Hormonal mechanisms for differentiation in plant tissue culture

Summary Cells possess extraordinary powers to organize their molecular processes not only to maintain a cell in a given steady state but also to recognize that state during differentiation. Regulation of these organizational forces appears to be under the control of chemical factors, and a hormonal concept of regulation has evolved. Hormones have been considered to act by reacting with a specific target site. This may be part of their mode of action, but I would like to suggest that a hormone enters and becomes part of a total molecular resonance system. In so doing, the entire molecular system of the cell is modified. Of the known plant hormones, the cytokinins, because of their role in experimentally induced cell division and differentiation, serve as a probe of hormonal involvement in differentiation. Cultured somatic cells of tobacco plants can be induced to undergo differentiation by addition of cytokinin and auxin to the medium. Studies of the cytokinin hormones show a series of diverse molecular involvements. The archetype cytokinin, N⁶-(Δ²-isopentenyl) adenosine (i⁶Ado), occurs in some molecular species of tRNA where it plays a vital role in the codon-anticodon interaction of tRNA and m-RNA. i⁶Ado under-goes extensive metabolism in the tobacco tissue. It is either degraded to adenosine or converted to derivatives that possess biological activity. It is perhaps, therefore, more correct to consider the hormone function as being derived from this total metabolic web. The normal somatic cells of tobacco cultures spontaneously change occasionally into an autonomous form that requires no external growth factors. This line of cells synthesizes i⁶Ado. The metabolic web of the hormone-dependent strain can be perturbed by added auxin but such is not the case in the autonomous strain. These data provide some insight into the altered state of cytokinin activity in which a cell line changes into an autonomous form. Curiously, in become independent of the requirement for exogenous cytokinin, the autonomous tissue becomes sensitive to added cytokinin. i⁶Ado also inhibits the growth of lines of mammalian cancer cells grown in culture. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Fine Structure of Haemoproteus columbae Kruse During Differentiation of the Ookinete*

Ookinete differentiation begins in vitro～1 hr after blood infected with mature gametocytes of Haemoproteus columbae is withdrawn from a pigeon. In the undifferentiated zygote, dense material accumulates at the point under the plasma membrane. The conoid and conoidal rings condense from this material. The nucleus is drawn out to a point with the intranuclear spindle (INS) at the peak. Atypical centrioles lie under the forming conoid in the cytoplasm next to the INS. Fibrous material under the inner membrane forms the polar ring from which subpellicular microtubules originate. One hr later the centrioles have disappeared and the nucleus has returned to the center of the organism. The conoidal complex forms the tip of a growing cytoplasmic projection, the anterior end of the ookinete. During this time an elaborate pellicle is differentiating antero-posteriorly; crystalloid formation begins with an extensive proliferation of rough endoplasmic reticulum (ER) continuous with the outer membrane of the nuclear envelope. Crystalloid particles are formed between the lamellae of the ER and collected in a sphere that is later partially surrounded by a small amount of ER. Ookinetes, differentiated 2 hr longer than the ookinetes in vitro, were obtained from the gut of the pigeon fly, Pseudolynchia maura. The differentiated pellicle of these ookinetes consists of a plasma membrane, an inner membrane layer composed of 2 appressed membranes, and in the anterior end, an electron-opaque lamina immediately under the inner membrane. Anterior to the polar ring, this lamina forms a canopy which, posteriorly, is drawn out into projecting ribs which diminish and disappear in the first third of the organism. Fifty to 60 subpellicular microtubules insert on the polar ring. Ookinetes differentiated in vitro were no more than 4 hr old. They lacked micronemes and retained a pellicular cytostome and “internal cytostomes.” The differentiation of micronemes probably occurs at a later time because they are visible after 6 hr in ookinetes in the fly gut. So many degenerating organisms appeared in vitro after 5 hr that this material was discarded.     

Integument and sensory nerve differentiation ofDrosophila leg and wing imaginal discs in vitro

Summary An ultrastructural analysis is presented of the cuticular and neural structures formed by the prothoracic leg and wing imaginal discs of maleDrosophila melanogaster larvae during culture in vitro with 0.2 g/ml of -ecdysone. A pupal cuticle, and subsequently an imaginal cuticle with a well-defined epicuticle and a laminated endocuticle is formed. The ultrastructure of the epidermis and of cuticular structures such as bristles, trichomes, apodemes, and tracheoles is very similar to that found in situ. Dendrites and nerve cell bodies are formed in vitro, and sensory axons form nerve bundles similar to those of normal appendages in situ, despite their isolation from the central nervous system. It is concluded that at the ultrastructural level, differentiation in vitro closely parallels the normal course of development.     

The role of nerve cell density in the regulation of bud production in hydra

Summary The role of nerve cell density in the regulation of bud production in hydra was examined. Animals with different rates of bud production were produced by altering the temperature, population density and illumination of their cultures. When the distribution of cell types was examined in animals with different rates of bud production, the density of nerve cells in those animals was found to be correlated with their rate of bud production. Transfer of animals from one environment to another resulted in immediate changes in the rate of differentiation of large interstitial cells into nerve cells. This suggests that the density of nerve cells may play a role in regulating the rate of bud production in hydra.     

Endopolyploidy inHelianthus annuus (Asteraceae), A scanning cytophotometric study

Endopolyploidy has been detected in some varieties ofHelianthus annuus L. (Asteraceae/Compositae) by means of scanning photometry of Feulgen-stained nuclei and analysis of nuclear structure. In the hypocotyl cells of seedlings, ploidy levels reach respectively 8 C and 16 C in different varieties, in the root cells 8 C and 16 C; in the cotyledons of ripening seeds 4 C to 8 C values have been found, while all nuclei of the inflorescence axis of one variety exhibit a DNA content of 4 C.—This is the first report of endopolyploidy in a non-succulentAsteraceae species. The characteristic distribution of the endopolyploidy levels in different varieties suggests a strong genetic and/or hormonal control of the final nuclear DNA content in differentiated cells.     

Alkaloids of callus tissues and redifferentiated plantlets in the papaveraceae

The callus tissues from 11 representative species of the Papaveraceae and the redifferentiated plantlets from four species were successfully derived and maintained. The alkaloids in the callus tissues and redifferentiated plantlets were examined in comparison with those of the original plants. All the callus tissues are similar in their alkaloid chemistry and contain benzophenanthridine, protopine and aporphine type alkaloids. By contrast, the plantlets have a more specific alkaloid pattern, being similar in content to the original plants.     

Membrane-fusions and cytoplasmic bridges in the cells of the developing cerebellum

Summary In the developing cerebellum of the neonate rats membranefusions and cytoplasmic bridges between cells were observed. These membrane-fusions were characterized by the presence of loops of membrane and cytoplasmic bridges between the two limits of the membrane-fusions. They were found between Purkinje cells, Purkinje cells and the migratory cells, mitotically potent cells of the external granular layer, and differentiating granule cells of the internal granular layer. The membrane-fusions were found to be a transient developmental phenomenon. Issues pertaining to the universality of membrane-fusions, their significance in the induction for cell differentiation, and the problem of fixation artifacts are discussed.This research was supported by N.I.H. Research Grants No. NS-08817 and CA-14650. Assistance of Mrs. Kunda Das in various aspects of electron microscopy is gratefully acknowledged     

Fetal mouse lung in circumfusion system cultures

Summary Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderately dense secretory granule in the center of the whorl of the endoplasmic reticulum. This work supported by Grant HL19684 from the National Heart and Lung Institute, National Institutes of Health. Pregnant Strong A mice were kindly supplied by Dr. Henry Browning of the Department of Anatomy.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.