Howard Holtzer

Formation of arrowhead complexes with heavy meromyosin in a variety of cell types.

The parasitic protozoan Toxoplasma gondii has been examined with the electron microscope in order to study the fine structure and the formation of the membranes surrounding the cell. The study of the ultrastructure of the membranes covering the parasite shows the existence of a three-membraned complex. Only the outer membrane is considered to be the plasma membrane; the two membranes below it form an inseparable whole of changeable molecular architecture (modifications in appearance depending on the methods of fixation, local differentiation). During reproduction, which takes place by fission or more often by endogeny, the membranes of the daughter individuals are formed from the membranes of the parent. At first the middle and inner membranes of the parent extend, separating the cytoplasm of the daughter cells from that of the parent. The three-membrane complex of the endozoites is completed at the time of their liberation; the external membrane of the parent covers the leaving endozoites; thus, the plasma membrane of the daughter cells derives also from that of the parent. These findings on the origin and role of limiting membranes during reproduction differ entirely from those described so far for other cells.

DNA synthesis and myogenesis

Abstract Differentiating muscle cells synthesizing myosin, the meromyosins, and actin do not concurrently synthesize DNA. Presumptive myoblasts which synthesize DNA do not concurrently synthesize myosin, the meromyosins or actin. The multinucleated skeletal muscle fiber is the product of cell fusion.

Intermediate filament proteins in the developing chick spinal cord.

Abstract The distribution of different intermediate filament (IF) proteins in the embryonic chick spinal cord was examined at several stages of development using immunohistochemical techniques, analytic gel electrophoresis, and electron microscopy. We have found that: (1) the fibroblast-type IF protein (vimentin) is present in virtually all of the replicating neuroepithelial cells of the early neural tube, as well as in radial glia, astrocytes, and Schwann cells in later stages of development; (2) the fibroblast-type IF protein is not detectable in definitive neurons; (3) the neurofilament proteins are first detectable in postmitotic neuroblasts at about the time of initial axon formation and they are restricted to neurons; (4) the astrocyte-type IF protein (glial fibrillary acidic protein) is in definitive astrocytes, but not in radial glia; (5) the prekeratin proteins are restricted to cells of the leptomeninges; and (6) the muscle-type IF protein (desmin) is restricted to vascular tissue in and around the developing spinal cord. These findings suggest that the fibroblast-type IF protein is the only IF protein in the early neuroepithelial cells and that the progeny of these cells will follow one of three different patterns of IF protein expression: (1) continued expression of only the fibroblast-type IF protein (radial glia); (2) expression of both the fibroblast-type IF protein and the astrocyte-type IF protein (astrocytes); or (3) expression of only the neurofilament proteins (neurons).

Chapter 6 The Cell Cycle, Cell Lineages, and Cell Differentiation*

Publisher Summary This chapter presents a general model for cell speciation based on the central roles of DNA synthesis and cell cycle. Two phases of differentiation can be recognized. The first involves the rapid commitment of cells to their respective lineages during the early cleavage stages, and in this regard there is no basic difference between the regulative and mosaic systems. The end product of the early cleavage stages is the “definitive stem cell.” The definitive stem cell retains the capacity to yield the daughter cells that are either replicate stem cells or terminally differentiated cells. The second phase of differentiation involves the biological conditions either in vivo or in vitro that manipulate these stable stem cells for growth, for morphogenesism, and for maintaining populations that turn over. During the early cleavage stage, these two phases are usually separable temporally. It is likely that they will prove to be separable mechanistically as well.

The relationship of cell division to the acquisition of adrenergic characteristics by developing sympathetic ganglion cell precursors

Abstract The relationship of the acquisition of defining characteristics by precursor cells of sympathetic ganglia to the withdrawal of these cells from the cell cycle was investigated in the developing chick. The characteristics studied included the ability to synthesize catecholamines (CA), the development of characteristic subcellular storage granules, and the specific uptake of norepinephrine (NE). All were present in presumptive sympathicoblasts and adrenal medullary precursors, which also became labeled after the injection of tritiated thymidine and so retained the ability to divide. These dividing CA-containing cells were found in both primary ganglia and secondary preand paravertebral ganglia. The developing sympathetic neuronal population was found to be a heterogeneous one. Some sympathetic precursor cells appeared to become postmitotic (or to enter a pause in division) early in ontogeny, while others continued to divide throughout the time of hatching. As embryogenesis proceeded, the proportion of CA-containing cells or their precursors which were dividing decreased. However, those cells which did divide probably divided repeatedly. It is concluded that some of the definitive characteristics of mature neurons are expressed by dividing precursor cells. The specific characteristic that marks the transition from immature dividing cells to mature postmitotic neurons has not yet been determined.

5-Bromodeoxyuridine: Effect on Myogenesis in vitro

Presumptive myoblasts, obtained by treating muscle from 11-day chick embryos with trypsin, multiply in vitro. On the 4th or 5th day in culture they abruptly fuse, form long multinucleated myotubes, and begin to synthesize myosin. Cultured cells exposed to 5-bromodeoxyuridine incorporate this analog of thymidine into their DNA. Cells with such falsified DNA are reversibly inhibited from forming myotubes and synthesizing myosin; such cells, however, continue to synthesize the various species of molecules required for cell multiplication.

An experimental analysis of the development of the spinal column: VI. Aspects of cartilage induction☆

Abstract Both the embryonic spinal cord and notochord induce the formation of cartilage in a population of chick somite cells. This inducing capacity is relatively specific—most other living and fixed tissues, including living and fixed cartilage, are ineffective. The cartilage promoting activity of the spinal cord appears to be mediated by a factor transmissible through mesenchymal or muscle tissue and through a millipore filter. The cartilage promoting activity of the notochord acts more locally and appears to be associated with the notochordal sheath. The time for the passage of the spinal cord cartilage promoting factor through the millipore filter is approximately eight hours.

Lineages, quantal cell cycles, and the generation of cell diversity

Most theories of determination or differentiation assume that embryonic cells differ from mature cells. Embryonic cells are thought to have metastable control mechanisms. These labile controls are believed to become progressively more stabilized as the cells differentiate. Zygote, blastula, neural plate, limb bud, somite, or ‘stem’ cells are conceived of as undifferentiated, totipotent, or multipotential cells. As such, these cells supposedly have available for activation a larger repertoire of phenotypic programmes than their progeny. A necessary corollary to this view is that the activation of one particular phenotypic programme out of the many available is a function of instructive exogenous inducing molecules.

Fibronectin alters the phenotypic properties of cultured chick embryo chondroblasts.

The state of chick embryo chondroblasts in culture was found to be sensitive to both fibronectin and another substance(s) (activity A) which could be extracted from chick embryo fibroblasts with 1 M urea or from conditioned medium. In the presence of either of these activities at concentrations of 25-150 micrograms/ml, chondroblasts, which normally grow as mixed cultures of floating and adherent cells, all immediately became attached to the tissue culture dish and spread. After several days, the morphology of these typically epithelioid cells became fibroblastic. This did not involve a selection process, since the effect was reversible. The synthetic program of these cells was also dramatically modified: the cultures no longer synthesized the chondroblast-unique type IV sulfated proteoglycan and began synthesizing alpha 2 collagen chains typical of fibroblastic or early limb bud cells. Fibronectin was resolved from activity A by gelatin affinity chromatography or gel filtration. Both activities were trypsin-sensitive. The two activities differed, however, on the basis of how the protein fractions in which they were found migrated in SDS-polyacrylamide gels, their specific activities and their effects on cell morphology and cell growth.

Transformation of chondroblasts by Rous sarcoma virus and synthesis of the sulfated proteoglycan matrix.

The presence of the extracellular matrix synthesized by chondroblasts provides a barrier to virus penetration. Chondroblasts can be infected and transformed following treatment with proteolytic enzymes. Using a temperature-sensitive transformation mutant of Rous sarcoma virus and rearing the cells at permissive temperature, we demonstrate that transformed chondroblasts stop synthesizing their cell-unique sulfated proteoglycan. If such transformed chondroblasts are shifted to nonpermissive temperature, the cells reinitiate the synthesis of their cell-unique sulfated proteoglycan.