Zoology 113 (2010) 175–183

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Zoology

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Skeletal muscle–melanocyte association during tadpole tail resorption in a tropical frog, Clinotarsus curtipes Jerdon (Anura, Ranoidea)

Lekha Divya a,1, Reston S. Beyo a,1, Parameswaran Sreejith a,1, Mohammad A. Akbarsha b,1, Oommen V. Oommen a,∗,1 a Department of Zoology, University of Kerala, Kariavattom, Thiruvananthapuram 695581, India b School of Life Sciences, Bharathidasan University, Tiruchirapally 620024, India article info abstract

Article history: We tested the hypothesis that melanin has a role as a molecule within the thyroid-mediated cascade. Received 17 June 2009 Light microscopic and ultrastructural changes in the skeletal muscle during tail resorption in tadpoles of Received in revised form 30 October 2009 the tropical frog Clinotarsus curtipes Jerdon (Anura: Ranoidea) were observed. Light microscopic analysis Accepted 6 November 2009 at metamorphic stage XVIII showed a melanized epidermis. A gradual migration of melanocytes from the epidermis to the dermis and filopodia of melanocytes pervading the skeletal muscle preceded tail Keywords: resorption. The invasion of melanocytes into the muscle bundles coincided with the breakdown of the Clinotarsus curtipes muscle bundles into sarcolytes and the arrival of macrophages at this site. This would suggest that the Melanin Filopodia melanocyte–sarcolyte association signals the arrival of macrophages at these sites as metamorphosis Sarcolytes progressed. Melanophages, macrophages with melanin granules, were observed at the climax stage of Metamorphosis XXIII. The sarcolytes and the melanin granules were phagocytosed by macrophages so as to completely cleanse the exocytic muscle debris and the melanin granules. The presence of large melanomacrophage centers in the tadpole liver at metamorphic climax suggests that these phagocytic macrophages were further processed in the liver and, likely, in the spleen. It is proposed that melanin, a byzantine molecule, has a role in the cascade of events leading to tail resorption in anuran tadpoles. © 2010 Elsevier GmbH. All rights reserved.

1. Introduction Anurans have been the focal organisms for understanding amphibian metamorphosis, primarily due to the dramatic nature The amphibian metamorphosis from tadpole to frog is one of of their metamorphosis and the easy handling of anuran species in the most enigmatic post-embryonic transformations. It encom- research. However, among the anurans, metamorphosis has been passes most, if not all, of the intra- and extra-cellular processes reasonably well studied only in three species, Xenopus laevis (South that are involved in vertebrate embryogenesis and organogenesis African clawed frog), Rana catesbeiana (bull frog) and Rana pipiens (Shi, 2000). The changes that take place in the tissues and organs (Northern Leopard frog), all temperate species. Although other anu- of the tadpole, as it transforms into a frog, have made it a model rans like Bufo and Rana species have also been studied, there is no for studying the post-embryonic organ remodeling and develop- report thus far on tail resorption in any tropical anuran. ment for nearly a century (Shi, 2000). Three primary morphological Clinotarsus curtipes Jerdon (1854) is widely distributed in the changes occur during metamorphosis: (i) resorption or regression Western Ghats of India. Known as the ‘leaf-litter frog’, it is found of tissues, organs or organ systems that have a primary function in a number of tropical forest types (evergreen to semi-evergreen only in the larva, (ii) remodeling of larval organs or organ systems moist and also dry deciduous forests). The tadpoles of C. curtipes into their adult form, and (iii) de novo development of tissues in the are the largest of any Indian tadpole known so far, although there adult that are not required by the larva. These changes are more are reports by Fabrezi et al. (2009) on giant tadpoles of Pseudis marked in anuran species and less obvious in urodeles and caecil- platensis Gallardo, 1961 having a prolonged metamorphic period ians. This entire panorama of events leads to the development of and also of Pseudis paradoxa by Emerson (1988). C. curtipes tad- an adult organism capable of living or surviving in a habitat very poles, which attain a length of nearly 10 cm, have an extended different from the one in which the tadpoles live. larval period lasting for about 6 months (Valamparampil, 1994) that makes them suitable for the study of stages in the developmen- tal changes. The tadpoles grow during the early stages of their life ∗ history and undergo metamorphosis with complete tail regression Corresponding author. Tel.: +91 471 2308906; fax: +91 471 2418906. E-mail address: [email protected] (O.V. Oommen). before turning into adults. C. curtipes tadpoles are darkly colored 1 All authors contributed equally to the manuscript. with dense melanophores in the skin and elsewhere. The tadpoles

0944-2006/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2009.11.002 176 L. Divya et al. / Zoology 113 (2010) 175–183

Fig. 1. Representative critical stages in the life cycle of Clinotarsus curtipes based on external morphology and morphometric index (left panel, photographs; right panel, line drawings). (A) Stage XVIII tadpole, (B) stage XXI, (C) stage XXIII, and (D) metamorphosed froglet. Scale bar = 1.3 cm. retain their black coloration from the early feeding stage to the cli- retention of strong tail muscle fibers until late during the life his- max of the metamorphic stage. Subsequently, they develop into the tory (Hiragond et al., 2001). These, at the end of metamorphosis, typical adult ‘bicolored frog’. suddenly collapse, giving rise to tail resorption. The process should The familiar black or brown coloration of amphibian tadpoles involve much more than the conventional apoptotic machinery. is usually attributed to melanin. Most life forms produce melanins, Resorption of the tail in the context of amphibian metamorpho- which are dark (usually black) and complex, poorly characterized sis involves degeneration of not only the skeletal muscle but also pigments, that are synthesized enzymatically or auto-oxidatively of the vertebral column/notochord, nerve cord, blood vessels, con- from a variety of cyclic, heterocyclic, phenolic or other resonance- nective tissue, etc. (Weber, 1964; Kerr et al., 1974; Kinoshita et al., stabilized precursor molecules. Melanins make up a heterogeneous 1985). Unlike the other tissues, which can be reabsorbed through class of natural pigments that have a myriad of biological functions the processes of apoptosis and/or lysosome-mediated necrosis, the (Nosanchuk and Casadevall, 2003). The presence of various kinds melanocytes of the skin do not succumb to either of these cell of melanins in almost every taxon suggests an evolutionary impor- death processes. A correlation has been reported between melanin tance. Up until now, however, there is no agreement concerning content and melanomacrophagic component during hibernation the primary or basic function of melanogenesis and its product in Rana esculenta (Barni et al., 2002). It is generally believed that melanin, or even concerning its character as a primary or a side the melanocytes of the tail skin provide melanin pigment for the effect (Plonka and Grabacka, 2006). post-metamorphic keratinization of the skin to the rest of the body. Anuran tail resorption has been used by several investigators as Purrello et al. (2001) reported that liver pigment cells containing an endpoint index for several morphological, histological, biochem- melanin underwent programmed cell death faster in comparison ical and molecular studies. The tail is genetically pre-determined with cells with low pigment content in R. esculenta. Therefore, to be resorbed, requiring only sufficient levels of thyroid hormones melanin itself might be involved in some way in the tadpole meta- to initiate the process (Valamparampil and Oommen, 1997). All morphosis. tissues that comprise the tadpole tail are resorbed during meta- morphosis, including the epidermis, connective tissue, skeletal muscle, blood vessels, notochord, etc. Kerr et al. (1974), in their 2. Materials and methods classic description of amphibian metamorphosis, suggested that the tail resorption occurs through a special kind of apoptosis. The Tadpoles of C. curtipes (Rana curtipes Jerdon) of various stages present paper describes the stage-wise morphological events asso- were maintained in the laboratory in fiberglass aquaria with aer- ciated with tail resorption in the tropical anuran, C. curtipes. The ated, dechlorinated tap water at conditions of ambient temperature so-called ‘prolonged metamorphosis’ of this species requires the (29 ± 2 ◦C) and photoperiod (approximately 12L:12D) and fed on L. Divya et al. / Zoology 113 (2010) 175–183 177

Fig. 2. Tail sections of Clinotarsus curtipes, embedded in paraffin and stained with nile blue sulphate. (A) Stage XVIII: showing epidermis (e), dermis (d), skeletal muscle (sm) and melanocytes (m); (B) stage XXI; (C) stage XXIII (*indicates melanin in deeper locations); (D) semi-thin tail section at stage XXIII showing sarcolytes (sl), migrating melanocytes (mg), blood vessel (bv) and empty space (es) surrounded by melanocytes. Toluidine blue O stain. Scale bars = 40 ␮m. boiled spinach (Amaranthus sp.) ad libitum. The tadpoles were Tadpoles were anesthetized by placing them on ice and trans- assigned to stages on the basis of external morphology (Taylor and verse slices of the tails of the metamorphic stages XVIII, XXI and Kollros, 1946) and the morphometric index (Valamparampil, 1994). XXIII (stages 41, 42 and 44 according to Gosner (1960), Fig. 1) were The tadpoles were broadly divided into premetamorphic (before fixed in 4% paraformaldehyde for 24 h at 4 ◦C. Samples were dehy- stage XI), prometamorphic (stages XII–XIX) and metamorphic cli- drated, infiltrated, embedded in paraffin and sectioned in a Leica max (stages XX–XXV). microtome (Leica, Wetzlar, Germany). The sections were stained The care and treatment of animals used in this study were in with hematoxylin and eosin for routine histological observations accordance with institutional and national guidelines (G.O. (Rt) No. and with nile blue sulphate for histochemical detection of melanin 240/07/F & Wld; IAEC-KU-2/05-06). (Lillie and Fullmer, 1976). 178 L. Divya et al. / Zoology 113 (2010) 175–183

Fig. 3. Light microscopy of semi-thin liver sections of Clinotarsus curtipes. (A) Stage XVIII. Arrows indicate sparsely distributed melanin. (B) Stage XXI, with large melanomacrophage centers (mmc) showing intense melanin pigmentation. (C) Stage XXIII. Arrows depict mmc undergoing melanin degradation. Toluidine blue O stain. Scale bars = 20 ␮m.

The sections of the tail were fixed in 2.5% glutaraldehyde in 3. Results cacodylate buffer (pH 7.2) at 4 ◦C for 4 h, washed in the buffer, dehydrated in grades of alcohol followed by propylene oxide and 3.1. Light microscopic observation of melanocyte embedded in resin. Semi-thin sections (1 ␮m thickness) were localization/distribution during regression of the tadpole tail at obtained with an ultra-microtome and stained in toluidine blue O stages XVIII, XXI and XXIII for light microscopic observation under an Axio-3 research micro- scope (Carl Zeiss AG, Oberkochen, Germany). Ultra-thin sections The tadpoles have well developed hind limbs with all the toes were stained in Reynold’s lead citrate and aqueous uranyl acetate well differentiated and a fully functional tail at stage XVIII (Fig. 1A). (Beyo et al., 2007). Electron micrographs were obtained in a trans- At this stage, the muscle bundles were prominent and intact and mission electron microscope (Philips 201C; Philips, Amsterdam, most of the melanocytes were located in the epidermis as a dense Holland) at 75 kV. Special attention was given to changes in the layer outside the dermis (Fig. 2A). The nile blue sulphate staining skeletal muscle and melanocytes. produced only a faint reaction. A few melanocytes were present L. Divya et al. / Zoology 113 (2010) 175–183 179 among the intact skeletal muscle bundles with no trace of dis- integration or even dissociation. The toluidine blue O sections of the liver at this stage showed sparse brown specks of melanin (Fig. 3A). The melanocytes reallocated gradually from the epidermis to the dermis with the regression of the tadpole tail. Both forelimbs of the tadpoles had emerged through the skin windows by stage XXI (Fig. 1B). The body became slender and could be well dif- ferentiated into head and trunk regions. The tadpole is an active swimmer at this stage. Although the tail is almost the same size as in stage XVIII, histological observation revealed condensation of the melanocytes towards the dermis (Fig. 2B). At this stage, the muscle fibers indicated some cytological changes reflective of degeneration but sarcolytes were not yet formed. Dense aggregates of melanin- containing cells grouped together and surrounded by a membrane [melanomacrophage centers (MMCs)] were noticed in the liver at this stage of metamorphic climax (Fig. 3B). Stage XXIII tadpoles developed prominent bulging eyes. The head was still broader than the trunk. The tail underwent a consid- erable reduction in size (Fig. 1C). The active swimmer of stage XXI turned into an almost sedentary tadpole. The froglets that emerged at the end of stage XXV had fully developed limbs and the remnant of the once elaborate tail as a small stub (Fig. 1D). These froglets were much smaller than the tadpole at its largest size. The tail of the tadpole at stage XXIII was much lighter than earlier (Fig. 1A–C) and had fewer melanocytes in the epi- dermis and even at the junction between the epidermis and dermis, but most were seen in the dermis (Fig. 2C). Subse- quently, there was intensified invasion of melanocytes among the muscle bundles, which in turn were disaggregated, form- ing the sarcolytes. Cells comparable to macrophages were also seen amongst the sarcolytes (Fig. 2D). The MMCs in the liver began to degrade their melanin content (lost their intense, heavy black pigmentation) and appeared to contain lipid-like granules (Fig. 3C).

3.2. TEM observations of stages XXI and XXIII tadpoles

The melanocytes that were located among the muscle fibers at these stages possessed an elongated and irregular nucleus (not shown) and a major portion of the cytoplasm of filopodia was closely packed with melanosomes (Fig. 4A). The melanosomes were of various sizes, but mostly oval in shape. Deeper in the dermis, the melanocytes existed as either individual migratory cells or cells producing long and branched processes, packed with melanosomes, pervading the muscle fibers (Fig. 4B). In addition to the melanosomes confined to the boundary of the melanocyte cell membrane (Fig. 5A), a few scattered melanin granules could be seen outside the boundary of melanocytes and lying scattered amidst Fig. 4. Cross-section of the tail in Clinotarsus curtipes. TEM showing melanosomes the skeletal muscle fibers (Fig. 5B and C). A closer examination of (ms) pervading deep into muscle fibers. (A) Filopodia (f) packed with melanosomes, these melanocytes revealed that the exocytosed melanin granules extending amidst sarcolytes and the nuclei (nu). (B) Melanocyte (mc) with cyto- were not delimited by a membrane. The contour of melanocytes plasm (cy) and irregular nucleus (nu) and packed with oval-shaped melanin granules (mg). Scale bars = 6 ␮m. with the melanosomes bulging out was suggestive of exocytosis of melanosomes or melanin granules (Fig. 5A). Invariably, there was a clear space between the bounding membrane and the gran- and (ii) lysosomal hydrolysis of the endosomes resulting in ule (Fig. 5c) in the intracellular melanosomes of such melanocytes. appearance of empty vesicles inside the melanosomes (Fig. 6C). In the vicinity of such granules the muscle fibers disaggregated as sarcolytes. 4. Discussion Another trend noticed was the accumulation of melanosomes/melanin granules in the cytoplasm of macrophages. This study, based on light and transmission electron micro- A few such macrophages were multinucleate (Fig. 6A). scopic observations on C. curtipes tadpoles during tail resorption, Macrophages that were totally free from melanosomes/melanin strongly suggests a link between melanocytes and phagocytosis granules in the cytoplasm arrived and accumulated around of sarcolytes during tail resorption in metamorphosing tadpoles. such multinucleate cells. Inside the macrophages the Amphibian metamorphosis is a complex process associated with melanosomes/melanin granules appeared to undergo disinte- the loss of some of the existing tissues and organs and the devel- gration in two ways: (i) transformation into diffuse bodies (Fig. 6B) opment and/or remodeling of other tissues and organs. However, 180 L. Divya et al. / Zoology 113 (2010) 175–183

Fig. 5. Cross-section of the tail in Clinotarsus curtipes. (A) TEM of a migratory melanocyte with the nucleus (n); melanosomes (m) showing apparent exocytosis of melanin granules (star). (B) TEM showing exocytosed melanosomes/melanin granules which are scattered (smg) among disintegrating muscle bundles (sm) and a few confined melanin granules within a macrophage (cmg). (C) TEM showing disintegration of muscle fibers (sm) in the vicinity of a melanocyte (mc) that is apparently disintegrating to release the melanosomes/melanin granules (mg). Arrows originate from melanin granules (mg) from which the bounding membrane is lifted off. Scale bar in A = 14.2 ␮m; in B=5␮m;inC=8␮m. as far as the tail is concerned, there is a complete resorption of the enzymes like proteases, nucleases and phosphatases in their latent tissues. This results in the disappearance of the tail from within, forms and also fail to induce tissue regression characteristic of concomitant with the transformation of the aquatic tadpole into a metamorphosis (Tata, 2003). Thus, a direct role of lysosomes in terrestrial frog. tissue degradation during tail resorption in amphibian metamor- Earlier studies that investigated the role of thyroid hormones in phosis does not appear to exist. the induction of tissue regression during metamorphosis suggested As is believed until now, the regression of the tail during such processes as macrophage infiltration, lysosomal expansion or amphibian metamorphosis is due to apoptosis or programmed cell activation of lytic enzymes (Weber, 1969; Tata, 1994; Yoshizato, death (Kerr et al., 1974). The bulk of the tissue mass of the tadpole 1996). However, lysosomal activators do not directly activate tail is skeletal muscle, which is a prominent cell type in growth pro- L. Divya et al. / Zoology 113 (2010) 175–183 181

Fig. 6. Cross-section of the tail in Clinotarsus curtipes. TEM of macrophages among disintegrating muscle fibers (mf) showing melanin granules in the cytoplasm and their fate. (A) A multinucleate (nu) macrophage (mp*) containing dense melanin granules (mg); macrophages free from melanin granules (mp) are shown aggregating around it. (B) Early and late (C) stages in the disintegration of the content of melanin granules (mg) in the cytoplasm of the macrophages with empty vesicles (ev) and diffuse bodies (db). For further details see text. Scale bar in A = 4.2 ␮m; in B = 13.5 ␮m; in C = 16.3 ␮m. grams of metamorphosis. The genes that express exclusively in the the switching on of some genes and switching off of others, fol- skeletal muscles are not direct response genes. It is estimated that lowed by the activation of caspases and endonucleases, resulting there might be as many as four waves of gene expression that pre- in the breakdown of chromatin into oligonucleosomal fragments. cede the activation of the muscle-specific genes (Buckbinder and This is followed by several cytological changes characteristic of Brown, 1992). apoptotic cell death. In the case of apoptosis in amphibian skele- The skeletal muscle is physiologically and cytologically unique tal muscles during tail resorption, the muscle fibers initially break in several respects. The large muscle bundles consisting of long apart as small blocks of sarcolytes which are described as exocy- striated muscle fibers are formed by myofibrils. The myofibrils are tosed skeletal muscle fibers. The sarcolytes are phagocytosed by repetitive units of sarcomeres, which along their length constitute macrophages and thus removed from the tail. It is only after this a syncytium and have peripherally located nuclei. The microscopic that the sarcolytes indicate the morphological features of apopto- features of tail muscle cell death are indicative of a special form sis. These ultrastructural observations of Kerr et al. (1974) in Litoria of apoptosis (Kerr et al., 1974). Apoptosis is a process of cell death glauterti have not been reported in any other amphibian species or from within. It involves a signal transduction cascade resulting in repeated in the same species. However, subsequent investigators 182 L. Divya et al. / Zoology 113 (2010) 175–183 have adopted molecular tools such as alterations in gene expression supported by a casual report of Yasutomi (1987) that epidermal and activation of caspases to substantiate occurrence of apopto- melanophores migrate to the dermis during metamorphosis in R. sis in sarcolytes (Sachs et al., 1997; Yaoita and Nakajima, 1997; japonica. Nakajima et al., 2005). The disintegration of skeletal muscle during tadpole tail resorp- Though this special form of apoptosis plays a crucial role in tion should involve apoptosis. Nishikawa and Hayashi (1995) the resorption of tail skeletal muscle in the tadpoles, the prin- described “apoptotic bodies” in tadpole tail muscle at metamor- ciples underlying the formation of sarcolytes are not adequately phic climax. The loose melanosomes/melanin pigment granules explained or traced. We have shown a migration of the melanocytes that are released/discharged in the vicinity of the skeletal mus- to the deeper regions and/or long processes of melanocytes per- cle have similarities with the lysosomes and may play a role in the vading the skeletal muscle fibers concomitant with or preceding rapid resorption of the tail within hours after the attainment of the breakdown of the latter as sarcolytes. These long processes metamorphic peak. may represent the filopodia (Scott et al., 2002) in vivo, which The second major observation in this study is the accumulation would deliver melanin to the site of muscle disintegration. These of melanosomes/melanin pigment granules in the macrophages dynamic cell extensions were first noticed in the 1960s (Gustafson (melanomacrophages) and the multinucleate nature of such cells. and Wolpert, 1961). Filopodia have been ascribed several func- Apparently, the multinucleate macrophages are produced due to tions such as (i) providing scaffolds for advancing cell protrusions fusion of several macrophages or fusion of loose melanocytes and (Goldberg and Burmeister, 1986), (ii) exerting tension as adhesive macrophages. Perhaps, sarcolytes and also melanocytes that arrive probes (Heidmann and Buxbaum, 1994), and (iii) allowing neuronal at the deeper locations and/or melanosomes that are exocytosed growth cones to cross otherwise unfavorable substrates by dilation from the melanocytes, as explained above, need to be removed. It (O’Connor et al., 1990). Filopodia have the ability to form synap- is logical to infer that the macrophages take on the task of cleans- tic contacts (Fiala et al., 1998) and, with receptors embedded in ing the debris (spent melanocytes or melanosomes and sarcolytes). their membranes, act as sensory devices to receive stimuli from This inference is substantiated by a few observations: the environment (Rehder and Kater, 1996). In this study, there is substantial ultrastructural evidence for the 1) The extracutaneous pigment system mostly consists of presence of melanin granules, exocytosed from the melanocytes, nodules formed by large aggregates of melanin-containing at deeper locations among the disintegrating muscle fibers during cells (melanophages) resembling macrophages. These nod- the process of sarcolyte formation and their subsequent phagocytic ules are thought to originate from the phagocytic activity of uptake by macrophages. We suggest that the increased abun- macrophages that accumulate melanin synthesized elsewhere dance and density of melanosomes in the migratory melanocytes (Agius, 1985; Zuasti et al., 1998). or their filopodia may reduce the cytoplasmic area available to 2) Melanosomes in the spleen of X. laevis result from aged the various other organelles. This may cause a mechanical dis- melanocytes that are engulfed by macrophages. Subsequent ruption of the cellular organization, resulting in exocytosis of the migration of melanized macrophages leads to melanin accumu- melanocytes/melanin granules. A unique cell extension process lation. This would represent a normal means of scavenging older was reported in turtle melanomacrophages placed into cell culture. pigment cells (Anders et al., 1980). Such a situation may exist These structures, referred to as cablepodia, are straight, narrow during tadpole tail resorption. and unbranching, forming cell networks. Dividing fibroblasts to 3) The role of melanin in tissue degeneration, as an aspect of devel- which cablepodia attached stopped cell division, suggesting that opmental changes, has been assumed earlier in connection with the interconnected networks of cell processes trap and process ocular development in ferrets, mice, hamsters and humans. It particulate matter, cells, etc., and also provide cell signal commu- was found that there is a constant relationship between zones nication (Johnson et al., 2005). of degeneration in the optic cup and the pigmented cells. It was Melanosomes are related to lysosomes, and both types of suggested that in several different mammalian species cellular organelles develop initially through the same pathway (Kushimoto degeneration and pigment granules are closely related. This was et al., 2001). The melanosomes that accumulate melanin form inferred from pigmented cells showing degenerative changes an enigmatic class of cell organelles that belong to the group within a quite limited region close to the optic disc. Within this of lysosome-related organelles (LROs). They store and synthesize region, the most obvious sign of cellular degeneration is the molecules that participate in everything from immunity to blood rosette formation in relation to lysosome-like structures resem- clotting in many cell types (Leslie, 2006). bling the melanolysosome (Strongin and Guillery, 1981). Lysosomal hydrolases are present in melanosomes and they are elevated in melanizing cells. Melanosomes may represent highly In the light of these observations and inferences it is pro- specialized members of the endolysosomal lineage of organelles. posed that the melanocytes/melanosomes, having fulfilled their It may be advantageous for the melanocyte to restrict pigment role as pigment cells in the skin, contribute to the attraction deposition to a lysosome-like organelle (Diment et al., 1995). of macrophages towards the site of metamorphic regression. The melanosomes could even be transferred from melanocytes Once having done this, they themselves are phagocytosed by to other cell types, as during keratinization in the skin (Zuasti macrophages and these macrophages in turn would become nod- et al., 1998; Pederzoli and Trevisan, 1990; Trevisan et al., 1991). ules through fusion among themselves. The resultant macrophage Melanosomes could be actively transferred from the cytoplas- nodules could be further processed in the liver/spleen. Our obser- mic processes of melanocytes to their surrounding cells (Zuasti vation of large MMCs in the tadpole liver at metamorphic climax et al., 1998). Our observations on the abundance of melanocytes supports this assumption. Therefore, the pigment melanin would and/or loose melanin granules in the tail of metamorphosing tad- play a crucial role in tadpole tail resorption, facilitating the poles, viewed together with these earlier studies, may suggest breakdown of skeletal muscle fibers into sarcolytes, a unique their lysosome-like role in the fragmentation of the skeletal mus- requirement for skeletal muscle apoptosis, and/or attraction of cle into sarcolytes. The similarity of melanosomes to lysosomes macrophages to the sites of muscle disintegration. Therefore, this may facilitate the removal of lysosomal components from matur- study suggests a possible link between melanocytes and tail skele- ing melanosomes. This would occur perhaps by a kiss-and-run type tal muscle breakdown into sarcolytes during tail resorption in of mechanism proposed for late endosome–lysosome fusion (Luzio amphibian metamorphosis. If the breakdown of muscle bundles et al., 2000). Our observation and inference are to a certain extent into sarcolytes and their subsequent phagocytosis by macrophages L. Divya et al. / Zoology 113 (2010) 175–183 183 and also the phagocytic removal of melanocyte/melanin granules Kerr, J.F.R., Harmon, B., Searle, J., 1974. An electron microscopic study of cell dele- were unrelated among themselves, there would be no reason why tion in the anuran tadpole tail during spontaneous metamorphosis with special reference to apoptosis of striated muscle fibres. J. Cell. Sci. 14, 571–585. the melanocytes should form filopodia to permeate into the core of Kinoshita, T., Sasaki, F., Watanabe, K., 1985. Autolysis and heterolysis of the epider- the tail and release the melanin granules. If phagocytic removal is mal cells in anuran tadpole tail regression. J. Morphol. 185, 269–275. the ultimate goal, the melanocytes could as well be phagocytosed Kushimoto, T., Basrur, V., Valencia, J., Matsunaga, J., Vieira, W.D., Ferrans, V.J., Muller, J., Appella, E., Hearing, V.J., 2001. A model for melanosome biogenesis based on by the macrophages at their location in the epidermis itself. the purification and analysis of early melanosomes. Proc. Natl. Acad. Sci. U.S.A. Since the majority of studies relating to pigmentation func- 98, 10698–10703. tions have dealt with cultured cells, a general concept linking Leslie, M., 2006. Melanin’s backups. J. Cell Biol. 175, 195. melanocytes with the disintegration of skeletal muscle into sar- Lillie, R.D., Fullmer, H.M., 1976. Histopathologic Technic and Practical Histochem- istry, vol. 4. McGraw Hill, New York. colytes and their subsequent cleansing by macrophages will require Luzio, J.P., Rous, B.A., Bright, N.A., Pryor, P.R., Mullock, B.M., Piper, R.C., extensive in vivo testing. 2000. Lysosome–endosome fusion and lysosome biogenesis. J. Cell Sci. 113, 1515–1524. Nakajima, K., Fujimoto, K., Yaoita, Y., 2005. Programmed cell death during amphibian Acknowledgments metamorphosis. Semin. Cell. Dev. Biol. 16, 271–280. Nishikawa, A., Hayashi, H., 1995. Spatial, temporal and hormonal regulation of pro- This study was funded by the University Grants Commission grammed muscle cell death during metamorphosis of the frog Xenopus laevis. Differentiation 59, 207–214. (UGC), New Delhi, Grant No. F 3-15/2007 SAP to the Department Nosanchuk, J.D., Casadevall, A., 2003. The contribution of melanin to microbial of Zoology, University of Kerala, Thiruvananthapuram; and UGC pathogenesis. Cell. Microbiol. 5, 203–223. Grant No. F.4-3/2006(BSR) RFSMS and Young Scientist Award by O’Connor, T.P., Duerr, J.S., Bentley, D., 1990. Pioneer growth cone steering decisions mediated by single filopodial contact in situ. J. 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