Embryology - Biology 104, Spring 2006 - Albert Harris and Corey Johnson   OUTLINE OF SEVENTH LECTURE: Jan 30, 2006, by Corey Johnson Multicullularity In the first lecture, we talked about the basics of how a single cell proliferates becoming more cells that moves, differentiates, proliferate, changes shape, and dies in order to produce a multicellular organism such as a frog, or sea urchin. Today, we turn our attention to the very "primitive" multicellular creatures. Why? Understanding the most primitive characteristics that enable cells to exist together, may (and does) provide us with a framework for understanding more complex interactions. Roux (1890s) often considered the father of experimental embryology (along with His) were among the first embryologists to articulate the idea that incomprehensibly complex processes can be reduced to smaller and smaller less complex units. From this paradigm, embryolgists (and other biologists) use simple organisms to gain a foothold in understanding more complex issues of interest. What are the characteristics of a multicellular organism? 1) Several/many cells : one or two parent cell(s) make a new individual 2) structure: cells organize into a reproducible form... the adult is reconstituted 3) specialization/differentiation: cells play different structural or functional roles 4) reproduction: a cell becomes a separate entity from parent, capable of its own reproduction Today we're talking about two kinds of progression from single cells to multicellularity: evolutionary development of multicellular forms from single-cell progenitors in the green algae (Chlamydomonas to Volvox), and the formation of multicellular structures from aggregated single cells within a culture (Dictyostelium). The unicellular green alga Chlamydomonas Mutations affecting flagella and photosynthesis are among the most important in Chlamydomonas. They can survive without being able to swim, and can use external carbon sources (other plants die without photosynthesis and Paramecia, etc. would starve if their cilia were paralyzed.) The life cycle of Chlamydomonas is interesting but odd. Instead of two sexes, they have two mating types called plus and minus (but not because of electric charge!) Plus and minus cells look identical at the light microscope level, but there are subtle differences when examined by electron microscopy. Imagine if human eggs and sperm had the same size & shape, or men & women were morphologically the same, but breeding required one of each of two kinds, like maybe if Republicans could only marry Democrats, but they looked the same, or IBM people could only marry Mac people, or UNC people only Duke people (stop this analogy!) Key points about the Chlamydomonas life cycle (see diagram) -- Plus and minus cells (haploid) recognize each other by proteins on their flagella -- The flagella stick to each other, bringing the anterior ends of the cell bodies together -- A cytoplasmic bridge forms between the two cells -- The cells then fuse to form a diploid zygote -- A hard wall is formed to create a zygospore that can withstand drought and extreme temperatures -- When environmental conditions improve, the zygote undergoes meiosis and germinates to release four haploid cells. Two of these haploid cells will be mating type plus and two will be minus, because mating type is controlled by a single genetic locus. The multicellular alga Volvox is genetically very close to Chlamydomonas. About 500 species of Chlamydomonas have been named based just on what you can see in a light microscope. DNA sequencing shows that they diverged into several different branches far back in evolutionary time. Multicellular forms (Gonium, Pandorina, Volvox) evolved in at least 3 of these branches. In the simpler multicellular forms, all the cells look alike (and look very much like individual Chlamydomonas cells). In Volvox, the surface cells look like Chlamydomonas, each with two flagella, but the interior cells differentiate into specialized reproductive cells. [PowerPoint slide shown in lecture, courtesy David Kirk of Washington University] The species of Chlamydomonas that is used as a model organism is closely related to one particular species of Volvox, much more closely than it is related to some other species of Chlamydomonas. Note: Volvox is not the direct ancestor of higher plants (which evolved from some other kinds of green algae). Another model of multicellularity: Dictyostelium discoideum This is a cellular slime mold, or in the preferred modern terminology, a "social amoeba". The cellular slime molds used to be regarded as fungi, but actually they are not very closely related. Began to be used by Raper, a former UNC student Has become a frequently used model of developmental biology The strain most people work with was isolated in North Carolina, near Mt. Mitchell. What could possibly be similar between the behavior of Dicty and that of Vertebrate Embryology??? Life cycle: Amoebae Ðaggregation--> Pseudoplasmodium ÐculminationÑstalk formation--> Fruiting body Ðspore formation, germination --> Amoebae What are the similarites between this life cycle (development) and embryogenesis? 1) Chemotaxis 2) Locomotion 3) Aggregation/adhesion 4) Regulation 5) Differentiation CHEMOTAXIS Amoebae feed on bacteria; when food runs out, the amoebae attract each other, forming masses of thousands of amoebae - a slug, also called a pseudoplasmodium or grex Aggregating Dictyostelium use chemotaxis to find each other, secreting pulses of the chemical cyclic AMP (cAMP - used by higher organisms as a second-messenger) And each amoeba re-orients its locomotion toward whatever direction the concentration of cyclic AMP is highest, attracting one another. The first evidence for this chemotaxis was in the 1940s; Dictyostelium were cultured on sheets of cellophane, through which only small molecules can diffuse Scientists noticed that when amoebae aggregated on one side of the cellophane, other amoebae aggregated exactly on the opposite side because the "attractant" molecule was diffusing through. Bonner (1940s) made water flow slowly past aggregating Dictyostelium, and found that the amoebae could only detect aggregation centers from the downstream side. This and related observations proved beyond doubt that chemotaxis was being used, but another twenty years of research were needed to find out that cAMP was the attractant. (1947 to 1967). * Embryos use chemotaxis as well. A classic example is NGF (Nerve Growth Factor) LOCOMOTION The locomotion used by Dicty is quite similar to that used by vertebrates (more so than other amoebae) Another good question is: do the slugs use the same mechanism of locomotion as the individual amoebae? And when the future spores crawl up the outside of the stalk: is that also the same propulsion mechanism? Mutant amoebae without myosin can still crawl, but can't cleave in the normal way. Much research has been done on this. And when the future spores crawl up the outside of the stalk: is that also the same propulsion mechanism? Another good question is: do the slugs use the same mechanism of locomotion as the individual amoebae? *Of the many kinds of amoebae, their locomotion is the closest to the mechanism of locomotion used by animal body cells. REGULATION A slug can differentiate into a fruiting body whether it has 5000 or 500,000 cells - an amazing feat of regulation. The slug is divided into 2 major parts, pre-stalk and pre-spore. The stalk forms from the anterior ~20% of cells and the spore forms from the remainder (there's a small section on the posterior end that forms part of the base). If that slug is cut into pieces, each piece can regulate to become a fruiting body. Mutations in Dictyostelium can change the proportions of spores versus stalk. Nobody has yet found the mechanisms. DIFFERENTIATION The amoebae at the front differentiate into stalk cells The rest of the amoebae differentiate into spores with hard shells. As this happens, the cells rearrange, like an inside-out fountain. The stalk cells form a tall stiff rod, (very similar to an amphibian notochord: swollen vacuolated cells, wrapped tightly in fibers -- cellulose instead of collagen, however) Spores later hatch out, if they are lucky somewhere there are bacteria to eat. There have been two hypotheses to explain how the two regions of the slug differentiate (similar to the two ideas about vertebrate development: regulative vs. mosaic) 1) Gradient hypothesis. This idea says there are gradients of substances that affect the cells of the slug which are said to be labile (developmentally flexible). 2) Cell autonomous differentiation. This idea says that the cell fate has already been specified by the time the amoebae form a slug. Both seem to be true! There is evidence that the stage of the cell cycle during which starvation was "encountered" determines (in part) which part of the slug, and hence the fruting body, to which they will contribute. This of course is not the whole story, because the can indeed regulate when the two cell types are separated. Morphogens (DIF and DIFase) are two compounds that can influence cell fate. link to Dictyostelium video ..and a few nice pictures

 

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