This is a movie showing C. elegans nematodes crawling on the surface of an agar plate in a Petri dish (the agar was first seeded with a nice thick lawn of E. coli bacteria, the food for C. elegans in laboratories). Note the larger adult hermaphrodites (you can see oval embryos inside them, near the middle part of the adult worms), and smaller larvae, and the small ovals on the plate which are embryos that have yet to hatch. C. elegans is a free living soil nematode, or roundworm, and is a self-fertile hermaphroditic species (essentially, the adults are females that make about 300 sperm prior to converting to oogenesis for the rest of their life).
A small number of males are made spontaneously, but most populations consist almost entirely of hermaphrodites (unless you single out males and hermaphrodites and set up crosses, in which case when a cross works, half the progeny are male and half are female, just as with people). We grow and maintain strains of C. elegans on plates like these. We use a platinum wire “spoon” to pick them up and transfer them from plate to plate, or to watch glasses where we cut them open with a scalpel to recover early stage embryos. Almost all of the work on C. elegans in the Bowerman lab focuses on using molecular genetics and other methods to study the function of the microtubule and microfilament cytoskeleton during early embryonic cell divisions. Some of the processes that we focus on currently include cell polarity, meiotic and mitotic spindle assembly and positioning, chromosome segregation, and cytokinesis.
Movie 2 Early Embryonic Mitosis
This is a time-lapse videomicrograph (aka movie) of the first two mitotic divisions during embryogenesis in C. elegans, beginning with a one-cell stage embryo that has just completed the second meiotic division. Meiosis I and II are completed after fertilization of an oocyte by a sperm; movies of meiosis must be made “in utero” inside live and anaesthetized worms). This movie is made using Differential Interference Microscopy (DIC), which relies on transmitted, polarized visible light. One can detect many events in live embryos using this simple form of microscopy. One can also use spinning disk confocal microscopy with GFP fusions to different proteins to image specific cellular structures in more detail. An example of this is shown on the home page for my lab, which uses GFP fusions to highlight both microtubules in the mitotic spindle and chromosomes during mitosis. The movie ends partway through the 4-cell stage. C. elegans embryos are about 50 microns in length, with a tough chitinous eggshell.
We isolate early stage embryos by cutting open an adult wild-type hermaphrodite in a watch glass (submerged in buffer). A mouth pipette is then used to transfer the embryo to a small agar pad on a glass slide, and the embryo then overlaid with a glass cover slip and mounted on a compound microscope with Differential Interference Contrast (DIC) optics.
Note the appearance of the oocyte and sperm pronuclei, which appear after the completion of meiosis II (at which time chromosomes decondense and nuclear envelopes form). The oocyte pronucleus is to the left (anterior pole) and the sperm pronucleus to the right (posterior pole). The sperm donates two centrosomes, in addition to chromosomes, and these centrosomes stay associated with the sperm pronucleus. These two haploid pronuclei migrate towards each other, meeting near the posterior pole. After they meet, they rotate and “centrate” with the spindle forming in the center of the embryo, aligned with long axis (the anterior-posterior axis). During anaphase of the first mitosis, the spindle moves slightly towards the posterior pole, and the posterior spindle pole rocks back and forth. This posterior displacement results in an unequal or asymmetric first mitotic cell division. Both daughters are born committed to distinct fates. Thus the first mitotic division provides an nice model for studying asymmetric cell division, a fundamental developmental process that generates cells with different fates during embryogenesis. The posterior daughter (called P1) is smaller than the anterior daughter (called AB), and divides later.
Notice that P1 divides more along the anterior-posterior axis, while AB divides more transversely. One focus of the research in the Bowerman lab is to understand how these different spindle positioning events are genetically programmed and controlled. These early embryonic events are all very reproducible and stereotyped in wild-type embryos. We study mutants in which these processes are defective; these time-lapse movies allow us to study in detail the nature of the defects we observe during cell division in mutant embryos. See Movie 6 for an example of an abnormal cell division in a mutant embryo.
Movie 3 C. elegans GFP::Histone 1-cell
This movie was made using an otherwise wild-type embryo obtained from a transgenic C. elegans strain that expresses a transgene encoding Green Fluorescent Protein fused in frame with a histone protein. This GFP::Histone fusion protein is expressed during oogenesis and incorporated into the nucleosomes that package the chromosomes in early embryonic cells. Thus GFP marks the chromosomes in these embryos.We used a spinning disk confocal microscope to make movies of the fluorescent fusion protein during early embryonic cell divisions, in order to visualize chromosome position and movement during these cell divisions.
Being able to watch cell division with different molecular structures marked with GFP-tagged proteins is an important tool for us in studying cell division in wild-type and mutant embryos. As in Movie 2, this movie shows the first two mitotic divisions. The bright objects at the periphery of the embryo, towards the anterior pole at the left, are the polar bodies, which contain chromosomes that were discarded during meiosis I and II. Note the differences in cell cycle timing at the 2-cell stage, the condensation of chromosomes as cells enter mitosis, and the different orientation and position of the chromosomes at metaphase during each division. These events are all very reproducible and stereotyped in wild-type embryos. We study mutants in which these processes are defective; these time-lapse movies allow us to study in detail the nature of the defects we observe during cell division in mutant embryos. See Movie 6 for an example of an abnormal cell division in a mutant embryo.
GFP Tublulin 1-cell
Movie 4 C. elegans GFP::Tubulin 1-cell
This movie was made using an otherwise wild-type embryo obtained from a transgenic C. elegans strain that expresses a transgene encoding Green Fluorescent Protein fused in frame with a tubulin protein. This GFP::Tubulin fusion protein is expressed during oogenesis and incorporated into the microtubules that are nucleated at centrosomes and form mitotic spindles during mitosis in early embryonic cells. We used a spinning disk confocal microscope to make movies of the fluorescent fusion protein during early embryonic cell divisions, in order to visualize microtubule dynamics and spindle positioning during these cell divisions.
Being able to watch cell division with different molecular structures marked with GFP-tagged proteins is an important tool for us in studying cell division in wild-type and mutant embryos. This movie shows only the first mitotic division. Note the rotation and centration of the mitotic spindle early in this division, which occur very early in mitosis.
This movie shows a 3-dimensional reconstruction using a “Z-stack” of images taken at several different focal planes in a fixed 2-cell stage embryo during mitosis (using a laser scanning confocal microscope). This embryo was stained with two different fluorescently labeled antibodies, one that recognizes tubulin and thus microtubules (green), and one that recognizes a centrosomal protein called SPD-5 (yellow). Note the orthogonal orientation of the mitotic spindles in a 2-cell stage embryo. P1 (the posterior cell) divides longitudinally and asymmetrically, while AB (the anterior cell—to the left) divides transversely and equally. AB is almost at metaphase (note the single lagging chromosome), while P1 is still in prophase, just before nuclear envelope breakdown.
Tracking microtubule plus ends with a GFP::EPB-2 fusion. This movie shows a one-cell embryo early in mitosis with the centrosomes and microtubule plus ends highlighted by a GFP fusion to the plus-end binding protein EBP-2. Note how the microtubules are growing out radially from each centrosome, with the centrosomes position along a transverse axis between the egg and sperm pronucle. This fusion allows one to analyze microtubule growth and dynamics in wild-type and mutant embryos.
Tracking microtubule plus end dynamics at the cortex in an embryo lacking the function of a gene that promotes microtubule catastrophe at the cortex. The plus ends are detected with a GFP::EBP-2 fusion (see Movie #6 for a wild-type example that focuses deeper in the cytoplasm, rather than at the cortex as in this movie). Note how in this movie one can see the microtubule plus ends as they move along the cortex. In wild-type embryos, microtubules undergo catastrophe and appear only as a single, relatively short-lived dot at the cortex. Chromosomes are also labeled in this movie with an mCherry:Histone fusion protein.
Microfilament cytoskeletal dynamics at the cell cortex during the first mitotic division of a one-cell C. elegans embryo.
A fusion of GFP to a domain from Drosophila moeisin is used to label microfilaments, which are highly enriched at the cell cortex in the early embryo. Note the highly dynamic nature of the microfilament cytoskeleton during the time period preceding and including cytokinesis.
Polarization of the one-cell C. elegans embryo, visualized by a GFP fusion to PAR-2 at the posterior cortex, and a fusion of mCherry to PAR-6 to mark the anterior cortex. PAR-2 and PAR-6 are required to polarize the zygote along its anterior-posterior (AP) axis and are themselves polarized in their distribution along the AP axis. By the time the egg and sperm pronuclei begin to migrate toward each other, this polarization is already largely accomplished (the leading edge of this polarization process is marked by the invagination called the pseudocleavage furrow which regresses later in mitosis). As a result, the first division is asymmetric, producing two unequally sized daughters cells that divide asynchronously and are born committed to distinct fates. After the first division, PAR-6 is present throughout the cortex in the anterior daughter (which divides equally), while PAR-2 and PAR-6 again become polarized in the posterior daughter, promoting its asymmetric division.
This is a DIC movie of the first mitotic division in a rfl-1(-) mutant embryo. This mutant embryo came from a mother that was -/- for a loss-of-function mutation in a gene called rfl-1. This mutant shows multiple defects during the first two rounds of mitosis. The first detectable defect occurs during pronuclear migration, when the cell membranes are hyper-active due to excess actomyosin contractility at the cortex (compare to DIC movie of a wild-type embryo in Movie 2). The next obvious defect is the abnormal positioning and small size of the first mitotic spindle. Late in mitosis, multiple cleavage furrows ingress (another abnormality), and eventually cytokinesis fails. Thus the wild-type rfl-1 gene is required for proper regulation of actomyosin contractility and microtubule stability during mitosis.
Thimo Kurz in my lab positionally cloned the rfl-1 gene and found that it encodes an E1 activating enzyme for the ubiquitin-like protein called Nedd8 in humans. Nedd8 is is a ubiquitin-like protein that is conjugated to Cullin proteins by a “Nedd8 conjugation pathway” that includes an E1 activating enzyme, an E2 conjugating enzyme and an E3 ligase). Cullin proteins themselves are the scaffolds for ubiquitin E3 ligases, which mediate the covalent modification of many different proteins by the highly conserved protein ubiquitin.Poly-ubiquitination targets proteins for degradation by the proteasome. One ubiquitin E3 ligase is required in the early C. elegans embryo to degrade a protein called MEL-26, which regulates actomyosin contractility. The same ubiquitin E3 ligase is also required to degrade prior to mitosis another protein called MEI-1/Katanin, which is part of a microtubule severing complex.
In the absence of RFL-1 function, MEL-26 accumulates to abnormally high levels and promotes extra furrowing during pronuclear migration and during cytokinesis. Also in rfl-1(-) mutant embryos, Katanin is NOT degraded before mitosis, leading to abnormally short microtubules (and defects in mitotic spindle positioning, assembly, and function). See Kurz et al (2002), Pintard et al (2003), and Kurz et al (2005) for more information on this topic of research in the Bowerman lab. This mutant provides one example of how we make discoveries about gene function by using molecular genetics to study cell division in the early C. elegans embryo.
Platynereis dumerilii male adult
This movie shows meiotic cell division in C. elegans filmed in a live worm immobilized in a microfluidic chamber constructed in our nanotechnology facility. To the left are three oocytes; the most mature one is at the right of these three. The chromosomes are labeled red with an mCherry::Histone fusion; microtubules are green with a GFP::beta-tubulin fusion. After the movie starts, you'll see the nuclear envelope break down and meiotic spindle assembly, followed by extrusion of chromosomes in the first polar body. The movie stops when this fertilized oocyte reaches metaphase of meiosis II. During meiosis I the fertilized oocyte passes through the spermatheca (you can see the sperm as small red dots within the spermatheca, which is invisible in this movie). To the right of the three oocytes is a one-cell zygote undergoing the first embryonic mitosis in the uterus. Note the very different size and morphology of the meiotic spindles compared to the mitotic spindles.