ELECTRON MICROSCOPE STUDIES ON SALIVARY GLAND CELLS I. The Nucleus of Bradysia mycorum

Salivary glands were fixed in cold 1 per cent osmium tetroxide in veronal-acetate buffer containing sucrose and embedded in methacrylate mixture or Araldite. The salivary gland nuclei of sclarids show a continuous production of nucleoli, which remain multiple and not consolidated into a single structure. The earliest recognizable nucleoli, which we call "elementary nucleoli," are aggregations of a few paired 40 A fibrils and a few 150 A particles, at many points within chromosome bands. Further development consists of the detachment of the elementary nucleoli from their points of origin and their subsequent mutual coalescence. As a result, dense patches of nucleolar material are formed which become large nucleoli at the surface of chromosomes, either attached to the band or free. The fully formed nucleoli have a characteristic dual structure with a narrow dense periphery and a broader less dense internum. Fibrils and particles are present in both regions, and the difference in density reflects differences in the packing of the two structural elements. The duality in structure is lost in later stages. The nucleolar fibrils appear to be similar to the chromosomal fibrils. The 150 A particles in nucleoli, chromosomes, and nuclear sap seem identical. The significance of these observations is discussed for nucleologenesis in general. I N T R O D U C T I O N The salivary gland nuclei of sciarids have multiple nucleoli (Fig. 1). Under the light microscope, these nucleoli are Feulgen-negative. Other cytochemical tests (I) reveal that these nucleoli contain two or three forms of ribonucleoproteins (RNP's) ; i.e., perichromosomal R N P and RNPs of the pars amorpha and of the parachromatin of the nucleolus described by other authors (I). The distribution of the ribonucleic acid (RNA) label in autoradiograms of a related sciarid nucleus (2) shows a close correspondence with the distribution of the nucleoli in electron micrographs of the present material. The work presented here deals with the origin, development, and later transformations of the nucleoli at the ultrastructural level, aspects which for nucleoli in general have not received all the attention they deserve. M A T E R I A L A N D M E T H O D S Bradysia mycorum Frey ( = Lycoriella solani (Winn), det. Tuomikoski), was first found in laboratory cultures of the midge Smittia sp. and has been thereafter maintained separately. The larval salivary glands of B. mycorum are strikingly differentiated into a short, 153 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom flattened, anterior portion and a much longer, tubular, posterior portion. As the cells in the anterior portion are much larger, they have been mainly used for the present investigation. The glands were dissected and fixed for 1 to 1}~ hours in cold (4 °) 1 per cent osmium tetroxide, buffered at pH 7.4 with aeetate-veronal (3), containing sucrose (4). After dehydration in ethanol, the tissue was embedded in n-butyl-methyl methacrylate mixture (5) or Araldite epoxyresin (6). Thin (400 to 800 A) and thick (up to 0.25 micron) sections were cut on a Porter-Blum Servall microtome with glass knives, and the sections were mounted on grids with collodion or formvar and carbon films. All Araldite sections were "stained" by floating the grids on a mixture of 1 per cent potassium permanganate and 2.5 per cent uranyl acetate (A. E. G. Dunn, unpublished data). Apart from the addition of uranyl acetate, the technique was as described by Lawn (7). According to Dunn, the uranyl acetate "stains" earlier than the permanganate and this accounts for the better preservation of general structure than in a plain permanganate-treated section. The mixture does not keep and was freshly prepared. The electron microscopes used were Philips E.M. 75 and Siemens Elmiskop I. The electron micrographs were made at original magnifications of 2000 to 20,000 and further enlargements were obtained photographically. O B S E R V A T I O N S In methacry la te -embedded tissue the electron opacity is highest in nucleoli, in termedia te in chromosome bands, and lower in in te rbands and nuclear sap (Fig. 1). In Ara ld i te -embedded and subsequent ly stained tissue, the resolution is generally bet ter and the same order of density prevails