THE DERMAL CHROMATOPHORE UNIT

JOSEPH T. BAGNARA, JOHN D. TAYLOR, and MAC E. HADLEY

From the Department of Biological Sciences, University of Arizona, Tucson, Arizona 85721

ABSTRACT

Rapid color changes of amphibians are mediated by three types of dermal chromatophores,xanthophores, iridophores, and melanophores, which comprise a morphologically andphysiologically distinct structure, the dermal chromatophore unit. Xanthophores, theoutermost element, are located immediately below the basal lamella. Iridophores, contain-ing light-reflecting organelles, are found just beneath the xanthophores. Under each irido-phore is found a melanophore from which processes extend upward around the iridophore.Finger-like structures project from these processes and occupy fixed spaces between thexanthophores and iridophores. When a frog darkens, melanosomes move upward from thebody of the melanophore to fill the fingers which then obscure the overlying iridophore.Rapid blanching is accomplished by the evacuation of melanosomes from these fingers.Pale coloration ranging from tan to green is provided by the overlying xanthophores andiridophores. Details of chromatophore structure are presented, and the nature of the inti-mate contact between the chromatophore types is discussed.

INTRODUCTION

Dermal melanophores are generally considered tobe responsible for the rapid color changes thatoccur in some amphibians, notably the hylids(see Parker, 1948). At first glance this conceptseems reasonable since dermal melanophores areprofoundly sensitive to intermedin, the majorchromatophore-regulating hormone. However,when the precise location of dermal chromato-phores in species exhibiting rapid color change istaken into account, serious doubt arises thatdermal melanophores alone are the principalpigmentary elements involved in the color-changemechanism. It has been clearly demonstrated bySchmidt's elegant observations (1920) on adultHyla arborea that these melanophores are founddeep in the dermis and are covered by a layer ofxanthophores and iridophores. Accordingly, inorder that dermal melanophores might becomemanifested fully it would seem that some arrange-ment must exist so that the masking effect of theoverlying chromatophores can be diminished.It is implicit in such a suggestion that chromato-

phores function together in a unified fashion. Theassociation of certain pigment cells with oneanother is consistent with this unity and is ex-emplified by the close apposition of xanthophoreswith iridophores to form the so-called xantho-leucosome (for discussion of chromatosomes andother pigment-cell terminology, see Bagnara,1966). The involvement of melanophores in suchan association is known from previously publishedliterature (Gaupp, 1904) and has been confirmedby Hadley (1966) who reported that the dendritesfrom underlying dermal melanophores terminateon the surface of iridophores in skin of Ranapipiens and who described the termination. Aphysiological demonstration of this unity is thefact that responses of both melanophores andiridophores are mediated by intermedin (Bagnara,1958, 1964; Hadley, 1966). Presumably, the actionof this hormone as well as the specific morphologyand location of the three dermal chromatophoretypes are the major factors responsible for thespecific shade of color manifested by the skin. In

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view of the unity of dermal chromatophores, with

respect to both structure and function, the present

study was undertaken to provide a clearer cytologi-

cal picture of the structural characteristics of

chromatophore interaction in color changeresponses. These studies have led to the develop-

ment of the concept of the "dermal chromatophore

unit," which provides the vehicle by which adult

amphibians can color-adapt rapidly.

MATERIALS AND METHODS

The majority of observations were made on skins ofthree hylids: Hyla cinerea which varies in color fromlemon yellow to dark green; Agalychnis dachnicolor, alarge, light to dark green species collected near Ala-mos, Sonora, Mexico; and H. arenicolor, a light tan tobrown-colored resident of southwestern canyonswhich was obtained in the vicinity of Tucson. Obser-vations were also made on skins of adult Rana pipiens.Some of the animals were injected with intermedin(porcine g-MSH) at a concentration of 1 X 10- 7

g/ml of Ringer solution. Because of their smaller size,H. cinerea and H. arenicolor received dosages of I mlwhereas A. dachnicolor and R. pipiens were injectedwith 2 ml of the intermedin solution. These frogswere sacrificed 2 hr after hormone administration.Observations were also made on control frogs whichwere not injected. Tissues used for electron micros-copy were fixed for 1-2 hr in 6% glutaraldehyde in0.1 M s-collidine buffer (pH 7.3) and postfixed in 2%OsO4 in the same buffer. After fixation, tissues weredehydrated in an ethanol series and embedded inEpon 812 (Luft, 1961). Sections were made on aPorter-Blum MT-2 ultramicrotome with a diamondknife. They were mounted on Formvar-coated grids,stained with either alkaline lead citrate (Reynolds,1963) or the double stain of saturated uranyl acetateand alkaline lead citrate, and examined in a PhilipsEM200 electron microscope. Thick sections (1-2 ,u)were also cut and then stained with toluidine bluefor light microscopy (Bennett and Radimska, 1966).Further observations were made from paraffinsections or from whole mounts of skins either fixed inFAA (two parts formaldehyde, one part acetic acid,17 parts 70% ethanol) or unfixed and mounted inclear Karo syrup (Bagnara, 1958). The latter methodis effective in preserving yellow pigments which aredissolved by fat solvents.

OBSERVATIONS

Consistent with light microscope observations, by

previous workers, on amphibians, electron micro-

graphs reveal clearly that, in the dermis, xantho-

Dhores (X) are outermost in location, just beneath

and in intimate contact with the basal lamella

(Figs. 1-4). In cross-section the xanthophore pre-

sents a flattened profile; its nucleus is usually cen-trally located, polymorphic in outline, and also

flattened. The most prominent pigmentary organ-elle is the pterinosome (PT) (Matsumoto, 1965).

These pteridine-containing structures are distrib-

uted uniformly throughout the cytoplasm. Inter-

spersed between them are droplets (CV) which

probably represent the carotenoid complement of

xanthophore pigments (Bagnara, 1966). Just be-

neath the xanthophore is found the iridophore (I).

The latter is often shaped like an inverted cone and

contains a rounded nucleus which is generallyfound near the base of the cell. The characteristic

feature of iridophores is a series of empty spaces

(RP), which are usually oriented in stacks. These

spaces contain reflecting platelets which Taylor

(1966, 1967) and Setoguti (1967) have shown to

be the pigmentary organelles of iridophores. In

species with many xanthophores, such as H.

cinerea or A. dachnicolor, a xanthophore is found

generally above every iridophore. The deepest

dermal chromatophore is the melanophore, the

body of which is found below the iridophore. In

H. cinerea and A. dachnicolor, the melanophore

body is in intimate contact with the lower surface

of each iridophore; however, in H. arenicolor,

melanophores are somewhat more deeply situated

and often lose this basal contact. In R. pipiens, a

relatively large space may separate the base of the

iridophore from the melanophore body. In all

four species, melanophore processes or arms

extend around the sides of each iridophore and

terminate in fingers which lie between the irido-

phore and its overlying xanthophore, as is shown

in Figs. 2 and 4 b.The arrangement of these three pigment cells

forms a discrete structural unit. The functional

relationships in this unit become obvious inintermedin-treated animals. In skins from these

animals, melanosomes migrate to the most distal

extensions of the melanophore. Altogether, these

structural and functional relationships have led

us to formulate a concept, the dermal chromato-

phore unit, which is depicted in Fig. 3 as a sche-

matic interpretation of the situation in those

species of amphibians that have been studied.

The salient feature of the unit is that each melano-

phore can modify the bright colors imparted by

iridophores above it by obscuring the light-

reflecting capacity of these cells. This color

modification is accomplished by migration of

68 THE JOURNAL OF CELL BIOLOGY VOLUME 38, 1968

FIGURE 1 Transverse section of H. cinerea skin from dorsal surface showing dermal chromatophore unitin white background-adapted state. Melanophore M; iridophore, I; xanthophore, X. Melanosomes (MS)are uniformly distributed in arms around sides of iridophore, but fingers (F) over iridophores are empty.BL, basal lamella; RP, reflecting platelets; CV, carotenoid vesicles; C, collagenous masses; PT, pterino-some. X 9,700.

FIGURE Same as Fig. 1, except treated with intermedin. Melanosomes now fill fingers over iridophoreat the expense of arms and body of melanophore. X 7,300.

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FIGURE 3 Schematic interpretation of the dermal chromatophore unit from several anurans. Adaptationto a dark background is represented.

melanosomes into melanophore fingers betweenthe xanthophore and the iridophore. In this way,not only is the iridophore obscured but thexanthophore-iridophore relationship is interruptedor modified (Fig. 4 e,f).

Melanophore fingers, which become obvious inintermedin-treated animals, apparently are fixedstructures. There is no indication of an actualmovement of fingers to and from the area betweenthe iridophores and xanthophores; instead, itseems that fingers remain static and that melano-somes actively migrate into and out of thesefingers. In white background-adapted animals inwhich melanosomes are concentrated in the bodyof the melanophore, the area between the irido-phore and the xanthophore contains numerousmembranes (Fig. 5). In all probability these

membranes are collapsed melanophore fingers

which are now devoid of melanosomes. By con-

trast, in intermedin-treated animals, these fingersare distended by an invasion of melanosomes (Fig.6). The relationship between cell membranes of

melanophore fingers and iridophores is complex.

Where the membranes of each of these two cells

are in intimate contact, as on the upper surface

or along the sides of the iridophore, they arefolded and give a rugose appearance (Figs. 7 and8). No such complex contact was found to existbetween xanthophores and melanophore fingers;although the membranes of these chromatophoresare in close apposition, the contact is smooth andwithout indication of foldings (Fig. 7). Thatmelanophore fingers remain permanently inplace is further suggested by indentations whichcan be seen around the edges of iridophores inH. arenicolor (Fig. 4 d). In this species, iridophoresare somewhat expanded in the absence of inter-medin so that the reflecting material is spreadthroughout the iridophore except for the indenta-tions which presumably represent vacant fingersthrough which melanosomes migrate frombeneath to the upper surface of the iridophore.In R. pipiens, iridophores are capable of consid-erable expansion, and indentations of the irido-phore surface are quite prominent. It appears thatthese indentations exist because the position of thefingers is permanent; thus, whenever the irido-phore expands, it must do so around these fingerswhich provide a localized barrier (Hadley, 1966).The external form of the iridophore is not visibly

J. T. BAGNARA, J. D. TAYLOR, AND M. E. HADLEY Dermal Chromatophore Unit 71

affected in H. cinerea or in A. dachnicolor; that is,iridophores exhibit no perceptible expansion orcontraction (Taylor, 1967). Accordingly, deepindentations were not found. Frontal sections ofiridophores of H. cinerea (Fig. 9) reveal thatfingers are uniformly distributed around theiridophore and that they do not actually indentits surface. These sections also demonstrate thatsome individual chromatophore units are oftenin contact with one another, whereas others areoften separated by collagenous masses (C in Fig.10) which extend from the basal lamella to thehypodermis (Figs. 1 and 2).

The nature of melanophore involvement in thedermal chromatophore unit is somewhat variableamong species. For example, there are fewerfingers over each iridophore in both H. cinerea andA. dachnicolor than in H. arenicolor in which asmany as 25 fingers are often found. In H. arenicolor,however, the fingers are considerably thinner thanthose in the other two hylids. But the net effect ofalmost completely obscuring the upper iridophoresurface is the same. The number of iridophoresserved by one melanophore is also variable. Indorsal skin of A. dachnicolor there appears to be amelanophore for every iridophore. This melano-phore seems primarily to serve its overlyingiridophore; however, it may also serve neighboringiridophores, as is especially obvious in the flankwhere single melanophores serve several irido-phores. Often an individual iridophore receivesdendritic processes from several melanophores.In the dorsal skin of both H. cinerea and H. areni-color there are about 10-15 iridophores for every

melanophore; this ratio is even higher in R.pipiens. Just as with A. dachnicolor, in the flank ofthese three species there are fewer melanophoresthan iridophores. Thus, individual melanophoreprocesses are more widespread, allowing forcontact with distant iridophores (Fig. 4 ).

Thus far, it has been implied that hormonalaction on the dermal chromatophore unit mainlyinvolves the movement of melanosomes into andout of melanophore fingers. Actually, intermedinaffects iridophores as well. Hadley (1966) hasshown that in the absence of intermedin, theiridophores of adult R. pipiens are dendritic. Thus,a maximum of reflecting surface is presented,unmasked by melanosomes which, under theseconditions, are concentrated toward the center ofthe melanophore beneath the iridophore. Asimilar situation prevails in H. arenicolor exceptthat the degree of iridophore expansion is far lessthan that in R. pipiens. Nevertheless, iridophoresare so numerous that they provide practically acontinuous sheet of reflecting material (Fig. 4 d).In intermedin-treated animals (Fig. 4 e) the skinbecomes dark not only because melanophorefingers partially mask the iridophore, but alsobecause the hormone causes iridophores to becomesomewhat punctate; this results in the exposure ofless reflecting surface. In H. arenicolor, evenxanthophore pigments are concentrated towardthe center of the cell (Fig. 4 d); however, in theintermedin-treated animal this yellow pigmentdisperses and covers a larger surface area (Fig.4 e). It was not possible to observe xanthophoreresponse in the other species that were studied.

FIGURE 4 a, Thick section (1 ,u) of skin from H. cinerea showing dermal chromatophoreunit during adaptation to a white background. X 800. b, Thick section (1 A) of skin cutparallel to dorsal surface, from H. cinerea treated with intermedin. Arms containingmelanosomes surround iridophores. Melanophore fingers are visible over some iridophores.SG, skin gland. X 1,240. c, Termination of melanophore fingers on iridophore in skin ofintermedin-treated male R. pipiens. X 1900. d, Whole mount of skin from H. arenicolorin the absence of intermedin, photographed with reflected light. Iridophores are expandedand are indented at their edges. Yellow spots over some iridophores are xanthophores inthe contracted state. Black circles represent openings of skin glands. X 50. e, Same asd,lexcept treated with intermedin. Iridophores are contracted, xanthophores are expandedand melanophore fingers are seen above iridophores. X 520. f, Whole mount of skin fromflank of H. arenicolor. Melanophore processes are widespread and show contact with severaliridophores. X 230. g, Whole mount of dorsal skin from H. cinerea adapted to light back-ground. Mounted in Karo syrup to preserve xanthophores. Green represents combinedeffect of xanthophores and iridophores. X 1040. h, Same as g, except treated with alcohol.Xanthophore pigments are removed and underlying iridophores blue (Tyndall effect).X 1040.

72 TuE JOURNAL OF CELL BIOLOGY VOLUME 38, 1968

J. T. BAGNARA, J. D. TAYLOR, AND M. E. HADLEY Dermal Chromatophore Unit 73

FIGURE 5 Transverse section of area between xanthophore and iridophore in skin of H. cinerea. Numer-ous membranes (arrow) are believed to represent collapsed melanophore fingers which are devoid ofmelanosomes. X 23,500.

FIGURE 6 Same as Fig. 5, except that frogs were darkened with intermedin. Melanophore fingers betweenxanthophore and iridophore are distended with melanosomes. X 23,500.

FIGURE 7 Same as Fig. 6, showing interdigitations between melanophore fingers and iridophore (arrow).

Contact between melanophore and xanthophore is smooth. X 18,000.

FIGURE 8 Tangential section through dorsal skin of H. cinerea demonstrating melanophore process be-

tween two adjacent iridophores. The rugose nature of contact between melanophore and iridophore is ob-

vious. X 23,500.

FIGURE 9 Same as Fig. 8, showing uniform distribution of melanophore processes around the iridophore.X 5,700.

FIGURE 10 Same as Fig. 9, demonstrating collagenous masses (C) extending between two dermal chro-matophore units. X 23,500.

76

It should be emphasized, also, that in the otherspecies of amphibians the number of xanthophoresis far greater; iridophores are covered by xantho-phores which form a more or less continuousyellow layer. In H. cinerea, the edges of adjacentxanthophores are always closely apposed to oneanother (Fig. 4 a).

DISCUSSION

The dermal chromatophore unit provides aremarkable example of the functional integrationof several morphologically distinct cells, xantho-phores, iridophores, and melanophores, to providea uniform gross effect. Before the gross effect canbe explained, the specific form and function ofeach chromatophore type must be understood.Xanthophores apparently are the least dynamicof the three chromatophore types. They containyellow pigments; in the species studied thesepigments presumably are carotenoid and pteridinein nature (Bagnara, 1966). Pterinosomes, thepteridine-containing organelles (Matsumoto,1965), were obvious in every xanthophore. Theseorganelles, together with small vesicles whichprobably contain carotenoid pigments, aredistributed uniformly throughout the cytoplasmand give the xanthophore a uniform yellow ap-pearance. Formerly, it was believed that xantho-phores of amphibians played no active part incolor change mechanisms because they werenever observed to expand or contract as a resultof hormonal or other stimulation. Our observa-tion that xanthophores of adult H. arenicolorexpand after administration of intermedin repre-sents the first example of an amphibian xantho-phore response to a hormone. It was not possibleto extend this observation to xanthophores of theother three species studied, because they areusually obscured by the underlying melanophorefingers.

The precise contribution of iridophores tocolor-change mechanisms is difficult to ascertainbecause of the complex nature of these cells.Clearly, the principal function of this chromato-phore is to reflect light. This is accomplished bythe many stacks of purine-containing reflectingplatelets which fill the iridophore. Taylor (1966,1967) explains that these platelets are so hard thatthey cannot be properly infiltrated and, conse-quently, they shatter during sectioning, leavingbehind their profiles in the form of empty spaces.The fact that stacks of platelets are often oriented

at different angles introduces the possibility thatlight-scatter might play an active role in pigmenta-tion. Whether or not this factor accounts for thered, green, or blue iridescence sometimes seen inindividual iridophores is unknown. In H. arenicolor,light emanating from the skin laden with irido-phores is white except at the location of a xantho-phore where the yellow color of the xanthophoreis expressed. Because there are relatively fewxanthophores in the dorsal skin of this species,white light reflected from the iridophores pre-dominates, and the xanthophore contribution isrepresented by a tinge of bronze or tan coloration.In H. cinerea and A. dachnicolor, light reflected fromthe iridophore layer of the dorsal surface appearsto be blue; in fact, each individual iridophoreappears to be blue. This blue is readily visible inwhole mounts of skin from which the overlyingyellow pigments have been leached (Fig. 4 h).Intact skin, either in the living frog or in wholemount preparations, appears green (Fig. 4 g). Inall probability this green results from a filteringeffect provided by the yellow of the overlyingxanthophore layer. A hypothesis of this type toexplain why some cold-blooded vertebrates aregreen was first made by Fox and Vevers (1960).

Whereas xanthophores and iridophores areresponsible for imparting pale coloration, themelanophore plays a role in darkening the animal.The migration of melanosomes from their basalposition within the melanophore to the fingersobscures the overlying iridophore and thus allowsthe animal to adapt readily to a dark-coloredbackground. Rapid blanching is accomplished bythe evacuation of melanosomes from these promi-nent fingers. In this way, the pigment is mobilewhile the chromatophore itself retains its perma-nent position. This observation is in agreementwith the early observation of Spaeth (1913) thatthe arms of the teleost melanophore are staticstructures. In the absence of melanosomes, thefingers collapse; thus, in white background-adapted frog the fingers are observed as mem-branes between the xanthophore and the irido-phore. An actual distension of the fingers occurswhen they are occupied by melanosomes. Asimilar observation has been made for melano-phores of larval H. regilla by Wise (1966). Themechanism for the migration of melanosomes intothe fingers is unknown. Other than melanosomes,few organelles are found in the fingers; there isevidence that only a few microtubules are present.

J. T. BAGNARA, J. D. TAYLOR, AND M. E. HADLEY Dermal Chromatophore Unit 77

Bikle et al. (1966) suggest that microtubules play arole in the concentration and dispersion of melano-

somes in fish melanophores.The close contact between the fingers of dermal

melanophores and the iridophores presents severalfacets of interest. The actual terminations of these

fingers were known previously in German litera-ture (see Gaupp, 1904); however, their functionalsignificance was not understood until the presentinvestigation. On the basis of our first observationof the melanophore-iridophore relationship, it

seemed attractive to suggest that in R. pipiens, atleast, iridophore contraction after intermedin

administration might be a secondary responseresulting from the action of the hormone on the

melanophore. In other words, the melanophore-iridophore junction (Fig. 4 c) might be analogousto a synapse which could act as a mechanism ofcommunication between the two chromatophores.This suggestion was rejected for several reasons,

the most important being that, from the stand-point of both absolute response and threshold ofresponse to various hormonal and pharmacologicalagents, melanophore reactivity and iridophorereactivity can be separated (Hadley, 1966).

Moreover, it is known that iridophores having noobservable morphological association with melano-phores respond to intermedin (Bagnara, 1958;

Hadley, 1966). At any rate, the suggestion of

communication between iridophores and melano-phores need not be of great consequence to thegeneralized concept of the dermal chromatophoreunit. In hylids, in which the unit is well developed,iridophores generally display no marked grossresponse to intermedin. They serve a pigmentary

function in a passive way, through their locationbeneath the xanthophore layer and above thedermal melanophores. The contact between themembranes of the melanophore fingers and theiridophore membrane gives no indication of a

communicating mechanism. The rugose mem-branes of these chromatophores appear to beinterdigitated, but the interdigitations have no

internal organization, such as that seen at synapticjunctions, which might be correlated with suchsites of contact. It appears that this intimatejunction serves to insure that melanophore fingerswhich are uniformly distributed over the irido-phore remain consistently in place, allowingefficient masking of the iridophore to occurwhenever the melanophore is stimulated todisperse its pigment. This interpretation is favoredby the large area of contact between the two

chromatophores that includes both the uppersurface and the sides of the iridophore.

The concept of two or more cell types function-ing as a discrete pigmentary unit is not a new one.In both cold-blooded vertebrates and mammalsthe epidermal melanocyte and its associatedepidermal cells function as the "epidermalmelanin unit" (Fitzpatrick and Breathnach, 1963;Hadley and Quevedo, 1966). Under appropriatestimulation, the epidermal melanocyte releasesmelanin granules into those surrounding epidermalcells which are serviced by dendrites from thatparticular epidermal melanocyte. With thedispersion into the epidermis of large numbers ofmelanin granules, the epidermis becomes dark.In contrast to the darkening effected by theresponse of the dermal chromatophore unit, thedarkening produced by the epidermal melaninunit is not readily reversible, for the animal canbecome pale again only after the deposited melaninhas been lost. Deposition of large amounts ofmelanin in the epidermis of animals with a well-developed epidermal melanin unit would obscurethe action of the dermal chromatophore unit. Itis noteworthy, therefore, that epidermal melano-cytes are absent or minimally functional in specieswhich exhibit rapid color changes. For example,the hylids used in the present study lack epidermalmelanocytes.

The principal purpose of this paper has been todescribe the dermal chromatophore unit found insome adult amphibians. The structural andfunctional arrangement of xanthophores, irido-phores, and melanophores in the dermis provides aremarkably efficient mechanism for color adapta-tion among amphibians and probably othercold-blooded vertebrates. Simple as the mechanismappears, it has some obvious complexities whichremain as a challenge for future investigation.Among the problems is the one of development ofthe unit during metamorphic climax. During thisperiod of rapid changes in the skin, the adultpigmentary pattern is established. Other im-portant problems to be solved include the mecha-nism of the movement of melanosomes in themelanophore fingers and the variation in formand function of each of the components of thedermal chromatophore unit among the variousvetrebrates which utilize it.

This research was supported in part by grantsGB-3681 and GB-4923 from the National ScienceFoundation.

78 THE JOURNAL OF CELL BIOLOGY VOLUME 38, 1968

We would like to thank Dr. Wayne Ferris for hisadvice in using the electron microscope facility whichis supported by National Science Foundation grantGB-3330. We would also like to thank Mr. GaryMonheit for preparing the whole mount preparations

of H. arenicolor skin and Mrs. Joanna McComb forpreparing the stereogram entitled dermal chromato-phore unit.

Receivedfor publication 15 December 1967, and in revisedform 20 February 1968.

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J. T. BAGNARA, J. D. TAYLOR, AND M. E. HADLEY Dermal Chromatophore Unit 79