensation (Fig. 13A), the growing element buds off posteriorly (Figs. 9 and 13B). Later this elongation also extends anteriorly with the adult cartilage still attached to the degenerating larval epibranchial (Fig. 13C). At midmetamorphosis the connection between the larval and adult cartilage breaks off (Fig. 13D). The growth of the adult epibranchial also varies with developmental stage. At the onset, the growth is by cell proliferation and apposition of cells detaching from the perichondrium. By early metamorphosis, however, an adult perichondrium is well developed and the elongation is “internally driven,” i.e., the result of division of chondrocytes at the tip of the adult epibranchial (Fig. 12). Comparisons of autoradiographs and standard histological sections indicate that the labeling found around the growing cartilage corresponds to the myoblasts of the surrounding muscle. We believe that the mid-ventral region of the perichondrium is the sole source of the progenitor cells of the adult epibranchial. This assertion is supported by

FIG. 13. Diagrammatic summary of our results on the genesis of the adult epibranchial in Eurycea bislineata. (A) A spatially restricted group of perichondral cells in the ventral region of the larval epibranchial begin to proliferate and to dedifferentiate as mesenchymal cells. (B) These cells undergo chondrogenesis and the adult element is seen as a branch off the larval cartilage. (C) The budding adult element elongates both posteriorly and anteriorly while stil retaining a point of attachment with the larval. (D) The connection between the larval and adult cartilages breaks off while the larval epibranchial degenerates and is reabsorbed.

the absence of any other localized area of high cell proliferation at the onset of the metamorphosis of the epibranchial cartilage. This hypothesis is further supported by experiments in which the animal is allowed to metamorphose after removal of the larval first epibranchial (Alberch and Gale, unpublished data). This is a very simple operation because of the superficial location of the larval epibranchials. The larval first epibranchial can be removed very cleanly, i.e., the cartilage and associated perichondrium are surgically removed with minimal damage to neighboring tissues. When the complete larval epibranchial is removed, no adult epibranchial differentiates. This result supports a perichondral origin of the adult chondrocytes, rather than one from more distant tissues. The fact that the source of cells involved in a secondary morphogenetic process is the perichondrium is not surprising. Nathanson et al. (1978) have shown fibrous connective tissue to be composed of an unusual type of cells since they retain the ability to differentiate as cartilage even after a state at which their differentiation is thought to be stabilized. We still have not answered,

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however, the key question of why the origin of the cells for the adult cartilage is restricted only to a specific region of the perichondrium rather than to all perichondral cells. Metamorphosis Evolves by Progressive Compartmentalization of Larval and Adult Developmental Processes In conclusion, our data support a metamorphic process characterized by irreversibility of differentiated larval states and by developmental compartmentalization. In the particular case studied, it appears that there are stringent limitations to how much a larval structure can be remodeled to adapt to a new adult function. This constraint has been circumvented by evolving redundant developmental systems. This is accomplished by the subdivision of cell populations into differential developmental fates that are expressed asynchronously. We propose that, early in development, neural crest cells are apportioned into prospective larval and adult chondrocytes. Larval cells undergo chondrogenesis while the small number of cells that will form the adult structures become sequestered in the perichondrium where they retain the potential to participate in further morphogenetic processes. At the time of metamorphosis these cells become “activated,” start rapidly proliferating and give rise to a population of mesenchymal cells that will form the adult cartilage. This contrasts with the death of the differentiated larval cells. Alberch (1986) has generalized these results to other aspects of amphibian metamorphosis and proposed, using comparative data, that the evolutionary trend toward increasing divergence of larval and adult morphology is accompanied by the appearance of developmental compartmentalization. For example, in primitive species of urodeles, such as Ambystoma, the larval first epibranchial is retained in a shortened and remodeled form (Alberch, 1986; Alberch and Gale, unpublished data). In the advanced family Plethodontidae independent larval and adult pathways evolved, allowing for the specialization of the adult epibranchial as part of a sophisticated tongue flipping apparatus (Lombard and Wake, 1976,1977). A characterization of the phylogenetic diversity of metamorphic patterns can provide insights into the mode of evolution of developmental systems in general. Subdivision of a single embryonic field into developmental regions may be a property of vertebrate embryogenesis and it is exemplified by metamorphic systems which we postulate to be characterized by an increasing degree of compartmentalization. This may be a prerequisite to allow morphological specialization and divergent evolution of structures in the same organism.

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117, 1986

We thank Dr. S. Tilley for his help in obtaining Eurycea larvae and Annette Coti! for her technical assistance. Laszlo Meszoly and Catherine McGeary assisted with the preparation of the manuscript. This research was supported by NSF grant PCM 83-08640.

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