Anat Embryo1(1992) 186:153-165

Anatomyand

Efnbryology

9 Springer-Verlag 1992

Regeneration in SMaria pavo (Blenniidae, Teleostei) Histogenesis of the regenerating pectoral fin suggests different mechanisms for morphogenesis and structural maintenance Bernhard Y. Misof 1'2 and Giinter P. Wagner 2

i Zoological Institute, University of Vienna, A-1090 Wien, Austria 2 Department of Biology,Yale University, Osborn Memorial Laboratories, P.O. Box 6666, New Haven, CT 06511-7444, USA Accepted April 24, 1992

Summary. The pectoral fin of blennies is differentiated

into a dorsal field and a ventral hook field. A histogenetic analysis of the regenerating pectoral fin was related to two questions. First, are histological specializations of the hook field responsible for the impairment of the regenerative capacity of pectoral fins of blennies? Second, can analysis of the temporal sequence of histogenetic events be used to make testable predictions about the tissue interactions required to re-establish the adult pattern? Regeneration of pectoral fins was examined in Salaria p a v o (Blenniidae, Teleostei). Approximately 80% of the length of the fin rays was amputated. Fin ray stumps were evaluated 7, 14, 24, 48 and 72 h after amputation, regenerates 4, 5, and 6 days after amputation and at length of about 30%, 50% and 60% regeneration of the original fin length. The regeneration process is subdivided into four stages: wound healing, blastema formation, fin ray formation and distal outgrowth and differentiation of hook characters. Analysis of the early events of regeneration, wound healing, blastema formation and distal outgrowth, yielded no profound differences from those of conventional fins in general. Impairment of regenerative capacity becomes manifested before histological differentiation of hook characters, and it is thus unlikely that their presence is the proximate cause of heteromorphic regeneration. The sequence in which the anatomical specializations characteristic of fin hooks (lepidotrichal cord, cuticle, fin web regression) appear was variable. Detailed analysis of older regenerates revealed a more regular pattern. In the first phase the characters appear to be largely independently organized, while they become locally correlated later. It is conAbbreviations: ac, actinotrichia; ahr, anterior hemi ray; cu, cuticle; dac, dense association of cells; phr, posterior hemi ray; fw, fin web;fb, fibroblasts; hr, hemi ray; hsb, hemi segment border; iwe, inner layer of the wound epithelium; ilt intralepidotrichal tissue; lac, loose association of cells; lc, lepidotrichal cord; me, mesenchyreal collumn; rowe, middle layer of the wound epithelium; pmc; posterior mc; seb, scleroblasts; sv, serous vesicles Correspondence to : B.Y. Misof (USA address)

cluded that the anatomical differentiation passes through two stages, initiation of anatomical differentiation, and then mutual adjustment of character expression leading to spatially correlated expression of the lepidotrichal cord, the cuticle and the fin web regression. Key words: Blenniidae- Pectoral fin regeneration - Fin

histogenesis - Developmental character adjustment

Introduction

The phenomenon of regeneration has not yet revealed most of its mystery. Several theoretical and experimental approaches have attempted to elucidate the mechanisms responsible for regeneration. This has been partly successful in invertebrates (for example Berill 1952; Bagunfi et al. 1988, 1989; Cummings and Bode 1984; Goss 1969; Sal6 and Bagun~t 1989), but less so in vertebrates (for recent reviews see Brockes 1989; Stocum 1991; Wagner and Misof 1992). A commonly circulated hypothesis is that regenerative capability is impaired with increasing organismal complexity (compare BagunA et at. 1989; Goss 1969), but without an operational definition of complexity (compare Kauffman 1969), this hypothesis lacks any applications (for a detailed review see Wagner and Misof 1992). In order to address this question in a reasonable way it is necessary to look for structures which exhibit differences of regenerative capacity in closely related species. Regeneration of fish fins appears to be one (see for example Blanc 1949; G~raudie and Singer 1977, 1985; Goss 1969) since it seems possible to study impairment of regeneration of homologous structures within a monophyletic group. Regenerative capability of fish fins has been studied from the end of the eighteenth century (mentioned in Morgan 1902; and Nusbaum and Sidoriak 1900). Morgan (1900, 1902) performed an elaborate series of regeneration experiments with the caudal fins of Fundulus heteroclitus and Carassius auratus to test the regenerative

154 potential of the fin. Under every experimental condition there was complete regeneration o f the fin. Morgan's study initiated a series of papers dealing with fin regeneration in various species (Banz 1935; Beigel 1910; Bogacki 1906; Eskin 1928; Morill 1906; Sauter 1934). It was concluded that a necessary condition for fin regeneration in Fundulus heteroclitus was the presence of ray stumps. If the fin rays are extirpated completely there will be no sign o f fin regeneration. Goss (1969) and Goss and Stagg (1957) confirmed Morgan's results by demonstrating that transplanted fin ray stumps induce the ectopic regeneration of fin rays. Resections of rays led to a total inhibition of ray regeneration in adult Fundulus. Not all of these results could be obtained with other species (Birnie 1934; Duncker 1905; Kemp and Park 1970; Nabrit 1929, 1931 ; Okada 1943). Even within the same species variable regenerative capability occurred, depending on the age o f the specimens (Scott 1909). Regeneration was obtained despite total resection of the fin rays in Syngnathus (Duncker 1905). Many experiments with larval fishes show comparable results (for example Nusbaum and Sidoriak 1900). Blanc (1949) summarized all available data on fin regeneration and presented evidence of variable regenerative capability in some fish families, especially in bottom-dwelling fishes. Heteromorphic regenerates, regenerates which are remarkably different from the original amputate, or total absence o f regeneration have been reported for pectoral fins of Blennius pholis and Cottus bubalis. For Blennius sanguinolentus, Eskin (1928) reported heteromorphic regeneration of the ventral and Caudal fins. A re-examination of non-regenerating species by G6raudie and Singer 1977, 1985) and Wagner and Misof (1992) yielded a different picture of regenerative capability in these species. An extensive study of pectoral fin regeneration in euteleosts (Wagner and Misof 1992) showed that heteromorphic regeneration of pectoral fins has originated at least three times independently within the euteleosts (Blennius, Cottus, Gob&, see Wagner and Misof 1992). In these three species an impairment o f fin regeneration appears at the stage of ray formation prior to any manifestation of the adult histological specializations. All these groups with heteromorphic regenerates are ground fishes, but some ground fishes do not regenerate their pectoral fins heteromorphically, e.g. Gobius, Misgurnus, Cobitis and Meiurus. The pectoral fin of blennies shows morphological specializations which are most probably adaptations to their life style as bottom dwellers of the intertidal zone (Brandst/itter et al. 1990; Laming 1982; Wickler 1960; Zander 1970, 1972). An analysis o f the fin anatomy was published by Brandst/itter et al. (1990). The fin displays a morphological and histological difference between a dorsal fan field and a ventral hook field. In the hook field a lepidotrichal cord (lc) is attached by collagenous connections to the anterior hemi rays. In proximal ray regions the lc is continuous with a column of less regularly organized loose mesenchyme. The Ic is covered by a specialized type of epidermis. In this region the superficial epidermal cells (sec) are prismatic and secrete an

dorsal

le a

ventral Fig. 1. First, second and third ray of a pectoral fin of Salaria pavo. Schematic drawing of the anatomical organized of the lepidotrichal cord, the cuticle and the asymmetric fin web regression in the hook field rays of the fin. Each fin ray (white) is composed of two symmetrically corresponding anterior and posterior hemi rays. A lepidotrichal cord (black) extends along the anterior hemi ray from base to tip. In the last distal third of rays of lc is most enlarged. The lc has sturdy collagenous conjunctions with the anterior hemi rays. In proximal positions the lc is replaced by a loose mesenchymal column with smaller diameter. In the schematic drawing lc and mesenchymal column are not discriminated, both are indicated in black. Above the le a differentiated epidermal part which produces an extracellular cuticle occurs. In this drawing the extension of the cuticle is shown stippled. Between the rays the fin web is stretched. Its shape is strongly asymmetric. The ventral (lower) edge of ray is devoid of a web in the distal region, and the dorsal (upper) edge is attached to the web. The described ray characters typically occur only in the first five rays of the pectoral fin. The asymmetric web regression exists in the first four fin web positions. Rey differentiations and asymmetric web shape constitute the hook field of the pectoral fin

extracellular cuticle of unknown chemical nature (Mittal and Banerjee 1979; Wagner, in preparation; Whitear 1986; Whitear and Mittal 1984). Between the specialized ventral fin rays an asymmetric fin web regression occurs. In Fig. l the situation is illustrated. In this paper a detailed histological analysis of the regeneration of the pectoral fin o f Salaria pavo is presented in order to address two questions: 1. Is the impairment of the regeneration process in blenny pectoral fins (Blanc 1949; Wagner and Misof 1992) related to the histological differentiation of the fin? 2. Does the temporal sequence of histogenetic events give some hints about the tissue interactions required to re-establish the adult pattern?

Material and methods For these experiments we used 52 specimens of Salaria pavo from the North Adriatic coast. Their average standard length was 74 ram, the maximum standard length 103 ram, the minimum standard length 56 mm. All fishes were mature. Males and females were used, but males prevailed, because they are easier to catch. Fishes were kept in sea water aquaria of 100 1 capacity at an average water temperature of 21~ C. Once a day they were fed ad libitum with dry food, shrimps and mussels. To avoid stress, fishes were provided with hiding-places in the aquaria. Prior to amputa-

155 tion, fishes were anaesthetized with MS 222 (Sandoz). Left pectoral fins were amputated with micro scissors so that ray stumps of approximately 2 mm remained. All rays were cut orthogonally. After operation the fishes were kept isolated in 30-1compartments within 100-1 aquaria. To examine fin regeneration fishes were killed by an overdose of MS 222. Regenerates were evaluated at 7, 14, 24, 48, 72 h and at 4, 5, 6 days post operationem (p~o.). Late regenerates were evaluated at approximately 30%, 40%, and 60% replication of the original fin length. For histology regenerates were fixed in Bouin's fixative or in buffered 3% glutardialdehyde (according to Gfiraudie 1977). Regenerates were decalcified with Titriplex III (Merck) for a period of 4 to 6 days. Paraffin sections, 6 ~tm thick, were made on a rotational microtome, and were stained with the trichrome technique after Goldner. Semi-thin Araldite sections, 1.5 gm thick, were prepared with regenerates fixed in Bouin and were decalcified with Titriplex III. Araldite sections were stained with toluidine blue after Richardson, with Stevenel's blue (DelCerro et al. 1980), and with Regaud's iron haematoxylin. PC3D software from Jandel Scientific, California, was used for three-dimensional reconstruction of the regenerates of 30%, 40% and 60% length of the original fin. A digitizer board was used to feed the computer with data from serial sections. Staining with neutral red was viewed to demonstrate possible cell death in the regressing fin web (after Hinchliffe and Griffiths 1986). Peritoneal injections of I% neutral red solution showed no staining in the regenerated fins. In a second trial, amputated regenerates of pectoral fins were incubated for 1 min in t % neutral red. With this method, staining of necrotic cell material was successful in controls of injured cichlid fins with artificially induced cell death, but again absent in regenerates of pectoral fins of blenhies of every stage. Results The regeneration process is subdivided into four stages: would healing, blastema formation, fin ray formation and distal outgrowth and the differentiation of hook characters (according to Wagner and Misof 1992). There are, of course, no discrete limits between these four stages. Especially, the distal outgrowth overlaps with the early differentiation phase.

In the connective tissue, the lc, the scleroblast ray coat, and intra-lepidotrichal tissue (ilt), fibroblast like cells appeared at the amputation level (Fig. 2 C). Under the basal membrane leucocytes were frequently recorded, indicating the process of wound healing. No mitotic cells could be observed in the wound epithelium, the lc, the iIt and in the scleroblast ray coat at this stage.

Blastema formation Blastema formation required 4 days, at 2i ~ C water temperature. At the beginning, fibroblast-like cells, in particular scleroblasts and cells from the connective tissue, entered the space beneath the wound epithelium (Figs. 2 C, 3 A, B). Frequently, cells from the connective tissue were closely attached to the basal membrane o f the wound epithelium. Scleroblasts of both hemi rays covered the amputation surface. Many of them were closely attached to the ray stumps forming a scleroblast cap over the amputated bony hemi rays. At the amputation level osteoclasts eroded the exposed ray surface (Figs. 2C, 3 C). These osteoclasts disappeared after blastema formation had finished. After the initial phase of blastema formation, (3 days p.o.) proliferative activity was observed in the connective tissue and frequently in the scleroblast ray coat. In the lc no proliferative activity was observed. Proximal to the amputation level proliferative cells were recorded in the scleroblast coat all the way down to the ray base. In late periods of blastema formation (4-5 days p.o.), mitotic cells were frequently found in the blastema (Fig. 4). In the epidermis proliferative activity was found from the ray base up to the amputation level. Distal to the amputation level no mitotic activity was observed. The blastema itself was composed of a histologically heterogeneous cell association. Distal to the hemi ray stumps cells with dense cytoplasm were concentrated. They appeared to be invading scleroblasts.

Wound healing Fin ray formation and distal outgrowth After 7 h post operationem (p.o.), at 21 ~ C water temperature, the first signs of wound healing appeared. The ventral part of the wound on the fin stump took more time to heal, probably because of the greater wound surface. Thus, the wound-healing process proceeded from the dorsal to the ventral part of the stump. A complete epithelial cover of the whole fin stump required nearly 24 h at 21 ~ C water temperature to be developed. The wound surface was first closed by a thin epithelial layer 15 cells thick, 24 h after operation (Fig. 2A). Three histologically different layers could be distinguished in the wound epithelium after 48 h p.o. (Fig. 2B, C): the innermost layer consisted of densely packed cuboidal cells. The middle layer was characterized by a loose cell association. The outer layer consisted again of compactly packed cells with densely stained cytoplasm. The cells of the outer layer were of squamous appearance. The wound epithelium was separated from the underlying mesodermal tissues by a thin basal membrane.

Distal outgrowth started with the formation of hemi rays that first became visible at the distal tip of the regenerative 5 to 7 days p.o.. In paraffin sections the first signs of hemi ray formation were in close apposition to the basal membrane of the epidermis. When ray formation started, the blastema was structured in a scleroblast cell mass, close to the basal membrane with histologically dense cytoplasm and an inner mass of fibroblastlike cells (Fig. 5A, B). This inner cell mass corresponded to the ilt of the ray stump. The dense collagenous character of the remaining ilt extended into the corresponding blastema portion, forming an obvious histological continuity between these two parts. This was recognizable prior to the first signs of hemi ray formation (compare Fig. 4). It was not possible to decide whether a primary or secondary cell lineage provided continuity of the stump scleroblasts and the blastema scleroblasts. In addition to ray formation in the distal tip, irregular bone

156

Fig. 2A-C. Longitudinal section through a fin ray stump after 24 h p.o. (A); 48 h p.o. (B, C). A A wound epithelium has closed the wound surface. Blastema formation has not started. Beneath the wound epithelium necrotic cell material is left. Bar 100 ~tm. B First invading fibroblast-like cells are visible above the amputation level.

This marks the beginning of blastema formation. The wound epithelium has three layers. Fibroblasts enter the space above the amputation level closely attached to the basal membrane. Bar 100 Ixm. C Detail of B. Bar 25 gm

157

Fig. 3A-C. Longitudinal sections through fin ray stump 72 h p.o. A Semi thin section of a ray stump 72 h p.o. Pronounced invasion of fibroblasts into the space beneath the wound epithelium. Serous vesicles above the amputated hemi rays occur temporarily. They

disappear during blastema formation. Bar 25 gm. B Detail of A. Bar 25 ~m. C Multinucleated osteoclast eroding the ray matrix at the amputation surface. Bar 10 gm

158

Fig. 4. Longitudinal section through a ray stump 4 days p.o. A blastema has developed. Mitoses occur in the blastema as well as in the stump. The blastema shows a heterogeneity between a central dense and a peripheral loose cell association. The dense

position is in direct correspondenceto the intralepidotrichaltissue. In the loose cell aggregation above the hemi ray, stump cells of dense basophiliccytoplasm(bc) are more frequent. Bar 50 gm

deposition above ray stumps occurred. These irregular bone deposits linked the ray stump to the reformed hemi rays of the distal tip. In the lc stump no mitotic cells were recorded, nor was autochthonous regeneration of this anatomical unit observable at this stage of regeneration. Although actinotrichia appear before ray formation in ontogeny (G~raudie 1977; G6raudie and Landis 1982; Wood 1982; Wood and Thorogood 1984) we never found a blastema with actinotrichia and without hemi rays. In fact, we found ray formation prior to the appearance of actinotrichia (compare Figs. 5B, 6B). This is in accordance with observations of Goss and Stagg (1957), Mari-Beffa et al. (1989) and Santamaria and Becerra (1991). In the epidermis proliferative activity was high even distal to the amputation level, but did not extend as far as to the distal tip.

Macroscopic description of the differentiation process

Differentiation of hook characters The differentiation phase overlapped to a high degree with the outgrowth phase. It started at 20% regeneration of the original amputate length in the ventral rays.

The reconstitution of the specialized ventral hook field started macroscopically with a strengthening of the first two rays. This was a result of/c-formation on the rays. This strengthening progressed to the third, fourth and fifth ray. Loosely correlated with the strengthening of rays, asymmetric fin web regression started between the first and the second ray and proceeded towards more upper fin web positions. In order to decide whether the changes of web shape were mediated by physiological cell death or were caused by differential growth rates in the web and the fin ray, neutral red staining was performed (after Hinchliffe and Griffiths 1986). There was no staining in regressing fin web parts. Macrophages, which typically eliminate dying cells and necrotic material, were also not observed in the web. This suggests that the asymmetric web regression depends on differential growth in the web relative to the fin rays rather than on physiological cell death. Morphometric observations on hook differentiation in blenny larvae (Pfizmfindi 1991) support this interpretation.

159

Fig. 5A, B. Longitudinal section through a regenerating fin ray, 6 days p.o. A About 1 m m has regenerated. Hemi ray development is pronounced. Bar 100 lam. B Detail. There exists a sharp distinction of a scleroblast cell mass and the ilt in the regenerated part.

The hemi segments are early manifested in the regenerating fin. Above the scleroblasts the inner epidermal cells become columnar. Note that no actinotrichia are visible in the regenerate. Bar 25 gm

160

Fig. 6A, B. Longitudinal section through another regenerating fin 6 days p.o. A Again about I mm has regenerated. The situation is comparable to that in Fig. 5 except the appearance of aetinotrichia in the ilt. Bar 50 gin. B Detail. In the distal tip the actinotrichia

are closely attached to the basal membrane. The actinotrichia never extend into the scleroblast cell mass of the regenerating hemi rays. Bar 10 ~tm

161 Histological description of the differentiation phase This section gives exemplary descriptions of three regenerates, after the time at which the strengthening of the rays was visible macroscopically. The degree of fin web regression was different in each specimen. Only the hook fields of the regenerates are discussed in detail, because the fan field regenerated without significant alterations to the histological pattern of the original fin part

First case: regenerate of 30% of the original amputate length (26 days p.o., 21 ~ C water temperature) The initial stages of fin web regression were evident only between fin rays 1-2 and 2-3 (Fig. 7 A, B). In the first ray a regularly organized and collagenous lc (according to the description in Brandst~itter et al. 1990) was developed. There existed no clear distinction between the lc of the stump and that of the regenerate, which was also the case for the bony hemi rays. The superficial epidermal cells (SEC) over the lc were columnar, and an extracellular cuticle was present. This cuticle was continuous with the cuticle of the stump. The distal extension of the cuticle reached the level of the ray formation zone. In the proximal direction the lc extended down to the ray base where no corresponding cuticle was visible. The first ray differed from the original one only in its relative shortness. The second ray of the regenerate showed neither Ic nor a cuticle in its distal part. Proximal to the level of fin web attachment a regularly organized lc of low diameter was developed. Distal to the amputation plane this regular cell organization disappeared. The collagenous connections to the anterior hemi ray were detached. This mesenchymal column of the proximal regenerated ray was linked by a thin joint to a histologically similar column in the stump. No cuticular differentiation of the epidermis existed on the second regenerated ray. In the third ray no signs of a cuticle were visible, nor was a regularly organized Ic expressed. A moderately prominent mesenchymal column extended along the anterior hemi ray. More distally along the regenerated ray, the anterior column was replaced by a short mesenchyreal column at the posterior hemi ray. This allotopic location of a mesenchymal column was rare, and never occurred in older regenerates or in the normal anatomy. Rays 4, 5, 6 and 7 possessed a moderately prominent mesenchymal column on the regenerated anterior hemi rays.

Second case: regenerate of 44% of the original amputate length (29 days p.o., 23~ C water temperature) The first ray resembled that described in the first case (Fig. 8A, B). Web regression between the first and the second ray was even less pronounced than in the first case. But here the second ray exhibited a regular lepidotrichal cord on the anterior hemi ray and a well-developed cuticular differentiation covering the lc. In the second ray, as in the first, the cuticle extended more distally than the lc, approximately 140 gm towards the level of

ray formation. In all other rays of the regenerate no signs of a cuticle, a Ic or a mesenchymal column were visible.

Third case: regenerate of 66% of the original amputate length (36 days p.o., 22~ C water temperature) The first three fin rays were exact replicates of the offgina state except for their relative shortness (Fig. 9A, B). Fin web regression in the first position was strongly pronounced, in the second position hardly initiated. The fourth ray exhibited a slightly developed mesenchymal column on the anterior hemi ray, as did the fifth and the sixth rays. Rays of the regenerate, other than the first three showed no clear histological differentiation of the epidermis or the lc.

MalJormed regenerates Regenerates of 12 specimens were examined after they had regenerated to approximately original fin length. This stage was reached about 8 weeks p.o.. Within this sample the frequency of malformations was 58% (n= 12%). Malformed regenerates were characterized by non-regenerated, growth-retarded, or bent rays. Histologically, non-regenerated or growth-retarded rays show partial bony fusions of corresponding hemi rays or fusions of neighbouring rays. These fusions may have prevented synchronous regeneration of affected rays, but they did not prevent their regeneration in all cases. Formation of hemi rays distal to the fused hemi rays was possible. These regenerating rays were then retarded in growth.

Discussion

Is the impairment of the regeneration process in blenny pectoral fins related to the histological differentiation of the fin ? The regeneration of the pectoral fin of Salaria pavo passes through two steps. First, the regenerate re-establishes the ray pattern of the original fin. In the second step regenerating rays of the hook field are progressively elaborated to express the characters specific for the hook field. The first step resembles the regeneration process of normal fins, e.g. those of Fundulus heteroclitus (reviewed in Goss 1969; Goss and Stagg 1957), Tilapia mossambica (Haas 1962; Kemp et al. 1968; Kemp and Park 1970), Tilapia melanopleura (Santamaria and Becerra 1991) or Carassius auratus (Maria-Beffa et al. 1989). Wound healing and blastema formation differ in no way from the processes described for these species. At blastema formation fibroblasts form a blastema above every ray stump. Thus it is worthwhile to distinguish blastemata above each ray stump, formed after the initial healing process and the distal growth zone of every regenerating ray. The formation of the hemi rays happens in the initial blastemata and is then followed by an elongation

162

7A

8A

~

FR3

7B

8B

FR2

-I IITIC

phr =qIC ahr

9B 9A

@ r/

~

~ ~ "

FR1

Fig. 7A, B. Three-dimensional reconstruction of three rays of the regenerate of 30% regeneration (Case 1). A Computer aided reconstruction from an anterior view. The lc and the m c are indicated grey. B Schematic interpretation of the reconstruction from a posterior view. The interpretation is shown from the posterior view because of the technical possibilities for demonstrating the spatial relationship among the anatomical parts. Rays are cut off and the epidermis is shown translucent so that the relation of lc, cuticle and fin web regression is clear. To the left is the first fin ray. Only the first fin ray shows a character combination comparable to the original one. The second fin ray has no cuticle. The lc is expressed only along a short section of the anterior hemi ray; proximal it is continous with a mesenchymal column. This mesenchymal column is constricted at the amputation level. The third ray has also developed a mesenchymal column. It is again constricted at the amputation level and is distally replaced by a short portion of a mesenchymal column on the posterior hemi ray. This character state combination is not found in the differentiated fin and represents a transitorial state of character differentiation during regeneration

Fig. 8A, B. Three-dimensional reconstruction of two rays of the regenerate of 44% regeneration (Case 2). A Computer aided reconstruction from an anterior view. The lc and the rnc are indicated grey. B Schematic interpretation of the reconstruction from a posterior view. lc and cuticle are well differentiated; in contrast fin web regression has not been initiated. This shows that lc and cuticle can occur without fin web regression

o f the h e m i rays. C o n s e q u e n t l y , the w o r d b l a s t e m a t a here relates o n l y to the s i t u a t i o n p r i o r to h e m i r a y form a t i o n . T h e a n a t o m i c a l s i t u a t i o n o f the tips o f regenera t i n g r a y s is c o m p a r a b l e to the s i t u a t i o n o f a d u l t fins (see G 6 r a u d i e a n d L a n d i s 1982; S a n t a m a r i a a n d Becerra 1991). We were n o t a b l e to find a c t i n o t r i c h i a before h e m i

r a y s a p p e a r e d in the b l a s t e m a t a , in fact, we o b s e r v e d h e m i r a y f o r m a t i o n p r i o r to the a p p e a r a n c e o f a c t i n o t r i chia. This is in s h a r p c o n t r a s t to the o n t o g e n e t i c develo p m e n t o f fins ( G 6 r a u d i e a n d L a n d i s 1982; W o o d 1982; W o o d a n d T h o r o g o o d 1984, 1987) b u t in a c c o r d a n c e with o b s e r v a t i o n s on r e g e n e r a t i n g fins ( G o s s a n d Stagg 1957; M a r i a - B e f f a et al. 1989).

Fig. 9A, B. Three-dimensional reconstruction of the first three rays of the regenerate of 66% regeneration (Case 3). A Computer-aided reconstruction from an anterior view. The lc and the m c are indicated grey. B Schematic interpretation of the computer-aided reconstruction of the regenerate. The situation of the first two fin rays closely resembles that of the amputate. Cuticle and lc appear in both rays, and between them there is a pronounced web regression. In the second fin web position no web regression is visible, but cuticle and lc are well established. That combination of character states is not found in differentiated fins. lc and cuticle expression precede fin web regression

163 Cytomorphological heterogeneities in the cellular composition of the blastemata (see Fig. 3C) were evident. Goss and Stagg (1957) also observed heterogeneities in the blastema of Fundulus. Goss interpreted this as possible cell lineage restrictions. Experimental results on allochthonous regeneration of transplanted rays convinced him that " I f this is true, then the cells of the blastema are not so fully dedifferentiated as their appearance might lead one to believe." (Goss et al. 1969; p. 123). In this case it is worth distinguishing cytomorphological differentiation and cytological determination. A blastema is comprised of cytomorphologically undifferentiated (dedifferentiated) cells by definition, but these cells need not have lost their state of determination. There are well-known examples where spatially separated domains of different gene expression, as molecular markers of cellular determination, lack cytomorphological differences, e.g. the blastoderm of the Drosophila embryo (Ingham 1988) or the developing hindbrain of vertebrate embryos (Lumsden and Keynes 1989; Lumsden 1990; Wilkinson and Krumlauf 1990). So what Goss really suggested was that the blastema cells might at least not be cytologically undetermined. Thus an understanding of cell lineage restriction in the blastema may shed light on the interplay of the cytomorphological dedifferentiation and cellular determination processes responsible for the reconstitution of regenerating rays. This distinction could be relevant for the explanation of the impairment of regeneration (see discussion below). SaIaria pavo specimens regenerated their pectoral fins heteromorphically in 58% of the experiments. Malformed regenerates show no disturbance of histological specializations of hook field rays. Disturbances occur at the ray formation stage of the regeneration, while the histogenetic elaboration of hook field rays occurs much later (see also Wagner and Misof 1992). Additionally the fifth ray, the least differentiated of the hook field rays (i.e. the extent of cuticularized epidermis and the size of the lc are least) is the ray that is most frequently affected by malformations (Wagner and Misof 1992). Hence, the differentiation of hook field-specific characters does not directly interfere with fin ray regeneration. Nevertheless the distribution of malformations among the regenerated pectoral fin rays clearly demonstrates a nonequivalence (in the sense used by Wolpert 1989) between the blastemata of hook field rays and those in the upper fin field. There are at least three conceivable ways in which this nonequivalence could be caused. The first is that all the blastemata are equivalent along the fin stump, but the vascularization, innervation and other possible tissue interactions which maintain and/or suffort blastema and hemi ray formation, are locally different. Then the differential capacity to regenerate between hook field rays and fan field rays would be caused by extrinsic factors and would not be due to intrinsic properties of the ray blastemata cells. A way to test this hypothesis experimentally would be mutual transplantations of ray blastemata. No profound shifts of the distribution of the malformations should occur along the fin stump. The second possibility is that all blastemata are equiv-

aleut early in regeneration, as in the first case, but a nonequivalence arises due to a pattern formation process which leads to a distinction of the upper and lower blastemata much earlier than Ic and cuticle differentiation. This hypothesis yields two experimentally testable predictions. First, it should be possible to perturb the generation of the nonequivalence of the fan and hook field cells prior to a critical time post operationem. Then the blastemata should exhibit a switch from early equivalence to an intrinsic nonequivalence in transplantation experiments. Second, fin rays ectopically transplanted after some critical time p.o. but before any morphological non-equivalence is evident, should differentiate like their neighbours in regenerates. Finally the third conceivable way is that the blastemata display an intrinsic nonequivalence from the beginning. This could be due to local recruitment of blastemal cells from already nonequivalent stump cells. Transplantation experiments of blastemata should clearly demonstrate these intrinsic properties in every possible experimental case. This interpretation of the nonequivalence can easily be linked to Goss's hypothesis of blastema differentiation. There is no simple answer to the question raised at the beginning of this section. The only certain conclusion that can be drawn from the present results is, that the differentiation of the hook field-specific tissue is not the direct cause of the malformations. Nevertheless there is a correlation between the location of the malformations and the extent of the hook field in the intact fin. Whether this correlation is due to differences in the fin ray stumps, or due to intrinsic cytological differentiations of the blastemal cells of the hook and fan field blastemata, can only be decided by transplantation experiments.

Does the temporal sequence of histogenetic events give some hints about the tissue interactions required to re-establish the adult pattern ?

In the adult Salaria pavo the hook field-specific differentiations are locally correlated. In addition, the lc and the cuticle occur always asymmetrically on the anterior hemi rays. A comparative study within the Blenniini demonstrated that the characteristics of hook field rays are a fixed trait for the Blenniini (Brandst/itter et al. 1990) despite variations in the shape of the see and their cuticle and variation in the relative size of the lepidotrichal cord and its collagenous attachment to hemi rays. In the regenerate, re-establishment of the hook field characters is a process proceeding from the first to the fifth ray. The elaboration of the hook characters of the fin (for basic anatomy compare Becerra et al. 1983; Lanzing 1976; Montes et al. 1982) starts in the first ray and the first fin web position. The subsequent course of differentiation in the second, third, fourth and fifth ray proceeds from the lower to the upper fin rays. This pattern of differentiation corresponds to that reported from the ontogeny of the pectoral fin of Salaria pavo (Pfizmfindi and Wagner, in preparation). Thus the pro-

164 cesses of elaboration during regeneration and development during ontogeny are comparable in this respect. During regeneration, deviations from this regular occurrence of hook characters are frequently observed. For instance, allotopic appearance of a loose mesenchymal column, which is the precursor of the regular lc, is observed in regenerates up to 60% regeneration. A mesenchymal column was found on fin rays outside the hook field or even on posterior hemi rays instead of anterior hemi rays. These observations suggest that there must be an active inhibitory mechanism suppressing the mesenchymal column dorsal to the hook field, and on all posterior hemi rays in later stages of regeneration and in the adult fin. A differentiation o f the sec's always occurs after a Ic is regenerated. This temporal succession of cuticle and lc formation is consistent with the hypothesis that the cuticle is induced and/or maintained by the lc (Wagner 1989). However, during regeneration the spatial correlation between cuticle and lc is much looser than in the final pattern. For instance, a cuticle occurred distal to the lc in regenerates (Figs. 7, 8, 9) i.e. in parts of the regenerating epidermis that were not in contact with the lc before. This distal portion of the cuticle was in continuity with the cuticular region covering the fully developed Ic, and may thus be due to a lateral spread of cuticle formation without direct local induction from the mesenchyme. Whether there is a direct mesodermalectodermal interaction has to be tested experimentally. Preliminary results show that an extirpation of the Ic leads to a dedifferentiation of the cuticular epidermis in Paralipophrys trigloides and Parablennius gattorugine (Wagner, unpublished observations). According to the induction model of fin hook development (Wagner 1989) the lc and the cuticle should depend on fin web regression. However, in two o f the three regenerates reconstructed from serial sections, a fully developed lc and cuticle were present despite the presence of a fin web. This shows that Ic differentiation can occur in the presence of a fin web, and falsifies this part of the cyclical induction model of hook development (Wagner 1989). In addition, it was found that the lc can occur in the absence o f a cuticle, which suggests that lc development may be the first step in hook differentiation. The relation o f lc, cuticle differentiation and asymmetric fin web regression is irregular at first. In cases two and three of the reconstructed regenerates a fully developed lc was found with no fin web regression. In case one, a slight fin web regression was already present, while only a weak mesenchymal column and no cuticle was seen in the second ray. The initiation of fin web regression seems to be independent of histological ray differentiation, but pronounced web regression always appears after differentiation o f the lc and cuticle. This shifting from irregular to regular relations may indicate a shift in mechanisms responsible for web regression. Preliminary experimental evidence that this could be true was presented by Wagner and Almeder (1991). In summary, during the reconstitution o f the hook characters the first step can either be the lc differentia-

tion or the first signs of a fin web regression. However, these events are spatially uncorrelated. Thus histogenesis does not establish the spatial correlation of the lc, cuticle and fin web regression which is typical for adult fins. On the other hand, in experiments with adult fins (Wagner and Almeder 1991, Wagner, in preparation) interdependences of character expression have been demonstrated to contribute to the mutual adjustment o f these characters. This is comparable to the mechanism that ensures the locally adjusted development of the retina and the lens in the vertebrate eye. Contrary to earlier suggestions, the eye cup does not induce the lens. Lens induction is caused by a series of inducers. It clearly starts prior to the formation of the optic vesicle. The eye cup only maintains the lens formation potential by excluding cephalic mesenchyme from contact with the presumptive lens material (Jacobson and Sater 1988). Thus these interactions which maintain the adult pattern must appear after, and the decoupled from, the histogenetic processes. It is therefore suggested that the final pattern o f hook character expression is caused by mutual stabilization which supersedes mechanistically different morphogenetic processes. One may thus distinguish between the morphogenetic origin of tissues, and morphostatic interactions causing the maintenance and stability of the final structure.

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