Differentiation occurs when cells change in structure and function within a period of time. As such, one of the major problems in research is the factor of time. Cells must be "competent" or primed for a differentiating change by being properly "induced" by some internal influence (gene, ion flux, metabolic alteration) or external factor (hormone, growth factor, cell-cell communication). Finally, they must be in the correct association with neighboring cells (position effect in embryos, contact inhibition or guidance) and strata for the change to occur. All of these factors combine to make an afternoon laboratory session on differentiation difficult to coordinate.
Consequently, the laboratory will use prepared slides of materials that have undergone differentiation, so that the time can be compacted into convenient laboratory periods. We will use some living cells by carefully choosing systems which can be closely monitored (slime mold development) or which are slower to develop and thus able to be "ready" at the time of a lab session (fern gametophytes).
We will observe specific molecular alterations indicating differential gene activity through indirect observation of the changes, primarily through the use of inhibitors of the basic DNA/RNA/Protein system.
A classical approach to the basic process of cellular differentiation has involved the primary formation of the three germ tissues of embryos, namely ectoderm, endoderm and mesoderm. The sea urchin provides readily available egg and sperm which can be fertilized in laboratory with ease. Further , there is little yolk within the egg, and the yolk is evenly distributed (i.e. an isolecithal egg). The embryo demonstrates "regulative" development (each embryo cell or blastomere has the ability to form a complete organism), it has several mutant strains available for genetic analysis, and it develops in a synthetic sea water environment, without the need for elaborate culture or "in utero" studies. The eggs are large enough to be studied with standard light microscopes and can be conveniently micro- manipulated for nuclear transplant and cytoplasmic injections.
The sea urchin embryo is thus a convenient model system for the complete analysis of early embryogenesis. For our purposes, we will limit the laboratory study to descriptions of the basic processes of cleavage, induction, migration and invagination of cells.
Caenorhabditis elegans is rapidly becoming a favorite organism for early embryogenesis. This invertebrate worm (nonparasitic nematode) reproduces as a self-fertilizing hermaphrodite. The genetics of the nematode is fairly simple; each contains a pair of sex chromosomes (XX = Female, XO = Male) and five pairs of autosomes. The male karyotype develops spontaneously in 1/700 developing embryos. Males can be mated to hermaphrodites, but hermaphrodites never mate with each other. Figure 13.1 presents the life cycle of C. elegans.
C. elegans has an asymmetric first cleavage which clearly establishes two distinctly different cells, which in turn are the progenitors of specific parts of the final organism. Starting from the single cell zygote, the first stage larva hatches in about 14 hours after fertilization with a total of 546 somatic nuclei and four primordial gonadal nuclei. The embryo is a classic "mosaic" where each cell carries the information for a piece of the whole. Blocking cell division as early as the two- cell stage will result in cells which are unable to form complete organisms, but which will demonstrate specific fates. The cells are each given labels indicative of their ultimate fate, and limited potential.1, 2 Figure 13.2 presents the general scheme for this cellular development.
The eggs of C. elegans are small, transparent, capable of developing outside of the mother, have relatively small genomes and have had each cell's development characterized. 3 With the isolation of mutants, and characterization of the specific gene loci (DNA sequencing), powerful probes of early gene control over embryogenesis have become available. 4
The sequence of events in the development of this nematode are observed by the formation of polar bodies and completion of meiosis following fertilization. At this time, we can see intensive cytoplasmic streaming which results in the observable segration of germ line specific granules to the posterior of the embryo. Subsequently, the pronuclei fuse and the first cleavage is initiated, giving rise to a large AB blastomere and a small P-1 blastomere. Antibodies can be made to the P granules and we can observe a clear mosaic pattern distribution can be observed with immunofluoresence.
The development of the blood and related cells has long been a subject of intensive study. One reason is that blood represents a type of differentiation which is continuous throughout the life of an organism, and demonstrates the role of stem cells in development. Stem cells are embryonic derivatives which retain the ability to form clones, yet remain relatively undifferentiated themselves. This is accomplished by the division of a stem cell into two cells, typically one of which will continue on to a highly differentiated role, while the other remains as a stem cell. It is possible for stem cells to divide into two stem cells, and on occasion, a stem cell will divide and form two differentiated cells (thus ceasing to be a stem cell). The hematopoietic system (blood forming) demonstrates each of these modes.
In adults, blood cells do not divide within the bloodstream, but are produced either within the bone marrow, lymph nodes, spleen or thymus. Thus, these organs make up the hematopoietic system, or blood forming system. Once the cells are formed through cellular division, they must mature before attaining their final differentiated state. Smears of bone marrow (and sometimes whole blood) will display many intermediate states of differentiation.
It is generally believed that all blood cells arise from a single type of cell (the "unitarian" theory), which is itself derived directly from embryonic mesenchyme. The cell is known as a hemocytoblast and is characterized by its large size (8-30 microns), minimal quantity of basophilic cytoplasm, lack of cytoplasmic granules, large nucleus and presence of 2-3 prominent nucleoli. They are fairly scarce within bone marrow (% of cells present), yet they are the progenitors of all other blood cells.
A hemocytoblast within bond marrow will give rise to granulocytes, megakaryocytes and erythrocytes. The same cell found in lymphoid tissues will give rise to lymphocytes. For the exercises used in this manual, we will limit our observations to those cells found in the bond marrow. The lymphocytic series is more difficult to study since it has fewer distinguishing marks (granules, basophilia). The erythrocyte, granulocyte and megakaryocyte series are clearly defined by observable light microscope characteristics. Collectively they represent a model series of cellular alterations leading toward differentiation from a stem cell population.
Photomorphogenesis of Ferns
Axenic growth of fern prothallia germinated from spores represents a ready sequence of cellular differentiation which has many of the standard characteristics. 5, 6, 7 In addition, the developing gametophyte is haploid (simplifying the genetics), and since the fern fronds grow asexually, usually all the spores from a given field are from the same plant, thus ensuring genetic homogeneity.
As the spore germinates and begins to develop a heart-shaped gametophyte, it will pass through several stages of recognizable development. The first is the formation and extension of a rhizoid, followed almost immediately by cellular division within a single plane. This division takes place within the "tip" cell and gives rise to a filamentous protonema. The tip cell will then alter the planes of division by 90° and subsequently, the growth will become two-dimensional. As this process continues, the typical shape of a young fern prothallus is established. The division continues forming a structure with a single layer of cells, with differentiated areas forming rhizoids, the body of the prothallus, antheridia (sperm) and archegonia (egg). Thus, there are a small number of easily recognizable differentiated states, within an organism that can be propagated from spores with genetic homogeneity and which can be grown easily under aseptic and axenic conditions.
The body of the fern gametophyte can be sliced into various components and a new prothallus will develop from each. The spores can be irradiated and will give rise to tumors, abnormal three-dimensional growths with complex structure and physiology. The cultures of developing gametophytes can, of course, be treated with any number of drugs for molecular analysis of function.
In addition, the developing gametophyte demonstrates a characteristic process of photomorphogenesis. This process is one in which light has an effect upon the structure and function of the plant, independent of photosynthesis. There are several examples of this process, most of which involve red light and far red light wavelengths and the germination and development of seeds. These systems involve the pigment phytochrome, and require somewhat difficult illumination control. The fern gametophyte, by comparison, alters its growth when grown in various components of visible light. Specifically, the shape of the gametophyte will differ when grown in red, blue and green light.
Under sufficient illumination with blue light, the gametophyte will develop as indicated above, that is, as though it were in white light. Under red illumination (corrected for the same energy intensity), the gametophytes will develop as filaments only. That is, the alteration in the plane of development which gives rise to the two-dimensional growth will not occur. Under green illumination, the gametophyte will grow filamentous, but will also develop and differentiate massive amounts of antheridia. Refer to Figure 13.4 for details of fern gametophyte development.
Cell Communication - Dictyostelium and cAMP
The development of the slime mold Dictyostelium discoideum has been chronicled for several decades, with the primary establishment of this important system accomplished by J.T. Bonner 8, 9, 10 and followed by too many investigators to list here. One would be remiss, however, to not acknowledge the extensive work of the Sussmans. 11 There has been a more recent review of the growth characteristics in Ashworth. 12
The basic developmental pattern of D. discoideum is given in Figure 13.5.
This organism is a cellular slime mold that lives most of its life cycle as a free amoeboid cell. The amoeba of D. discoideum function as simple protists, yet when nutrition becomes limited, they can aggregate and form a multicellular slug, with many of the characteristics of a true multicellular organism.
Within its life cycle, the amoeboid cell emerges from a spore, is strictly parasitic on bacteria and divides by simple binary fission. When the number of amoeboid cells increases, and there is a slight drying of the environment, the amoeba will gather, form protective slime sheaths around themselves and begin differentiating. The cells arrange themselves into positions, and the location a cell has will in turn determine its ultimate fate, whether it will become a part of the stalk (prestalk) or will be involved in the sexual reproduction of the species (prespore).
The aggregated amoeba first form a slug or pseudoplasmodium which travels about as though it were a multicellular organism. Eventually, it will settle down on the agar plate (or a leaf if in a lake) and form a base, stalk, and fruiting body (sorocarp). Spores are formed within the fruiting body and the process begins again.
It has long been known that the primary induction of this phenomenon is a pulsed level of the nucleotide cAMP. 13 The level of cAMP that a cell is exposed to is fundamental to its differentiation into either stalk cell or spore cell. 14 Cells can be isolated, which are thus prestalk or prespore, after cAMP is administered.
If agar cultures of the amoeba are supplemented with 10 M cAMP, and the amoeba are placed in small drops onto the surface of the media (3-8 mm diameter), the cells will form a ring around the drop as they migrate from the center. 15 Within 24 hours, some of the advancing cells within the ring will differentiate into separate stalk cells. By contrast, in the absence of cAMP, the cells aggregate and form normal pseudoplasmodia.
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