Studies on the development of melanophores in in vitro cultured amphibian neural plates

Various parts of neural plates of Japanese newt (Cynops pyrrhogaster) neurula embryos were cultured alone in drops of culture media (Niu-Twitty's balanced salt solution or modified Leibovitz L-15 medium) with or without fetal calf serum (FCS). Although none of the parts gave rise to melanophores in a medium without FCS, some produced melanophores in a medium with FCS. The localization of melanophore-producing areas in the neural plates corresponded to that of Tada's (1944) findings. The assumption that FCS affects survival and development of melanophores is excluded, because neural fold cells do not require FCS to develop into melanophores. Therefore, there may be in FCS some factor which acts on the specialization of neural plate cells into melanophores. The results of this experiment suggest that this factor may be heat labile. The findings also indicate that FCS does not induce melanophores in gastrula ectoderm, but only affects neurula neural plate cells so as to give rise to melanophores.     

The spermatogenesis of ichthyophis glutinosus (Linn.)

Summary The primary spermatocytes which are the products of division of the spermatogonia embark on the prophase of the first division after a brief period of rest. The leptotene filaments are formed soon after and the polar orientation of the filaments results in a leptotene bouquet. Parallel conjugation of the leptotene filaments gives rise to the thicker pachytene threads, the conjugation beginning at the proximal pole. When the fusion between the apposing filaments is more or less intimate, the polarisation is lost and immediately after, splits appear along the threads at intervals, giving rise to the diplotene stage. The diplotene is followed by a pronounced and conspicuous diffuse stage where all individuality of the chromosomes is temporarily lost, the nucleus itself becoming a faintly staining reticulum, in its greatest development. Soon, the chromosomes emerge from the diffuse mass and the bivalents which now appear more or less clear, twist about each other giving rise to the strepsinema. A condensation and contraction of the chromosomes give rise to the diakinesis where 21 deeply staining tetrads of different forms are seen arranged peripherally inside the nuclear membrane. The spindle is formed between the centrioles, the nuclear membrane is lost and the tetrads take their place on the spindle. 21 tetrads can be clearly seen in the metaphase plates of the division, the four components of each tetrad being seen clearly for the first time in anaphase. A conspicuous interkinesis separates the first and second divisions, when the nucleus resumes the appearance of a faintly staining network. The second division follows quickly separating each dyad into two monads. 21 dyads are seen in the metaphase plates of this division while 21 monads can be counted in the anaphase plates. The resulting cells are the spermatids.     

Development of the cell covering in the dinoflagellate Scrippsiella hexapraecingula (Peridiniales, Dinophyceae)

The organization and development of cell coverings in two alternate phases of the life cycle in a marine dinoflagellate, Scrippsiella hexapraecingula Horiguchi et Chihara, were investigated by thin sectioning and freeze‐fracture electron microscopy. In one of these phases, the motile phase, cells have an outermost plasma membrane that is lined with flattened amphiesmal vesicles. Groups of microtubules lie beneath these vesicles. In mature motile cells, thecal plates are completely enclosed in individual amphiesmal vesicles. After settling, the cells enter the second, non‐motile phase. Here, ecdysis occurs, resulting in several steps including formation of the first pellicle layer (PI), fusion of the inner amphiesmal vesicle membranes to form the new plasma membrane, deposition of the second pellicle layer (PM) under PI, and the appearance and fusion of juvenile amphiesmal vesicles to form new territories, which eventually give rise to new thecal plates in the next motile phase. Thus, the pattern in which thecal plates are arranged in motile cells is determined at the time when the amphiesmal vesicles develop into non‐motile cells.     

Somatic chromosomal reduction in yeast

Summary An abnormal type of segregation by which endopolypoid cells give rise to buds having the normal diploid complement of two chromosomes, is described in five-day old fermenting liquid cultures.Microphotographs are presented to show that the above phenomenon is not confined to liquid cultures alone but occurs also in endopolyploid cells from agar slants.It is suggested that the mode of segregation described above is a type of somatic reduction in chromosome number by which the yeast strain can survive in an unfavourable environment wherein the population consists predominantly of endopolyploid cells of varying complexity.     

When 2+2=5: the origins and fates of aneuploid and tetraploid cells

Aneuploid cells are frequently observed in human tumors, suggesting that aneuploidy may play an important role in the development of cancer. In this review, I discuss the processes that may give rise to aneuploid cells in normal tissue and in tumors. Aneuploid cells may arise directly from diploid cells through errors in chromosome segregation, as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint. A second route to formation of aneuploid cells is through a tetraploid intermediate, where division of tetraploid cells can yield very high rates of chromosome missegregation as a consequence of multipolar spindle formation. Diploid cells may become tetraploid through a variety of mechanisms, including endoreduplication, cell fusion, and cytokinesis failure. Although aneuploid cells may arise from either diploid or tetraploid cells, the fate of the resulting aneuploid cells may be distinct. It is therefore important to understand the different pathways that can give rise to aneuploid cells, and how the varied origins of these cells affect their subsequent ability to survive or proliferate.     

Isolation and Characterization of Bacteriophage T4 Base Plates

A method for isolating bacteriophage T4 base plates from lysates of Escherichia coli B cells infected with the ts mutant in gene 19, ts B31 has been developed. By electrophoresis in polyacrylamide gel with sodium dodecyl sulfate the base plates have been shown to contain five to seven protein components with molecular weights of 36,000, 53,000, 66,000, 81,000, 87,000, and probably about 100,000. Electron microscope studies have demonstrated that base plates may occur in two structural states: in the form of hexagons or stars. Star rays and short fibrils are not radial or elongated and are turned sideways at an angle to the radius. Base plates do not complement in vitro with free tail cores isolated after disintegration of particles of the wild-type bacteriophage.     

When 2 + 2 = 5: The origins and fates of aneuploid and tetraploid cells

Aneuploid cells are frequently observed in human tumors, suggesting that aneuploidy may play an important role in the development of cancer. In this review, I discuss the processes that may give rise to aneuploid cells in normal tissue and in tumors. Aneuploid cells may arise directly from diploid cells through errors in chromosome segregation, as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint. A second route to formation of aneuploid cells is through a tetraploid intermediate, where division of tetraploid cells can yield very high rates of chromosome missegregation as a consequence of multipolar spindle formation. Diploid cells may become tetraploid through a variety of mechanisms, including endoreduplication, cell fusion, and cytokinesis failure. Although aneuploid cells may arise from either diploid or tetraploid cells, the fate of the resulting aneuploid cells may be distinct. It is therefore important to understand the different pathways that can give rise to aneuploid cells, and how the varied origins of these cells affect their subsequent ability to survive or proliferate.     

Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin

Increasing evidence suggests the existence of osteoclast diversity. Here we investigated whether precursors obtained from marrow of the mandibula or long bone could give rise to phenotypically different osteoclasts. Formation of multinucleated cells was assessed after culturing mouse marrow cells of the two bone types with macrophage colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL) for up to 10 days on plastic, bone or dentin. Two times more osteoclasts formed from long bone marrow cells on bone compared to dentin, whereas higher numbers of jaw osteoclasts formed on dentin. Resorption of dentin or bone was similar for osteoclasts formed from both types of precursors. In contrast to jaw marrow derived osteoclasts, long bone osteoclasts predominantly had a multi-compartmented shape, with at least two nuclei containing compartments per cell. Osteoclasts on bone contained two times more actin rings than osteoclasts on dentin, regardless of their precursor origin. However, the area per osteoclast covered by actin rings was similar (20%) for both substrates. This study suggests that marrow cells obtained from different bones give rise to different osteoclasts. The substrate on which the osteoclasts are generated plays a role in steering their formation rather than their resorption.     

Fates of the blastomeres of the 32-cell-stage Xenopus embryo

A detailed fate map of all of the progeny derived from each of the blastomeres of the 32-cell-stage South African clawed frog embryo (Xenopus laevis), which were selected for stereotypic cleavages, is presented. Individual blastomeres were injected with horseradish peroxidase and all of their descendants in the late tailbud embryo (stages 32 to 34) were identified after histochemical processing of serial tissue sections and whole-mount preparations. The progeny of each blastomere were distributed characteristically, both in phenotype and location. Most organs were populated largely by the descendants of particular sets of blastomeres, the progeny of each often being restricted to defined spatial addresses. Thus, the descendants of any one blastomere were distinct and predictable when embryos were preselected for stereotypic cleavages. However, variations among embryos were common and the frequencies with which one may expect organs to contain progeny from any particular blastomere are reported. The differences in the fates of the 16-cell-stage blastomeres and their 32-cell-stage daughter blastomeres are outlined and can be grouped into three general categories. The two daughter cells may give rise to equal numbers of cells in a particular organ, one daughter cell may give rise to many more of the cells in an organ derived from the mother blastomere, or one daughter cell may give rise to all of the progeny in an organ derived from the mother blastomere. Thus, cell fates are segregated during cleavage stages in both symmetric and asymmetric manners, and the lineages exhibit a diversification mode (G. S. Stent, 1985, Philos. Trans R. Soc. London Ser. B 312, 3-19) of cell division.     

Poisson-like Fluctuation Patterns of Revertants of Leucine Auxotrophy (Leu-500) in Salmonella Typhimurium Caused by Delay in Mutant Cell Division

Leu(+) mutants from Salmonella typhimurium leu-500 strain MA412 arise at high frequencies and mutant colonies appear over a broad range of time on selective plates. This observation suggested that these Leu(+) mutants might be induced or ``directed.' If such a mechanism was responsible, mutants should originate on selective plates rather than in the preceding culture in nonselective conditions and should give rise to Poisson-like fluctuation curves upon plating of sister cultures on selective medium. Poisson-like distribution profiles were indeed observed for Leu(+) mutants of S. typhimurium MA412. However, an explanation for the observed Poisson-like fluctuation patterns without a need for selection-induced mutations was found. Microscopical analysis and cell mass/viable count measurements showed that the size of Leu(+) mutant cells was often much larger than those of nonmutants. This size difference was a stable characteristic of a large proportion of Leu(+) mutants, was observed both in stationary and growing culture and did not measurably affect the division rates of the cells in nutrient broth. As the transition from normal-sized nonmutant to oversized mutant cells during the nonselective culture phase of the fluctuation experiment may have been accompanied by a period with no or few completed cell division cycles, the number of mutant offspring may have been smaller than that of sibling nonmutants. Such underrepresentation of mutants in the final culture is expected to give rise to Poisson-like fluctuation patterns without invoking ``directed' mutations.     

Microtubule and actin filament organization during acentral divisions in potato suspension culture cells

Summary Microtubule and filamentous(F)-actin organization in the potato suspension culture line HH260 was studied by fluorescence microscopy in double-labelled cells. During interphase, microtubules and F-actin were randomly arrayed in isodiametric cells but were aligned transversely to the direction of growth in elongated cells. Microtubules and F-actin coaligned in preprophase bands which were, however, comparatively rare and diffuse. Interestingly, more than half of the cells in telophase contained phragmoplasts that were either horseshoe-shaped or straight, instead of being round. We traced the cause of this difference to preprophase, where misplaced nuclear localization away from the central axis of cells may give rise to acentrally placed spindles and, subsequently, to acentrally placed phragmoplasts and cell plates. Further, we hypothesize that it is the uneven fusion of the expanding cell plates with the parent plasma membrane, and the accompanying depolymerization of those parts of the phragmoplasts, that gives the incomplete phragmoplasts observed.Abbreviations DAPI 4,6-diamidino-2-phenylindole - MBS 3-maleimidobenzoyl-N-hydroxy-succinimidester - PMSF phenyl-methylsulfonyl fluoride - SB stabilization buffer     

Migrating neural crest cells in the trunk of the avian embryo are multipotent

Trunk neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. Previously, we demonstrated that many premigratory trunk neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. The results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after leaving the neural tube either during their migration or at their sites of localization. Here, we test the developmental potential of migrating trunk neural crest cells by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells as they migrate through the somite. By two days after injection, the LRD-labelled clones contained from 2 to 67 cells, which were distributed unilaterally in all embryos. Most clones were confined to a single segment, though a few contributed to sympathetic ganglia over two segments. A majority of the clones gave rise to cells in multiple neural crest derivatives. Individual migrating neural crest cells gave rise to both sensory and sympathetic neurons (neurofilament-positive), as well as cells with the morphological characteristics of Schwann cells, and other non-neuronal cells (both neurofilament-negative). Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. Our data demonstrate that migrating trunk neural crest cells can be multipotent, giving rise to cells in multiple neural crest derivatives, and contributing to both neuronal and non-neuronal elements within a given derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of cells combining features of two different cell types in the colonic crypts and pyloric glands of the mouse.

A small number of epithelial cells which combine features of two cell types were observed in the descending colon and pyloric stomach of the mouse. In the descending colon, where the base of the crypts is mainly composed of poorly differentiated "vacuolated" cells, a few of these cells contain, besides the characteristic "vacuoles," mucous globules identical to those in mucous cells or, less frequently, dense granules such as are found in entero-endocrine cells. Because there is evidence that the poorly differentiated vacuolated cells give rise to the other cells of the epithelium, those which also contain mucous globules or dense granules are likely to be differentiating into mucous cells or entero-endocrine cells respectively. In the pyloric stomach, where the glands are mainly composed of mucous cells, some of which are poorly differentiated, a few of the latter exhibit, besides the mucous globules, entero-endocrine type granules or features of caveolated cells. It is likely that the poorly differentiated mucous cells give rise to the other gland cells; and, therefore, those mucous-containing cells which also display dense granules or caveolated cell features are taken to be differentiating into entero-endocrine or caveolated cells respectively. Most of the cells containing two kinds of secretory materials are believed to be stem cells which initially contain a few vacuoles (colon) or mucous globules (pylorus) but are differentiating into a cell containing a different type of secretion. Rare observations of two kinds of secretory materials in a mature cell suggest that the transitional period may be prolonged, perhaps indefinitely.     

Rod photoreceptor development in vitro: intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina

We describe a reaggregated cell culture system in which retinal neuroepithelial cells from embryonic rats proliferate extensively and give rise to rod photoreceptors on the same schedule in vitro as they do in vivo. Both the proliferative potential of the embryonic neuroepithelial cells and the timing of their differentiation into rods are not changed by the presence of a 50-fold excess of neonatal neural retinal cells, although many more of the embryonic cells develop into rods in these circumstances. In such mixed-age cultures, dividing neonatal cells proliferate much less and give rise to rods much sooner than do dividing embryonic cells, suggesting that the dividing cells at the two ages are intrinsically different. These and other findings suggest that both cell-cell interactions and an intrinsic program in neuroepithelial cells determine cell fate in the developing rat retina.     

Morphology and ultrastructure of the organ of Bellonci in the marine amphipod Gammarus setosus

The three-dimensional structure of the organ of Bellonci in the marine amphipod Gammarus setosus and the relationship between its sensory cells and concretion are described using light, transmission, and scanning electron microscopy, with chemical treatment for cell lysis, calcium chelation, glycogen staining, and lanthanum labelling. The organ is encapsulated and has three units called fuselli. Each is enclosed by two fusellar cells which generate and release calcium granule strands into the cores of the fusellar concretions, which are united in the center of the organ. The surface of each fusellus is traversed by spiral dendrites entering dorsally and ending ventrally. The spiral dendrites arise from sensory neurons contained in a palm-shaped ganglion in the center of the capsule, beyond which they are twisted like a rope before reaching the concretion. The spiral dendrites are linked in pairs by gap and tight junctions and each gives origin to two pairs of 9+0 sensory cilia 30 μm apart. The ciliary distal segments give rise to long tubules which are in contact with the calcium granule strands. The ciliary proximal segments are expanded by many long mitochondria which interdigitate with the branched striated ciliary rootlets. The concretion is suspended in the capsule cavity by axons originating from four neurons of a remote mechanoreceptor. The structure of the organ suggests that it is a sensory organ involved in the reception and integration of a variety of stimuli.     

Evidence for an internal component of the bacteriophage T4D tail core: a possible length-determining template.

The length of the T4 tail is precisely regulated in vivo at the time of polymerization of the tail core protein onto the baseplate. Since no mutations which alter tail length have been identified, a study of in vivo-assembled tail cores was begun to determine whether the structural properties of assembled cores would reveal the mechanism of length regulation. An assembly intermediate consisting of a core attached to a baseplate (core-baseplate) was purified from cells infected with a T4 mutant in gene 15. When core-base plates were treated with guanidine hydrochloride, cores were released from baseplates. The released cores had the same mean length as cores attached to baseplates. Electron micrographs of these cores showed partial penetration of negative stain into one end, and, at the opposite end, a modified tip which often appeared as a short fiber projecting from the core. When cores were purified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two minor proteins and the major core protein were detected. One minor protein, the product of gene 48 (gp48), was present in at least 72% of the amount found in core-baseplates, relative to the amount of the major core protein. These findings suggest that cores contain a fibrous structure, possibly composed of gp48, which may form a "ruler" that specifies the length of the T4 tail.     

Interstitial stem cells in Hydra: multipotency and decision-making

Interstitial stem cells in Hydra constitute a population of multipotent cells, which continuously give rise to differentiated products during the growth and budding of Hydra polyps. They also give rise to germ cells in animals undergoing sexual differentiation. Cloning experiments have shown that interstitial stem cells are multipotent. In vivo tracing of stem cell lineages has revealed that stem cells divide symmetrically to yield two stem cells or asymmetrically to yield one stem cell daughter and one daughter cell which initiates nerve or nematocyte differentiation. Following commitment, some nerve cell precursors migrate from the body column into the head or foot region, thus giving rise to the high density of nerve cells observed in these regions. Stem cell proliferation is regulated by changes in the self-renewal probability and is controlled by stem cell density. Nerve cell commitment is controlled by several peptides including the Head Activator. Factors affecting nematocyte commitment are not known, but wnt and notch signaling are both required for differentiation of committed precursors.     

Wood Anatomy and the Development of Interxylary Phloem of Ipomoea hederifolia Linn. (Convolvulaceae)

In Ipomoea hederifolia Linn., stems increase in thickness by forming successive rings of cambia. With the increase in stem diameter, the first ring of cambium also gives rise to thin-walled parenchymatous islands along with thick-walled xylem derivatives to its inner side. The size of these islands increases (both radially and tangentially) gradually with the increase in stem diameter. In pencil-thick stems, that is, before the differentiation of a second ring of cambium, some of the parenchyma cells within these islands differentiate into interxylary phloem. Although all successive cambia forms secondary phloem continuously, simultaneous development of interxylary phloem was observed in the innermost successive ring of xylem. In the mature stems, thick-walled parenchyma cells formed at the beginning of secondary growth underwent dedifferentiation and led to the formation of phloem derivatives. Structurally, sieve tube elements showed both simple sieve plates on transverse to slightly oblique end walls and compound sieve plates on the oblique end walls with poorly developed lateral sieve areas. Isolated or groups of two to three sieve elements were noticed in the rays of secondary phloem. They possessed simple sieve plates with distinct companion cells at their corners. The length of these elements was more or less similar to that of ray parenchyma cells but their diameter was slightly less. Similarly, in the secondary xylem, perforated ray cells were noticed in the innermost xylem ring. They were larger than the adjacent ray cells and possessed oval to circular simple perforation plates. The structures of interxylary phloem, perforated ray cells, and ray sieve elements are described in detail.     

Reversible, cell-heritable changes during the development of tobacco pith tissues

Cytokinin requiring cells of Nicotiana tabacum L. cv "Havana 425" can be induced in culture to become cytokinin autotrophic. This process is known as cytokinin habituation. Earlier we showed that pith parenchyma tissue consists of inducible cells, which habituate at high rates when treated with cytokinin, and noninducible cells, which remain cytokinin requiring under these conditions. The inducible and noninducible phenotypes are determined states that arise during the development of the tobacco plant and are inherited by individual cells. Here we show that pith tissue of plants regenerated from cloned lines of noninducible cells exhibits the inducible phenotype indicating that noninducible cells, or their descendants, can become inducible. This change in competence for habituation appears to have an epigenetic basis; it is reversible, occurs at high rates, and depends on the developmental state of the cells. The habituated state occurs in two forms that can be distinguished by their difference in developmental potential. Habituated cells derived from inducible pith cells give rise to normal plants whose leaf and pith tissues require cytokinin for growth in culture. In contrast, habituated cells obtained by transferring noninducible cells on media with progressively lower cytokinin concentrations give rise to plants whose leaf and pith tissues exhibit a cytokinin-habituated phenotype in culture.     

Medaka cleavage embryos are capable of generating ES-like cell cultures

Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.