The ultrastructure of differentiating iridophores and xanthophores in Aqulychnis dacnicolor

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Authors Rothstein, Jeffrey, 1950-

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Link to Item http://hdl.handle.net/10150/348162 THE ULTRASTRUCTURE OF DIFFERENTIATING IRIDOPHORES

AND XANTHOPHORES IN AGALYCHNIS DACNICOLOR .

"by

Jeffrey Rothstein

A.Thesis Submitted to the Faculty of the

DEPARTMENT OF CELL AND.DEVELOPMENTAL BIOLOGY

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE WITH A MAJOR IN BIOLOGY

. Ih the Graduate College • . - '

THE UNIVERSITY OF ARIZONA

1 9 7 7

Copyright 1977 Jeffrey Rothstein STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of re­ quirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder.

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

7/ ' Joseph T . Bagnara Professor of Biology AGKNOESpaMEMTS

I wish to thank Dr. Joseph T. Bagnara for his help, suggestions and constructive criticism during this investigation, particularly durlhg the time I'was. writing this thesis..

. I am deeply indebted to Dr. Wayne Ferris for his time and effort in teaching me the use of the electron microscope and in helping with the interpretation of micrographs. The,author is also: grateful to Dr.

Konrad Keck for reading and Commenting on this thesis and to Mardi

Wolford and Sam Semoff for their time and technical assistance in the

■ . . « ' „ ' ' . ERi lab. TABLE OF GOMTENTS

Page :

LIST OF ILLBSTBAilOMS '. ■. . . . \ . . .. „ ...... , vi

ABSTRACT # v . . . V . V. . X

IMTROWCflON . , , , , . . . \ 1

LITERATURE REVIEW , . , ...... * ...... 3

Xanthophores and Erythrophore s ...... 3 . PterlnosQme Chemistry . . . . »• . . . 3 Pteridlne Chemistry and Pterinosome Ultrastructure . .... 3 Pterlnosome Ultrastructure ...... 4 Pterinosome Development ...... 6 Origin of Pterinosomes ...... 7 . Carotenoid Vesicles ...... 8 Iridophores ...... 9 Reflecting Platelet Chemistry . . , ...... 9 Platelet Chemistry and Ultrastructure ...... 9 Reflecting Platelet Ultrastrueture . 10 Reflecting Platelet Origin and Development ...... 12

MATERIALS AMD METHODS ...... 15

RESULTS ...... * . 17

. Light Microscopic Description ...... 17 Representative Stages ...... 17 Electron Microscopic Description ...... 49 Xanthohlasts ...... 49 Xanthophores ...... 70 Pterinosome Development ...... ' . . . 72 Iridohlasts . . , ...... 75 Reflecting Platelet Formation ...... ,^4 ...... Ill Maturing Iridophores ...... 115 Blast Cells of Indeterminant Type ...... 121 The Mixed Ghromatophore Layer ...... ' . . . 122 Mature Iridophores ...... 123 ■ Melanophore Development...... , ...... 124 V

TABLE OF C0NTENTS--Cdntime4

Page

DISCUSSION ...... 130

Similarities in Chromato'blast Differentiation .. . , , . . . . . 130 Nuclear -BlebMng ...... 130 Riliosome^assoeiated Vesicles ...... , . . . . . 131 The Primordial Vesicle ...... 132 . Indeterminant Blast Cells ...... 133 Evidence of a Stage-specific Increase in Hormone Levels . . . 134 Definition of Terms ...... 134 Dermal Melanophore Proliferation During Stage 21 ...... ' 135

SUMMARY ...... 137

Light Microscopy ...... , . . 137 Electron Microscopy ...... 138

REFERENCES CITED . . .' ...... 140 LIST OF ILLUSTRATIONS

Figure Rage

1. Section of stage 9 skin lacking a diseernable ’basement iseitbi^aiie « ». « * » * o « « * « = ^ « « » » « _» c » o o © » IS

2. Section of stage 9 skin with a basement membrane . , 19

3. . : Section of stage 14 skin ...... „ . ' „ ... . 20

4. . Vesicular appearance of zanthoblast cytoplasm ...... 21

5. Section of stage 16 skin . .. . . , V...... « 22

6. Stage 16 zanthoblast layer ...... 23

7. Iridophore identification . . . . 4 ...... 24

8. Section of stage 18 skin ...... 25

9. ., Polarized light view of section of stage 18 skin ..... 26

10. Secretion of the skin glands ...... 27

11. ’• Section of stage 21 skin ...... 28

12. Mixed chromatophore layer of stage 21 skin 29

13. Dermal chromatophores in stage 21 . „ ...... - . . 30

14. Section of stage 22 skin ...... 31

15. - Distribution of chromatophores in stage 22 ...... 32

16. The skin gland layer of stage 23 ...... 33

17. Chromatophore distribution in stage 23.'...... , . 34

18. Polarized view of stage 23 chromatophores ...... 35

1 9 . Section of stage 25 skin . . .• ...... 36

20. Longitudinal section through a skin gland duct ...... 37

' vi , ■" vli

' LIST OF ILLUSTRATIONS— Continued

Figure Page

21. The dermal chromatophore unit ...... 38

22. EM overview of. stage 18 dorsal dermis ...... 51

. 23. Barrier separating xanthohlast from Basement membrane , . 52

24. Establishment of the xanthophore layer' ...... 53

25. Reduction of the collagen barrier ...... 54

26. Nuclear blebbing in a xanthoblast . . ' ...... • 55

27. Ribosome associated vesicles in a xanthoblast ...... 56

. 28. Ribosome associated vesicles in a -melanophore ...... 57

29. Characteristic vesicles of xanthoblasts . . ., ...... 58

30. Coalescing smooth vesicles . . , ...... 59

31. Coupling of smooth vesicles ...... 60

32. Cell shape, of a typical xanthophore ...... 61.

33. Pterinosome.. development ...... 62 .

34. Characteristic pterinosome types ...... 63

35. Pterinosomes-of stage 23 xanthophores ...... 64

36. Pterinosomes at high magnification . . . . '. . . , . . . 65

37. Possible association of pterinosomes and rough ER . . . , . 66

38. Unknown structures associated with rough ER ...... 67

39. Early iridoblasts ...... 76

40. Type I iridoblast .- ...... , , . , . . . . , . , . , 77

41. Overview of stage 20 dermis ...... 78

42. Early type III iridoblast,■ ...... , ...... , 79

' 43. Overview-of differentiating. Iridophores . ;. 80 viii

LIST OF ILLUSTRATIONS— Continued

Figure Page

44. A hypodermal iridoblast ...... 81

. 45. Fragmentary smooth vesicles ...... 82

46. Pre-reflecting platelet morphology ...... 83

47. Reflecting platelet formation ...... '84

48. Possible reflecting platelet fusion ...... 85

49. Reflecting platlet formation in a stage 21 iridophore . . 86

50. Reflecting platelet development . '..... 87

51. Cytoplasmic inclusions of a stage 22 iridophore ..... 88

52. Iridophore differentiation ...... 89

53. Cytoplasmic projections of iridophores ...... 90

54. Stage 22 iridophore ...... 91

55. Typical organelles of stage 21 iridophores ...... 92

56. Typical stage 21 iridophore ...... 93

57. Iridophore approaching xanthophore ...... 94

58. Chromatophore contiguity...... 95

59. Endocytotic vesicle ...... 96

60. Cytoplasmic cahaliculi...... 97

■ 61. Melanophore encircling an iridophore ...... 98

62. Platelet-free region of cytoplasm ...... 99

63. Multi-vesicular bodies in platelet free cytoplasm . . . . 100

64. Unusual multi-vesicular bodies ...... 101

65. Stage 22 multi-vesicular bodies ...... 102

.66, Unusual multi-vesicular body within rough ER ...... 103 ix

LIST OF ILLUSTRATIONS— Coiitlmed .

Figure Page

67. Granular vesicles ...... 104

68. The indeterminant "blast cell ...... ' 105

69. The mixed chromatophore layer , . '...... , . . . 106

70. Stage 25 iridophore ...... 107

71. Melanosome development- ...... 125

72. ' Stage 21 melanophore ...... 126

73. : Melanin deposition ...... 127 ABSTRACT

The dorsal skin of specimens of Agalychnis d.acnicolor in vari- ous metamorphic stages was studied using both light and electron micro- ■ scopy. light microscopy emphasized the histological development of the skin while the electron microscopy concentrated on the differentiation of dermal chromatophores.

Of special interest in the development of the skin was the early establishment of the xanthoblast layer, which in some instances appeared to precede establishment of the basement membrane. Skin gland primordia were first noted during stage 14. Their epl-integumental product was observed in all stages subsequent to stage 20.

At the EM level, chromatoblasts were the predominate form of dermal pigment cell during stages 18-20. During stage 21, all three types of chromatoblasts were observed to differentiate into chromato­ phores . The origin of both pterinosomes and reflecting platelets were traced to ribosome covered vesicles derived from 'blebhihg' of the outer nuclear membrane of appropriate chromatoblasts.. Three distinct types Of ' iridoblasts were observed and cursory observations were made of melano- some differentiation.

The significance of the similarities in the mechanisms of dif^ ferentiation of all three chromatoblasts was discussed. Evidence was also presented indicating an increase in the levels of circulating hormones during stage 21.

X INTRODUCTION

Chromatophores have been shown to originate in the neural crest

of amphibian embryos (DuShane 1935, 1943; Niu 1947, 1954). Because the : nomenclature for non-melanophore pigment cells was inconsistent in early

studies, a convention was established (Bagnara 1966) in which pigment

cells were named with respect to their colors. Accordingly, yellow pig­ ment cells were defined as xanthophores, red pigment cells were defined as erythrophores, and Iridescent or white pigment cells were called iridophores.

Generally, chromatophores of amphibians have been observed to appear in a developmental sequence,.1.e., melanophores, xanthophores, iridophores and erythrophores, respectively (Hama 1963, Bagnara 1966).

These chromatophores, in the adult anuran integument, are arranged in a distinctive pattern, the dermal chromatophore unit (Bagnara, Taylor and

Hadley 1966), which functions in the adaptive coloration displayed by many poikilotherms. Numerous investigators have accumulated a great deal of data about the control mechanisms of chromatophores and other aspects of animal pigmentation (for review see Bagnara and Hadley 1973).

' ' What is of special interest in the present study is the differen­ tiation of xanthophores and iridophores, and the ontogeny of their re­ spective pigmentary organelles, pterinosomes and reflecting platelets.

Agalyohnis dacnicolor was selected for this investigation to further ,

1 the analysis of pigmentation of this species (Taylor and Bagnara 1969).

Recent research in this area has resulted in the identification of a pigment, rhodomelanochrome (Bagnara, Taylor and Prota 1973), which was . subsequently localized in an unusual type of melanosome within the melanophores of A. dacnicolon: (Bagnara and Ferris 1974). Investigation, of other members of the Phyllomedusinae have shown that this unusual melanophore is a characteristic specific of this subfamily and a possible evolutionary link may exist between AgalyChnis and Phyl- • lomedusa in America and at least some members of Litoria in Australia

(Bagnara and Ferris 1975). LITERATURE REVIEW

. Xanthopiiores and Erythrophores

Pterinosome Chemistry -

The association of pteridines with xanthophores has "been demon­

strated (Bagnara 1961) and it was shown that they are definitive pigments

, of amphibians (Obika 1963). Pterinosomes are pteridine containing

organelles characteristic of xanthophores and erythrophores (Matsumoto

1965) Bagnara 1966,• Eamei-Takeuchi, Eguchi and Hama 1968; Obika and

Matsumoto 1968; Ide and Hama 1969; Obika 1969; Berns and Narayan 1970).

While it is certain the pterinosomes of lower vertebrates contain un- •

conjugated pteridines, it is not clear whether these organelles are the

site of pteridine biosynthesis (for review see Obika 1969). Pterinosomes

have been shown to contain tyrosinase activity in goldfish (Ide and Hama

1969) but not in amphibians (Obika and Matsumoto 1968). Pterinosomal

pigment may also possibly contain uric acid (Hama 19-70).

Pteridine Chemistry and Pterinosome Ultrastructure

The chemistry of pteridines per se is beyond the scope of the

present study. However, it is important to realize that a relationship

between the chemical component of pterinosomes and their ultrastructural

development probably exists. A definite sequence of pteridines have

3 been shown to parallel the development of amphibian larvae (Hama 1963) . and pteridines have been found in the adults of all anurans and the adults of most plethodontids (for a comparative study see Bagnara and

Obika 1965). It was demonstrated that a fundamental set of flarval pteridines’ exists in the skin of larval amphibians, which at the time of metamorphosis may disappear completely, partially, or remain the same depending on the species. In those species where some or all of the larval pteridines were lost, new adult pteridines were presumed to have been produced de novo (Hama 1963).

It was suggested (Hama 1970 ) that the deposition of pteridines in pterinosomes may be involved with the architecture of the inner structure of pterinosomes. since treating pterinosomes with an alcohol- ammonia solution causes dramatic ultrastructural change (Hama and Fukuda

1964). It has also been demonstrated that pteridine interconversions may occur in anuran skin (Stackhouse 1966). More recently, in a study of FleurQdeles waltlii (Potter 1970), a definite relationship was estab­ lished between empty-looking, larval, pterinosomes which were character­ istic of pteri dine-carotenoi d xanthophores and the fibrillar pterinosomes which are characteristic of adult Fleurodeles skin which was found to contain riboflavin. Other evidence also suggests that riboflavin is responsible for the yellow pigmentation of other urodele forms

(Bagnara 1966).

'Pterinosome Ultrastructure

Two morphological types of pterinosomes have been described, a

'•filamentous type' and a 'lamellar type' (Obika 1969). Goldfish ■ ' - ; ■ ' ■ . : - v 5 erythrophores contain pterinosomes which are ellipsoidal (long diameter

0.6u,/ shorter diameter 0.4a) and contain irregularly arranged 40A fila- - ments (Matsumoto and Obika 1968). Xanthophores of adult Fleurodeles and

AmbyStoma (axolotl) contain ovate pterinosomes, 0.5-0.7u in diameter, which contain irregularly arranged filaments similar to those in gold­ fish (Potter 1970).

Lamellar pterinosomes are generally oval, 0.7-0.8u long, and contain a distinctive, concentric arrangement of internal, lamellae

(Obika 1969). The pterinosomes of the swordtail were diagrammed as spheroid (Matsumoto 1965), (long axis 0.7u, short axis 0.5u) with at least ten, concentric, internal lamellae 120A thick. The limiting mem­ brane was distinct and 160A thick. Lamellar pterinosomes have also been described in a teleost (Kamei-Takeuehi et al. 1968, Kamel-Takeuchi and .

Hama 1971), several species of anurahs (Kawaguti, Kamishima and Sato

1965> Obika and Matsumoto 1968; Taylor 1969; Yasutomi and Hama 1971, \

1972) and reptiles (Breathnach and Poyntz 1966, Taylor and Hadley 1970>

Rohrlick and Porter 1972).

A third- type of pterinosome has recently been described in axolotl (Dunson 1974) which reportedly differed from, those previously described. This pterinosome was thought to be unique in that it appeared to be an empty vesicle. From the description presented, this reportedly new pterinosome is probably identical With the empty pteri-- nosomes of neotenic Ambystoma and larval Pleurodeles.. It was suggested that these empty pterinosomes of larval Pleurodeles may develop into the filamentous type found in adults of these salamanders (Potter 1970). Similarly, larval jrterinosome of Hyla arborea were reported to be devoid

of Inner structure. . These empty pterinosomes also proved to be larval

forms which in this case developed into 1 lamellar type1 ' adult pterino­

somes typical of othef anurans (Obika 1969).

Pterlndsome Development.

A series of developmental stages have been demonstrated for the

.differentiation of pterinosomes in Oryzias latipes (Kamei-Takeuchi and .

Hama 1971), Rana japonica (Yasutomi and Hama 1971, 1973), Xenopus .

laevis (Kawaguti, Kamishima and Sato 1965j Yasutomi and Hama 1972) and

Hyla arbprea japonica (Kawaguti et a!, 1965). All of these studies '

showed remarkable similarity in the development of pterinosomes of

xanthbphores and/or erythrophores of the various species investigated.

The initial stage of pterinosome development was represented by single-

membraned vesicles which were usually empty or contained occasional

filaments or amorphous material. The intermediate stages were repre­

sented by single-membraned vesicles with filamentous strands or in- ■

distinct lamellae. The final stages were represented by a pterinosome

which consisted of many concentric lamellae. Mature pterinosome# some­

times lacked clearly defined limiting membranes (Yasutomi and Hama 1971),

and it was common to find a single xanthophore or erythr©phore which

contained all these developmental stages (Kamei-Takeuchi and Hama 1971,

Yasutomi and Hama 1971).

That these various forms of pterinosomes do represent a sequen­

tial change in pterinosome structure was verified by counting the number of pterinosomes of each stage, from representative micrographs of the .

.ohromatophores of larvae of different metamorphic stages (Kamei-Takeuchi

and Hama 1971; Yasutomi and Hama 1971, 1972 ). The.ob servation that in

larval xanthophores, just prior to metamorphosis, ten’percent of the .

pterinosomes counted were immature stages, while in post-metamorphic

xanthophores over 40 percent of the pterinosomes counted were immature

stages, caused speculation that a different type of differentiation may

exist between larval and adult pterinosomes (Yasutomi and Hama. 1971, /' '

1972). An alternative hypothesis is suggested in the present study.

Origin of Pterinosomes .

. . While the development of lamellar pterinosomes has been defined

in terms of a generalized sequence of ultrastructural events, no evi- \

dence has been presented demonstrating the origin of the initial stage

of pterinosome development, i.e., the 'empty' single-membraned vesicle. .

Because rough endoplasmic reticulum was scarce and smooth endo­

plasmic reticulum common in the erythrophore of the swordtail fish, it

was speculated "that the pterinosomes of swordtail erythrOphores are .

closely related in their origin to the smooth-surfaced membrane"

(Mat sumo to 1965:, p. 503). Pleurodeles pterinosomes were thought to have

originated from the'rough endoplasmic reticulum (Potter 1970). During

the metamorphosis of H. arborea, pterinosomes were, frequently' observed

to become multivesicular bodies by "infoldings of the outer membrane".

(Obika 1969? p. 415), which suggested that other cellular components

were contributing to the formation of adult pterinosomes. The present study suggests that the initial, single-meiribraned-vesicle> stage of

: pterinosome development originates from nuclear hlehhing through a de­

velopmental sequence different from the above hypotheses.

; Carotenoid Vesicles : .

The bright colors of xanthophores and erythrophores result from

the presence of pteridines and/or other pigments such as earotenolds

(Matsumoto: 1965, Obika 1969). One, two or three pigments may be re­

sponsible .for xanthophore color. These pigments .appeared sequentially -

rather than simultaneously (Potter 1970). ' Pteridines generally occur at

the time of xanthophore differentiation (Hama and Obika I960; Hama,

Matsumoto and Obika I960; Hama 1963; Obika 1963; Hama and Fukuda 1964; :

Matsumoto 1965; Hama and Hasawega 1967; Hama 1969). Carotenoids are not

synthesized by animals and must therefore be obtained from their food;

. Consequently, carotenoids have not been detected as pigments until after

feeding had begun (Potter 1970). That carotenoids are pigmentary com­

ponents of xanthophores and erythrophores has been based on the prepon­

derance of these pigments in skin of fishes and vertebrates which contain

these cells, and the extractability of these compounds with various or­

ganic solvents (Bagnara 1966), The presence of carotenoids, associated

with smooth membranous vesicles, has been established in Hyla'cinerea

(Bagnara, Taylor and Hadley 1968; Bagnara and Hadley 1969) and R.

japonica (Obika and Matsumoto 1968). That carotenoids are contained

within carotenoid vesicles has been demonstrated in Pleurodeles

(Potter. 1970). In A. mexicanum, a variant which lacks carotenoids was

found to also lack carotenoid vesicles (Potter 1970). 9 Iridophores

Reflecting Platelet Chemistry

Reflecting platelets are membrane-bound, organelles that contain •

purines (Bagnara 1966; Setoguti 1967; Taylor 1967, 1969; Rohrlick and.

Porter 1972; Ide and Hama 1972a) and are characteristic of iridophores

found in the integument, and iris of numerous species (for review see

Bagnara and Hadley 1973). In amphibian integument the most common

purines are adenine, guanine and hypoxanthine (Bagnara 1966).

- The incorporation of labeled ( % ) guano sine and ( ^ C ) adenosine

into guanine and hypoxanthine, respectively, in skin isolated from

anuran tadpoles suggested the presence of purine nucleoside phosphorylase

in iridophores (Ide and Hama 1972a). This enzyme subsequently was demon­

strated in iridophohes (ide and Hama 1972b). The appearance of irido­ phores was found to have no effect on pteridine levels in amphibians

either quantitatively or qualitatively (Hama 1963). Similarly, pteri-

dines were found to be Very scarce in the iridophores of goldfish

(Hama, Matsumoto and Obika. I960).

Platelet ' Chemistryand ' Ultrastructure

Changes in the chemical constituents of reflecting platelets can

dramatically effect the ultrastructure of these organelles in the

iridophores of anurans (Taylor 1967, 1969). Discussion of the physio­

logical regulation of iridophores is beyond the scope of the present

study (see Bagnara and Hadley 1969, Hadley and Goldman 1972, • 10

Bagnara and Hadley 1973, Ide 1974). It should he noted;, however, that

the absence of MSH in hypophyseetomized larvae can cause iridophore

differentiation in regions of the skin where they would not normally be

found (Bagnara 1957). '

Reflecting Platelet Ultrastructure

Electron micrographs of untreated thick sections of .'Hyla arbprea '

japonica (Kawaguti, Kamishima and Sato 1965; Setoguti 1967) and fish

(Kawaguti and Kamishima 1966b) indicate that the cytoplasm of irido- ,

■phores are filled with numerous needle or plate-like electron ,dense In.-

elusions called reflecting platelets. Depending on the species, reflect­

ing platelets vary considerably in length and width, however they retain

a remarkable inter-specific uniformity of thickness of approximately

1000A (Kawaguti and Ohgishi 1962; Kawaguti and Kamishima 1964a; Kawaguti

1965; Kawaguti, Kamishima and Sato 1965; Kawaguti and Kamishima 1966a,

1966b). The spaces between reflecting platelets in Rana japonica

(Setoguti 1967) and a cuttlefish (Kawaguti and Ohgishi 1962) have been

measured to be 1000A. The average width of iridophore reflecting plate­

lets in untreated skin of A. dacnicolor is 0.14u=0.15u (Taylor 1967,

1969). •

One study of reflecting platelet crystals reported them to be*

composed of sub-units about 250A wide (Kawaguti and Kamishima 1966a).

Other workers have failed to substantiate the reported sub-structure of

reflecting platelet crystals (Setoguti 1967, Taylor 1969). Most investi­

gations of the ultrastructure of reflecting platelets however cannot

report on the possible sub-structure of reflecting platelets because the platlets w s e © not direetly observed.' What was observed are the holes

or empty spaces left.behind when the crystalline material contained in

the reflecting platelet had been removed, Iridophores therefore were .

reported as having a fenestrated appearance (Kawaguti, Kamishima. and

Sato 1965).

Explanations of the disappearance of reflecting platelet cry­

stals include the possibility that: (a) crystals are shattered or other­

wise removed mechanically during thin sectioning (Setoguti 1967, Taylor

1969), (b) crystals are dissolved during staining of the sections with

.alkaline lead stains (Kawaguti et al. 1965, Setoguti 1967) and/or (c)

crystals are damaged by exposure to electron beams (Kawaguti et al.

1965, Ferris 1976). It has been demonstrated that reflecting platelet.

crystals of Anolis carolinensis are dissolved out of sections prepared

for electron microscopy by alkaline lead staining (Rohrlick and Porter

1972).

The membranous nature of the reflecting platelet is certain but

the exact structure of this membrane is unclear. In By lid frogs re­

flecting platelets have been observed to be surrounded by a double

membrane (Kawaguti et al. 1965, Taylor 1969). A similar observation-was

made" of the reflecting platelets, of a cuttlefish (Kawaguti and Ohgishi

1962). All of the other descriptions of iridophores cited in this sec­

tion do not explicitly state that a double membrane surrounds the

reflecting platelets of the respective species investigated. Recently

(Rohrlick and Porter 1972), a detailed description of the reflecting

platelets of A. carolinensis showed that the reflecting platelets in 12

this species are composed of a semi-fused double-membraned vesicle.

Observations of structures similar to those described by Rohrlick and - J . ' Porter are reported in the present study. The iridophores described in

opisthobranchiate mollusks (Kawaguti and Kamishima 1964b) differ so

radically from the vertebrate iridophores■mentioned above, no resem­

blance was apparent j hence they are not discussed.

■Reflecting Platelet Origin . and 'Development

• Iridophores are of neural crest origin and differentiate in

response to environmental factors (Stevens 1954). It has been specu­

lated (DuShane 1943), on the basis of what was termed "considerable

evidence, 'r that chromatoblasts require some substance or substances i.'from

the surrounding tissues in order to synthesize pigment.

A single reference (Kamei-fakeuchi et al. 1968) was found which

explicitly suggested a developmental sequence of ultrastructural events

for the development of reflecting platelets in the "leueopbore" of a

(teleost) vertebrate. Three types of "granules,” representing a de­

velopmental sequence were shown to exist ■ in the same "leucophore" of

Oryzias. The type a granule appeared to be a single-membraned Vesicle — — — t 0,5-0.6u in diameter. The type b granule appeared to be a double-

membraned vesicle filled with an amorphous homogeneous material of the

same electron density as the cytoplasm. The type c granule differed

from the type b granule in that numerous clear spaces appeared in the

granule, usually at the inner margin of the membrane.. It was

speculated that these empty spaces "may be caused by purine-like sub­

stances such as the presence of:guanine within the iridophore," 1 3 The formation of membrane bound "iridosomal platelets" has been

demonstrated in .cephalopod iris and skin (Arnold 1967) but the chemical

composition of these structures is unknown. Iridophores have also been

described in the skin of the fiddler crab Uca pugnax (Green and Neff •

1969). A generalized scheme or sequence of ultrastructural events re­

sulting in the formation of reflecting platelets has been described in a cephalopod (Arnold 1967) but not in any vertebrate forms. However, cer­ tain common principles did emerge from observations of the iridophores of various species.

1. Granule filled vesicles have been described in the iridophores

' Lacerta vivipera (Breathnach and Poyntz 1966), Ambystoma

mexicahum (Dunson 1974) and in A. dacnicolor in the present study.

In Lacerta vivipera these membrane-bound granules were thought to

contain crystalline material, suggesting the possibility that

these structures may represent formative stages„ Similar ob­

servations and conclusions were made in the present study.

2. In Hyla arborea japonica guanine platelets were found within the

cisternae of the endoplasmic reticulum and the thin double

membranes which separated adjacent reflecting platelets were

observed to be part of the endoplasmic reticulum (Kawaguti

et al. 1965). The role of the endoplasmic reticulum in the

formation of pigmentary organelles has been demonstrated in the

melanophore of the goldfish (Turner, Taylor and Tchen 1975)

and in the iridosomal platelet of cephalopoda (Arnold 1967).

3. The idea'that iridosomal platelet formation was dependent upon

the exclusion of other regions of the cytoplasm was stated u explicitly in the case of cephalopods (Arnold 1967) and is im­

plicit in the descriptions of all vertebrate reflecting plate­

lets described.

4. Mlerotubules have been observed in association with reflecting

platelets (Dunson 1974) in AmbyStoma. In H. arborea 60A diame­

ter filaments were observed in bundles > especially in cytoplasm

around the periphery of each.group of reflecting platelets

(Setoguti 1967). In Av oafolinensis a complex series of micro-

filaments, forming a filament lattice, was demonstrated and

thought to.function in the production of iridescence and possi­

bly physiological color change (Rohrlieh and Porter 1972).

The only observations relative to amphibian reflecting platelet formation found in the literature were in urodele forms. Pre-reflecting platelets, similar to those described in the present study, were identi­ fied as such in the peritoneal iridophores of the axolotl (Dunson 1974).

Structures identified as "undamaged reflecting platelets" in an electron micrograph of the iridophores of hypophysioprivic Pleurodeles larvae appear identical to structures identified as pre-reflecting platelets in the present study. The mature reflecting platelets of Pleurodeles were described in the same study (Potter 1970) as similar to those reported in anurans.. MATERIALS AND METHODS

• A. dacnicolor larvae were raised from natural spawnings that

occurred in a greenhouse at The University of Arizona. Because a stag­

ing sequence for A. dacnicolor tadpoles is not available, that of Rana . pipiens (A. 0. Taylor and Kollros 1946) was adopted to compare the rela­ tive degree of. metamorphic development of individual tadpoles. After the

tadpoles were anesthetized with MS-222, dorsal skin was'removed with

iridectomy,scissors and forceps from tadpoles of various stages and

fixed in three percent glutaraldehyde buffered with 0.1 M cacodylate

(Dawes 1971). During this biopsy procedure the dermis was unavoidably

damaged. Fixation was usually overnight at 4°C. The skins were washed

twice in 0.1 M cacodylate and postfixed in two percent osmium tetroxlde, buffered with cacodylate, for 2-4 hours. They.were then dehydrated

through a graded series of ethanol and propylene oxide and were flat

embedded in Epon 812 (Luft 1961). Thin sections were cut on a Porter-

Blum MT-2 ultramicrotome with glass knives. Sections were mounted On

formvar coated, 75 mesh, copper grids. These sections were double

stained with saturated uranyl acetate (Watson 1958) and alkaline lead

citrate (Reynolds 1963).

Thick sections (.0,. 5-1 .Qu). were also cut with the ultramicrotome

and stained with Basic Fuchsin, Methylene Blue or Toluidine Blue. Light micrographs were taken with a Zeiss, polarizing, light microscope.

15 ;/ ’ ; : . •' 16 Electron micrographs were taken with a Phillips 200 at 60 Kv. Light microscopic observations were made on thick sections of skin from stages

9, 14? 16, 18-23? and 25 while electron microscopic observations were restricted to stages 18-23. Consequently, the identification of cell types made at the light microscopic level was verified•ultrastruc- turally only for the latter metamorphic stages, stages 18-23„ RESULTS

Light mi oroseopi c oh servatiort of the dorsal skin of Agalychnis dacnieolor showed it to he composed of epidermis, chromatophores, a mucous gland layer, compact connective tissue and sub-collagenous (sub­ cutaneous) tissues. The present description compares very favorably with observations of the dorsal skin of H. arborea (Kawaguti et al.

1965). V :/

Light Microscopic Description

Stage 9 . In the earliest developmental stage observed, stage 9

(Figure 1), the epidermis consisted of three relatively well-defined layers, while the dermis consisted primarily of collagen. (Figures 1-21 show representative stages of skin development and are therefore grouped together.) The most superficial epidermal layer was a single layer of cuboidal.Lor squamous cells with densely staining cytoplasm. Beneath this single^cell layer was another layer 2-4 cells thick. Cells in this intermediate layer were generally larger, irregularly shaped, and their cytoplasm .appeared more 'transparent. This intermediate layer was the only epidermal region in which melanophores were observed. The most basal epidermal layer was the most densely stained. These basal cells were

17 18

Figure 1. Section of stage 9 skin lacking a discernible basement mem­ brane. — An EM thick section showing the epidermis (E ), epidermal melanophores (eM), collagen of the dermis (D), and spaces between the superficial collagen fibers which contain xanthoblasts (Xb). Beneath the collagen of the dermis is the hypodermis (H). 400 X. 19

Figure 2. Section of stage 9 skin with a basement membrane. — An EM thick section shov/ing the epidermis (E ), epidermal melano- phores (eM), and the basement membrane (BM). The dermis (D) contains xanthoblasts (Xb) and dermal melanophores (dM). Beneath the collagen of the dermis is the hypodermis (H). 400 X. 20

Figure ]. Section of stage 14 skin. — An EM thick section showing the early development of the skin glands (G), and the formation of a distinct layer of xanthoblasts (Xb). The hypodermis (H) contains a capillary with blood cells. 400 X. 21

Figure 4. Vesicular appearance of xanthoblast cytoplasm. — An EM thick section of stage 14 skin demonstrating the vesicular appear­ ance of the cytoplasm of xanthoblasts (Xb), dermal melano- phores (dM), and a developing skin gland (G). 1,024 X. 22

Figure 5. Section of stage 16 skin. — An EM thick section showing the location of the skin glands (G) relative to the basement mem­ brane (BM) and the epidermis (E). As skin glands grew they moved dorsally separating the overlying dermal elements from the main body of the collagen layer (C ) of the dermis. 400 X 23

Figure 6. Stage 16 xanthoblast layer. — Two layers of cells with vesicular cytoplasm are shown just ventral to the basement membrane (BM). Strands of collagen (C) appear to divide these cells horizontally into two separate populations. It was suggested that the dorsal population consists of xantho- blasts (Xb) while the ventral population may contain irido- blasts (lb) and/or blast cells of indeterminant type. An iridophore (I ) is present among the ventral population of cells. 1,600 X. 24

Figure 7. Iridophore identification. — Cells which demonstrated bright cytoplasmic regions when viewed with polarized transmitted light were identified as iridophores (I ). These bright regions resulted from the birefringence of the crystalline material within the reflecting platelets (RP) of iridophores. This section is a polarized view of Figure 6. 1,600 X. 25

Figure 8. Section of stage 18 skin. — Two morphologically distinct skin glands (G) were routinely observed during, and subse­ quent to, stage 18. Small spaces containing cells were common in the ventral part of the collagen layer. The hypo- dermis of this section appeared to have been removed during biopsy. 400 X. 26

Figure 9. Polarized light view of section of stage 18 skin. — Irido- phores (I ) were found in the region formerly occupied by the ventral population of cells with vesicular cytoplasm. Occasional dermal melanophores (dll) were also observed in this region. 1,024 X. 27

Figure 10. Secretion of the skin glands. — During stage 20 the appearance of an epi-integumental secretion (S ) was first observed. The proximal end of the duct of one of the skin glands (G) has been cut at an oblique angle. 400 X. Figure 11. Section of stage 21 skin. — An EM thick section showing an epi-integumental layer of secretory product (S ), epidermis (E), and basement membrane (EM). The xanthophores (X) were more compressed than in previous stages and distributed in a single cell layer just ventral to the basement membrane. Beneath the xanthophores was a mixed chromatophore layer of iridophores (I ) and melanophores (M). An unknown cell type (U) was located in the space beneath the mixed chromatophore layer and the abutting skin glands (G). 400 X. 29

Figure 12. Mixed chromatophore layer of stage 21 skin. — A substan­ tial increase in the number of melanophores (M) was noted during stage 21. Melanophores (M) and iridophores (I) shared the same horizontal stratum just ventral to the xanthophore (X) layer. Unstained reflecting platelets (RP) are observable in iridophores. 1,024 X. 30

Figure 13. Dermal chromatophores in stage 21. — Two types of melano- phores were observed in the dermis beneath the xanthophore (X) layer. Dermal melanophores (dM), which contained small melanosomes (sm), and iridophores (I ) comprised the mixed chromatophore layer. Ventral to this mixed chromatophore layer a melanophore with large melanosomes (im), character­ istic of epidermal melanophores (eM) was observed. 4,320 X. 31

Figure 14. Section of stage 22 skin. — Stage 22 appeared very similar to stage 21. Beneath the secretory coat (S) was the epi­ dermis (E ) which was separated from the dermis by a basement membrane (BM). The dermis consisted of a xanthophore (X) layer, a mixed layer of melanophores (M) and iridophores (I), skin glands (G), and collagen (C). Capillaries and an unknown cell type (U) were usually found between adjacent skin glands. 400 X. Figure 15. Distribution of chromatophores in stage 22. — Xanthophores Distribution ofchromatophores instage 22. — Figure 15.

4 t ‘* ewe w kngad () 1,024X. between twoskin glands (G). themelanophores (M)andiridophores (l)remained inter­ spersed. A capillary A (CAP) containing cells blood spersed.isshown (X)thewere superficialmost chromatophores beneath which

32

33

Figure 16. The skin gland layer of stage 23. — During this stage the skin glands (G) formed a continuous barrier of secretory structures which completely separated the dermal chromato- phores from the collagen layer (CL) of the dermis. Within the epidermis (E) the distal end of a secretory gland duct (GD) was observed. 400 X. 34

Figure 17. Chromatophore distribution in stage 23. — The xanthophores (X) melanophores (M) and iridophores (I) remained distributed as a single cell layer of xanthophores overlying a mixed melanophore-iridophore layer. Between this mixed chromato­ phore layer and the underlying skin glands (G) were cells of unknown type (U). 1,600 X. 35

Figure 18. Polarized view of stage 23 chromatophores. — The reflecting platelets (RP) of iridophores (I) are birefringent and thus appear as bright areas when viewed with polarized light. This section is a polarized view of Figure 17. 1,600 X. 36

Figure 19. Section of stage 25 skin. — At low magnification only one major difference was apparent between skin of this stage and that of stage 23; the skin glands (G ) were not contigu­ ous and did not form a continuous barrier between the dermal chromatophores and the deep collagen layer (CL). A skin gland duct (GD), filled with secretory product (S), was observed to connect a skin gland with the external surface of the skin. 4-00 X. 37

Figure 20. Longitudinal section through a skin gland duct. — Section of stage 25 skin showing continuity between the dermal skin gland (G) and the epidermis (E) via a skin gland duct (GD) which is filled with secretory product (S). 1,024 X. 38

Figure 21. The dermal chromatophore unit. — High magnification of stage 25 skin demonstrated that a functional re-orientation of some of the chromatophores had occurred. The xanthophores (X) retained their original position just beneath the base­ ment membrane (BM). The melanophores (M) appeared to have dropped to the ventral side of the iridophores (I ) but re­ tained cytoplasmic processes, containing melanosomes (m), which embraced the iridophores and usually terminated be­ tween the iridophores and the overlying xanthophores. 1,600 X. 39 :

variable in appearance, some- appeared triangular while others were ir­

regularly shaped. In some, instances basal cells appeared to be resting

directly on the underlying dermal collagen while in others a distinct . basement.membrane was observed (Figure 2).

' Within the dermis a limited amount of differentiation was

apparent. The most superficial collagen strands were often less dense­

ly packed than the more basal strands. These superficial strands were

separated by spaces that were sometimes filled with cells, usually melanophores and xanthoblasts. Two criteria were used to identify

xanthoblasts and xanthophores. The major criterion was.the proximity

of a given cell to the ventral surface of the basement membrane. A ,

secondary criterion was a vesicular appearance of the cytoplasm when

viewed at high magnification. These criterion were shared by both

xanthoblasts and xanthophores of every Stage studied. Consequently

xanthoblasts could not be distinguished from xanthophores solely on the basis of light •microscopy.- However, cells which fit these criteria were

verified to be xanthoblasts (or xanthophores) by electron microscopy.

Xanthoblasts were observed in stage 9 skin in which no discernible base-' ment membrane could be found (Figure 1). On the basis of this observa­

tion it is suggested that xanthoblasts may line up in the dermal collagen

prior to the formation of the basement membrane.

The deep collagen had a very solid appearance and no inter­

strand spaces were observed. Sub-collagenous cells, including melano­

phores and cells which may have been blast cells, were observed. This

sub-collagenous region has been termed the hypodermis (Kawaguti et al,

1965). 40

Stage 14. Dorsal skin of stage 14 (Figure 3) differed in several ways from stage 9. -

1. A distinct basement membrane was consistently observed. •

2. The dermis had differentiated into a superficial collagen layer,

perforated with xanthoblast-filled inter-strand spaces, while '

the deep collagen remained tightly khit. It was determined that

these spaces were filled with xanthoblasts by the criteria

described above (Figure 4).

3. Solid balls of cells were observed in the dermis for the first

time. These- solid balls of cells were interpreted to be im­

mature skin glands because they were located.in the same dermal

position as skin glands (Figure 3). Maturing skin glands were

occasionally observed in sections of stage 14 skin which also

/ contained the solid balls of cells just described.

4. There appeared to be an increase in the variety of hspodermal

cells observed, including capillaries containing nucleated blood

cells. ; . r

Stage 16. At stage 16 (Figure 5) the epidermis had a more homo­ geneous appearance. All the epidermal cells tended to be more uniform in size and staining properties. The most superficial cells were cuboidal while the underlying cells were irregular and slightly larger.

The basal population showed no unique properties other than its position immediately overlying the basement membrane, which was distinct. The epidermis varied in thickness from 3-7 cells, being thickest were skin glands were absent. Occasionally epidermal melahophores were observed. ; . ; . ; : ■ - , 41 ' The dermis of stage 16 showed considerable differentiation.,

Superficially it consisted of a xanthohlast layer which appeared to he

1-2 cells thick. The collagen strands which persisted within this, popu­ lation of xanthoblasts often seemed to divide the xanthohlast layer = horizontally into two separate, single cell layers (Figure 6). it may he the case that the more basal of these two 'xanthohlast' layers actually consists of blast cells of indeterminant type. EM observations of stage 18 skin suggest that other blast, cells, such as iridohlasts, had cytoplasm which would probably have appeared vesicular when viewed at the light microscope level. Underlying this layer of blast cells was a layer of dermal melanophores. Occasionally, melahophores were found among these indeterminant blast cells, but more often they were basal to these vesicular cells and appeared separated from them by collagen strands.

Of several sections studied, only a single iridophore was ob­ served (Figure 7). Mature iridophores (i.e., cells containing reflect­ ing platelets) do appear infrequently during stage 16, but they were not considered to be representative cells of stage 16 dermis. Presumably iridohlasts were present, but they were not identified because no reliable criteria exist for the identification of iridoblasts at the light microscopic level.

Identification of iridophores was based on the birefringence that mature reflecting platelets demonstrate when viewed with transmitted, . polarized light (Shanes and Nigrelli 1941, Bagnara 1969). This 42 Mrefringe.nce was the sole criterion for the identifioation of mature iridophores (Figure 7).

The skin glands observed during stage 16 were situated in the superficial collagen layer and were not contiguous with one another. '•

These glands may be similar to those reported elsewhere (Blaylock,

Ruibal and Platt-Aloia 1976). Occasionally a gland was situated such that its .diameter coincided with the horizontal axis of the surrounding basement membrane. Overlying xanthophores were never observed to be con­ tiguous with the skin glands since an intervening collagen layer was always present."

The deep collagen layer contained no chromatophores and only a few inter-strand spaces were noted. Few hypodermal structures were observed, but this region was subject to variable mechanical stress when the skin was removed during biopsy. In sections which contained some remnant of the hypodermis, hypodermal iridophores were observed. The development of these hypodermal iridophores seemed.to parallel the de­ velopment of the dermal iridophores (Figure 44). .

Stage 18. Stage 18 epidermis could be characterized as a bi­ layer (Figure 8). The more superficial layer was approximately three . cells thick.. These squamous cells had a clear, near-transparent appear­ ance. The underlying layer, including the germinal cell population, was

1-3 cells thick. > These basal cells were cuboidal or irregularly shaped and their cytoplasm stained more intensely than the overlying cell layer. ' 43

. Beneath the basement membrane was a single-cell.xanthophore layer whieh was•superficial to a layer of blast cells of indeterminant type. Some of these blast cells were presumed to be chromatoblasts be­ cause within this layer of blast cells iridophores were observed.

Basal to this layer were melanophores. Chromatophores of all types were most abundant in those areas of the dermis directly overlying the skin glands (Figure 9).

Two types of skin glands were routinely observed in stage 18 skin. One type of skin gland contained a granular material while the other appeared to contain vesicles. These glands may be similar to the granular and mucous glands described in the skin of this species Blaylock et al. 1976). Intermittant juxtaposition of skin glands resulted in the separation of the dermis into a superficial, chromatophore-containing region and a deep.) relatively undifferentiated dermal region which con­ sisted primarily .of collagen. This deep dermal region,_ however, did show signs of differentiation at this stage. Small inter-strand spaces were observed which usually contained nuclei.

Stage 19. The epidermis of stage 19 differed little from the epidermal bi-layer described. above.. Only minor variations in cell shape were observed. The dermis consisted of a well-defined xanthophore layer in which some collagen strands remained. This layer had developed from the differentiated superficial collagen layer, described in stage 14, • into a distinctive chromatophore layer which contained only occasional

strands of collagen, iridophores were immediately ventral to the xanthophores, as they had been in stage 18, and seemed to contain variable numbers of reflecting platelets. The nuclei of these chromato- phores had apparently moved, from their central position typical of , stage 16, to the basal part of the cell. This asymmetry has been noted by other observers (Setoguti 1967, Taylor 1969). Melanophores of stage

19 were usually situated beneath iridophores. However in some instances melanophores Were also found within the iridophore layer. Thus at stage 19 and stage 20 (Figure 10) the xanthophores seemed to overlie a ' mixed melanophore-iridophore layer. The dermal chromatophore unit - . described by Bagnara, Taylor and Hadley (1968) was not typical of stage

19 or 20 dorsal skin.

The skin glands of stage 19 continued to show the development described in stage 18, as did the deep collagen layer. The histologi­ cal significance of this continued skin gland development will be dis­ cussed in the description of stage 23 integument.

Stage 20. Stage 20 (Figure 10) differed little from stages 18 and 19. A figure is presented to demonstrate a section with a partial view of a skin gland duct and the presence of epi-integumental granular material which will be explained below.

/

Stage 21. The most striking observation of stage 21 (Figure 11) was the presence, of a new superficial skin component. This component appeared to be non-cellular in. nature and was interpreted to be a pro­ duct of the skin glands and delivered to the skin surface Via ducts.

It is speculated that this non-cellular material may be related to the 45

mucous secretion reported in a study of Phyllomedusine skin structure

and wiping behavior (Blaylock et al, 1976).

The xanthophore layer in stage 21 appeared more compressed than

that of stages 19 and 20. Individual xanthophores were thought to be

.bi-convex cells because of their spindle-shaped appearance when viewed

in tangential section. The long axes of these 1 spindle shaped' cells

paralleled the basement membrane (Figure 12).

Underlying these compressed xanthophores was a mixed chromato-

phore layer of melanophores and iridophores. This melanophore-iridophore

layer had by stage 21 become more fully developed than that described in

stages 19 and 20. ' One major change within this mixed chromatophore

layer was a substantial increase in the number of melanophores. While a

count of the respective chromatophores was not made, inspection of the

figures representative of stages 18-21 will support this observation.

From the figures presented•it is apparent that in stage 21 the ratio of

•iridophores to melanophores is roughly equal while in prior stages

there were more iridophores than melanophores. Furthermore it was

pointed out in stage 18 that the iridophores, identified under polarized

light, were located in a population of blast cells of indeterminant type.

It is suggested that these blast cells were chromatoblasts, some of which

differentiated into iridoblasts while others differentiated into melano-

blasts which underwent melanin deposition during stage 21.

It is speculated, on the basis of a single EM Observation

(Figure 13), that two different melanophore types may exist in the

dermis. The 1 dermal1 melanophores described in the previous stages were ' . 46 identified as dermal melanophores on the basis of the location, and not

thbir. xiltrastructure. , These 1 dermal1 melaiiophores were invariably, lo­

cated ventral to the layer of indeterminant blast cells and were usually

separated from this layer by collagen fibers (Figures 1-10). It may be

the case that some of the melanophores found in the dermis prior to

stage 21 are ultrastructurally of the epidermal type (i.e., contain I-.cr-•

large melanosomes) and that most of the true dermal melanophores (i.e.,

containing relatively small melanosomes) differentiate and deposit . melanin.during stage 21. It may also be the case that the epidermal melanosome observed may simply 'represent a rare instance in which a

single epidermal melanophore failed to migrate to the epidermis. . It

should be emphasized that since melanophores were, not the object of the present investigation melanophores were not subject to careful observa­

tion and consequently no data were collected on the frequency of this phenomenon.

The iridophores of stage 21 also demonstrated continued develop­ ment relative to those of previous stages. There was a noticeable in­

crease in the number, of reflecting platelets observed in each cell when viewed with polarized light. Whereas these structures had originally been distributed on the superficial side of the nucleus (Figure 9),

during this stage they had a more perinuclear distribution. By stage

23 the reflecting platelets would completely surround the nucleus which would concomitantly assume a more central location (Figure 18). This process was not easily defined in terms of development stages and

consequently no stage-specific description of reflecting platelet 47 distribution would be accurate. At best it can be said.that during stage 21 the distribution of reflecting platelets heavily favored the apical end of Iridophores. . '

During stage 21 a new cell type was observed for the first time

(Figure 11, 14, 17, and.20)..These cells filled the spaces between the . skin glands and the overlying mixed chromatophore layer. The function of these cells is unknown. The deep collagen layer remained unchanged - from that described in stage 19.

. . Stage 22. Stage 22 (Figure 14) .§ppeared very similar to stage 21

(Figures 11 and 12). The basement membrane, while still discernable, was not conspicuous, The chromatophores remained distributed as a xanthophore layer overlying a mixed chromatophore (melanophore- iridophore) layer. This mixed chromatophore layer was located in the same region formerly occupied by the ventral population of cells with vesicular cytoplasm. It is suggested that these vesicular cells were chromatoblasts which underwent differentiation during stage 21 and re^'luV suited in.the mixed chromatophore layer.

Capillaries were routinely observed in both transverse and tangential section for the first time in the spaces between skin glands

(Figure 15). These spaces also contained the unknown cell type which first appeared during stage 21.

Stage 23. The completion of the skin gland layer was the major new feature observed in stage 23 (Figure 16). The skin glands which had shown.consistent growth since their appearance in stage 14 formed a " . .• ■ 4^ continuous layer in stage 2 3 . This visually discrete skin gland layer

formed.a continuous harrier of secretory structures completely separating

the dermal chromatophore from the underlying deep collagen. During this

■ slow process of growth and development, the superficial collagen de- :

■ scribed in stage 14 was almost completely displaced.

It is apparent from the figures presented of stages 21 and 23

(Figures 11 and 16) that the completion of the skin gland layer cannot

be strictly ascribed to stage 23. This feature has been ascribed to

stage 23 because every section of this stage studied had a. complete skin

gland layer while the skin gland layers of other stages were not always

complete. . What is of greater importance than the exact stage number is

that the development of skin glands climaxed at the time when this spe­

cies leaves its larval aquatic existence and enters into terrestrial

adulthood. The function of these glands in reducing the amount of, water

lost through evaporation from the skin has recently been described

(Blaylock et al. 1976).

With respect to the dermal chromatophores no major changes were

observed from the descriptions of these cells in stages 21 and 22. The

absence of the dermal chromatophore unit (Bagnara, Taylor and Hadley

1968) was the Only major difference between stage 23 skin and adult

skin..

Stage 25. Skin from an adult, specimen, stage 25+ (Figure 21)

was found to contain dermal chromatophore units. . These units were not

routinely observed in previous stages. The xanthophores retained their

original position just beneath the basement membrane. However, the 49

mixed chpomatophore layer had changed substantially. The cell bodies of

the melanophores had moved to' the ventral surface of the iridophores and

no longer occupied the same horizontal stratum. Melanophore processes,

which contained melanosomes, extended dorsally and embraced the over-

lying iridophores. These processes usually terminated between the

iridophores and the overlying xanthophores.

It is assumed that the ventral movement of the melanophores

occurred during stage 24 since evidence of such movement was infre- •

quently found in stage 23 skin. Melanophores were not easily measured

because their numerous processes and melanosomes obscured cell boundaries

between adjacent melanophores. Xanthophores of mature skin, which re-

• tained their flattened appearance had cell bodies of variable length .

which measured 12-l8u along their long axis. Iridophores at stage 25+

were roughly spherical and measured 12u in diameter.

■ The skin gland layer described during stage 23 was not apparent

in stage 25+. This may have been due- to-..the region from which the skin

was biopsied or may reflect the growth of the skin> to accommodate the

increased size of the adult, without a corresponding increase in the

number of skin glands.

Electron Microscopic Description

Xanthoblasts

Two stages.of xanthophore development were observed: xantho-

blasts and xanthophores. Xanthoblasts were distinguished from mature

xanthophores on the basis of two criteria: (l) the absence of , 50 well-formed multi-lamellar pterinosomes, and (2) general cell shape and position within the dermis. Xanthoblasts in early metamorphic stages had extensive, cytoplasmic processes which penetrated between strands of collagen which surrounded the cell (Figure 22), :(Figures 22-38 show ultrastruetural aspects of xanthoblast differehtiation and are therefore ' grouped together.) While the long axis of the cell did roughly parallel the basement membrane, the contours of the dorsal aspect of these cells often did not closely follow the contours of the overlying basement membrane (Figure 22).

Presumptive xanthophores or xanthoblasts were characteristic of the dermal xanthoblast layer of stages 18 through 20. Xanthoblasts did appear during stages 21-23 but with decreasing frequency as metamorpho­ sis progressed. Mature xanthophores, while characteristic of stages 21,

22, and 23 also appeared in earlier metamorphic stages.

Xanthoblasts were the most superficial pigment cells observed and thus were closest to the basement membrane. However, an often sizeable barrier of loosely-packed collagen separated the xanthoblasts from the overlying basement membrane as well as from one another

(Figure 23). • Consequently, in earlier metamorphic stages xanthoblasts did not form a contiguous cell layer. However, as metamorphosis pro­ gressed, processes of adjacent xanthophores did.touch more frequently.

As individual xanthophores abutted one,another the inter-cellular spaces became negligible except where collagen strands persisted (Figure 24-).

With metamorphosis the collagen barrier which had previously separated the xanthoblasts from the basement membrane was reduced to a fraction Figure 22. EM overview of stage 18 dorsal dermis. — Low magnification of region where dermal chromatophores will eventually be situated. Beneath the basement membrane (BM) are processes of xanthoblasts (Xb) which are surrounded by collagen fibers (CF) and large inter-cellular spaces. An iridoblast (lb ) is located between the xanthoblast layer and a skin gland (G). 4,900 X. 52

Figure 23. Barier separating xanthoblast from basement membrane. — Collagen fibers (CF) were always observed between the base­ ment membrane (BM) and the underlying xanthoblasts (Xb). This barrier of collagen fibers was of variable thickness and tended to decrease in thickness as xanthoblasts matured. Also shown are the nucleus (N) of a stage 19 xanthoblast, smooth vesicles (SV) of variable diameter, and carotenoid vesicles (CV). 14,040 X. 53

Figure 24. Establishment of the xanthophore layer. — Typical stage 23 showing two contiguous xanthophores (X) which contain multi-lamellar pterionsomes (P). The inter-cellular spaces between adjacent xanthophores, melanophore processes (MP), and iridophores (I ) have become negligible and contain only small numbers of collagen fibers (CF). 14,040 X. Figure 25. Reduction of the collagen barrier. — Stage 20 xanthoblast (Xb) moving dorsally. The result of such movement was the eventual displacement of many of the overlying collagen fibers (CF) and the close approximation of the dorsal sur­ face of this cell to the ventral surface of the basement membrane (BM). Also shown are iridoblasts (To). 5,265 X. 55

Figure 26. Nuclear blebbing in a xanthoblast. — The outermost membrane of the nucleus (N ) of this stage 19 xanthoblast had ribo­ somes (R) associated with its cytoplasmic surface. The separation of this outer membrane of the nuclear envelope resulted in perinuclear spaces (PS) which contained fila­ mentous material. Ribosome associated vesicles (RV) were also observed at this time. 14,040 X. 56

Figure 27. Ribosome associated vesicles in a xanthoblast. — Irregu­ larly shaped ribosome associated vesicles (RV) and mito­ chondria (M) were observed in a stage 18 xanthoblast (Xb). This cell was identified as a xanthoblast because it con­ tained smooth vesicles (SV) identical to those in the xanthoblasts immediately dorsal to it (smooth vesicles in portion of cell not shown). Iridoblasts (lb) were also present. 11,550 X. Figure 28. Ribosome associated vesicles in a melanophore. — A stage 18 melanophore which contained ribosome associated vesicles (RV) similar to those observed in xanthoblasts. This melanophore also demonstrated a blebbed outer membrane of the nucleus (N ) which had ribosomes (R) associated with its cytoplasmic surface. 36,540 X. $8

Figure 29. Characteristic vesicles of xanthoblasts. — Stage 19 xanthoblast (Xb) containing ribosome associated vesicles (RV), smooth vesicles (SV), carotenoid vesicles (CV), microfilaments (mfs) and free ribosomes (R). 14,040 X. 59

Figure 30. Coalescing smooth vesicles. — Stage 19 xanthoblast showing the breakdown of smooth vesicle limiting membranes and the resultant coalescence of smooth vesicles (SV). One smooth vesicle appears to contain an irregular concentric membrane (CM). This membrane was interpreted to be the result of a transverse section through two adjacent 1 coupled' vesicles (see Figure 31). 14,04-0 X. 60

Figure 31. Coupling of smooth vesicles. — Stage 21 xanthoblast showing a longitudinal view through two adjacent smooth vesicles in a male-female coupling. An iridophore (I) is present just ventral to the xanthoblast (Xb). 14,040 X. 61

Figure 32. Cell shape of a typical xanthophore. — Stage 21 xantho- phore (X) containing occasional multi-lamellar pterinosomes (P ). When viewed in transverse section xanthophores appeared spindle shaped, contained a roughly ellipsoidal nucleus (N ) and had their long axis parallel to the basement membrane (EM). 8,280 X. 62

Figure 33. Pterinosome development. — Stage 21 xanthophore containing pterinosomes in various stages of development. Earliest stages are represented by a smooth vesicle (SV), type la pterinosome (la), and the type lb pterinosome (lb), re­ spectively. Later stages in pterinosome development are represented by type II pterinosomes II and type III (III) pterinosomes, respectively. Also shown in this micrograph are carotenoid vesicles (CV), swirls of microfilaments (mfs) an " ribosome associated vesicles (RV) of very small diameter. 14,04-0 X. 63

Figure 34. Characteristic pterinosome types. — Stage 21 xanthophore (X) showing a typical variation in the arrangement of in­ ternal pterinosomal lamellae (PL). Stages of development indicated are identical to those identified in Figure 33 (see text). 14,040 X. 64

Figure 35. Pterinosomes of stage 23 xanthophores. — Stage 23 xantho- phore showing its pterinosomes to be identical to those found in earlier stage xanthophores. Pterinosomal stages indicated are identical to those identified in Figure 33 and defined in text. 14,040 X. 65

Figure 36. Pterinosomes at high magnification. — Stage 21 pterinosomes shown at high magnification. Smooth vesicle (SV) appears to have an intact limiting membrane while pterinosomes with internal lamellae have fragmentary or no discernible limit­ ing membrane. Smaller diameter structures are carotenoid vesicles (CV). 45,920 X. iue3. PossibleStage association ofpterinosomes andFigure37.rough — ER. .rm erltdt trnsms 14,040X. berelated to pterinosomes. 21xanthophore (X)showing roughendoplasmic reticulum (ER) in association largewith diameter vesicles (V) which may

66

67

Figure 38. Unknown structures associated with rough ER. — High magni­ fication view of identical structure shown in Figure 37. Large vesicular structures (V) appear to have at least two and possibly more elements comprising their circumference. 52,650 X. ' - ; - 68

of.its original thickness. This reduction in thickness was accomplished^

at least in part, by the ameboid movement of the xanthoblasts toward the basement membrane until their superficial surface, very closely followed

the contours of the overlying basement membrane (Figure 25).

The nuclei of xanthoblasts varied in size and shape, sometimes

appearing as.simple ellipsoids, while in other cells they were polymor­

phic to the degree that extensive processes of cytoplasm seemed to perforate the nucleus ( Figures 26 and 57 ). The limiting .membrane of the nucleus was frequently observed to 'bleb1 away from the main body of the nucleus, resulting.in the formation of a number of perinuclear spaces which usually contained fibrous material. This 'biebbed' membrane usu­

ally had ribosome-like structures.associated with its cytoplasmic

surface (Figure 26). This 'blebbing' is Similar to that observed in

MSH treated melanophores and in control melanophore.s observed in the

present study (Figure 28).

With the exception of occasional mitochondria, no cytoplasmic .

organelles were observed in xanthoblasts. A variety of cytoplasmic

vesicles, however, did fill these cells. Carotenoid vesicles were most •

abundant in the earlier stages; their number seemed to decrease with

metamorphosis. These vesicles were usually spherical, varied from

0.4-0.6u in diameter and contained an amorphous, electron dense sub.-*

stance (Figures 29 and 30).

Ribosome-associated vesicles were observed in xanthoblasts

containing actively 'blebbing' nuclei. These vesicles had ribosome-like

structure associated with their cytoplasmic surfaces in much the same ■ 69 manner as the 'hlehbed1 nuclear membrane. These vesicles also contained a fibrous material morphologically similar to the material observed in the. perinuclear spaces described above. These vesicles may represent structures which 'blebbed' off the nuclear envelope or they may repre­ sent transverse sections through tubules which may have retained a .. connection With the nuclear membrane (Figures 26 and 27).

Smooth-membraned, ribosome-free, vesicles were also observed in the cytopiasm of xanthoblasts.- These ribosome-free ■ vesicles were ir^ regular spheres 1.0-1.2u in diameter and were usually larger than their ribosome covered counterparts. These smooth vesicles contained a fibrous material similar to that contained in the ribosome-associated vesicles and the perinuclear spaces which resulted from nuclear 'blebbing'

(Figure 29).

Irregular concentric membranes were frequently observed within these smooth vesicles (Figure 30)• Further observation indicated that these irregular concentric membranes were actually protrusions of an ad­ joining vesicle which had 'pushed in' the side of the first: vesicle

(Figure 31). This male-female coupling, when viewed in transverse section gave the appearance of a smooth vesicle containing irregular concentric, membranes (Figure 30). This phenomenon has also been de­ scribed in the xanthophores of salamanders (Potter 1970). Such inter? actions of smooth vesicles were not limited to pairs of vesicles and often included three or more vesicles (Figure 31).

Smooth vesicles also interacted.in another way, they coalesced.

The ruptured membranes of adjoining vesicles appeared to fuse forming ' 70

nrulti-lo'bed. vesicular units and allowed the contents of respective

vesicles to mix freely (Figures 23, 29 and 30).

The three types of cytoplasmic vesicles described above consti­

tuted the major part of the non-organellar cytoplasmic inclusions of

xanthohlasts. - Multi-lamellar, mature pterinosomes were' completely

absent from these cells. Microfilaments and free cytoplasmic ribosomes

were common but their distribution.had no discernable patterh. Moderate

. numbers of similar particles, termed "BNP particles" have been observed

in the erythrophores of the swordtail (Matsumoto 1965).

The ribosomes found in xanthoblasts can be characterized by their

distribution into three groups: (!) those particles attached to the

cytoplasmic surface of the 'blebbed' nuclear membrane, (2) particles

associated with the cytoplasmic surface of the rough or ’ribosome

coated' vesicles., and (3) free cytoplasmic ribosomes. Each of these

three groups of ribosomes were found to measure 220 A. This compares

with a diameter of 220 A described fOr the ribosomes of eukaryote cells

(Lehninger 1970).

Xahthdphores

Xanthophor.es were distinguished from, xanthoblasts primarily by •

the. presence of multi-lamellar pterinosomes. A less characteristic

difference was the presence of cytoplasmic organelles, i.e., endoplasmic

reticulum and golgi apparatus:;, structures not commonly observed in

xanthoblasts. Xanthophores usually formed a contiguous layer just be­

neath the basement membrane, the contours of which the superficial

surfaces of the xanthophores closely paralleled. The stellate nature of ; • ' ■' 71 xanthoblasts was retained by the xanthophores but was less obvious be­ cause of the lack of intercellular spaces, making the boundaries between adjacent xanthophores and processes,. at times, indistinct (Figure 24).

A similar, stellate shape was attributed to the erythrophores of the goldfish based upon light microscopic observation of these cells in the fin web (Matsumoto and Obika 1968),

The main body of a xanthophore was usually a biconvex disc from which cytoplasmic processes^ paralleling the axis of the basement mem­ brane, radiated. When viewed in transverse section, the cell body appeared spindle shaped, the long axis varied from 12-l8u, The nucleus in such transverse sections was located at the widest part of the apparent spindle and was characteristically ellipsoidal, its long axis paralleling that of the cell (Figure 32).

Small, spherical vesicles which were presumed to be slightly modified carotenoid vesicles were found in the cytoplasm of xanthophores

(Figures 24 and 33). The carotenoid vesicles of xanthophores differed from those of xanthoblasts in a number of ways: (1) their content, while still amorphous, showed greater variation in electron density; generally being less electron dense'than those of xanthoblasts; (2) xanthophore carotenoid vesicles showed greater variation in diameter than those of • xanthoblasts. Carotenoid vesicles representing intermediate stages of this modification were also observed in xanthoblasts (Figure 31).

Vesicles similar to those described here have been described in the xanthophores of salamanders (Potter 1970). These vesicles, which were approximately 0.4u in diameter, were demonstrated to be carotenoid 7 2 vesicles on the basis of electron microscopic and chromatographic evidence. '

Ribosome-associated vesicles were usually absent from xantho- phores or reduced to a fraction of their original diameters (Figure 33).

Single and multi-lbbed, smooth-membraned vesicles of approximately - .the same diameters as those observed in xantboblasts persisted in

.xanthophores;. Three types of pterinosomes, representing a developmental sequence, were observed within a single xanthophore and were often in close proximity to these smooth-membraned vesicles»

Pterinosome Development

Type I pterinosomes closely resembled the smooth-membraned vesicles described in xahthoblasts. Morphologically, the difference between these two vesicular structures was quantitative; pterinosomes x contained more fibrous material. There is reason to speculate that this morphological distinction is trivial and that these two vesicular struc­ tures are chemically identical. "Empty looking pterinosomes" have been described (Dunson 1974> p.. 261) which are very similar to the smooth- membraned vesicles discussed here. Furthermore, vesicles which are morphologically indistinguishable from those described in the present study and those reported elsewhere (Dunson 1974) have been shown to be pterinosomes (Potter 1970).

Two variations of type I pterinosomes were observed: type la contained fibrous material distributed randomly within the pterinosome, and type lb contained fibrous material distributed in a concentric 73 sphere under the limiting membrane. The limiting membrane of both.vari­

ations of the type I pterinosome were often indistinct and fragmentary

in appearance, the fibrous contents merging imperceptibly with the membrane (Figure 33). '

. Type II pterinosomes differed from type I pterinosomes quali­

tatively; they contained distinct, fibrous lamellae and were not bounded by a discemable membrane (Figures 33 and. 34). A similar observation was. made of the pterinosomes of R . japonica (Yasutbmi and Hama 1971).

These fibrous lamellae were often fragmentary, sometimes concentrically

distributed and often'associated with an amorphous, electron dense material. The limiting 'membrane’ of type II pterinosomes appeared to . be a spherical boundary layer of overlapping fibers which were in­

distinguishable from the fibrous material of which the internal lamellae were composed. These fibrous strands were of variable length,'thickness, and electron density. They were often detached from the surrounding boundary layer at one or both ends. Detached strands were observed inside type II pterinosomes in the same region where concentric lamellae

appeared. There was only a quantitative difference in the morphology of

these detached fibers and the concentric lamellae, the latter being more substantial and presumably having formed from the former (Figures

34 and 36). ' ' '

Variation in the distribution of complete concentric lamellae within type II pterinosomes was observed. Some type II pterinosomes had complete lamellae aggragated in the center of the pterinosomal

cavity with fragmentary lamellae or fibers spiralling, out from the 7 4 ; center of aggregation to the fibrous boundary layer (Figures 34 and 36).

Other type II pterinosomes had complete lamellae restricted to the peri­ phery of the pterinosomal cavity while their centers were composed of

fragmentary fibers and associated, amorphous, electron dense material

(Figures 35 and 36).

Type . Ill pterinosomes differed from type II only in that they ■ consisted of at least ten complete concentric lamellae„ This criterion was used in order to pattern the present developmental sequence of pterinosomes after the most applicable and generalized sequence already established in the literature (Kamei-Takeuchi and Hama 1971)„ Occa­

sionally type 111 pterinosomes were, observed to contain electron opaque areas of variable size. These regions of electron opacity were thought to result from adjacent internal lamellae touching or fusing, some of these opacities were the result of the lamellae being sectioned at an oblique angle (Figures 35 and 36).

Pterinosomes of each type were found.to be consistent in size

(Figures 33# 34, 35 and. 36) and measured approximately 1.4^1.7u in diameter. In a single instance (Figures 37 and 36) pte'rinosome-like vesicular structures were found to be associated with rough endoplasmic reticulum. Cytoplasmic inclusions other than pterinosomes consisted mainly of free ribosomes and microfilaments which were often observed in the vicinity of pterinosomes. • Microfilaments were observed to form

swirling patterns in the cytoplasm (Figure 33) but a general pattern of

distribution of either microfilaments or free ribosomes was not evident. ' 75

.Iridoblasts

Two stages of iridophore development were observed; irldotilasts

and iridophores. Iridoblasts were identified by comparing blast cells

with mature iridophores. The primary criteria for the identification of

iridoblasts were (l) their location immediately inferior to the xantho-

blast (or xanthophore) layer, (2) the absence of mature reflecting

platelets, and (3) the presence of intra-cellular structures suggestive

of presumptive reflecting platelets. Those blast cells which met these

• criteria are.discussed in this section while indeterminant blast cells

will be discussed in a subsequent section. Iridophores were identified

by the presence of reflecting platelets.

Iridoblasts were predominant during stages 18-20 while irido­

phores were characteristic of stages 21-23- Ifidophores were also ob­

served as early as stage 18, their numbers increasing as metamorphosis

■progressed. Iridoblasts, however, were not observed in stages 21-23«

Three morphologically distinct types of iridoblasts were

observed. Type I iridoblasts were characteristically flat cells, with a .

single, prominent, centrally located nucleus (Figures 22 and 39).

(Figures 39-70 show ultrastructural aspects of iridoblast differentiation

and are therefore grouped together.) When viewed in transverse section

the cytoplasm of these cells appeared disproportionately distributed on

either end of the nucleus, forming what appeared as cytoplasmic pro­

jections which paralleled the long axis of the nucleus. These processes

varied in length which made determining cell size difficult. It was

estimated that type I iridoblasts were flattened discs 20u in diameter 76

Figure 39. Early iridollasts. — This micrograph is an enlarged view of a selected portion of Figure 22. A type II iridoblast (Ibg) is shown just beneath a discontinuous layer of xantho- blasts (Xb) in stage 20 dermis. Ventral to these cells and separated by a large inter-cellular space is a type I iridoblast (Ib^). The cytoplasm of this cell is filled with irregular ribosome-associated vesicles (RV) which contain a filamentous material. Also included in the cytoplasm of this cell are mitochondria (M) and free ribosomes (R). 14,040 X. 77

Figure 40. Type I iridoblast. — This micrograph shows a type I irido- blast (Ib-^) which has moved dor sally and is no longer separated from the overlying xanthoblasts (Xb) by a large intercellular space. Ventral to this iridoblast is a dif­ ferentiating dermal melanophore (dM) which contains ribosome associated vesicles (RV). 15,054 X. 78

Figure 41. Overview of stage 20 dermis. — Beneath the basement mem­ brane (BM) are processes of xanthoblasts (Xb) which contain carotenoid vesicles (CV). Ventral to this xanthoblast layer are two types, or stages, or iridoblasts. On the right is a type II iridoblast (Ib2 ) containing both smooth (SV) and ribosome associated vesicles (RV). On the left is a type III iridoblast (Ib^) which contains vesicles which appear oval in transverse section (0V). Note the separation of each layer of chromatoblasts by collagen fibers (OF). 4,900 X. 79

Figure 42. Early type III iridoblast. — Ventral to the xanthoblast (Xb) processes is an early type III iridoblast (Ib^) which contains fragmentary smooth vesicles (SV) and free ribosomes (R). These elongate, and usually discontinuous, vesicles were interpreted to be an intermediate between the smooth vesicles observed in type II iridoblasts and the cylindrical vesicles observed in other, slightly more mature, type III iridoblasts. 15,054 X. 80

Figure 43. Overview of differentiating iridophores. — Ventral to the xanthoblast (Xb) processes is the earliest recognizable stage of iridophores (I). These cells correspond to type III iridoblasts in both their location within the dermis and their cytoplasmic inclusions, e.g., ribosome associated vesicles (RV), fragmentary smooth vesicles (SV). The only major difference between these iridophores and type III iridoblasts is the presence of spaces (S) which are indica­ tive of purine deposition. A skin gland (G) is located in the lower right hand corner of this micrograph. This is a micrograph of stage 18 skin. 5,040 X. 81

Figure 44. A hypodermal iridoblast. — The hypodermal iridoblast (HI) shown above was found ventral to the sheets of collagen fibers (CF) of the deep dermis of stage 18 skin. This cell contained a lobed nucleus (N) and pre-reflecting platelets (pRP) identical to those observed in dermal iridoblasts. 12,315 X. 82

Figure 45. Fragmentary smooth vesicles. — This micrograph is a high magnification view of a selected portion of Figure 44. The cytoplasmic inclusions of the hypodermal iridophore (HI) included smooth vesicles (SV) in various stages of fragmentation, small diameter ribosome associated vesicles (RV), and the earliest recognizable pre-reflecting platelet (pRP) observed. Note the ribosomes (R) associated with the cytoplasmic surface of the outer nuclear membrane (OM), and the nuclear surface of the inner nuclear membrane (IM). 44,333 X. 83

Figure 46. Pre-reflecting platelet morphology. — This micrograph is a high magnification view of a selected portion of Figure 44. A pre-reflecting platelet (pRP) is shown which is identical to those observed in dermal iridoblasts. Fragmentary mem­ branes (FM) were interpreted to be derived from previously existing smooth vesicles. These fragmentary membranes in­ completely enclosed a roughly cylindrical region of cytoplasm which contained filamentous material which appeared to fuse into a distinct longitudinal fiber (LF). 44,333 X. 84-

Figure 47. Reflecting platelet formation. — This micrograph is a high magnification view of a selected portion of Figure 43. What is of special interest are the three reflecting platelets (RP) each of which contain the characteristic empty space into which crystalline purine(s) is tbrought to be deposited. The reflecting platelet in the upper right hand corner demonstrates that the longitudinal fiber (IF) is probably the initial site of purine deposition within the developing reflecting platelet. 8,900 X. 8$

Figure 48. Possible reflecting platelet fusion. — Stage 18 iridophores (I) containing pre-reflecting platelets (pRP) and developing reflecting platelets (RP) with characteristic empty spaces. Since early reflecting platelets are several times smaller than more mature reflecting platelets some device must exist to permit 1 growth' of reflecting platelets. It is specu­ lated that the two reflecting platelets in the iridophore on the left may represent one device for the 'growth * of reflecting platelets via fusion of these structures at an early stage. 23,330 X. 86

Figure 49. Reflecting platelet formation in a stage 21 iridophore. — The numerous cytoplasmic inclusions found in a stage 21 iridophore included pre-reflecting platelets (pRP), reflect­ ing platelets (RP) in various stages of development, ribosome associated vesicles (RV), fragmented smooth surfaced vesicles (SV), and free ribosomes (R). 14, 040 X. 87

Figure 50. Reflecting platelet development. — A high magnification view of a group of reflecting platelets in a stage 22 iridophore. Each platelet consists of a fragmentary limiting membrane (LM), an empty space (ES) which contained crystalline purine in vivo, and a halo of amorphous material (AM) which sur­ rounded the purine crystal space. Surrounding these de­ veloping reflecting platelets are granular vesicles (GV), multi-vesicular bodies (MV) and free ribosomes (R). 52,650 X. 88

Figure 51. Cytoplasmic inclusions of a stage 22 iridophore. — Typical structures of developing iridophores include reflecting platelets (RP), pre-reflecting platelets (pRP) with large, distinctive, longitudinal fibers (LF), multi-vesicular bodies (MV), granular vesicles (GV), and smooth membrane fragments (MF). 52,560 X. 89

Figure 52. Iridophore differentiation. — A stage 19 iridophore which contains all those structures found in iridoblasts and iridophores which contribute to the formation of reflecting platelets, i.e., ’blebbed’ nuclear membrane (NM), ribosome associated vesicles (RV), smooth vesicles (SV), pre­ reflecting platelets (pRP), and reflecting platelets (RP). 14,040 X. 90

Figure 53. Cytoplasmic projections of iridophores. — Stage 22 irido- phores with cytoplasmic projections (CP) which are associated with an extra-cellular filamentous material (FM). The con­ figuration of these cytoplasmic 'fingers1 relative to the filamentous material often suggested that this material was about to be engulfed, or had just been released, by the iridophores. 14,040 X. 91

Figure 54. Stage 22 iridophore. — This micrograph represents the same cell shown on the right side of Figure 53. This iridophore demonstrates reflecting platelets (RP) of typical size for stage 22 iridophores and the basal position of the nucleus (N). The linear array of mitochondria (M) just dorsal to the nucleus is not typical. 14,040 X. 92

Figure 55. Typical organelles of stage 21 iridophores. — The parallel array of mitochondria (M) flattened saccules of rough endo­ plasmic reticulum (ER), granular vesicles (GV) and reflect­ ing platelets (RP) shown were frequently observed in iridophores of this stage. Other iridophore characteristics shown include free ribosomes (RP), cytoplasmic projections (CP) and extra-cellular filamentous material (FM). Note the apposition of one of the saccules ot the plasma membrane. 45,450 X. 93

Figure 56. Typical stage 21 iridophore. — Stage 21 iridophore showing the nucleus (N), reflecting platelets (RP), rough endo­ plasmic reticulum (ER), and cytoplasmic projections (CP). 11,900 X. 94

Figure 57. Iridophore approaching xanthophore. — Stage 23 chromato- phores showing an iridophore (I) extending cytoplasmic projections (CP) and containing reflecting platelets (RP). An overlying xanthophore (X) is shown containing multi- lamellar pterinosomes (P). An atypically large barrier of collagen fibers (CF) exists between the xanthophore and the basement membrane (BM). 14,040 X. 95

Figure 58. Chromatophore contiguity. — Stage 23 chromatophores abutting one another in typical fashion. The xanthophore (X) con­ tains multi-lamellar pterinosomes (P) in various stages of development. Between the xanthophore and the underlying iridophore (I) is a melanophore process (MP). 8,580 X. 96

m # s

Figure 59. Endocytotic vesicle. — Section of stage 21 iridophore show­ ing invagination of the plasma membrane (PM) interpreted to be an endocytotic vesicle (EV) containing extra-cellular filamentous material (FM). A cytoplasmic projection (CP) appears to be in the process of closing the vesicle. Also shown are mitochondria (M), free ribosomes (R) and reflect­ ing platelets (RP). 52,650 X. 9 ri

Figure 60. Cytoplasmic canaliculi. — During stage 21, iridophores (I ) were infrequently observed containing what appeared to be deep channels or a tubule network, which permeated their cytoplasm. These spaces were termed cytoplasmic canaliculi (CC) because they differed markedly from endoplasmic reticulum typically observed in stage 21 iridophores. The iridophores shown contain reflecting platelets (RP) and are encircled by melanophore processes (MP). 8,580 X. 98

Figure 61. Melanophore encircling an iridophore. — The iridophore (I) shown in this micrograph is identical to the iridophore in the lower left hand corner of Figure 60. This irido­ phore shows the flattened saccules of rough endoplasmic reticulum (EH) that are typical of stage 21 iridophores. 8,580 X. 99

Figure 62. Platelet-free region of cytoplasm. — Cytoplasmic inclusions of the platelet-free region of stage 21 iridophores included granular vesicles (GV), mitochondria (M), microfilaments (infs), and free ribosomes (R). On the right hand side of this micrograph are parallel arrays of ’hollow' granular vesicles (HV), and reflecting platelets (RP), some with distinct longitudinal filaments (LF). 52,650 X. 100

Figure 63. Multi-vesicular bodies in platelet free cytoplasm. — Inclusions of platelet-free cytoplasm of stage 22 iridophores included multi-vesicular bodies (MV) of various types, smooth vesicles (SV), rough vesicles (RV), and free ribo­ somes (R). 14,040 X. 101

Figure 64. Unusual multi-vesicular bodies. — Unusual structures, which technically fit the working definition of multi- vesicular body (MV) used in this thesis were observed within the lumen of the endoplasmic reticulum of stage 22 iridophores. 14,040 X. 102

Figure 65. Stage 22 multi-vesicular bodies. — This micrograph repre­ sents a high magnification view of a selected portion of Figure 63. A large multi-vesicular body (MV) is shown containing smaller vesicles with fine granular contents (FG) and coarse granular contents (CG). Also shown are rough vesicles (RV) and granular vesicles (GV). 51,480 X. 103

Figure 66. Unusual multi-vesicular body within rough ER. — This micro­ graph represents a high magnification view of a selected portion of Figure 63. An unusual structure which fits the definitiion of multi-vesicular body (MV) used in this text is shown within what appears to be a rough vesicle. From Figure 63 this apparent rough vesicle was interpreted to be a saccule of rough endoplasmic reticulum (ER) cut in trans­ verse section. Also shown is a granular vesicle (GV). 105,300 X. 104

Figure 67. Granular vesicles. — This micrograph represents a high magnification view of a selected portion of Figure 62. Some of the granular vesicles (GV) shown have a solid appearance while others of roughly the same diameter seem to have hollow interiors (HI) which have the same lack of electron density as the empty space (ES) of a nearby de­ veloping reflecting platelet (RP) with the distinct longi­ tudinal fiber (LF). Also shown is a multi-vesicular body (MV). 52,560 X. 105

Figure 68. The indetenninant blast cell. — This overview of stage 18 skin shows a blast cell of indetenninant type (B) between a dermal melanophore (dM) and type III iridophores (ib^). These indeterminant blast cells were distinguished from the type III iridoblasts by their respective cytoplasmic con­ stituents. The cytoplasm of indeterminant blast cells were predominantly filled with rough endoplasmic reticulum (ER) while the cytoplasm of the type III iridoblasts were filled with fragmentary smooth membranes (FM) which resulted from the fragmentation of smooth vesicles. A high magnification view of the iridoblast located in the center of this figure was presented as Figure 4-8. 5,040 X. Figure 69. The mixed chromatophore layer. — During stages 21-23 iridophores (!) and melanophores (M) occupied the same horizontal stratum directly beneath the xanthophore (X) layer. This is a section of stage 21 skin. A comparison of chromatoblast size relative to the respective chromatophore can be made by turning back to Figure 4-3 which was taken at the same magnification. 5,04-0 X. 107

Figure 70. Stage 25 iridophore. — Iridophore of mature animal showing the basally located nucleus (N) surrounded by a thin margin of platelet-free cytoplasm (PC) and reflecting platelets (RP) which were oriented perpendicular to the horizontal axis of the skin. (Courtesy of Dr. Wayne Ferris, University of Arizona, Tucson.) 7,980 X. 108

and contained ellipsoidal nuclei (long diameter 4.On, short diameter

2.On). : \ " V ■

Nuclear 'blehblng' was routinely observed In type I Irldoblasts.

The 'blebbed1 membrane had ribosome-like (220 A diameter) particles associated: with its cytoplasmic surface while the perinuclear space,

defined by the inner surface of the 'blebbed' membrane and the main body of the nucleus contained an amorphous, sometimes filamentous substance.

Ribosome associated vesicles were characteristic of type I iridoblasts.

While similar to those rough vesicles described in zanthoblasts, the rough vesicles of iridoblasts were more irregular in size and shape, often forming large chambers.- These vesicles also contained a fila- ; mentous material morphologically similar to that described in xantho- blast rough vesicles.

A second characteristic of type I iridoblasts was the greater

electron density of their cytoplasm relative to that of neighboring

cells. This greater electron density proved, at high magnifi'cation, to be the result of large numbers of free ribosomes and polysomes which were often associated with a filamentous material. These cytoplasmic, ribosome-like structures were found to be 220 A in diameter. Mitochon­

dria were the only cytoplasmic organelles observed in these iridoblasts.

On the basis Of micrographs from several■animals type 1 irido­ blasts were interpreted to move superficially, toward the xanthoblast

layer (Figure 40). In doing so, these cells were interpreted to lose

their characteristic, flattened appearance and become more ellipsoidal

type II iridoblasts. . Type II iridoblasts retained the cytoplasmic 109

organelles and inclusions described in the earlier,; flattened form.

Nuclear 'blabbing' identical to that described for the flatter, type I

iridoblast was also common in type II irldoblasts (Figure 41).

In addition to the ribosome-associated vesicles, smooth vesicles were also found in type II iridoblasts.' These smooth vesicles were ir­

regular, sometimes fragmentary, and contained less filamentous material

than their ribosome-associated counterparts. It is suggested, because

of the presence of intermediate forms (Figure 41), that these smooth vesicles resulted from the dissociation of ribosomes from the rough vesicles. These newly dissociated ribosomes would presumably have been

added to the existing free ribosome population.

Type II irldoblasts were interpreted to have continued the dorsal migration toward the xanthoblast layer which was noted of type I irido­ blast s. As this ascent continued, the type II iridohlast conformed to

the surfaces of the overlying collagen fibers and xanthoblasts. until the

intercellular spaces which had existed were minimised and, in most cases,

filled with collagen fibers. Once the type II iridoblasts had assumed

their final position in the dermis, they began to form pre-platelet

structures and develop into irldophores (Figure 43).

During stage 20 (exclusively) type III irldoblasts were noted

in the same horizontal strata as were type II irldoblasts (Figure 41).

These blast cells differed from type II irldoblasts both in-general

cell shape and cytoplasmic content. Type III irldoblasts (Figures 41

and 42) were roughly ellipsoidal with a single, central nucleus of

similar shape. In transverse section these cells appeared roughly oval ' . . H O with prominent, oval nuclei. Both the long axis of the nucleus (3.0u)

and the cell (9.0u) roughly paralleled the basement membrane. The most

distinctive feature of type III iridoblasts was the 'crowded' appearance

of their cytoplasm. The endoplasm of these cells appeared to be filled with roughly cylindrical vesicles. These vesicles were- smooth-membraned

and appeared to contain a- filamentous substance. Furthermore, these

cylindrical vesicles evidenced a pattern of discontinuity and coalescence

similar to that described in xanthoblast differentiation. Multivesicular bodies were infrequently observed among these endoplasmic, cylindrical

vesicles.

The ectoplasm of type III iridoblasts was equally 1 crowded' but with ribosome-like structures (220 A) which"were preferentially distri­ buted under the dorsal regions of the plasma membrane. These dorsal

surfaces also evidenced numerous cytoplasmic projections which were

filled with ribosome-like structures (Figures 41 and 42). These struc­

tures differed morphologically from those to be described in iridophores

in that they were polymorphic rather than uniformly 'finger-like.1

Nuclear 'blabbing' was not pronounced in type 111 iridoblasts.

However, where 'blebbed' nuclear membrane was observed, the membrane had

ribosome-like structures (220 A) associated with its cytoplasmic surface,

as in the previously described■iridoblasts. The perinuclear cytoplasm

in type H I iridoblasts was usually dense with a thin margin of free

ribosomes. Rough vesicles, similar to those described above, were ob­

served, but only in a single cell. Ill

It is suggested that, type III iridablasts were derived directly

from type II irlddblasts. This suggestion is based on several similari­ ties between these two iridoblasts and the presence of an early type III

iridoblast (Figure 42) which contained cytoplasmic structures inters mediate to those observed in type II and type III iridoblasts (Figure 41).

Reflecting Platelet.Formation

By definition type III iridoblasts that demonstrated empty cytoplasmic spaces characteristic of reflecting platelets have been .. termed iridophores„ Type III iridoblasts'which contained these spaces . were therefore considered the earliest iridophore. observed"(Figure 43).

The cytoplasm of these early iridophores was usually filled with frag­ mentary membranes. These fragmentary membranes were frequently observed to be arranged in an elongate configuration which enclosed filamentous material and occasional small granules. It is suggested that these . ' . ■ , ' . - fragmentary membranes resulted from the continued. fragmentation of the

smooth vesicles characteristic of type II iridoblasts. This suggestion was supported by the absence, or greatly reduced number, of smooth vesicles in type III iridoblasts which contained these fragmentary membranes (Figure 43).

Often a single, distinctive, central fiber was longitudinally

arranged in the area incompletely enclosed by the fragmentary membranes „

It is speculated, on the basis of the intermediate forms presented

(Figures 44, 45 and 46), that these distinctive fibers formed from the more wispy filaments usually associated with the encapsulating, fragmen- tary membranes. It is suggested that these structures were the 112 primordial reflecting platelet, the pre-reflecting platelet, because the distinctive central fiber they contained seemed to be the initiation site for the formation of purine crystals which were thought to result in the formation of the empty space(s ) along the Central fiber (Figures

47 and 48).

It is therefore suggested that the sequence of ultrastructural events: which resulted in the eventual formation of reflecting platelets and which constituted the differentiation of iridoblasts into the iridophores, was as follows: (1) ’blebbing' of portions of the ribosome associated outer nuclear membrane, (2) formation of ribosome associated . vesicles from the 'blabbed' portions of the nuclear membrane, (3) for­ mation of smooth membraned vesicles from the ribosome associated ^vesicles by the dissociation of the ribosomes from the cytoplasmic surface of the rough vesicles, (4) fragmentation of these smooth vesicles, (5) formation of pre-reflecting platelets from fragmented smooth vesicles, (6) ini­ tiation of purine deposition along the central fiber.of the pre-v' reflecting platelets. Morphologically, this last step was identified by the presence of one or more small, empty looking spaces along the central fiber (Figure 47). These spaces were presumed to be the sites of purine crystals in vivo„

Additional support that the ultrastructural sequence of events described above was the probable route of reflecting platelet formation was found by the observation of the entire sequence outlined above in a single cell (Figure 52). ' 113 Pre-reflectlng platelets similar to those described in type III

iridoblasts were characteristically found in cells which were unambigu­

ously identified as iridophores (Figure-49). • These pre-reflecting

platelets differed from those observed in type III iridoblasts only in

a quantitative way; they were usually longer. Becuase of the variable

iSngth of pre-reflecting platelets, especially in stages 21-22, .and the

juxtaposition of numerous, small, smooth-surfaced vesicles it is specu­

lated that pre-reflecting platelets 'grow' longitudinally by membrane

fusion with adjacent pre-refiecting platelets or other smooth membranes.

/ This speculation was supported by the observation, at high magnification / - . \ ■ : ■ ■ - ' of two adjacent pre-reflecting platelets undergoing, what appeared to be.

end-to-end fusion (Figure 48).

Subsequent to the initiation of purine deposition reflecting

platelets continued to 'grow.' It is speculated that this continued

'growth' may have involved•fusion with a totally different structure,

granular Vesicles.: A detailed description of these granular vesicles

and a discussion of their possible role in reflecting platelet elonga-r

tion will be presented in a paragraph entitled "Granular Vesicles" which

is contained in the subsection "Maturing Iridophores."

Reflecting Platelet Development. ; When viewed at high magnified.-

. tion the reflecting platelets of developing iridophores could be

described as consisting of three components, the reflecting platelet

space, limiting membranes, and an intermediate region of amorphous ma-

' terial (Figures 50 and 51). As mentioned in the literature review,

reflecting platelet crystals per se are not usually visible in thin sectioned material prepared for electron microscopy. That crystalline

purines actually exist within reflecting platelets has been demonstrated

(Taylor 3:967).. What can he directly observed are empty spaces which are

thought to represent the in vivo sites of purine crystals that were re* moved during preparation of the material for electron microscopy (see

,Literature Review for discussion)«

The limiting membranes of developing reflecting platelets are

often discontinuous (Figures 50 and 51) arid did not exhibit the well- V .

defined double membranes characteristic of mature reflecting platelets.

It is speculated that the incompleteness of these membranes in developing

reflecting platelets.functions to accommodate the elongation of the

purine crystal and that new membrane may be added by the fusion of

reflecting platelet membranes with the membranes of adjacent fragmented

vesicles. '

An amorphous material was routinely observed between the empty

reflecting platelet space and the fragmentary limiting membrane of

developing reflecting platelets (Figures 50 and 51). A similar region

of amorphous material, described as a ’halo,’ has been induced in mature,

anuran reflecting platelets by the injection of intermedin. It was also

observed that this 'halo1 developed at the expense of the purine crystal

which it surrounded (Taylor 1967, 1969). It is speculated that this

’halo,1 or the surrounding inner membrane, may be the site of one or

more of the enzymes necessary for intermediary purine metabolism, e.g.,

nucleoside phosphorylase, which has been demonstrated in iridophores

(Ide and Hama 1972b). Maturing Irldophores

While maturing irldophores continued to demonstrate some of. the

intracellular structures characteristic of iridohlasts (i.e., pre­

reflecting platelets, a distinctively electron dense cytoplasm caused by

free ribosomes, nuclear 'blebbing') several new features, some of which

were unique, were also observed.

'ciliary Cell Membrane. Cilia-like cytoplasmic projections were

routinely observed on the periphery of irldophores of stages 21 and 22,

and to a lesser degree in stages 23 and 25. These projections were

filled with free ribosomes and were often associated with a filamentous,

electron-dense extra-cellular material (Figures 53, 55, 56 and 57).

The projections differed from the polymorphic projections of type III

iridohlasts in that they were uniformly 'finger-like.' Similar projec­

tions and the extra-cellular filamentous material were not observed in

any other chromatophore type.

•Iridophore Movement. The appearance of these cytoplasmic pro-

- j actions coincided with a change in cell shape. The elongate polymor­

phic shape assumed by type III iridoblasts was not retained by irido­

phore s in stages 21-23, instead iridophores became more spherical as

they matured. Concurrent with this change in cell shape, iridophores of

stages 21-23 also seemed to move closer to the overlying xanthophore.

population (Figure 57). In previous stages dermal chromatophores,

especially xanthoblasts and iridoblasts, had almost invariably been

separated by collagen fibers and intercellular spaces. By stage 23 ' '-■■■■ 0 i 116 direct contact among all three types of chromatophores was routinely observed (Figure 58).

It is speculated that a possible function of the cytoplasmic projections observed in iridophores during stages 21 and 22 was to assist the Ifidophore in its superficial migration and in contracting its previously polymorphic cell body into its adult, spherical, shape by

'sweeping' it around obstructing collagen fibers.

Endocytosis. Some of the cytoplasmic projections described in the previous section were interpreted to have an endocytotic function.

These projections were routinely observed encircling extracellular, fila­ ments ( Figure . 53 ) . Actual images of endocytosis were not observed as frequently, as were the cytoplasmic projections. However, images of cell ' membrane activity (invaginations), interpreted to be endocytosis of extra-cellular filaments and vesicles containing these filaments, were observed (Figure 59).

■ Cytoplasmic Cdnaliculi. During stage 21 (exclusively) irido­ phores were observed which contained what appeared to be channels or a tubule network, which permeated the entire cytoplasm. Cells of this type were not frequently encountered and it was not possible to determine the three-dimensional structure of these ihtra-eeliular spaces (Figure

60). It is not known whether these cytoplasmic canaliculi are (as the term would imply) indentations or grooves which deeply penetrate the cytoplasm or a tubule network similar to what might be expected if the^ lumena of the endoplasmic reticulum of a cell were to become swollen. 117

It is believed that these intra-cellular spaces d o ■communicate with the

extra-cellular environment. Evidence of the openings, or pores, such a hypothesis would require are presently limited to isolated observations.

The origin of these spaces remains open to speculation.

Iridophores with'a highly developed rough'endoplasmic reticular

system were present in the same thin section which contained iridophores with cytoplasmic canaliculi. Flattened saccules were the typical form of endoplasmic reticulum of stage 21 iridophores (Figures 55, 5.6 and 61).

It is speculated that the cytoplasmic canaliculi described above may

.have given rise to the extensive endoplasmic reticulum observed in iri­ dophores of stages 21-23. The evidence for.this conversion is at best circumstantial. Of the iridophores studied thus far,'canaliculi and rough endoplasmic reticulum have been mutually exclusive within any given cell.

If frequency of observation, and the metamorphic stages in which a given structure is observed, are indications of the permanence (or transitional nature) of that structure, then logically, Canaliculi were the most likely cytoplasmic structures to have given rise to the flattened saccules of rough endoplasmic reticulum typical of irido­ phores of stages 21-23.

Distinguishable Cytoplasmic-Regions. The cytoplasm of maturing iridophores can be described as being divided into platelet-packed regions and relatively platelet-free regions. The platelet-free cyto- ■ plasm usually demonstrated a perinuclear distribution with 'peninsulas' which radiated out and penetrated between platelet-packed regions : 118

(Figure 62), Platelet-free cytoplasm Invariably contained mitochondria and other cytoplasmic organelles and inclusions,

It was inferred from the presence, within a single iridophore, of multiple platelet-packed regions, that several points within the cytoplasm had been initiation sites for platelet formation. This agrees with the random distribution of pre-reflecting platelets observed in type III iridoblasts (Figure 44) and multiple sites of platelet forma­ tion in the cytoplasm basal to the nucleus,

' 'Nohpigmentary Cytoplasm!c Organelles;.. - The presence of mitochbn- dria and rough endoplasmic reticulum in the platelet-free regions of maturing iridophores has already been mentioned and similar observations appear in the literature (Setoguti 1967),. With respect to mitochondria it might, be added that these structures showed a striking increase in number during stage 21 (Figures 54 and 56). A similar increase in the number of mitochondria was noted in A. dacnicolor iridophores subsequent to treatment with intermedin (Taylor 1967). The average size of mitochondria in this species was reported to be 1.9u and control cells contained 2-10 mitochondria per cell (Taylor 1967).

Rough endoplasmic reticulum displayed a pronounced spacial arrangement relative to forming reflecting platelets. Parallel arrays of rough endoplasmic tubules and forming reflecting platelets were ob­ served (Figure 55). The significance of this phenomenon is unclear.

Golgi apparatus was infrequently'observed in iridophores. 13.9 Cytoplasmic Inclusions. Observations at high 'magnification of the platelet-free regions of cytoplasm revealed diverse cytoplasmic inclusions in addition to those already described (i.e., mierofilaments and free ribosomes). A definite function could not be assigned to these structures.

.Multi~vesicular Bodies. In this study, a multi-vesicular body was defined as any vesicle which contained within its lumen two or more vesicles, or a combination of a vesicle (or vesicles) and granules•

Multi-vesicular bodies observed in maturing iridophores exhibited a variety of morphological types (Figures 63, 64? .65 and 66). The limits ing membrane ■ was smooth and varied in shape from spherical to irregular.

Fragmentary limiting membranes were also observed. The contents of these structures included vesicles with fine,granular contents, vesicles with coarse granular contents,, fine granules and coarse granules. Combi­ nations of all of the above structures were observed in the lumena of multi-vesicular bodies.

Transverse and oblique sections through rough endoplasmic tubules in the platelet-free region which adjoined developing clusters of re­ flecting platelets, 'revealed that the lumena of these tubules contained an unusual structure which technically fit the working definition of a multi-vesicular body (Figures 63 and 64). These intra-reticular multi- vesicular bodies appeared to be small (O.lSu diameter) spheres of moderate electron density which was surrounded by a limiting membrane.

Embedded within this matrix of moderate electron density was an array of smaller (475 A diameter) electron opaque spheres which were located just beneath the limiting, membrane and at the very center of the multi-

vesicular body. In transverse section these electron opaque spheres

appeared distributed in a pattern reminiscent of that of micro-tubules

viewed in a transverse section of a flagellum.

Granular Vesicles. Platelet-free cytoplasm of maturing irido- phores also contained vesicles which contained a granular material

(Figure 62). These granular vesicles often appeared electron opaque at low magnification (Figures 62 and 63). However., at higher magnification these apparently opaque vesicles were resolved into roughly spherical . vesicles (0.27u diameter) which Were packed with small (190 A diameter)

granules (Figure 67). Many of these granular vesicles contained hollow

or empty looking interiors. Granular vesicles were observed to have

empty cores of variable diameter and some of these vesicles exhibited

empty cores of.such large diameter that almost nothing of the granular

cortex was apparent (Figure 67).

.These granules were the third component of the unusual parallel

array of organelles described previously (Figure 55) in connection with reflecting platelet formation. It is speculated that these granules may

contribute to the 1 growth,' or parallel orientation of reflecting plate­

lets since they ...were predojitinately..at' the'interface'of platelet-packed •and platelet-free- regions of cytoplasm (Figure 62).. Granules similar to

those described were also observed in stage 23 iridophores (Figure 58).

Similar membrane-bound granules varying in sectional diameter

from 0.1-0.2um were observed in the 1guanophores1 of L. vivipara

(Breathnach and Poyntz 1966). These vesicles were reported to have a . 121 . uniformly granular internal structure, while others possessed an addi­ tional inner membrane enclosing a relatively more electron dense area.

Homogeneous membrane-bound granules were also described recently in the iridophores of axolotl (Dunson 1974). .

The variable diameter of the hollow core displayed by these granular vesicles may represent a 'filling up' or an 'emptying out' of these structures in vivo. Because they are electron transparent, as are the empty spaces of reflecting platelets, it is speculated that these : granules may also contain purine crystals. If this is the* case, then these vesicles may function as a means by which iridophores enlarge their reflecting platelets once they have been formed. The hypothesized crystalline core of these vesicles would be added to the crystalline core of. the developing reflecting platelet while their membrane would fuse with the membranes of the reflecting platelet. Given the hypo­ thetical existence of purine crystals within these vesicles, it might also be the case that these vesicles fuse with one another as a secondary source of new reflecting platelets.

It might also be speculated, based on the coincidental appearance of these granular vesicles with the structures in Figure 55, that these granules are in some way contributing to the parallel alignment of the structures shown in Figure 55.

Blast Cells of Indeterminant Type

During stages 18-20 an indeterminant blast cell type was ob-.... served (Figure 68).. These blast cells were intermingled with type III iridoblasts during stage 18. These blast cells differed from type III 122 irido"blasts in that their predominant cytoplasmic.constituent was a well-defined rough endoplasmic reticulum while the major cytoplasmic constituent of type III iridoblasts were smooth, fragmentary membranes which formed pre-reflecting platelets. Since hoth cell types had mitochondria, polymorphic cell bodies and the same type of minimal e nuclear 'blebbing,' the sole criterion for distinguishing between these two.cell types, at least those observed thus far, were their respective, characteristic cytoplasmic inclusions, i.e., rough endoplasmic reticulum in the indeterminant blast cells and smooth fragmentary membranes and pre-reflecting platelets in type III iridoblasts.

The Mimed Chromatophore Layer

The appearance of a mixed chromatophore layer during stage 21 was reported in the light microscopic section and confirmed during electron microscopic investigation (Figure 69). , This mixed chromato- phore layer consisted of intermingled melanophores .and; iridophores": which appeared to be in a period of continuing maturation. Iridophores during stage- 21, when viewed in transverse section, exhibited a fairly equal distribution of cytoplasmic volume between regions that were platelet-packed'and regions that were platelet-free. Melanophores during stage 21 appeared to be divided into two distinct populations, cells which contained large numbers of melanosomes (Figure 69) and cells which contained only occasional melanosomes but extensive amounts of rough endoplasmic reticulum (Figure 70). Melanophores of this latter type were interpreted to be less mature than those which contained large numbers of melanosomes. It should be emphasized that the . ; 123 melanosomes mentioned in this section do not constitute the final,.adult

melanosome which has been described for this species (Bagnara, Taylor

and Prota 1973; Bagnara and Ferris 1974, 1975) and appears after meta­

morphosis has been completed,

While melanophore development was not - the primary goal of this

study, incidental observations, coupled with the necessity to distin­

guish respective chromatoblasts from the mixed chromatoblast layer found

in stages prior to stage 21, necessitated at least a cursory study of the

ultrastruotural details of melanoblast development..

' Mature irldophores ■

Unlike immature irldophores, mature irldophores were firmly sur­

rounded by other dermal chromatophores, i.e., empty intercellular spaces

were not observed. Superficially, mature iridophores always abutted the

overlying xanthophores, with the exception of melanophore processes

which were routinely observed between the.iridophore and its overlying

xanthophore (Figure 70). Mature (stage 25) iridophores were typically

spherical cells (l4.0u in diameter) and evidenced few, if any, of the

cytoplasmic 'cilia', and other cell membrane activity ascribed to less

mature stages. The nucleus of stage 25 iridophores was usually basal

and when viewed in transverse section, appeared to be encircled by a

narrow margin of platelet-free cytoplasm. /

Except for a narrow margin of platelet-free cytoplasm just be­

neath the cell membrane, all of the cytoplasm distal to the nucleus was

completely packed with platelets. These platelets were not uniform in

general size and shape. Generally, reflecting platelets which were relatively superficial had a 1 swollenf appearance and appeared to be

.. several times the diameter of the more basally loeated refleeting

platelets. .This tendency was routinely observed within iridophores and

. for this reason is presumed not to be artifactual.' It was also noted

that reflecting platelets, especially the larger ones, were often ori­

ented perpendicular to the horizontal axis of the skin.

Melanophore Development

Cells which were interpreted to be early dermal melanophores

were observed in stage 21 skin (Figures 71, 72 and 73). (Figures 71-73

show ultrastruetural aspects of melanoblast differentiation and are there­

fore grouped together.) These cells were identified as early dermal

melanophores because they contained only a few electron opaque struc­

tures which were interpreted to be the smaller melanosomes character­

istic of dermal melanophores. These dermal melanophores were interpreted

to be in a developmental stage analogous to type III iridoblasts (or

• the earliest iridophores observed) because they contained only a few

identifiable pigmentary organelles and numerous Vesicular structures

which were suggestive of presumptive melanosomes. .

These pre-melanosomes were bounded by a double membrane (Figures

.72 and 73). They differed from mitochondria because their internal

structure usually appeared amorphous, granular, or filamentous but did

not contain cristae. The speculation that these structures represented

pre-melanosomes was supported by observations that these structures

were (1) roughly the same diameter as the electron-opaque dermal

melanosomes, (2) they were associated with the rough endoplasmic 125

i y<

Figure 71. Melanosome development. — High magnification view of stage 21 cell thought to be a dermal melanophore because it con­ tained electron opaque bodies thought to be dermal melano- somes (M). On the right side of this micrograph is a granular vesicle (GVn ) of moderate electron density with a localized region of high electron density. To the immediate left of GV]_ is a granular vesicle of high electron density (GV2 ) which was interpreted to be an intermediate form be­ tween GVj and the dermal melanosome (M) on the far left. Also shown are the nucleus (N) and a well-developed endo­ plasmic reticulum (ER). 116,620 X. 126

Figure 72. Stage 21 melanophore. — The well-developed rough endoplasmic reticulum (ER) was characteristic of this cell type as were isolated melanosomes (M) and numerous pre-melanosomes (pM). In addition to these structures smooth membraned tubules (T) were also present. 58,310 X. 127

Figure 73. Melanin deposition. — This micrograph is another portion of the same cell shown in Figure 72. In the upper right hand corner is a pre-melanosome (pM) with variable electron density suggestive of melanin deposition. 58,310 X. ,128

reticulum In the same manner as dermal melanosomes, (3) some of these

pre-melanosomes had localized Internal regions of high electron density

which were suggestive of melanin deposition (Figures 72 and 73).

Early dermal melanophores shared several characteristics with

other differentiating chromatophores, e.g., they were located in the.

same dermal stratum, they evidenced minimal nuclear 'blebbing,1 riho- .

somes were associated with the cytoplasmic surface of the outer nuclear membrane, only a relatively small number of randomly distributed identi­

fiable pigmentary organelles were apparent.

' In addition to pre-melanosomes, early melahophores contained

delicate, barely discemable smooth tubuleS (Figure 73) which were in

close proximity to the well-developed rough endoplasmic reticulum. It

is speculated that these tubules may be ribosome-free diverticulae. of

' the rough SR. and that they may contribute to melanosome differentia­

tion.

These observations on melanophore development are, at best,

cursory and require further study. However, a working hypothesis for'

detailed study has been suggested by observations made thus-far. It is

hypothesized that the double membrahed vesicles observed are part of the I sequence of ultrastruetural events which culminate in melanosome forma­

tion. Those double membraned vesicles with amorphous or filamentous

contents may represent the first stage (observed thus far) of pre-

melanosome differentiation. The second stage of differentiation may be

represented by vesicles with granular contents. . The third stage may be

represented by vesicles with granular contents which contain localized 129 regions of increased electron density. This increased electron density may he indicative of melanin deposition and would ultimately result in

electron opaque vesicles, i.e.> melSnosomes (Figure 71). 'DISCUSSION

The major events which constitute chromatoblast differentiation

are biochemical processes which result in changes in protein synthesis

and accessory metabolism (Wilde 1961). Concomitant with these changes

• in synthetic pathways are ultrastructural changes which result in the

formation of pigmentary organelles. =In the previous sections the de­

velopment of specific pigmentary organelles within their respective

■ ehromatophores was described. In reviewing this data patterns of

. differentiation emerged which were common to all three ehromatophores.

Similarities in Chromatoblast Differentiation

Nuclear•Blabbing

Nuclear 'blabbing' was observed in xanthoblasts, iridoblasts and

melanophores. It is suggested that this phenomenon is a prerequisite

for chromatoblast differentiation in A. dacnicolor. While the mechanism

Of nuclear 'blabbing' remains unclear, its morphological consequences

are obvious and prompt speculation about its function. At least three

morphological entities make.their initial appearance, or increase dra­

matically, during periods of nuclear 'blabbing': (1) ribosome-like

particles, (2) cytomembranes, in the form of vesicles, and (3) fila­

mentous material.which these vesicles contain.

130 131

As all aside, it should be noted that this list is not considered

to be exhaustive of the nucleus-derived products which are produced at

the time of 1 blobbing.' It was apparent from observations made of the

filamentous contents of both rough and smooth-membraned vesicles that

•individual filaments were easily discernable from one another, One

could speculate from this observation that these fine filaments were

supported in an electron transparent, perhaps aqueous, matrix which did

not stain with the heavy metals used.

It is.suggested that the association of these three entities in

' ' ' morphologically identical ribosome-associated vesicles in all three

chromatophores is significant. Significance is also given to the simi­

larities in origin and morphology between the ribosome-associated

vesicles and rough endoplasmic reticulum.

RibbSOme-aSSOciated Vesicles

If one considers the organizational principles inherent in the

ultrastructure of rough vesicles and endoplasmic reticulum, 'routine'

. cellular details begin to become .more significant. Simplistically,

■ ■ i ribosome-like granules are associated with one side.(the cytoplasmic

side) of a cytomembrane while on the opposite side of this cytomembrane

an electron dense, usually filamentous, material was invariably found.

Furthermore, the space which contained this filamentous material was

1 outside' the intracellular milieu. This principle was observed, without

exception, in numerous instances, i.e., the perinuclear space which re­

sulted from the 'blebbing' of the outer membrane of the nuclear envelope,

the ribosome-associated vesicles, endoplasmic reticulum and even the 132 extra-cellular filamentous material characteristic of iridophores of stages 21 and 22. On the cytoplasmic side of the plasma membrane (which separated this filamentous material from the intracellular milieu) were large numbers of free rlbospmes, some.of which may have become tempos - rarily 'associated' with the cytoplasmic surface of the plasma membrane. •

Based on these observations, it is suggested that these rough vesicles share the synthetic capabilities usually attributed to the endo­ plasmic reticulum of fully differentiated cells. ' In chromatoblasts these rough vesicles would function as a transitional (chromatoblast) form of endoplasmic reticulum which would permit the translation of whichever part of the chromatoblast genome a particular blast cell had been 'determined' to express. It is further suggested that the mechanism by which the appropriate differential protein Synthesis is accomplished may include (1) the type or types of ribosomes associated with the cyto­ plasmic surface of the rough vesicle, the sequence of the ribosome-like structures (assuming a biochemically heterogeneous population of ribo­ somes), the nature of the cytomembrane itself, available substrates, etc. This speculation is supported by the suggestion of several authors

(for citations see Poletti and Castellano 1967), that the nuclear mem—

.brane may play a formative role in the origin of the endoplasmic reticu­ lum of other eukaryote cell types.

.. , The Primordial Vesicle •.

In a recent review of the occurrence of chromatophores with multiple types of pigmentary organelles the suggestion was made.(Bagnara

1972, p. 178 ) that there may exist a.'."primordial organelle that is 133 competent to form any of the three basic pigment containing organelles and- that, depending upon the specific developmental cues it receives*, it differentiates into melanosomes, pterinosomes, or reflecting platelets."

Such a primordial organelle would* by definition, be a'relatively un- . differentiated structure with, synthetic capabilities. It is suggested v that these ribosome associated vesicles are a likely candidate for the primordial vesicle hypothesized above.■ It is not being.suggested that \ the ribosome-associated vesicles differentiate directly into pigmentary organelles. The results of this investigation would indicate just the contrary to be true; rough vesicles apparently contribute their constitu­ ents to the formation of pigmentary organelles. While these constituents

(i:..e. .ribosomes, cytomembranes and filamentous material) may appear identical* biochemically they may have already differentiated into the precursors appropriate for respective pigmentary organelles.

The developmental cues which theoretically initiate differentia­ tion undoubtedly cause biochemical modifications within the toti­ potent ial, completely undifferentiated chromatoblast. It may be the case that these modifications are restricted ultrastructurally to the rough vesicles, or they may'be incorporated into other structures, e.g.* the golgi^derived vesicles responsible for melanosome formation in gold­ fish. (Turner et al. 1975), or some amorphous constitutent of the cytoplasm.

Indeterminant Blast Cells

It is, speculated that the indeterminant blast cells described previously may represent melanoblasts since the cytoplasm of these ■ ■ 134 ■

Indeterptinant blast cells and cells identified as early melanophores were both characterized by pronounced endo-reticular systems „ However, the differentiation of melanoblasts was not intended to be the major thrust of the present study and the cursory observations obtained thus far are subject to verification by a detailed ultrastructural investi­ gation. ' '

■ Evidence of a;Stage-specific Increase in . ~ 4'HQ^one X evels

Several pieces of.eyidence suggest that there is an increase in homone levels during stage 21. One. of the developmental effects attributed to hormones is an increase in mitotic activity of existing melanophores (Bagnara and Hadley 1972, p. 70). A single mitotic figure was observed in the present study in a stage 21 melanophore.

Stage 21 skin generally showed more variation with respect to • the degree of differentiation of all three chrpmatoblasts. While excep­ tions were noted it can be stated as a general, 'rule of thumb' that dorsal skin of animals of stages. 18-20 contained more dermal chromatoblasts than dermal chromatophores. During stage 21 the majority of the dermal pig­ ment cells differentiated sufficiently to be properly termed chromato­ phores. .(.It. is suggested that the distinction between 1 chromatoblast' and 1 chromatophore' be defined by the absence or presence of at least a single developing pigmentary organelle.)

Definition'of Terms / -

XanthoblastS' should continue to be termed xanthoblasts until they are observed to contain at least a single, multi-lamellar ■ 135

pterlnosome. Irldoblasts should continue to he termed 'hlasts’ until

' they are observed to contain at least one reflecting platelet with a i characteristic empty space indicating the presence in vivo of crystal­

line purine.• 'Melanophore' should he used to designate a cell with at

least one electron-opaque melanosome

An increase in the number of mitochondria has been described as

an effect of intermedin injection of A. dacnicolor. Another change in

. iridophores attributed to experimentally increased hormonal levels was

the appearance of a 'halo' of amorphous material within reflecting

platelets (Taylor 1967). Both of these phenomena were observed during ' •

the present investigation in control animals of stage 21. Because each

of the events listed above have been attributed to increased hormonal

levels it is suggested that during stage 21 intact A. dacnicolor have

increased levels of circulating hormones.

Dermal Melanophore Proliferation During Stage. 21 -

One of. the most striking changes during stage 21 was the rapid

increase in the number of dermal melanophores in the mixed chromatophore'

layer. This increase may have been caused by the deposition of melanin

within melanoblasts which had already migrated into the mixed chromato- '

blast (iridoblast-indeterminaht blast) layer prior to stage 21. Both

iridobiasis and indeterminant blast cells (which in all probability were

melanoblasts) were observed in this dermal stratum during stages 18-20.

Thus the dramatic increase of melanophores noted during stage 21 may

have resulted from the deposition of melanin within melanoblasts which ' 136

had already established themselves in the same layer as iridoblasts.

Another possibility is that at least some part of the dermal melano- .

phores observed in the mixed chromatophore layer of stage 21 were fully

differentiated dermal melanophores which had formerly been situated in

the deeper dermal layer and migrated to dorsally during stage 21 in re-

■sponse to increased hormonal levels. ■ \ • SUMMARY

.Light Microscopy

Ohservatioh of stage 9 dorsal skin indicated that xanthohlasts were already established in the superficial collagen layer of stage 9 A. dacnicolor tadpoles.

The basement membrane was apparent in some, but not all, sec­ tions of stage 9 dorsal skin observed. It was therefore con­ cluded that the establishment of the basement membrane was not a prerequisite to the establishment of a xanthoblast layer in the superficial collagen of stage 9 dermis.

Skin gland•primordia were first observed in stage 14 dorsal skin.

Iridophores were first identified in stage 16 skin but were not found with sufficient frequency to be considered representative . of any metamorphic stage prior to stage 18.

Granular, epi-integumental material, which was thought to be the secretory product of the skin glands, was first observed during stage 20.

This epi-integumental material formed a compact layer in all dorsal skin observed of stages 21-23 and 25.

Some of the melanophores observed in the dermis proved to contain large, epidermal type melanosomes, when viewed with the electron microscope. 138

A mixed chromatophore layer, consisting of iridophores and :

melanophores, was observed in' stage 21, 22 and 23.

The dermal chromatophore unit was observed in stage 25+ skin

but not in stage 23 skin. • It was concluded that the dermal

chromatophore unit developed during stage 24 or 25. .

Electron Microscopy /

Nuclear 'blabbing' was observed in xanthoblasts, iridoblasta

and melanophores.

Ribosome associated vesicles were observed in all three types of

chromatoblasts. These vesicles were thought to have been de­

rived from 'blebbed' portions of the outer nuclear membrane.

Smooth membraned vesicles were observed, in both xanthoblasts and

iridoblasts. These smooth vesicles were thought to have been

formed by the dissociation of ribosomes from the rough vesicles

described above.

In xanthoblasts, these smooth vesicles developed into pterlno-

somes. Three types of pterinosomes, representing a develop­

mental sequence, were observed in xanthophores.

In iridoblasts, these smooth vesicles became pre-reflecting

.platelets. These pre-reflecting platelets were elongate struc­

tures with a fragmentary limiting membrane and a central,

longitudinal fiber along which purine crystallisation was

initiated. 139 6. Elongate structures with fragmentary limiting membranes and a

central fiber which evidenced purine crystallization were termed

reflecting platelets..

7. Subsequent to their formation, reflecting platelets 'grew'

' longitudinally; possibly by end-to-end fusion with one another

and/or by fusing with granular vesicles.

8. During the course of differentiation of iridoblasts three mor­

phologically distinct stages, representing a developmental

sequence, of iridoblasts were identified.

9. Ultrastructural criteria for distinguishing between the three

types of chromatoblasts observed, and their respective chromato-

phores, were established.

" - : • , . «• . 10. A possible sequence of ultrastructural events was suggested for

the differentiation of melanoblasts into melanophores.

11. Ultrastructural evidence was presented to support the hypothe­

sis that an increase in circulating hormonal levels•occurred

during stage 21. REFERENCES CITED

Arnold, J. M. 1967. Organogenesis of the cephalopod iridophore: cyto- membranes in development. J. Ultrastruct. Res. 20:410-421.

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Bagnara, J. T. 1961. Chromatotropic hormone, pteridines, and amphibian pigmentation. Gen. Comp. Endocrinol. l(2):124-133.' '

Bagnara, J. T. 1966. Cytology and cytophysiology of non-melanophore pigment cells. In Bourne and Danielli.(eds.) International Review of Cytology. New York: Academic Press, pp. 173^205.

• Bagnara, J. T, 1969. Responses ofpigment cells of amphibians to intermedin. In La specificite.' zoologique de hormone hypophysaires et de leurs activites, Collogues .Intemationaux du Centre National de la Recherche Scientiflque, No. 177,, pp.

Bagnara, J. T. 1972. Interrelationships of melanophores, iridophores, and xanthophores. In V. Riley (ed.) Pigmentation: its genesis and control. New York: Appleton-Century-Crofts, pp. 171-180.

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