- Email: [email protected]
Studies on amphibian yolk
J. ULTRASTRUCTURE RESEARCH
9, 225-247 (1963)
Studies on A m p h i b i a n
225
Yolk
5. Electron Microscopic Observations on the Utilization of Yolk Platelets during Embryogenesis SHUICHI KARASAKI
Biology Division, Oak Ridge National Laboratory, z Oak Ridge, Tennessee Received February 8, 1963 The closely related phenomena of yolk platelet utilization and the progressive differentiation of cells in the developing amphibian embryos have been studied with the electron microscope. The first change in the yolk platelets is a decrease in thickness and a subsequent disappearance of the superficial layer in cells undergoing primary differentiation. At later stages, the surface of the crystalline structure begins to decompose into separate units. Further disintegration of the platelet is coupled with the formation of laminar or vesicular membranes, fine particles, and fibrils. The ultrastructure of the membranous components is very similar to that of cytomembranes. It is not improbable that these membranes represent an intermediate step in the formation of cytomembranes at the expense of yolk platelet material. During the decomposition of yolk platelets, mitochondria, pigment granules, and lipoid bodies are found in a close association with the yolk platelets. In Rana species (as opposed to other amphibian species), crystalline structures typical of yolk platelets are frequently located within the mitochondria throughout embryonic development. The significance of this observation is discussed. It is well established that the yolky material in an amphibian egg constitutes a reservoir which is utilized during some phase of ontogeny as a source of energy, of building materials or of both, but at just what phase of development and for what processes they are utilized have received only slight attention (however, see 5, 15). Previously it has not been possible with light microscopy to describe in any detail the morphological changes in the utilization process. Indeed, when the yolk platelets contained within embryonic cells are examined in this manner they seem to remain in much the same condition until their disappearance at the larval stage (5). With the electron microscope, however, it has been possible to pursue some of the morphological changes in fine structure which yolk platelets undergo during the progressive differentiation of the embryonic cell. The yolk platelets of young embryos z Operated by Union Carbide Corporatien for the United States Atomic Energy Commission.
226
s. KARASAKI
and ripe eggs of all amphibian species contain a superficial layer of granular or fibrillar materials, a central main body displaying a crystalline lattice structure, and an enclosing membrane of approximately 70 • thickness (16, 19). The first visible changes in the platelet are observed in the superficial layer, which decreases in thickness and finally disappears completely during the early stages of differentiation (16). After the disappearance of the superficial layer, the main body begins to decompose and to form concentric laminae in Triturus (16), suggesting the possibility that cytoplasmic membranous structures may be directly derived from the yolk platelets. Lanzavecchia and Le Coultre (24), studying similar laminar structures formed from yolk platelets in Rana embryos, believe that it is possible to follow step by step the transformation of yolk platelets into mitochondria. The time of alleged transformation of yolk platelets coincides approximately with the stage during which the mitochondria increase in number and complexity and the endoplasmic reticulum system becomes more elaborate. Subsequently the embryonic ceils gain the specialized organelles which characterize the differentiated cells. Since it is likely that the yolk platelet is intimately involved in the genesis and elaboration of cytoplasmic organelles, it is important to study the yolk platelet with regard to the embryonic region in which its utilization first begins and the extent of its relationship with the previously mentioned membranous structures.
MATERIALS AND METHODS The embryos used were mainly those of Triturus pyrrhogaster and Rana pipiens, from early cleavage to hatching stage. Developmental stages were numbered according to Okada and Ichikawa (28) for T. pyrrhogaster and according to Shumway (34) for R. pipiens. A number of observations were also made on selected stages of Diemictylus viridescens, Bufo vulgaris, Rana nigromaculata, and Microhyla carolinensis. Various embryonic tissues of known prospective significance were isolated from the
Figs. 1-16 are electron micrographs of osmium-fixed material embedded in Epon, Figs. 1, 2, 6, and 11 are from sections stained with lead acetate; the rest are from sections stained with uranyl acetate. FIG. 1. Part of a presumptive ectoderm cell in the blastula embryo (stage 9) of Rana pipiens. Each yolk platelet consists of a superficial layer (S) and a central main body (B), both enclosed within a single membrane (Y). Lipoid bodies (L), mitochondria (M), vesicles (V), and RNP-particles (R) can be identified, x 15,000. FIG. 2. Part of an epidermal cell at the tail-bud stage (stage 18) of R. pipiens. A yolk platelet (B)oin which the superficial layer has disappeared shows a moir6 pattern with wide spacing (average 570 A). Four mitochondria (M) have elaborate cristae and contain small yolk platelets. The endoplasmic recticulum is partly smooth surfaced and partly rough surfaced at E. Free RNP-particles (R) are abundant. Lipoid bodies (L) are less dense than those found in earlier embryos. Pigment granules (P) can be identified. × 20,000.
228
S. KARASAKI
developing embryos at each successive stage. For a quantitative analysis of yolk platelet utilization, the frequency of types of the yolk platelet configurations was examined in electron micrographs of the developing ventral ectoderm. Care was taken to have samples of the cell population belonging to the same cell lineage arranged in a sequence of developmental stages. In particular, the samples of the developing ventral ectoderm from R. pipiens were taken from the same egg batch (except those of stage 18). Details of the preparation for electron microscopy have been previously described (19). Isolated pieces of embryonic tissues were fixed for 30-60 minutes with 2% OsO4 in balanced saline buffered with Veronal acetate at pH 7.4. Following dehydration in an ethanol series, specimens were embedded in Epon 812. Sections were cut with a diamond knife on a Porter-Blum microtome, stained with uranyl or lead acetate, and examined with an RCA EMU 3E electron microscope.
OBSERVATIONS
Basic structure of the yolk platelet As described previously (19), the yolk platelets of young amphibian embryos are composed of three basic parts: an internal main body with a crystalline structure, a superficial layer with granular or fibrillar materials, and a limiting membrane of approximately 70 A thickness (Fig. 1). These three basic components are present in the yolk platelets of the mature egg and are usually retained until the blastula stage or later, depending upon the embryonic region and the species.
Disappearance of the superficial layer The first visible change in the yolk platelet begins in the superficial layer. In the developing ectoderm cell, some of the small yolk platelets measuring 1-2/z in diameter start to lose their superficial layer at the blastula stage (stage 9) in R. pipiens (Chart l) and at the gastrula stage (stage 11) in T. pyrrhogaster (Chart 2). The superficial layer in the larger platelets progressively decreases in thickness during development and subsequently disappears almost completely in the ectodermal region at the tail-bud stage [stage 18 in R. pipiens (Fig. 2 and Chart 1) and stage 27 in T. pyrrhogaster (Chart 2)]. The time of complete disappearance varies with the embryonic region. It occurs first in the dorsal mesoderm, at the late gastrula stage (stage 12) in R. pipiens, and at the neurula stage (stage 17) in T. pyrrhogaster, and then in the ectoderm. The platelets of the ventral endodermal region, however, remain unchanged even at the tail-bud stage in both species (stage 18 in R. pipiens and stage 27 in T. pyrrhogaster).
UTILIZATION OF YOLK PLATELETS
229
100 9O 8070.
o-- 0
60. ¢r-
50 2 40-
502010 12
21 26 54 42
62
( ~ 0 0 Q Q 8 9 10 11 12 428 425 520 492 741
~ 14 550
82 (~ 17 420
96 AGE IN ~.~.-....HOURS " ° r ~ . ~ AT '8°C 18 STAGENO. 722 TOTALNO. OF PLATELETS
CHART 1.
lOO 9O
o
8o 7o
~
w
60
o 504030" 2010•
,e
2
4
O© 10 410
11 656
............ --'' 7
® 17 702
10
~ 24 450
15 AGE IN Df~FScAT 27 STAGE NO. 701 TOTAL NO. OF PLATELETS
CHART 2.
CHARTS I and 2. The frequency of yolk platelet configurations in the developing ventral ectoderm of Rana pipiens (Chart 1) and Triturus pyrrhogaster (Chart 2). The frequency of yolk platelets with the following configurations are shown: those with a superficial layer ( - o -), those associated with laminar or vesicular membranes (-- - • -- -), and those enclosed within mitochondria ( - - ± - -). The frequency of these various configurations is expressed as a percentage of the total n u m b e r of yolk platelets (given at b o t t o m of the figure) examined in numerous electron micrographs of each stage.
230
s. KARASAKI TABLE I
FREQUENCY OF YOLK PLATELET CONFIGURATIONS IN THE PROSPECTIVE ECTODERM OF THE EARLY GASTRULAa Percentage of total number of platelets
Species
Number of Platelets Examined
within Superficial Layer
within Laminar Membranes
636 520 734
79 % 63 % 35 %
0% 4% 20 %
Triturus pyrrhogaster Rana pipiens Mierohyla carolinensis
within Mitochondria
0% 3% 0%
a The stage of these three species is at the time of the first indication of the dersal lip.
A difference can also be observed in the rate at which various amphibian species lose the superficial layer of their yolk platelets (Table I). In earlier stages, parts of the cytoplasm are engulfed by deep indentations of the nuclear surface. In this cytoplasmic region, yolk platelets are frequently observed without their superficial layer.
Decomposition of the main body Most electron micrographs show separate fine particles, about 40 to 60/~ in diameter and extremely electron dense, at the periphery of the crystalline main body after the disappearance of the superficial layer (Figs. 3 and 5). Frequently these particles are very numerous, forming a dense layer without ordered structure (Fig. 4). Very near to the crystalline lattice, they seem to show a regular alignment giving a gradual transition from the crystalline to the disordered pattern (Fig. 4). Some main bodies of larger platelets disintegrate into small fragments which are subdivided by a transverse fissure (Fig. 12). Irregularly shaped bodies with deep indentations are also observed (Fig. 13). Although the peripheral decomposition or fragmentation in the main body was sometimes quite striking, most main bodies retained a clear indication of their crystalline pattern (Figs. 4, 5, and 6). Periodic
Fro. 3. Presumptive neural ceils in Triturus neurula (stage 17). A large yolk platelet lacking the superficial layer but still retaining the limiting membrane (Y) intact around the main body (B) is present. Two small platelets, at close proximity to the protruded nuclear membrane (N), show decomposition of the main body coupled with the formation of laminated membranes. The laminated body at the center of the figure (arrow) may have been derived at the expense of the main body. × 25,000. FIG. 4. Part of a yolk platelet associated with laminated membranes in the neuroblast of a Triturus embryo (stage 18). The main body (B) displays a parallel fringe system of 68 ~ spacing. Peripherally (arrow) the regular band pattern changes into a region without ordered structure which seems to be composed of dense particles (40-60/~ in diameter). × 150,000.
UTILIZATION OF YOLK PLATELETS
231
232
s. KARASAKI
patterns with wide spacing (from 200 to 1000 A) were sometimes observed in the main body during the decomposing process (Fig. 2). This is referred to as the moir6 pattern, indicating some degree of shifting within the crystalline lattice.
Types of yolk platelet configuration during decomposition In most cases, disintegration of the main body was coupled with formation of various configurations as follows: (1) Laminar type (Figs. 3-6, 10, and 16); (2) Vesicular type (Figs. 7, 8, and 16); (3) Granular type (Figs. 8 and 16); and (4) Fibrillar type (Fig. 9). (1) Laminar type. This is the most frequently observed configuration. As seen in Figs. 3-6, laminated membranes are stacked around the main body. These are parallel sheets between two and twenty in number. Though they are in general arranged concentrically in the form of partial or complete rings, their outlines are frequently irregular. The thickness of a single sheet is 50-100 A, with an average 70/~i. The least distance between two adjacent sheets is 50 A. At the periphery of the decomposed main body, the fine particles are aligned suggesting a gradual transition into membranes. Some membranes show a triple-layered structure which corresponds to Robertson's (30) "unit membrane" (Fig. 6). These lamellae either terminate with a free edge or form flattened cisternae. Laminar components appear very similar to cytomembranes in general (Fig. 5). A fine laminar appearance is at first restricted to the periphery, and later the whole main body frequently seems to give rise to myelintype figures, in which a concentric arrangement of membranes is often apparent (Fig. 10). In T. pyrrhogaster and M. carolinensis, concentric lamellae are frequently observed around the main body of platelets larger than 3 #. In R. pipiens, on the other hand, stacks of lamellae are located in restricted areas near the surface of the larger main bodies (Fig. 6). In the developing ectoderm cells the concentric lamellae are first observed in the smaller yolk platelets (1-2 #) at the blastula stage (stage 9) in R. pipiens (Chart l) and at the neurula stage (stage 15) in T. pyrrhogaster (Chart 2)i The laminar type configuration appears in the large yolk platelets and increases in frequency during succeeding stages, especially at the tail-bud stage. Differences among species can be observed in the rates or stages at which laminar membranes appear around the main body (Table I, Charts 1 and 2). (2) Vesicular type. This type occurs usually in platelets larger than 3 # at stages later than tail-bud stage. It consists of many small vesicles, varying between 20 and 200 F~a. 5. Laminar configuration surrounding the main body (B) of a yolk platelet in a Triturusembryonic explant. The opaque particles (40-60 A) indicated by arrows have the same dimensions as the particles hexagonallyarranged in the main body. The band spacing is 70 ]k in the crystalline pattern of the main body. x 100,000,
UTILIZATION OF YOLK PLATELETS
233
234
S. KARASAKI
FI~. 6. Part of a yolk platelet associated with laminar type configurations in the epidermal cell of a R. pipiens embryo (stage 18). Layered membranes, measuring about 70 ~ in thickness, are arranged concentrically. Some membranes (arrow) show a unit membrane. These lamellae either terminate with a free edge or form cisternae. A remnant of the main body (B) shows a fringe pattern with a spacing of 73 ~. x 80,000.
m/z in size (Figs. 7 and 8). The vesicular bodies are found either in the matrix of granular type components or lying against the laminar type components. Most of the vesicles appear to have a clear homogeneous interior; others contain various structures, such as granules and granular or cloudy masses. (3) Granular type. A granular substance, opaque and with large granules (100-200 A), can sometimes be found in the area surrounding the main body. The granules, usually accompanied by laminar or vesicular membranes (Figs. 8 and 16), are difficult to distinguish in size and shape from the RNP-particles which are found free in the cytoplasm. This type is observed in all embryonic regions at the tail-bud stage. (4) Fibrillar type. The close attachment of tufts of faint, fibrillar material to the granular matrix on the surface of the platelet is observed during the later stages of platelet decomposition in the mesodermal cells of R. pipiens (stage 18) (Fig. 9) and T. pyrrhogaster (stage 27). Under high magnification these tufts consist of oriented
UTILIZATION
OF Y O L K P L A T E L E T S
235
FIG. 7. Part of a neuroblast in the brain of a R. pipiens embryo (stage 18). A vesicular and granular matrix surrounds main bodies (B) of the yolk platelet. A pigment granule (P) is embedded in the matrix region. × 8000. FI~. 8. High magnification of the area indicated by an asterisk (*) in Fig. 7. The size of the vesicles varies from 0.05 to 0.5/~. The dimension of the particles in this region are similar (100-200 A) to those of the RNP particles on the outer surface of the nuclear membrane (arrow). x 55,000.
arrays of fine, straight, often discontinuous fibrils in nearly parallel order, each of which measures in the range of 40 to 100 • in width. These materials are closely similar to myofibrils, which are f o u n d in the myoblast at later stages.
Limiting membrane of the decomposing yolk platelets Following the disappearance of the superficial layer, the limiting m e m b r a n e of yolk platelets seems to disintegrate or f o r m a series of small vesicles. Such decomposition is the more p r o n o u n c e d the later the developmental stages, although some yolk platelets retain their limiting membranes intact even after the disappearance of the superficial layer (Fig. 3). Occasionally within the intact investing membranes, the disintegrating main b o d y with its derivatives is observed (Figs. 3, 6, 8, and 16). Sometimes pigment granules are surrounded by such membranes (Figs. 15 and 16). 16 - 631831 J . Ultrastrueture Research
236
S. KARASAKI
FIG. 9. Fibrillar-type matrix at the surface of a decomposing platelet in a myoblast of a R, pipiens embryo (stage 18). The decomposed yolk material seems to be continuous with the tuft of fine filaments, extending out into the cytoplasmic area (arrow). In the same cell, tufts or individual filaments are scattered throughout the cytoplasm. × 50,000. FIG. 10. Several profiles of laminated bodies in a rnyoblast of a R. pipiens embryo (stage 18). These bodies seem to arise from the small yolk platelets. × 30,000.
Relation to mitochondria
T h r o u g h o u t the embryonic development of R. pipiens, small yolk platelets measuring 0.5 to 1 # in size, are found within mitochondria (Fig. 2 and Chart 1). Profiles of these yolk platelets are usually rectangular rods of varying length and width. Only rarely does one see the hexagonal shape that results f r o m sectioning n o r m a l to the flat face of a hexagonal plate (Figs. 2 and 11). At high magnification, hexagonal bodies display a hexagonal net pattern with a spacing of about 70 A. On the other hand, rectangular bodies show a parallel band pattern or no distinct pattern. Studies of m a n y sections of these configurations suggest that the main bodies are located between the two layers of a single cristae of mito-
UTILIZATION OF YOLK PLATELETS
237
chondria as mentioned by Ward (44) in the oocyte. None of these crystals have been seen within the mitochondrial matrix. This type of configuration was found in all embryonic regions during all embryonic stages of R. pipiens and R. nigromaculata. However it was never found in any region from the embryos of T. pyrrhogaster, D. viridescens, B. vulgaris, and M. earolinensis. During the differentiation of the ventral ectoderm of R. pipiens, the frequency of this configuration appeared to increase from about 1% of the total number of yolk platelets at the mid-cleavage stage to about 5-8 % during late gastrula through tail-bud stage (Chart 2). In the earlier stages, mitochondria containing the crystalline inclusions are spherical in shape and reveal only a few cristae arranged irregularly, while in the later stages mitochondria with numerous cristae and a dense matrix contain the crystalline structures. Throughout all stages, mitochondria increase in number and size and develop simultaneously a more complex internal structure. Thus, most of the mitochondria containing the crystals are very similar in structure to those without crystals but present in the same cells (Fig. 11). In R. pipiens a number of mitochondria lie in close proximity to those yolk platelets which are in a state of extensive decomposition (Fig. 12). In some cases, mitochondria are embedded in the granular matrix of the main body. In T. pyrrhogaster, the association is not so evident.
Relation to pigment granules A definite structural difference is observed between embryonic pigment granules of urodeles (T. pyrrhogaster, D. virideseens) and those of anurans (R. pipiens, R. nigromaculata, B. vulgaris, M. carolinensis). In the urodeles the spherical granules, measuring 0.4 to 0.6 #, are composed of a number of subunits measuring about 500 A which appear extremely dark. In the anurans the granules are single spherical bodies of about 0.4 to 0.6/z, in which subunits are not apparent. In both types of embryonic pigment granules a fine investing membrane is present. The embryonic pigment granules are abundantly present in the ectodermal cells, being preferentially distributed in the cortical layer and perinuclear zone of these cells during the earlier embryonic stages. During yolk decomposition in later stages, embryonic pigment granules can sometimes be found attached to the surface of decomposing platelets (Figs. 7 and 15). In Fig. 15, a pigment granule is included together with the main body of the platelet, within a single membrane. Sometimes they are embedded in the membranous complex or granular matrix of the decomposing yolk platelets (Figs. 7 and 16). The pigment granules within these portions seem to be partially disorganized. During development, embryonic pigment granules apparently diminish in number and cannot be identified in any tissues of adult amphibia.
FIG. 11. High magnification of a part of cytoplasm indicated in the lower left (*) of Fig. 2. Yolk platelet main bodies are located in the interspaces of mitochondria. The hexagonal body displays a crystalline hexagonal net pattern with spacings of 70 ~ . The other two bodies do not show distinct patterns, x 70,000.
FIG. 12. Part of a myoblast in the tail-bud of a R. pipiens embryo (stage 18). Yolk platelets are subdivided by a transverse fissure, suggesting the disintegration into smaller fragments. Many mitochondria (M) lie closely associated with the yolk platelets, x 27,000. Fic. 13. Part of a myoblast in the tail-bud of R. pipiens (stage 18) showing an irregular shaped platelet with a deep indentation. This type of decomposition is rather rare. Note the close apposition of many mitochondria (M) to a lipoid body (L). x 30,000.
240
S. KARASAKI
FIG. 14. Part of an epidermal cell of a R. pipiens embryo (stage 18). Lipoid bodies (L) are found within the decomposing yolk material (B) and in close association with the yolk platelet, x 20,000. FIc. 15. Part of a neuroblast of a Triturus neurula (stage 17). A pigment granule (P) is included together with main body of a platelet within a common membrane. The pigment granule appears to be partially dispersed. × 50,000.
Relation to lipoid bodies
In all species studied, lipoid bodies a p p e a r as r o u n d lobes, varying f r o m 0.5 to 3 # in size, a n d are usually very dense in the earlier stages (Fig. 1). D u r i n g the succeeding stages of development, they become less dense a n d usually coalesce (Fig. 2). Occasionally lipoid bodies larger t h a n 5 # in d i a m e t e r are found. D u r i n g the earlier stages, lip o i d bodies are distributed t h r o u g h o u t the c y t o p l a s m without any special relationship to the y o l k platelets. D u r i n g the stages of y o l k resorption, lipoid bodies b e c o m e FIG. 16. A complex body derived from the yolk platelet, in a neuroblast of a Triturus embryo (stage 24). A limiting membrane encloses various configurations consisting of laminated membranes (1), vesicles (2), and granules (3). Fine particles, 40-60 ~ in diameter and extremely electron dense, are scattered throughout the inside of the complex (arrow). Two typical pigment granules (P) are also observed within the complex. A yolk platelet (B) below still displays a crystalline pattern with an average lattice spacing of 69 &. x 80,000.
UTILIZATION OF YOLK PLATELETS
241
242
s. KARASAKI
located closer to the yolk platelets (Figs. 13 and 14). Sometimes they become embedded in the decomposed yolk matrix (Fig. 14). Occasionally large complex structures, 5 to 30 # in size, can be seen to contain yolk crystalline structures and lipoid bodies as well as mitochondria and vesicular components. DISCUSSION Yolk platelets during early developmental stages undergo characteristic morphological changes which are closely related with the progressive differentiation of cells and possibly with the processes of embryonic determination. Several authors (5, 8, 15, 22, 32), using light microscopy, have observed the decomposition of the yolk platelets during the embryonic development of amphibians. The stages and regions of greatest yolk utilization, however, still are not known clearly. According to Bragg (5), no appreciable decomposition of yolk platelets occurs until the late neurula stage in anuran embryos, and it then takes place mainly in the dorsal tissues. On the other hand, Daniel and Yarwood (8) observed histological signs of yolk breakdown as early as the zygote stage in newt embryos. However these views have never been supported by any clear quantitative observations. The present investigations by electron microscopy clearly show that there is actually a significant alteration of yolk platelets as early as the gastrula stage in all of the species studied, although the time of alteration differs in each species. The superficial layer of the platelets may be utilized at the time when primary morphogenesis takes place, although the time of utilization depends on the embryonic region. Disappearance of the superficial layer begins in the dorsal mesodermal cells earlier than in other embryonic regions. This finding is of interest especially in connection with the organizer phenomena associated with primary embryonic induction. During experimental mesodermalization of Triturus ectoderm under the influence of specific proteins, the disappearance of the superficial layer of platelets seemed to be one of the initial changes in the ectodermal cells (20, also cited in 48). After the subcytolytic treatment which causes neural induction in Triturus ectoderm, a similar phenomenon can be observed in the treated ectodermal cells (17, also cited in 47). In both cases, an alteration of developmental pathways occurs. The chemical and structural nature of the components of the superficial layer may have a significant bearing on primary processes of differentiation. Frequently the yolk fraction of amphibian embryos has been claimed to contain an appreciable amount of RNA (31, 40). Lanzavecchia and Le Coultre (24) have indicated that the particles in the superficial layer are ribonucleoprotein granules, and they suggest that the cytoplasm obtains a supply of ribonucleoprotein when the platelets lose their superficial layer. In a previous study (19), however, we have presented two observations which seem to
UTILIZATION OF YOLK PLATELETS
243
contradict these ideas. The particles in the superficial layer are much smaller than the ribosome particles in the same cells. Furthermore, they sometimes seem to resemble threadlike materials rather than granules. These were also confirmed by the electron micrographs of Ward (45). Recent cytochemical and biochemical studies on yolk platelets have provided no evidence for the presence of RNA in either the superficial layer or the main body (27, 39, 42). Rather, a localization of polysaccharide (27, 39) in the superficial layer was suggested. Apparently in the earlier embryonic cells the main bodies of the platelets are protected from enzymatic actions by the presence of a superficial layer. If this is the case, the removal of this layer either during the normal course of development or by some experimental treatment may create a new situation in the embryonic cell. Then the utilization of the main body can result from the action of enzymes such as cathepsin (7, 9) or phosphoprotein phosphatase (1, 12, 13, 26), whose presence in amphibian embryos has been demonstrated. After the disappearance of the superficial layer, the platelet begins to show modifications of its crystalline structure: crystalline arrangements of dots or fringes interiorly, separate particles (40-60 A in size) at the edge of the crystal, and various characteristic configurations more exteriorly (laminar and vesicular membranes, granules, and fibrils). This arrangement poses two questions: (a) Is there a relationship between the fine particles and the crystalline structures? and (b) What becomes of the peripheral components? It appears that the crystalline main body first decomposes into separate fine particles at its periphery. There are two arguments in favor of this hypothesis: (a) The particles appear very similar in dimension to the components of the crystalline structure, i.e., 40 ~ (19, 43), and (b) very near to the crystalline lattice, particles show a regular alignment making a gradual transition from a crystalline to a disordered pattern. It is possible, then, that the particles may be derived from the main body itself by a process through which the crystalline structure becomes disordered. Various configurations, including lamellae, fibrils, vesicles, and granules, appear around the crystalline structure. This observation is in general agreement with the earlier observations by Karasaki (16) made on T. pyrrhogaster, and by Lanzavecchia and Le Coultre (24) made on R. esculenta, and by Sung (38) made on R. nigromaculata. In the embryonic cells of early chick blastoderm, similar configurations were also shown in the electron micrographs of Bellairs (2). The presence of the membranous components near or within the matrix of the fine particles suggests that they are possibly formed from these fine particles, but at this time it is impossible to say whether or not the particles are directly transformed into membranous components. However, when isolated main bodies of the yolk platelets of T. pyrrhogaster were exposed to a solution of 0.2 % sodium deoxycholate, they gave rise to laminated membranes which often paired (18). Each single membrane meas-
244
s. KARASAKI
ured 50-100 A in thickness. This is the same thickness as that of the membranous structure which appears around the main body during normal development. From this experiment one might assume that the membranous structure is formed by the association of macromolecules from the crystalline structure under the influence of the physical and chemical forces of their environment. During cellular differentiation in general, the cytomembrane system becomes very extensive and elaborate; a continuous new formation of cytomembranes must, therefore, be granted (3, 11, 14, 41). This has been specifically shown in amphibian embryonic development (10, 16). The stages of elaboration and increase of various cytomembranes coincide approximately with the stages at which yolk platelets decompose and laminated membranes are formed. One possible speculation is that the membranous structures around the main body represent an intermediate step in he formation of cytomembranes at the expense of the yolk platelets. In this connection, it is interesting that these membranous structures can often be seen to have a triple-layered structure, with a total width of about 70 A. This dimension corresponds to Robertson's (30) electron micrographs of the unit membrane found in many cells. Many of the cytomembranes or physiologically semipermeable membranes in general have this appearance (30). The laminated figures, which form from yolk platelets, bear some resemblance to the myelin figures described by Stoeckenius (35). According to his electron micrographs, laminar systems of pure phospholipids have dark bands which are around 20 A in thickness. By the addition of basic protein (globin from beef erythrocyte) to phospholipids (from human brain), he observed an increase to 50 A in the width of these bands on the outer surface of the myelin figures. The membranous structures around the yolk platelets could, then, be constructed of a film of a protein plus the polar groups of a .leaflet of phospholipids, which appear dense after osmium fixation. Lipovitellin, which is a main component of the crystalline structure of yolk platelets, consists mostly of protein and phospholipid (33, 43), and could be available for the myelin formation of the lipoprotein membrane mentioned above. In the yolk of the unincubated chicken egg, Bellairs (4) found the laminated-membranes which have a "unit membrane" structure of 75 A thickness. The phospholipids from chick yolk readily form myelin figures by hydration (29) and the phospholipids react with proteins to produce salt-linked compounds (6). Recently Luzzati and Husson (25), using X-ray diffraction, have demonstrated several new liquid-crystalline structures in phospholipid-water systems for which only a lamellar structure had been assumed in the past. Stoeckenius (36) has studied these phospholipid-water systems with the electron microscope; under certain conditions, he has found hexagonal array of cylinders whose structure and dimensions are in excellent agreement with the X-ray observations. The crystalline structure of
UTILIZATION OF YOLK PLATELETS
245
yolk platelets has also been suggested to comprise a simple hexagonal packing of cylindrical structural units containing a lipoprotein (19, 43). Thus a transformation from the crystalline structure to the laminated membranes may be explained in part by the high polymorphism of the lipid-water systems. Interpretation of the membranous systems remains speculative as long as no biochemical data on the cytomembranes are available. Throughout the embryonic stages in R. pipiens, the crystalline structure of yolk platelets was found in the interspaces of mitochondria. This can be seen in some electron micrographs accompanying this paper, which are in general agreement with those of Lanzavecchia and Le Coultre (24) made on R. esculenta and of Sung (37) made on R. nigromaculata. The two former workers interpret their observations as an indication of direct transformation of yolk platelets into mitochondria during development, although they admit that not all yolk platelets are directly transformed into mitochondria. However, before any definite conclusions on this important issue may be reached, the following points should be carefully considered: 1. This type of configuration is observed in Rana species [R. pipiens, R. nigromaculata (also in 34), R. esculenta (24)], but in no other species of amphibia (T. pyrrhogaster, D. viridescens, Xenopus laevis (10), B. vulgaris, and M. carolinensis). 2. Throughout the embryonic development of R. pipiens, these figures exist continuously in all embryonic regions from the matured egg to the hatching stages and with a low degree of incidence. 3. Most of the yolk platelets in amphibian species including those of Rana are probably converted into laminar or vesicular membranes which do not show the morphological features characteristic of mitochondria. 4. It is doubtful that yolk platelets contain enzymatic systems characteristic of mitochondria (46). From chemical and physical viewpoints, a direct conversion of macromolecules making up yolk platelets into macromolecules of mitochondria is rather improbable. In Rana species as well as in other amphibian species, the main pathway of morphological transformation of yolk platelets is not through mitochondria, but through nonmitochondrial membranes. The "yolk-mitochondria" which occur in a low incidence in all embryonic stages may be interpreted in one of the following ways: (a) Yolk platelets are taken up or engulfed into preexisting mitochondria through a process involving the chance coalescence of both limiting membranes. The mitochondria then enzymatically degrade the crystalline structure and utilize the products for protein synthesis and phosphorylation. This interpretation is rendered unlikely, however, by the fact that crystalline material within mitochondria is not observed in the embryos of T. pyrrhogaster, B. vulgaris and M. carolinensis, and since a random coalescence of yolk platelets and mitochondria should also occur in these species.
246
s. KARASAKI
A possibility which cannot be ruled out, however, is that specific mutual affinity of these structures is only present in species of Rana. (b) During oogenesis, yolk platelets have been observed to originate within the interspaces of mitochondria specifically in Rana (23, 44, 45) but not in Triturus (21). One may postulate that this phenomenon is continually taking place, even in the embryo. The small increase in the number of "yolk-mitochondria" during embryogenesis might be interpreted as an expression of a dynamic state in which the normal yolk platelets, continually being broken down to a small extent during early development, would provide the raw material for a resynthesis within empty mitochondria. In stages after hatching, however, the catabolic processes of yolk breakdown become so accelerated and generalized as to affect even the "yolk-mitochondria", and all yolk structures are rapidly utilized. This interpretation is thus directly opposed to the view of Lanzavecchia (cf. 24), who assumes that his "yolk-mitochondria" pictures represent a direct transformation of yolk platelets into mitochondria, rather than an abortive formation of yolk within mitochondria during early embryogenesis. An interesting point is that a close relationship seems to exist between pigment granules and yolk platelets. Barth and Barth (1), Flickinger (12), and Nass (26)have described an enzyme (phosphoprotein phosphatase) in pigment granules which liberates phosphate from the phosphoprotein of the yolk platelets. In Fig. 15, it looks as if a pigment granule might be participating in the digestion of the main body of a yolk platelet. Finally, some electron micrographs in R. pipiens and T. pyrrhogaster embryos show a yolk complex which closely resembles the Bellairs' "complex yolk drop" (2) (large yolk drops surrounded by membranes containing all the typical forms of cellular components in the granular matrix). The direct conversion hypothesis (2) also seems improbable in this case. There is a possibility that mitochondria and other components migrate into the granular matrix of decomposing yolk platelets. I wish to express my sincere thanks to Drs. Tuneo Yamada and David Prescott for their advice and encouragement. I am also greatly indebted to Drs. Robin Wallace and Oscar Miller, Jr., for their helpful discussion and criticism. REFERENCES 1. BARTH, L. G. and BARTH, L. J., The Energetics of Development, Columbia Univ. Press, New York, 1954. 2. BELLAIRS,R., Or. Embryol. Exptl. Morphol. 6, 149 (1958). 3. - ibid. 7, 94 (1959). 4. - J. Biophys. Biochem. Cytol. 11, 207 (1961). 5. BRAGG,A. N., Biol. Bull. 77, 268 (1939). 6. COOK,W. H. and MARTIN,W. G., Can. J. Biochem. Physiol. 40, 1273 (1962). 7. D'AMELIO,V. and CEAS, M. P., Experientia 13, 152 (1957).
UTILIZATION OF YOLK PLATELETS
247
8. DANIEL, J. F. and YARWOOD, E. A., Univ. Calif (Berkeley) Publ. Zool. 43, 321 (1939). 9. DEUCHAR, E. M., J. Embryol. Exptl. Morphol. 6, 223 (1958). 10. EAKIN, R. M. and LEHMANN, F. E., Arch. Entwicklungsmech. Organ. (Roux' Arch.) 150, 177 (1957). 11. FAWCETT, D. W., in RUDNICK, D. (Ed.), Developmental Cytology, XVI Growth Symposium, p. 161, Ronald Press, New York, 1959. 12. FLICKINGER,R. A., J. Exptl. Zool. 131, 307 (1956). 13. HARRIS, D. L., J. Biol. Chem. 165, 541 (1946). 14. HAY, E. D., in RUDNICK, D. (Ed.), Regeneration, XX Growth Symposium, p. 177, Ronald Press, New York, 1962. 15. HOLTFRETER,J., J. Exptl. ZooL 103, 81 (1946). 16. KARASAKI,S., Embryologia (Nagoya) 4, 247 (1959). 17. - Electron-Microscopy (Tokyo) 7, 96 (1959). 18. - in HIGASHI, N. (Ed.), The World through the Electron Microscope, Biology, p. 43, Japan Electron Optics Laboratory Co., Tokyo, 1961. 19. - - - - J. Cell Biol. 18, 33 (1963). 20. - in preparation. 21. - in preparation. 22. KEDROWSKI, B., Z. ZellJbrsch. Mikroskop. Anat. 26, 21 (1937). 23. LANZAVECCHIA, G., Proc. European ConJ[ Electron Microscopy, Delft, 2, 746 (1960). 24. LANZAVECCHIA,G. and LE COULTRE,A., Arch. Ital. Anat. Embryol. 63, 445 (1958). 25. LUZZATI, V. and HussoN, F., J. Cell Biol. 12, 207 (1962). 26. NASS, S., Biol. Bull. 122, 232 (1962). 27. OHNO, S., KARASAKI,S. and TAKATA,K., Exptl. Cell Research in press. 28. OKADA, Y. K. and ICHIKAWA,M., Ann. Rept. Exptl. Morphol. (Tokyo) 3, 1 (1947). 29. REVEL, J. P., ITO, S. and FAWCETT, D. W., J. Biophys. Biochem. CytoL 4, 495 (1958). 30. ROBERTSON,J. D., in BOYD, J. D., JOHNSON, F. R. and LEVER, J. D. (Eds.), Electron Microscopy in Anatomy, p. 74, Williams & Wilkins, Baltimore, 1960. 31. ROUNDS, D. E. and FLICKINGER, R. A., J. Exptl. Zool. 137, 479 (1958). 32. SAINT-HILAIRE,C., Zool. Yahrb. Abt. Allgem. Zool. Physiol. Tiere 34, 107 (1914). 33. SCHJEIDE,O. A., LEVI, E. and FLICKINGER,R. A., Growth 19, 297 (1955). 34. SHUMWAY, W., Anat. Record 78, 139 (1940). 35. STOECKENIUS,W., Y. Biophys. Bioehem. Cytol. 5, 491 (1959). 36. - J. Cell Biol. 12, 221 (1962). 37. SUNG, H. S., ExptI. Cell Research, 25, 702 (1961). 38. - Embryologia (Nagoya) 7, 185 (1962). 39. TAKATA, K., and OHNO, S., personal communication. 40. VAns, W., Arch. Entwicklungsmech. Organ. (Roux' Arch.) 153, 504 (1962). 41. WADDINGTON,C. H., New Patterns in Genetics and Development. Columbia University Press, New York, 1962. 42. WALLACE, R. A., Biochim. Biophys. Acta in press. 43. - ibid. in press. 44. WARD, R. T., J. CellBiol. 14, 303 (1962). 45. - ibid. 14, 309 (1962). 46. WEBER,R. and BOELL,E. J., Develop. BioL 4, 452 (1962). 47. YAMADA, T., it/ABERCROMBIE, M. and BRACHET, J. (Eds.), Advances in Morphogenesis, Vol. 1, p. 1, Academic Press, New York, 1961. 48. - J. Cellular Comp. Physiol. 60, Suppl. l, 49 (1962).
9, 225-247 (1963)
Studies on A m p h i b i a n
225
Yolk
5. Electron Microscopic Observations on the Utilization of Yolk Platelets during Embryogenesis SHUICHI KARASAKI
Biology Division, Oak Ridge National Laboratory, z Oak Ridge, Tennessee Received February 8, 1963 The closely related phenomena of yolk platelet utilization and the progressive differentiation of cells in the developing amphibian embryos have been studied with the electron microscope. The first change in the yolk platelets is a decrease in thickness and a subsequent disappearance of the superficial layer in cells undergoing primary differentiation. At later stages, the surface of the crystalline structure begins to decompose into separate units. Further disintegration of the platelet is coupled with the formation of laminar or vesicular membranes, fine particles, and fibrils. The ultrastructure of the membranous components is very similar to that of cytomembranes. It is not improbable that these membranes represent an intermediate step in the formation of cytomembranes at the expense of yolk platelet material. During the decomposition of yolk platelets, mitochondria, pigment granules, and lipoid bodies are found in a close association with the yolk platelets. In Rana species (as opposed to other amphibian species), crystalline structures typical of yolk platelets are frequently located within the mitochondria throughout embryonic development. The significance of this observation is discussed. It is well established that the yolky material in an amphibian egg constitutes a reservoir which is utilized during some phase of ontogeny as a source of energy, of building materials or of both, but at just what phase of development and for what processes they are utilized have received only slight attention (however, see 5, 15). Previously it has not been possible with light microscopy to describe in any detail the morphological changes in the utilization process. Indeed, when the yolk platelets contained within embryonic cells are examined in this manner they seem to remain in much the same condition until their disappearance at the larval stage (5). With the electron microscope, however, it has been possible to pursue some of the morphological changes in fine structure which yolk platelets undergo during the progressive differentiation of the embryonic cell. The yolk platelets of young embryos z Operated by Union Carbide Corporatien for the United States Atomic Energy Commission.
226
s. KARASAKI
and ripe eggs of all amphibian species contain a superficial layer of granular or fibrillar materials, a central main body displaying a crystalline lattice structure, and an enclosing membrane of approximately 70 • thickness (16, 19). The first visible changes in the platelet are observed in the superficial layer, which decreases in thickness and finally disappears completely during the early stages of differentiation (16). After the disappearance of the superficial layer, the main body begins to decompose and to form concentric laminae in Triturus (16), suggesting the possibility that cytoplasmic membranous structures may be directly derived from the yolk platelets. Lanzavecchia and Le Coultre (24), studying similar laminar structures formed from yolk platelets in Rana embryos, believe that it is possible to follow step by step the transformation of yolk platelets into mitochondria. The time of alleged transformation of yolk platelets coincides approximately with the stage during which the mitochondria increase in number and complexity and the endoplasmic reticulum system becomes more elaborate. Subsequently the embryonic ceils gain the specialized organelles which characterize the differentiated cells. Since it is likely that the yolk platelet is intimately involved in the genesis and elaboration of cytoplasmic organelles, it is important to study the yolk platelet with regard to the embryonic region in which its utilization first begins and the extent of its relationship with the previously mentioned membranous structures.
MATERIALS AND METHODS The embryos used were mainly those of Triturus pyrrhogaster and Rana pipiens, from early cleavage to hatching stage. Developmental stages were numbered according to Okada and Ichikawa (28) for T. pyrrhogaster and according to Shumway (34) for R. pipiens. A number of observations were also made on selected stages of Diemictylus viridescens, Bufo vulgaris, Rana nigromaculata, and Microhyla carolinensis. Various embryonic tissues of known prospective significance were isolated from the
Figs. 1-16 are electron micrographs of osmium-fixed material embedded in Epon, Figs. 1, 2, 6, and 11 are from sections stained with lead acetate; the rest are from sections stained with uranyl acetate. FIG. 1. Part of a presumptive ectoderm cell in the blastula embryo (stage 9) of Rana pipiens. Each yolk platelet consists of a superficial layer (S) and a central main body (B), both enclosed within a single membrane (Y). Lipoid bodies (L), mitochondria (M), vesicles (V), and RNP-particles (R) can be identified, x 15,000. FIG. 2. Part of an epidermal cell at the tail-bud stage (stage 18) of R. pipiens. A yolk platelet (B)oin which the superficial layer has disappeared shows a moir6 pattern with wide spacing (average 570 A). Four mitochondria (M) have elaborate cristae and contain small yolk platelets. The endoplasmic recticulum is partly smooth surfaced and partly rough surfaced at E. Free RNP-particles (R) are abundant. Lipoid bodies (L) are less dense than those found in earlier embryos. Pigment granules (P) can be identified. × 20,000.
228
S. KARASAKI
developing embryos at each successive stage. For a quantitative analysis of yolk platelet utilization, the frequency of types of the yolk platelet configurations was examined in electron micrographs of the developing ventral ectoderm. Care was taken to have samples of the cell population belonging to the same cell lineage arranged in a sequence of developmental stages. In particular, the samples of the developing ventral ectoderm from R. pipiens were taken from the same egg batch (except those of stage 18). Details of the preparation for electron microscopy have been previously described (19). Isolated pieces of embryonic tissues were fixed for 30-60 minutes with 2% OsO4 in balanced saline buffered with Veronal acetate at pH 7.4. Following dehydration in an ethanol series, specimens were embedded in Epon 812. Sections were cut with a diamond knife on a Porter-Blum microtome, stained with uranyl or lead acetate, and examined with an RCA EMU 3E electron microscope.
OBSERVATIONS
Basic structure of the yolk platelet As described previously (19), the yolk platelets of young amphibian embryos are composed of three basic parts: an internal main body with a crystalline structure, a superficial layer with granular or fibrillar materials, and a limiting membrane of approximately 70 A thickness (Fig. 1). These three basic components are present in the yolk platelets of the mature egg and are usually retained until the blastula stage or later, depending upon the embryonic region and the species.
Disappearance of the superficial layer The first visible change in the yolk platelet begins in the superficial layer. In the developing ectoderm cell, some of the small yolk platelets measuring 1-2/z in diameter start to lose their superficial layer at the blastula stage (stage 9) in R. pipiens (Chart l) and at the gastrula stage (stage 11) in T. pyrrhogaster (Chart 2). The superficial layer in the larger platelets progressively decreases in thickness during development and subsequently disappears almost completely in the ectodermal region at the tail-bud stage [stage 18 in R. pipiens (Fig. 2 and Chart 1) and stage 27 in T. pyrrhogaster (Chart 2)]. The time of complete disappearance varies with the embryonic region. It occurs first in the dorsal mesoderm, at the late gastrula stage (stage 12) in R. pipiens, and at the neurula stage (stage 17) in T. pyrrhogaster, and then in the ectoderm. The platelets of the ventral endodermal region, however, remain unchanged even at the tail-bud stage in both species (stage 18 in R. pipiens and stage 27 in T. pyrrhogaster).
UTILIZATION OF YOLK PLATELETS
229
100 9O 8070.
o-- 0
60. ¢r-
50 2 40-
502010 12
21 26 54 42
62
( ~ 0 0 Q Q 8 9 10 11 12 428 425 520 492 741
~ 14 550
82 (~ 17 420
96 AGE IN ~.~.-....HOURS " ° r ~ . ~ AT '8°C 18 STAGENO. 722 TOTALNO. OF PLATELETS
CHART 1.
lOO 9O
o
8o 7o
~
w
60
o 504030" 2010•
,e
2
4
O© 10 410
11 656
............ --'' 7
® 17 702
10
~ 24 450
15 AGE IN Df~FScAT 27 STAGE NO. 701 TOTAL NO. OF PLATELETS
CHART 2.
CHARTS I and 2. The frequency of yolk platelet configurations in the developing ventral ectoderm of Rana pipiens (Chart 1) and Triturus pyrrhogaster (Chart 2). The frequency of yolk platelets with the following configurations are shown: those with a superficial layer ( - o -), those associated with laminar or vesicular membranes (-- - • -- -), and those enclosed within mitochondria ( - - ± - -). The frequency of these various configurations is expressed as a percentage of the total n u m b e r of yolk platelets (given at b o t t o m of the figure) examined in numerous electron micrographs of each stage.
230
s. KARASAKI TABLE I
FREQUENCY OF YOLK PLATELET CONFIGURATIONS IN THE PROSPECTIVE ECTODERM OF THE EARLY GASTRULAa Percentage of total number of platelets
Species
Number of Platelets Examined
within Superficial Layer
within Laminar Membranes
636 520 734
79 % 63 % 35 %
0% 4% 20 %
Triturus pyrrhogaster Rana pipiens Mierohyla carolinensis
within Mitochondria
0% 3% 0%
a The stage of these three species is at the time of the first indication of the dersal lip.
A difference can also be observed in the rate at which various amphibian species lose the superficial layer of their yolk platelets (Table I). In earlier stages, parts of the cytoplasm are engulfed by deep indentations of the nuclear surface. In this cytoplasmic region, yolk platelets are frequently observed without their superficial layer.
Decomposition of the main body Most electron micrographs show separate fine particles, about 40 to 60/~ in diameter and extremely electron dense, at the periphery of the crystalline main body after the disappearance of the superficial layer (Figs. 3 and 5). Frequently these particles are very numerous, forming a dense layer without ordered structure (Fig. 4). Very near to the crystalline lattice, they seem to show a regular alignment giving a gradual transition from the crystalline to the disordered pattern (Fig. 4). Some main bodies of larger platelets disintegrate into small fragments which are subdivided by a transverse fissure (Fig. 12). Irregularly shaped bodies with deep indentations are also observed (Fig. 13). Although the peripheral decomposition or fragmentation in the main body was sometimes quite striking, most main bodies retained a clear indication of their crystalline pattern (Figs. 4, 5, and 6). Periodic
Fro. 3. Presumptive neural ceils in Triturus neurula (stage 17). A large yolk platelet lacking the superficial layer but still retaining the limiting membrane (Y) intact around the main body (B) is present. Two small platelets, at close proximity to the protruded nuclear membrane (N), show decomposition of the main body coupled with the formation of laminated membranes. The laminated body at the center of the figure (arrow) may have been derived at the expense of the main body. × 25,000. FIG. 4. Part of a yolk platelet associated with laminated membranes in the neuroblast of a Triturus embryo (stage 18). The main body (B) displays a parallel fringe system of 68 ~ spacing. Peripherally (arrow) the regular band pattern changes into a region without ordered structure which seems to be composed of dense particles (40-60/~ in diameter). × 150,000.
UTILIZATION OF YOLK PLATELETS
231
232
s. KARASAKI
patterns with wide spacing (from 200 to 1000 A) were sometimes observed in the main body during the decomposing process (Fig. 2). This is referred to as the moir6 pattern, indicating some degree of shifting within the crystalline lattice.
Types of yolk platelet configuration during decomposition In most cases, disintegration of the main body was coupled with formation of various configurations as follows: (1) Laminar type (Figs. 3-6, 10, and 16); (2) Vesicular type (Figs. 7, 8, and 16); (3) Granular type (Figs. 8 and 16); and (4) Fibrillar type (Fig. 9). (1) Laminar type. This is the most frequently observed configuration. As seen in Figs. 3-6, laminated membranes are stacked around the main body. These are parallel sheets between two and twenty in number. Though they are in general arranged concentrically in the form of partial or complete rings, their outlines are frequently irregular. The thickness of a single sheet is 50-100 A, with an average 70/~i. The least distance between two adjacent sheets is 50 A. At the periphery of the decomposed main body, the fine particles are aligned suggesting a gradual transition into membranes. Some membranes show a triple-layered structure which corresponds to Robertson's (30) "unit membrane" (Fig. 6). These lamellae either terminate with a free edge or form flattened cisternae. Laminar components appear very similar to cytomembranes in general (Fig. 5). A fine laminar appearance is at first restricted to the periphery, and later the whole main body frequently seems to give rise to myelintype figures, in which a concentric arrangement of membranes is often apparent (Fig. 10). In T. pyrrhogaster and M. carolinensis, concentric lamellae are frequently observed around the main body of platelets larger than 3 #. In R. pipiens, on the other hand, stacks of lamellae are located in restricted areas near the surface of the larger main bodies (Fig. 6). In the developing ectoderm cells the concentric lamellae are first observed in the smaller yolk platelets (1-2 #) at the blastula stage (stage 9) in R. pipiens (Chart l) and at the neurula stage (stage 15) in T. pyrrhogaster (Chart 2)i The laminar type configuration appears in the large yolk platelets and increases in frequency during succeeding stages, especially at the tail-bud stage. Differences among species can be observed in the rates or stages at which laminar membranes appear around the main body (Table I, Charts 1 and 2). (2) Vesicular type. This type occurs usually in platelets larger than 3 # at stages later than tail-bud stage. It consists of many small vesicles, varying between 20 and 200 F~a. 5. Laminar configuration surrounding the main body (B) of a yolk platelet in a Triturusembryonic explant. The opaque particles (40-60 A) indicated by arrows have the same dimensions as the particles hexagonallyarranged in the main body. The band spacing is 70 ]k in the crystalline pattern of the main body. x 100,000,
UTILIZATION OF YOLK PLATELETS
233
234
S. KARASAKI
FI~. 6. Part of a yolk platelet associated with laminar type configurations in the epidermal cell of a R. pipiens embryo (stage 18). Layered membranes, measuring about 70 ~ in thickness, are arranged concentrically. Some membranes (arrow) show a unit membrane. These lamellae either terminate with a free edge or form cisternae. A remnant of the main body (B) shows a fringe pattern with a spacing of 73 ~. x 80,000.
m/z in size (Figs. 7 and 8). The vesicular bodies are found either in the matrix of granular type components or lying against the laminar type components. Most of the vesicles appear to have a clear homogeneous interior; others contain various structures, such as granules and granular or cloudy masses. (3) Granular type. A granular substance, opaque and with large granules (100-200 A), can sometimes be found in the area surrounding the main body. The granules, usually accompanied by laminar or vesicular membranes (Figs. 8 and 16), are difficult to distinguish in size and shape from the RNP-particles which are found free in the cytoplasm. This type is observed in all embryonic regions at the tail-bud stage. (4) Fibrillar type. The close attachment of tufts of faint, fibrillar material to the granular matrix on the surface of the platelet is observed during the later stages of platelet decomposition in the mesodermal cells of R. pipiens (stage 18) (Fig. 9) and T. pyrrhogaster (stage 27). Under high magnification these tufts consist of oriented
UTILIZATION
OF Y O L K P L A T E L E T S
235
FIG. 7. Part of a neuroblast in the brain of a R. pipiens embryo (stage 18). A vesicular and granular matrix surrounds main bodies (B) of the yolk platelet. A pigment granule (P) is embedded in the matrix region. × 8000. FI~. 8. High magnification of the area indicated by an asterisk (*) in Fig. 7. The size of the vesicles varies from 0.05 to 0.5/~. The dimension of the particles in this region are similar (100-200 A) to those of the RNP particles on the outer surface of the nuclear membrane (arrow). x 55,000.
arrays of fine, straight, often discontinuous fibrils in nearly parallel order, each of which measures in the range of 40 to 100 • in width. These materials are closely similar to myofibrils, which are f o u n d in the myoblast at later stages.
Limiting membrane of the decomposing yolk platelets Following the disappearance of the superficial layer, the limiting m e m b r a n e of yolk platelets seems to disintegrate or f o r m a series of small vesicles. Such decomposition is the more p r o n o u n c e d the later the developmental stages, although some yolk platelets retain their limiting membranes intact even after the disappearance of the superficial layer (Fig. 3). Occasionally within the intact investing membranes, the disintegrating main b o d y with its derivatives is observed (Figs. 3, 6, 8, and 16). Sometimes pigment granules are surrounded by such membranes (Figs. 15 and 16). 16 - 631831 J . Ultrastrueture Research
236
S. KARASAKI
FIG. 9. Fibrillar-type matrix at the surface of a decomposing platelet in a myoblast of a R, pipiens embryo (stage 18). The decomposed yolk material seems to be continuous with the tuft of fine filaments, extending out into the cytoplasmic area (arrow). In the same cell, tufts or individual filaments are scattered throughout the cytoplasm. × 50,000. FIG. 10. Several profiles of laminated bodies in a rnyoblast of a R. pipiens embryo (stage 18). These bodies seem to arise from the small yolk platelets. × 30,000.
Relation to mitochondria
T h r o u g h o u t the embryonic development of R. pipiens, small yolk platelets measuring 0.5 to 1 # in size, are found within mitochondria (Fig. 2 and Chart 1). Profiles of these yolk platelets are usually rectangular rods of varying length and width. Only rarely does one see the hexagonal shape that results f r o m sectioning n o r m a l to the flat face of a hexagonal plate (Figs. 2 and 11). At high magnification, hexagonal bodies display a hexagonal net pattern with a spacing of about 70 A. On the other hand, rectangular bodies show a parallel band pattern or no distinct pattern. Studies of m a n y sections of these configurations suggest that the main bodies are located between the two layers of a single cristae of mito-
UTILIZATION OF YOLK PLATELETS
237
chondria as mentioned by Ward (44) in the oocyte. None of these crystals have been seen within the mitochondrial matrix. This type of configuration was found in all embryonic regions during all embryonic stages of R. pipiens and R. nigromaculata. However it was never found in any region from the embryos of T. pyrrhogaster, D. viridescens, B. vulgaris, and M. earolinensis. During the differentiation of the ventral ectoderm of R. pipiens, the frequency of this configuration appeared to increase from about 1% of the total number of yolk platelets at the mid-cleavage stage to about 5-8 % during late gastrula through tail-bud stage (Chart 2). In the earlier stages, mitochondria containing the crystalline inclusions are spherical in shape and reveal only a few cristae arranged irregularly, while in the later stages mitochondria with numerous cristae and a dense matrix contain the crystalline structures. Throughout all stages, mitochondria increase in number and size and develop simultaneously a more complex internal structure. Thus, most of the mitochondria containing the crystals are very similar in structure to those without crystals but present in the same cells (Fig. 11). In R. pipiens a number of mitochondria lie in close proximity to those yolk platelets which are in a state of extensive decomposition (Fig. 12). In some cases, mitochondria are embedded in the granular matrix of the main body. In T. pyrrhogaster, the association is not so evident.
Relation to pigment granules A definite structural difference is observed between embryonic pigment granules of urodeles (T. pyrrhogaster, D. virideseens) and those of anurans (R. pipiens, R. nigromaculata, B. vulgaris, M. carolinensis). In the urodeles the spherical granules, measuring 0.4 to 0.6 #, are composed of a number of subunits measuring about 500 A which appear extremely dark. In the anurans the granules are single spherical bodies of about 0.4 to 0.6/z, in which subunits are not apparent. In both types of embryonic pigment granules a fine investing membrane is present. The embryonic pigment granules are abundantly present in the ectodermal cells, being preferentially distributed in the cortical layer and perinuclear zone of these cells during the earlier embryonic stages. During yolk decomposition in later stages, embryonic pigment granules can sometimes be found attached to the surface of decomposing platelets (Figs. 7 and 15). In Fig. 15, a pigment granule is included together with the main body of the platelet, within a single membrane. Sometimes they are embedded in the membranous complex or granular matrix of the decomposing yolk platelets (Figs. 7 and 16). The pigment granules within these portions seem to be partially disorganized. During development, embryonic pigment granules apparently diminish in number and cannot be identified in any tissues of adult amphibia.
FIG. 11. High magnification of a part of cytoplasm indicated in the lower left (*) of Fig. 2. Yolk platelet main bodies are located in the interspaces of mitochondria. The hexagonal body displays a crystalline hexagonal net pattern with spacings of 70 ~ . The other two bodies do not show distinct patterns, x 70,000.
FIG. 12. Part of a myoblast in the tail-bud of a R. pipiens embryo (stage 18). Yolk platelets are subdivided by a transverse fissure, suggesting the disintegration into smaller fragments. Many mitochondria (M) lie closely associated with the yolk platelets, x 27,000. Fic. 13. Part of a myoblast in the tail-bud of R. pipiens (stage 18) showing an irregular shaped platelet with a deep indentation. This type of decomposition is rather rare. Note the close apposition of many mitochondria (M) to a lipoid body (L). x 30,000.
240
S. KARASAKI
FIG. 14. Part of an epidermal cell of a R. pipiens embryo (stage 18). Lipoid bodies (L) are found within the decomposing yolk material (B) and in close association with the yolk platelet, x 20,000. FIc. 15. Part of a neuroblast of a Triturus neurula (stage 17). A pigment granule (P) is included together with main body of a platelet within a common membrane. The pigment granule appears to be partially dispersed. × 50,000.
Relation to lipoid bodies
In all species studied, lipoid bodies a p p e a r as r o u n d lobes, varying f r o m 0.5 to 3 # in size, a n d are usually very dense in the earlier stages (Fig. 1). D u r i n g the succeeding stages of development, they become less dense a n d usually coalesce (Fig. 2). Occasionally lipoid bodies larger t h a n 5 # in d i a m e t e r are found. D u r i n g the earlier stages, lip o i d bodies are distributed t h r o u g h o u t the c y t o p l a s m without any special relationship to the y o l k platelets. D u r i n g the stages of y o l k resorption, lipoid bodies b e c o m e FIG. 16. A complex body derived from the yolk platelet, in a neuroblast of a Triturus embryo (stage 24). A limiting membrane encloses various configurations consisting of laminated membranes (1), vesicles (2), and granules (3). Fine particles, 40-60 ~ in diameter and extremely electron dense, are scattered throughout the inside of the complex (arrow). Two typical pigment granules (P) are also observed within the complex. A yolk platelet (B) below still displays a crystalline pattern with an average lattice spacing of 69 &. x 80,000.
UTILIZATION OF YOLK PLATELETS
241
242
s. KARASAKI
located closer to the yolk platelets (Figs. 13 and 14). Sometimes they become embedded in the decomposed yolk matrix (Fig. 14). Occasionally large complex structures, 5 to 30 # in size, can be seen to contain yolk crystalline structures and lipoid bodies as well as mitochondria and vesicular components. DISCUSSION Yolk platelets during early developmental stages undergo characteristic morphological changes which are closely related with the progressive differentiation of cells and possibly with the processes of embryonic determination. Several authors (5, 8, 15, 22, 32), using light microscopy, have observed the decomposition of the yolk platelets during the embryonic development of amphibians. The stages and regions of greatest yolk utilization, however, still are not known clearly. According to Bragg (5), no appreciable decomposition of yolk platelets occurs until the late neurula stage in anuran embryos, and it then takes place mainly in the dorsal tissues. On the other hand, Daniel and Yarwood (8) observed histological signs of yolk breakdown as early as the zygote stage in newt embryos. However these views have never been supported by any clear quantitative observations. The present investigations by electron microscopy clearly show that there is actually a significant alteration of yolk platelets as early as the gastrula stage in all of the species studied, although the time of alteration differs in each species. The superficial layer of the platelets may be utilized at the time when primary morphogenesis takes place, although the time of utilization depends on the embryonic region. Disappearance of the superficial layer begins in the dorsal mesodermal cells earlier than in other embryonic regions. This finding is of interest especially in connection with the organizer phenomena associated with primary embryonic induction. During experimental mesodermalization of Triturus ectoderm under the influence of specific proteins, the disappearance of the superficial layer of platelets seemed to be one of the initial changes in the ectodermal cells (20, also cited in 48). After the subcytolytic treatment which causes neural induction in Triturus ectoderm, a similar phenomenon can be observed in the treated ectodermal cells (17, also cited in 47). In both cases, an alteration of developmental pathways occurs. The chemical and structural nature of the components of the superficial layer may have a significant bearing on primary processes of differentiation. Frequently the yolk fraction of amphibian embryos has been claimed to contain an appreciable amount of RNA (31, 40). Lanzavecchia and Le Coultre (24) have indicated that the particles in the superficial layer are ribonucleoprotein granules, and they suggest that the cytoplasm obtains a supply of ribonucleoprotein when the platelets lose their superficial layer. In a previous study (19), however, we have presented two observations which seem to
UTILIZATION OF YOLK PLATELETS
243
contradict these ideas. The particles in the superficial layer are much smaller than the ribosome particles in the same cells. Furthermore, they sometimes seem to resemble threadlike materials rather than granules. These were also confirmed by the electron micrographs of Ward (45). Recent cytochemical and biochemical studies on yolk platelets have provided no evidence for the presence of RNA in either the superficial layer or the main body (27, 39, 42). Rather, a localization of polysaccharide (27, 39) in the superficial layer was suggested. Apparently in the earlier embryonic cells the main bodies of the platelets are protected from enzymatic actions by the presence of a superficial layer. If this is the case, the removal of this layer either during the normal course of development or by some experimental treatment may create a new situation in the embryonic cell. Then the utilization of the main body can result from the action of enzymes such as cathepsin (7, 9) or phosphoprotein phosphatase (1, 12, 13, 26), whose presence in amphibian embryos has been demonstrated. After the disappearance of the superficial layer, the platelet begins to show modifications of its crystalline structure: crystalline arrangements of dots or fringes interiorly, separate particles (40-60 A in size) at the edge of the crystal, and various characteristic configurations more exteriorly (laminar and vesicular membranes, granules, and fibrils). This arrangement poses two questions: (a) Is there a relationship between the fine particles and the crystalline structures? and (b) What becomes of the peripheral components? It appears that the crystalline main body first decomposes into separate fine particles at its periphery. There are two arguments in favor of this hypothesis: (a) The particles appear very similar in dimension to the components of the crystalline structure, i.e., 40 ~ (19, 43), and (b) very near to the crystalline lattice, particles show a regular alignment making a gradual transition from a crystalline to a disordered pattern. It is possible, then, that the particles may be derived from the main body itself by a process through which the crystalline structure becomes disordered. Various configurations, including lamellae, fibrils, vesicles, and granules, appear around the crystalline structure. This observation is in general agreement with the earlier observations by Karasaki (16) made on T. pyrrhogaster, and by Lanzavecchia and Le Coultre (24) made on R. esculenta, and by Sung (38) made on R. nigromaculata. In the embryonic cells of early chick blastoderm, similar configurations were also shown in the electron micrographs of Bellairs (2). The presence of the membranous components near or within the matrix of the fine particles suggests that they are possibly formed from these fine particles, but at this time it is impossible to say whether or not the particles are directly transformed into membranous components. However, when isolated main bodies of the yolk platelets of T. pyrrhogaster were exposed to a solution of 0.2 % sodium deoxycholate, they gave rise to laminated membranes which often paired (18). Each single membrane meas-
244
s. KARASAKI
ured 50-100 A in thickness. This is the same thickness as that of the membranous structure which appears around the main body during normal development. From this experiment one might assume that the membranous structure is formed by the association of macromolecules from the crystalline structure under the influence of the physical and chemical forces of their environment. During cellular differentiation in general, the cytomembrane system becomes very extensive and elaborate; a continuous new formation of cytomembranes must, therefore, be granted (3, 11, 14, 41). This has been specifically shown in amphibian embryonic development (10, 16). The stages of elaboration and increase of various cytomembranes coincide approximately with the stages at which yolk platelets decompose and laminated membranes are formed. One possible speculation is that the membranous structures around the main body represent an intermediate step in he formation of cytomembranes at the expense of the yolk platelets. In this connection, it is interesting that these membranous structures can often be seen to have a triple-layered structure, with a total width of about 70 A. This dimension corresponds to Robertson's (30) electron micrographs of the unit membrane found in many cells. Many of the cytomembranes or physiologically semipermeable membranes in general have this appearance (30). The laminated figures, which form from yolk platelets, bear some resemblance to the myelin figures described by Stoeckenius (35). According to his electron micrographs, laminar systems of pure phospholipids have dark bands which are around 20 A in thickness. By the addition of basic protein (globin from beef erythrocyte) to phospholipids (from human brain), he observed an increase to 50 A in the width of these bands on the outer surface of the myelin figures. The membranous structures around the yolk platelets could, then, be constructed of a film of a protein plus the polar groups of a .leaflet of phospholipids, which appear dense after osmium fixation. Lipovitellin, which is a main component of the crystalline structure of yolk platelets, consists mostly of protein and phospholipid (33, 43), and could be available for the myelin formation of the lipoprotein membrane mentioned above. In the yolk of the unincubated chicken egg, Bellairs (4) found the laminated-membranes which have a "unit membrane" structure of 75 A thickness. The phospholipids from chick yolk readily form myelin figures by hydration (29) and the phospholipids react with proteins to produce salt-linked compounds (6). Recently Luzzati and Husson (25), using X-ray diffraction, have demonstrated several new liquid-crystalline structures in phospholipid-water systems for which only a lamellar structure had been assumed in the past. Stoeckenius (36) has studied these phospholipid-water systems with the electron microscope; under certain conditions, he has found hexagonal array of cylinders whose structure and dimensions are in excellent agreement with the X-ray observations. The crystalline structure of
UTILIZATION OF YOLK PLATELETS
245
yolk platelets has also been suggested to comprise a simple hexagonal packing of cylindrical structural units containing a lipoprotein (19, 43). Thus a transformation from the crystalline structure to the laminated membranes may be explained in part by the high polymorphism of the lipid-water systems. Interpretation of the membranous systems remains speculative as long as no biochemical data on the cytomembranes are available. Throughout the embryonic stages in R. pipiens, the crystalline structure of yolk platelets was found in the interspaces of mitochondria. This can be seen in some electron micrographs accompanying this paper, which are in general agreement with those of Lanzavecchia and Le Coultre (24) made on R. esculenta and of Sung (37) made on R. nigromaculata. The two former workers interpret their observations as an indication of direct transformation of yolk platelets into mitochondria during development, although they admit that not all yolk platelets are directly transformed into mitochondria. However, before any definite conclusions on this important issue may be reached, the following points should be carefully considered: 1. This type of configuration is observed in Rana species [R. pipiens, R. nigromaculata (also in 34), R. esculenta (24)], but in no other species of amphibia (T. pyrrhogaster, D. viridescens, Xenopus laevis (10), B. vulgaris, and M. carolinensis). 2. Throughout the embryonic development of R. pipiens, these figures exist continuously in all embryonic regions from the matured egg to the hatching stages and with a low degree of incidence. 3. Most of the yolk platelets in amphibian species including those of Rana are probably converted into laminar or vesicular membranes which do not show the morphological features characteristic of mitochondria. 4. It is doubtful that yolk platelets contain enzymatic systems characteristic of mitochondria (46). From chemical and physical viewpoints, a direct conversion of macromolecules making up yolk platelets into macromolecules of mitochondria is rather improbable. In Rana species as well as in other amphibian species, the main pathway of morphological transformation of yolk platelets is not through mitochondria, but through nonmitochondrial membranes. The "yolk-mitochondria" which occur in a low incidence in all embryonic stages may be interpreted in one of the following ways: (a) Yolk platelets are taken up or engulfed into preexisting mitochondria through a process involving the chance coalescence of both limiting membranes. The mitochondria then enzymatically degrade the crystalline structure and utilize the products for protein synthesis and phosphorylation. This interpretation is rendered unlikely, however, by the fact that crystalline material within mitochondria is not observed in the embryos of T. pyrrhogaster, B. vulgaris and M. carolinensis, and since a random coalescence of yolk platelets and mitochondria should also occur in these species.
246
s. KARASAKI
A possibility which cannot be ruled out, however, is that specific mutual affinity of these structures is only present in species of Rana. (b) During oogenesis, yolk platelets have been observed to originate within the interspaces of mitochondria specifically in Rana (23, 44, 45) but not in Triturus (21). One may postulate that this phenomenon is continually taking place, even in the embryo. The small increase in the number of "yolk-mitochondria" during embryogenesis might be interpreted as an expression of a dynamic state in which the normal yolk platelets, continually being broken down to a small extent during early development, would provide the raw material for a resynthesis within empty mitochondria. In stages after hatching, however, the catabolic processes of yolk breakdown become so accelerated and generalized as to affect even the "yolk-mitochondria", and all yolk structures are rapidly utilized. This interpretation is thus directly opposed to the view of Lanzavecchia (cf. 24), who assumes that his "yolk-mitochondria" pictures represent a direct transformation of yolk platelets into mitochondria, rather than an abortive formation of yolk within mitochondria during early embryogenesis. An interesting point is that a close relationship seems to exist between pigment granules and yolk platelets. Barth and Barth (1), Flickinger (12), and Nass (26)have described an enzyme (phosphoprotein phosphatase) in pigment granules which liberates phosphate from the phosphoprotein of the yolk platelets. In Fig. 15, it looks as if a pigment granule might be participating in the digestion of the main body of a yolk platelet. Finally, some electron micrographs in R. pipiens and T. pyrrhogaster embryos show a yolk complex which closely resembles the Bellairs' "complex yolk drop" (2) (large yolk drops surrounded by membranes containing all the typical forms of cellular components in the granular matrix). The direct conversion hypothesis (2) also seems improbable in this case. There is a possibility that mitochondria and other components migrate into the granular matrix of decomposing yolk platelets. I wish to express my sincere thanks to Drs. Tuneo Yamada and David Prescott for their advice and encouragement. I am also greatly indebted to Drs. Robin Wallace and Oscar Miller, Jr., for their helpful discussion and criticism. REFERENCES 1. BARTH, L. G. and BARTH, L. J., The Energetics of Development, Columbia Univ. Press, New York, 1954. 2. BELLAIRS,R., Or. Embryol. Exptl. Morphol. 6, 149 (1958). 3. - ibid. 7, 94 (1959). 4. - J. Biophys. Biochem. Cytol. 11, 207 (1961). 5. BRAGG,A. N., Biol. Bull. 77, 268 (1939). 6. COOK,W. H. and MARTIN,W. G., Can. J. Biochem. Physiol. 40, 1273 (1962). 7. D'AMELIO,V. and CEAS, M. P., Experientia 13, 152 (1957).
UTILIZATION OF YOLK PLATELETS
247
8. DANIEL, J. F. and YARWOOD, E. A., Univ. Calif (Berkeley) Publ. Zool. 43, 321 (1939). 9. DEUCHAR, E. M., J. Embryol. Exptl. Morphol. 6, 223 (1958). 10. EAKIN, R. M. and LEHMANN, F. E., Arch. Entwicklungsmech. Organ. (Roux' Arch.) 150, 177 (1957). 11. FAWCETT, D. W., in RUDNICK, D. (Ed.), Developmental Cytology, XVI Growth Symposium, p. 161, Ronald Press, New York, 1959. 12. FLICKINGER,R. A., J. Exptl. Zool. 131, 307 (1956). 13. HARRIS, D. L., J. Biol. Chem. 165, 541 (1946). 14. HAY, E. D., in RUDNICK, D. (Ed.), Regeneration, XX Growth Symposium, p. 177, Ronald Press, New York, 1962. 15. HOLTFRETER,J., J. Exptl. ZooL 103, 81 (1946). 16. KARASAKI,S., Embryologia (Nagoya) 4, 247 (1959). 17. - Electron-Microscopy (Tokyo) 7, 96 (1959). 18. - in HIGASHI, N. (Ed.), The World through the Electron Microscope, Biology, p. 43, Japan Electron Optics Laboratory Co., Tokyo, 1961. 19. - - - - J. Cell Biol. 18, 33 (1963). 20. - in preparation. 21. - in preparation. 22. KEDROWSKI, B., Z. ZellJbrsch. Mikroskop. Anat. 26, 21 (1937). 23. LANZAVECCHIA, G., Proc. European ConJ[ Electron Microscopy, Delft, 2, 746 (1960). 24. LANZAVECCHIA,G. and LE COULTRE,A., Arch. Ital. Anat. Embryol. 63, 445 (1958). 25. LUZZATI, V. and HussoN, F., J. Cell Biol. 12, 207 (1962). 26. NASS, S., Biol. Bull. 122, 232 (1962). 27. OHNO, S., KARASAKI,S. and TAKATA,K., Exptl. Cell Research in press. 28. OKADA, Y. K. and ICHIKAWA,M., Ann. Rept. Exptl. Morphol. (Tokyo) 3, 1 (1947). 29. REVEL, J. P., ITO, S. and FAWCETT, D. W., J. Biophys. Biochem. CytoL 4, 495 (1958). 30. ROBERTSON,J. D., in BOYD, J. D., JOHNSON, F. R. and LEVER, J. D. (Eds.), Electron Microscopy in Anatomy, p. 74, Williams & Wilkins, Baltimore, 1960. 31. ROUNDS, D. E. and FLICKINGER, R. A., J. Exptl. Zool. 137, 479 (1958). 32. SAINT-HILAIRE,C., Zool. Yahrb. Abt. Allgem. Zool. Physiol. Tiere 34, 107 (1914). 33. SCHJEIDE,O. A., LEVI, E. and FLICKINGER,R. A., Growth 19, 297 (1955). 34. SHUMWAY, W., Anat. Record 78, 139 (1940). 35. STOECKENIUS,W., Y. Biophys. Bioehem. Cytol. 5, 491 (1959). 36. - J. Cell Biol. 12, 221 (1962). 37. SUNG, H. S., ExptI. Cell Research, 25, 702 (1961). 38. - Embryologia (Nagoya) 7, 185 (1962). 39. TAKATA, K., and OHNO, S., personal communication. 40. VAns, W., Arch. Entwicklungsmech. Organ. (Roux' Arch.) 153, 504 (1962). 41. WADDINGTON,C. H., New Patterns in Genetics and Development. Columbia University Press, New York, 1962. 42. WALLACE, R. A., Biochim. Biophys. Acta in press. 43. - ibid. in press. 44. WARD, R. T., J. CellBiol. 14, 303 (1962). 45. - ibid. 14, 309 (1962). 46. WEBER,R. and BOELL,E. J., Develop. BioL 4, 452 (1962). 47. YAMADA, T., it/ABERCROMBIE, M. and BRACHET, J. (Eds.), Advances in Morphogenesis, Vol. 1, p. 1, Academic Press, New York, 1961. 48. - J. Cellular Comp. Physiol. 60, Suppl. l, 49 (1962).