Ronald R. Cowden ¹ and H. E. Lehman ²

Bermuda Biological Station for Research, Inc., St. George's West, Bermuda
¹ Department of Pathology, J. Hillis Miller Health Center, Gainsville, Florida, USA
² Department of Zoology, University of North Carolina, Chapel Hill, North Carolina, USA

^aContribution No. 311 of the Bermuda Biological Station.
^b This research was supported in part by grants from the National Institutes of Health RG-10003 and
GM-K3-6176-R1  (R.R.C.), the National Science Foundation G18666 (R.R.C.), and G9886 (H.E.L.).

Echinoderm development has attracted the attention of embryologists, cytologists, and cellular physiologists for almost a century, and more recently has been the focus of a great deal of work on the biochemistry of development. Fertilization and the cortical events attending fertilization in echinoderms have received particular attention (see Lord Rothschild, 1956 and R. D. Allen, 1958). A number of workers have investigated certain developmental stages in detail using either cytological or biochemical methods (Agrell, 1958; Immers, 1957; Markman, 1957; and Olsson, 1961). The incorporation of labeled precursors into nucleic acids, proteins, or mucopolysaccharides during development have been performed by Agrell and Persson (1956) and by Backstrom (1959); Kavanau (1954) has investigated protein synthesis.

Although the past decade has seen considerable advance in cytochemical methodology, a general survey of the topological distribution of nucleic acids, proteins and mucopolysaccharides during echinoderm development using tinctorial cytochemical methods has not appeared. Consequently, the objective of this study has been to survey the distribution of the above mentioned classes of macromolecular substances during development to the 48 hour pluteus stage in two echinoderm species. Attempts have been made to determine the stage in development at which nucleoli first appear, the usual cytological manifestation of new nuclear RNA synthesis; to investigate the distribution of nucleohistone during development; and to follow morphological and chemical alterations in the primary mesenchyme. The primary mesenchyme is a conspicuous derivative of a primary germinal layer which is morphologically recognizable in early cleavage and which achieves finite structure within the first 48 hours of development (Wolpert and Gustafson, 1961). Whereas embryos of the sea urchin, Lytechinus verigatus, have formed the principal material used in this investigation, a developmental series of the sand dollar, Melita quinquesperforata, was also examined for comparison.

Materials and Methods:

Adult Lytechinus verigatus were collected in the vicinity of the Bermuda Biological Station, St George's West, Bermuda. Gametes were obtained by injection of 0.53 M KCl into the coelom. After suitable washing, small groups of eggs were inseminated with sperm in slightly alkaline sea water (ca. pH 8.4). After fertilization membranes appeared, the eggs were again washed with filtered sea water and allowed to develop to the desired stage. Melita quinquesperforata were obtained in the vicinity of the Sea Horse Key Marine Biological Laboratory of the University of Florida, and were handeled in a similar fashiom. Unfertilized eggs, 2 cell stage, 16 cell stage, prehatching blastulae (ca. 4.5 hr.), mesenchymal blastulae (5-10 hr.), early gastrulae (12 hr.), prism gastrulae (18 hr.), early plutei ( 24 hr.) and 48 hr. plutei were selected for this study.

Eggs or embryos were fixed in ethanol-acetic acid (3:1), Bouin, or 10 percent formalin with 2 percent sodium acetate added. After conventional paraffin embedding, serial sections at 5 m were prepared. Ethanol-acetic acid fixed material was stained for RNA by Flax and Himes' (1952) pH 4.0 azure B method after pretreatment with DNase. Similarly fixed material was stained for DNA by the Feulgen reaction or by Einarson's (1949) gallocyanin-chromalum after pretreatment with RNase. Bouin fixed sections were stained for reactive groups of proteins by Mazia, Brewer and Alfert's (1953) mercuric bromphenol blue method. Basic nucleoproteins were stained by Alfert and Geschwind's (1953) pH 8.1 method in formalin fixed sections. Acid mucopolysaccharides and neutral polysaccharides or substances with adjacent glycol groups were demonstrated by the periodic acid-Schiff-alician blue (PAS--A.B.) sequence of Crawley et al (1956) using formalin fixed material.

Observations:

In the unfertilized ovum, the cytoplasmic distribution of RNA, protein, and PAS-positive material was essentially uniform, and this condition was maintained through the first three cleavage divisions. As Immers (1957) reported, there was a reduction in the intensity of cytoplasmic PAS staining after fertilization. The ova were surrounded by an acid mucopolysaccharide membrane which was stained by alician blue. During the early cleavage divisions, this material extended into the cleavage furrows and was presumably continuously elaborated by the dividing cells. After the fourth cleavage (16 cells), a quartet of micromeres are produced by unequal cytoplasmic division. These cells later form the primary mesenchyme. At interphase, the cytoplasm of these cells contained a markedly higher concentration of RNA than the other blastomeres (Figure 1). As Agrell (1958) reported, the high concentration of cytoplasmic RNA was no longer apparent after the micromeres entered prophase, and neither nucleoli nor basophilic chromocenters were observed in any of the embryonic nuclei up to this stage. The cytoplasmic concentration of protein demonstrable by mercuric bromphenol blue did not increase in the micromeres and was generally comparable to levels in the macromeres (Figure 2). Mitotic synchrony was also upset at this point in development as may be seen in Figure 2.

The cytoplasmic distribution of RNA, protein and PAS-positive material was uniform within all cells of the prehatching blastulae (4.5 hrs). Local increases in cytoplasmic basophilia were not noted and no nucleoli or basophilic chromocenters were observed within any blastula cell nuclei (Figure 3). In embryos of this stage, interphase nuclei as well as chromosomes were deeply stained by the pH 8.1 fast green method for histones (Figure 4), and this pattern of nuclear staining was maintained throughout the further course of development. A conspicuous reduction in nuclear volume as compared to the volume of early cleavage blastomere nuclei was noted in prehatching blastulae. In the mesenchymal blastulae (8-10 hr.) the vegetal surface was somewhat thickened, and cells of the primary mesenchyme formed a loosely organized cell heap inside the vegetal wall of the blastocoel (Figure 5). Again the usual manifestations of new RNA synthesis were absent; neither nucleoli nor cytoplasmic accumulations of basophilic material were observed in any cells at this stage of development. The cytoplasm of the primary mesenchymal cells appeared to be somewhat deficient in basophilic material (Figure 6).
Protein and PAS positive material were also uniformly distributed within the cytoplasm of all cell types in the mesenchymal blastulae.

In early gastrulae (ca. 12 hr.) the acid mucopolysaccharide membrane which surrounded the whole embryo up to this point was carried inward with the invagination and lay in contact with the gastrocoel surface of the endoderm. Nucleoli were observed for the first time in development in all nuclei. Usually two small basophilic nucleoli were present in each nucleus (Figure 7). The cytoplasm of the primary mesenchyme cells was more uniformly basophilic than in the mesenchymal blastuae, indicating an increase in cytoplasmic RNA concentration. Cytoplasmic concentrations of protein and PAS positive material were not detectably altered at this point in development. There was, however, considerable mitotic activity in the early gastrula, probably contributing to the consolidation and elaboration of the invaginated material. Coincident with the construction of the larval skeleton in the prism gastrula (ca. 18 hrs.) cytoplasmic concentrations of protein in primary mesenchymal cells increased (Figure 8). There was also a coincident increase in PAS positive material in these cells (Figure 9) suggesting the elaboration of mucoprotein, probably related in the construction of the larval skeleton. Cells of all three primary layers were still either cuboidal or rounded, contained uniformly basophilic nuceloli within their nuclei, and displayed about the same level of cytoplasmic basophilia as encountered in the early gastrulae.

In the 24 hour plutei, the cytoplasm of primary mesenchymal cells was packed with PAS positive material (Figure 10). This material was not stained by alician blue. Cytoplasmic protein concentrations were also elevated in these cells (Figure 11), whereas cytoplasmic protein levels in the invaginated endoderm and endomesoderm were considerably reduced. During the next 24 hours, the arms of the plutei were extended and considerable morphological differentiation of cell types was accomplished. Cells became more extended and lost the cuboidal or rounded shape characteristic of morphologically undifferentiated embryonic cells as they took part in the formation of larval tissue.Some of the mesenchymal cells assumed the shape of fibroblasts, while cells lining cavities were more flattened. Some of the mesenchymal cells still contained large PAS positive inclusions (Figure 12). Neucleoli were present in nuclei of cells of all primary layers and the general impression was one of continuing synthetic activity at 48 hours.

Developmental events up to the 48 hour pluteus were essentially identical in both Lytechinus and Melita.

Discussion:

Using biochemical methods, Backstrom (1959) reported that a pulse of synthesis of new RNA precedes hatching and that another occurs during gastrulation in sea urchin development. Cytologically, the usual indications of RNA synthesis were absent in echinoderm development up to the gastrula stage, and it is consequently necessary to consider the possibility that RNA synthesis may occur in development before nucleoli are actually formed. Recent cytochemical studies of Wallace (1962) on anucleolate Xenopus embryos indicate that some RNA synthesis does occur in these embryos. Further studies on the incorporation of tritiated uridine into the RNA of developing embryos of the tunicate, Ascidia nigra, indicated that limited RNA synthesis occurred in the late pre-hatching larvae, while nucleoli did not appear until metamorphosis was initiated (Cowden, unpublished observations). Consequently, it appears that some RNA synthesis may occur in development at stages prior to the appearance of nucleoli. It should be pointed out, however, that in the two species in which the developmental effects of the anucleolate condition have been studies; i.e., Xenopus (Wallace, 1962) and Chironomus (Beermann, 1960), the condition was eventually lethal. These considerations, nevertheless, form a compatible explanation for Ficq et al. (1963) and Backstrom's (1959) findings and those reported in the present study concerning the synthesis of RNA prior to the appearance of nucleoli in post blastulae. An animal-vegetal gradient of RNA distribution in preblastulae such as that reported by Markman (1957) was not observed.

Upon division of the eight cell stage, a quartette of micromeres are produced which eventually give rise to the primary mesenchyme. This is a determinate step in cleavage since Horstadius (1939) has demonstrated that the balance of the embryo is incapable of regulation if the micromeres are removed. Micromere formation is also attended by a break-down in mitotic synchrony and an animal-vegetal mitotic gradient is established. In development of the snail, Nassarias (Ilynassa) which is determinate, the posterior blastomeres which eventually produce mesodermal structures, contain a higher RNA concentration than the anterior blastomeres (Collier, 1960). However, it is difficult to place the pronounced increase in cytoplasmic RNA concentration in echinoderm micromeres in the same category. As Agrell (1958) first reported, and as this study has confirmed, this increase in cytoplasmic RNA concentration is transitory and does not persist beyond prophase of the next division. It is not accompanied by a corresponding increase in protein which, according to Rasch and Woodard (1959), should occur since RNA usually exists in the form of a conjugated nucleoprotein. Agrell (1958) suggested that this differential segregation of RNA into micromeres prior to the division of vegetal blastomeres between the third and fourth cleavage may have come about by some form of cytoplasmic condensation. From a developmental point of view this would implicate ooplasmic segregation as a mechanism by which selective movement of cytoplasmic material could endow daughter blastomeres with different developmental specificities. This could provide a chemical basis for determinate development. It is, however, not possible to draw this inference from the data now available. The possibility of selective synthesis of RNA in the micromeres seems to be ruled out by Ficq et al. (1963) who only found evidence of nuclear RNA synthesis in preblastulae, probably "messenger RNA".

Telephoros (1959) reported that histones and protamines were present in the cytoplasm of sea urchin ova, and that interphase nuclei were not stained by Alfert and Geschwind's (1953) pH 8.1 fast green method in cleavage nuclei. Staining of interphase nuclei did not occur until later in development, and at this point staining disappeared. Similar reports have appeared concerning the tinctorial behavior of histones in the development of the snail, Helix (Bloch & Hew, 1960 and 1961), and Horn (1962) has reported the same pattern of histone distribution in amphibian development. Horn (1962) particularly noted that condensed cleavage chromosomes were stained by the pH  8.1 fast green method while interphase nuclei were not stained. The results of the current investigation support these general observations, but strong cytoplasmic staining in ova and cleavage embryos was not observed. Cleavage nuclei were very faintly stained but this could have been due to a concentration effect as recently suggested by Moore (1963). Huang & Bonner (1962) have proposed that histones may function in differentiation by blocking specific sites in the DNA molecule and suppressing the activity of these specific loci. This suggests that the unstained interphase nuclei may have unrestricted developmental capacities which become restricted as soon as histone appears in the nucleus. While staining of condensed chromosomes during cleavage should be relatively unimportant to their conception of histone function since DNA is metabolically inactive during mitosis, it does indicate that chromosomal histone is present in early cleavage. The possibility that histone is diluted in the larger cleavage nuclei should not be ignored. As a corollary to chemical differentiation of the nuclei, nuclear volume decreases to a considerable extent during the course of differentiation. This concentrates the chromophores used in demonstrating histones and increases their sensitivity to visual observation.

The observations concerning intracellular protein distribution during echinoderm development failed to indicate any striking alterations during the early course of development. In the prism gastrula and 24 hour pluteus, there was an obvious increase in protein concentration in primary mesenchyme cells which at these stages were actively engaged in the construction of the larval skeleton and larval connective tissues. Similarly with the differentiation of the larval endoderm in preparation for the commencement of feeding, there was a noticeable decrease in the cytoplasmic protein concentration. According to Markman (1961) and Guidice et al. (1962) who studied the incorporation of labeled amino acids into developing sea urchin proteins, protein synthesis follows or coincides with stages in which RNA synthesis is demonstrable. Kavanau's (1954) biochemical study of protein synthesis in sea urchin development supports this general assumption which is in agreement with current notions of the role of RNA and protein synthesis in development (see Brachet, 1957).

Using S ³⁵, Immers (1961 b) demonstrated that the sea urchin synthesized sulfated mucopolysaccharides to a considerable extent, particularly in the primary mesenchyme in the stages immediately preceding and during larval skeletal formation. Immers (1961 c) also reported that while staining of the mesenchymal mucopolysaccharides with basic metachromic dyes was "masked",  the incorporation of S ³⁵ into these mucopolysaccharides implied that they were sulfated and would normally react with basic metachromatic dyes. In his studies and the present one, only the ectodermal cortical membranes were stained by alician blue at low pH or by basic metachromatic dyes. Since PAS staining of the mesenchymal mucopolysaccharide was quite intense, it is possible that the SO₄ groups of the acid mucopolysaccharides may have been blocked by basic proteins. This represents an instance where the usual histochemical criteria for differentiating between acid and neutral mucopolysaccharides have broken down, and points to the importance of using independent methods to assess the validity of tinctorial cytochemical methods when these are available.

Summary:

1. The initial cytological manifestations of new RNA synthesis ( nucleoli and increased cytoplasmic basophilia) were first observed in early gastrula. Since previous chemical studies by others have indicated that RNA synthesis  occurs in the prehatching blastula, and since it has been demonstrated that RNA synthesis can occur in anucleate Xenopus larvae, it is suggested that limited RNA synthesis during development may take place before nucleoli are formed.

2. A considerable increase in the cytoplasmic concentration of RNA in micromeres formed after the fourth cleavage (16 cells) was observed; this was restored to normal by the ensuing prophase and was not accompanied by a corresponding increase in cytoplasmic protein concentration. This may occur by some form of cytoplasmic segregation of RNA, or less probably by de novo synthesis of RNA. The significance of these two possibilities are discussed.

3. Cleavage interphase nuclei were weakly stained by a method for histones but condensed (mitotic) chromosomes were clearly stained. In prehatching blastulae and all succeeding stages, interphase nuclei as well as condensed chromosomes were deeply stained by a method for histones.

4. The cytoplasmic concentration of protein was uniform in all cells up to the prism gastrula stage. Primary mesenchyme cells then accumulated cytoplasmic protein and levels were further increased in the 24 hour pluteus. Endodermal cells, however, displayed a lower cytoplasmic concentration in the 24 hour pluteus.

5. After gastrulation, cells of the primary mesenchyme progressively accumulated PAS positive materials. It was presumed that this material was elaborated in conjunction with the construction of skeletal elements. While it was not stained by methods for acid mucopolysaccharides, autoradiographic evidence (Immers, 1961 b & c) indicated that these are sulfated.

Acknowledgments:

We should like to acknowledge the assistance of Dr. W. H. Sutcliffe, director of the Bermuda Biological Station, for his many courtesies during our stay in Bermuda, and the technical assistance of Miss Yolanda Pagen and Mrs. Sally Borelli. Our thanks also to Dr. E. Lowe Pierce, director of the University of Florida Sea Horse Key Marine Biological Laboratory, for his assistance in obtaining sand dollars.

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