Yolk utilization in the notochord of newt as studied by electron microscopy

When cells of amphibian embryos differentiate they begin to synthesize the structural proteins characteristic of the cell type, and this is immediately preceded by the beginning of the absorption of the yolk platelets. These events may be studied in cultured cells (Jones & Elsdale, 1963) or in sections of fixed embryos. When amphibian platelets are utilized by the cell, they undergo certain well-marked changes in ultrastructure that have been studied by electron microscopy, for example by Karasaki (1959, 1963b) and Sung (1962). The details of these changes depend in part upon the species, but also upon the particular tissue concerned. However, it is possible to generalize to the extent of saying that the utilization of a yolk platelet involves a decrease in the volume of the regularly-ordered crystalline core, which before differentiation occupied the bulk of it. This is accompanied by a corresponding increase in the volume of its outer shell of irregularly-packed granular matrix. In some cases a yolk platelet at such a stage is found to be bounded by an envelope consisting of a few layers of thin lamellae. The present paper demonstrates that in the notochord tissue of newt at stages during the utilization of the yolk, the platelets are surrounded by an exceptionally thick and well developed lamellar zone.

The present observations were made in the course of experimental work by Selman & Jurand (1964) on the effect of ultrasonic vibrations upon the endoplasmic reticulum of notochord cells. The appearance of the yolk to be described here was identical in both control and experimental material, and it was clear that none of the ultrasonic treatments had any effect upon the yolk structures.

Larvae of Triturus alpestris were selected between developmental stages 34 and 41 according to the normal table of Glaesner (1925) for Triturus vulgaris. The tail tips were fixed in 1 per cent, osmium tetroxide in sucrose solution with a Veronal-acetate buffer at pH 7·2, after Caulfield (1957) but using 3 per cent, sucrose. The fixation was for 15 min. in a refrigerator at 0°C. and then for a further 15 min. outside, slowly warming to room temperature. After dehydration in graded ethyl alcohols (35, 70, 95 and 100 per cent.) and having replaced the absolute alcohol by two changes of 1:2-epoxy-propane, the material was embedded in Araldite (Glauert & Glauert, 1958) in gelatin capsules and polymerized at 58°C. overnight. The ultra-thin sections were cut with a Porter-Blum microtome transversely in the region roughly between the anus and the tail tip. For increased contrast the sections were stained by floating the grids face downwards for 20 min. on an aqueous solution of 1 per cent, potassium permanganate together with 2·5 per cent, uranyl acetate. Excess stain on the sections was then removed by floating them successively on distilled water (a few minutes), 0·25 per cent, citric acid (30 sec.) and distilled water again (a few minutes). Other sections were stained with 0·5 per cent, phosphotungstic acid in absolute alcohol for 10 min. followed by a short rinse in absolute alcohol (Pease, 1960). Examination and photography were done using a Siemens Elmiskop I electron microscope.

Paraffin embedded sections were also prepared for light microscopy by fixing whole larvae in 5 per cent, trichloro-acetic acid with 1·37 per cent, lanthanum acetate and staining with methyl green and pyronin.

The notochord tissue at these stages (free-swimming larvae) was found to contain yolk platelets whose morphology was characteristic of all stages of the utilization process. There was also a number of yolk platelets whose form was unchanged from that in the earliest developmental stages. Unchanged yolk and the forms associated with yolk utilization have been observed in the same cell. Light microscopy indicates that at stages later than these used here for electron microscopy the number of platelets is at first markedly diminished and then falls to zero.

Unchanged yolk platelets are ovoid and vary in size. In notochord they may have a longitudinal diameter of between 1 μ and 5 μ. In section the interior and bulk of the early-stage yolk platelet appears homogeneous at low magnification of the electron microscope (Plate 1, A), but at higher magnifications a line-and-dot pattern emerges which suggests that the macromolecular units are precisely ordered in a crystalline lattice (Plate 1, E). The ultrastructure of this crystalline zone is unchanged during yolk utilization while its extent is progessively reduced (Plate 1, D). The dotted pattern is often clearest near the edge of the zone and consists of a hexagonal array of electron-dense spherical units. Further towards the middle of the crystalline zone the dotted pattern may merge gradually into a parallel line pattern, the lines of which are spaced at approximately 70Â and always run parallel to a side of the dotted hexagon, so that within one yolk platelet the lines are either parallel or within 52° to 66° of each other (Plate 1, E). In one instance the crystalline zone showed a line pattern in which the line systems intersected at 90° and 75° in different regions of the same yolk platelet (Plate 1, B). The nature of the line-and-dot pattern observed probably depends in part upon the direction of sectioning with respect to the crystalline lattice. If more than one yolk platelet shows structure within the same photographed field of view there is no relationship between the directions of the lines observed in different platelets, so that the line pattern is not due to astigmatism. It is probably caused by superimposed images of spherical units in several adjacent lattice layers.

The fact that the interior zone of yolk has a crystalline lattice structure is supported by an observation made when attempting to stain Araldite sections with a 0·5 per cent, solution of phosphotungstic acid in absolute alcohol. Small crystals of the acid formed preferentially against the sectioned face of the yolk (Plate 2, G). The only other part of the sections where this occurred was against the outer part of the notochordal sheath. The phosphotungstic acid did not, however, appear to stain the crystalline zone of yolk, as its electron density was not increased by this treatment. The long axes of the phosphotungstic acid crystals, as shown in Plate 2, G, for instance, were found to show no detectable direction of preferred orientation with respect to the crystalline lattice of the yolk surface. It is known that potassium phosphotungstate can crystallize as needles which are triclinic (see Dawson, 1963).

The irregular granular zone in unchanged yolk extends in a thin layer round the crystalline zone although it commonly forms a considerably wider band round its smallest circumference. On utilization, the granular zone consisting of granules about 50 Å in diameter is much extended, and eventually it appears to replace all the crystalline zone. In yolk utilization within the notochord, the granular zone is almost invariably surrounded by layers of lamellae. In a few cases the lamellar zone was observed to enfold the yolk platelet on all sides except for small polar regions, and in one case the lamellar zone was situated immediately to one side of the platelet (Plate 2, G). .

Towards the end of the process of yolk utilization the crystalline zone is absent and pockets of the irregular granular material, with rarely a few vesicles included, are left in small, rather irregularly-rounded envelopes of lamellae in the cytoplasm (Plate 1, F; Plate 2, M & N). Sometimes in these later stages a few ribosomes are found amongst the granular yolk material within the lamellar envelope. It is noteworthy that where both granular material and ribosomes are present, the two may be distinguished without difficulty (Plate 1, F; Plate 2, L). A body has also been photographed consisting of the lamellar envelope with only normal ribosome-containing cytoplasm and no granular or crystalline yolk material within it (Plate 2, N). This suggests that at least during the later stages of yolk utilization the envelope is often incomplete, so that portions of cytoplasm may be engulfed.

Serial sections cut through a single yolk granule have established that the outer zone does, in fact, consist of layers of lamellae which are commonly photographed in section, and does not, for instance, consist of aggregates of linear or threadlike units (Plate 2, H-K). Each lamella is approximately 60 Å thick and about fifty approximately parallel lamellae have in some cases been estimated to form the outer envelope to the granular zone. These lamellae are not found associated with yolk platelets at earlier developmental stages when no utilization occurs.

When paraffin-embedded stained sections of notochord of Triturus alpestris were studied by light microscopy, after fixation with the trichloro-acetic acid and lanthanum acetate solution to give good preservation for proteins and nucleic acids, the superficial lamellar zone could not be detected. This fixation seems unsuitable for this structure although general preservation was excellent. Selman & Pawsey (in preparation) have demonstrated that for yolk platelets in Xenopus notochord of corresponding developmental stages fixed in Smith’s fixative, prepared according to Rugh (1948), all three zones may be clearly distinguished (Plate 1, C), and they possess individual and distinctly different staining properties.

Yolk platelets of Triturus alpestris have an oval outline (occasionally round or spindle-shaped) in thin sections so that here the word platelet is perhaps misleading although it is commonly used to indicate that one is not concerned with the irregularly-shaped lipid inclusions called lipochondria. The yolk platelets in early stages of development of this species have an ultrastructure which appears not to differ from the account given by Karasaki (1959, 1963a) for Triturus pyrrho-gaster. Karasaki (1959) and Sung (1962), the latter author using Rananigromacu-lata, have demonstrated the parallel line pattern at approximately 70 Å spacing which is associated with the central region of the yolk, and although these papers do not depict the hexagonal dotted pattern of ultrastructure, this has been clearly recorded for yolk in oocytes of Rana pipiens by Ward (1962), and for yolk in embryos of Rana piepiens and Triturus pyrrhogaster by Karasaki (1962, 1963a). There seems at present to be insufficient evidence to specify with certainty the precise crystalline form in which macromolecular units are packed. Either form of regular close-packing of spheres (face-centered cubic or hexagonal) as well as the particular hexagonal but not close-packed form favoured, from mainly biochemical considerations, by Wallace (1963), could each give the line-and-dot pattern as shown in our Plate 1, E, for instance. Yolk platelets are necessarily sectioned at random orientation and one may hope to deduce the most probable structure by comparing the frequency with which particular linear or dotted patterns are found in a large number of good high-resolution electron-micrographs with the frequencies predicted from consideration of the various models. A good alternative, although technically-demanding, approach would be to compare high-resolution electron-micrographs from sections cut from the same yolk granule in two planes with a known angle between then.

It is perhaps worth noting that workers using species of Rana, for instance Karasaki (1963b) and Perry & Hibbard in unpublished work carried out in this Institute, have found two different types of yolk present. Most of the yolk platelets in this genus appear to be similar in general form and ultrastructure to the form invariably found in Triturus, but in Rana there is also a lesser number of a somewhat smaller type of yolk platelet having a rectangular profile and found within mitochondria, and these clearly seem to have a different ultrastructure. This second type of yolk seems to be the same as the crystalline body described by Yamada (1959). It is possible that the presence of this type of body may in some degree affect the interpretation of biochemical studies made on yolk, as for instance in the work of Panijel (1951) who found that the smaller platelets from Rana fusca contained a higher proportion of nucleic acid than the larger platelets.

Yolk utilization is commonly regarded as providing the differentiating cell with a supply of energy and protein-rich organic material. The notochord of these stages is both increasing the vacuolization of its cells and the thickness of the notochordal sheath (see Waddington & Perry, 1962). The present account of our observations has assumed, partly for sake of clarity, that a particular sequence of developmental stages takes place in the notochord yolk platelet. The assumption seems justified by the fact that our electron micrographs form a well-documented single sequence of stages with very gradual intermediate steps, but it is also supported by light microscope observations and the electron microscope studies of other authors using different amphibian material.

It is tempting to assume further that these morphological changes represent an interconversion of material from the crystalline to the granular and then to the lamellar form, or at least from the crystalline to the granular form. Subsequently the yolk material must be broken down to very small units and dispersed in the cytoplasm. We know little of the activity of the cell in all this. The yolk platelet during its utilization does not seem to be intimately associated with other cell organelles such as mitochondria or endoplasmic reticulum. The units of the granular zone appear different to ribosomes. This observation has also been made by Karasaki (1963a). It is possible that the lamellar zone is synthesized by the cytoplasm from material other than that derived from the yolk platelet. The only piece of evidence that seems at first sight against the direct interconversion hypothesis for the origin of the lamellae, is our finding of a single case of a pigment granule within the granular zone of a yolk platelet during the middle phase of the utilization process, while the granular zone itself was bounded by a well-developed lamellar zone. Karasaki (1963b) has also observed pigment granules in close association with yolk platelets.

It seems to be established that the morphological changes associated with yolk utilization can be different in the different tissues within a single species. Lamellar zones are certainly not confined to notochord, but they do appear to be more common there than in other tissues for the species we have studied. In certain tissues, yolk appears to be utilized by a process involving conversion between the crystalline and the granular zones without there being any lamellae present at all. It it not known whether the yolk utilization that is associated with different morphological changes is also associated with the presence of different enzyme systems.

On a étudié au microscope électronique l’ultràstructure des plaquettes vitellines dans les cellules chordales de Triturus alpestris au cours des stades larvaires, quand le vitellus est utilisé par les cellules en cours de différenciation. Le noyau cristallin interne de la plaquette, qui en occupe le plus gros volume aux stades précoces, est remplacé progessivement au cours de ces processus par une zone graunulaire irrégulièrement ordonnée. Cette zone est limitée par une enveloppe consistant en multiples assises de lamelles. La zone lamellaire est un caractère spécial des plaquettes vitellines dans la chorde pendant leur utilisation.

The authors wish to thank Professor C. H. Waddington, F.R.S., for discussion and encouragement. We are indebted to Mr D. E. Bradley and the staff of the Zoology Department, University of Edinburgh, for maintenance work on the Siemens electron microscope.

Our thanks are also due to Dr M. M. Harding of the Chemistry Department, University of Edinburgh, for discussion on the crystallographic aspects of the work.

The scale-line in each figure represents 0·25μ, except for Plate 1, C, where the scale represents 5μ. All figures except C are electron micrographs made as described in the text from notochord tissue of larval stages of Triturus alpestris.