Cynthia J. Kenyon
Vice President, Aging Research
Calico Labs, LLC
1986 Searle Scholar
Former Member of Advisory Board (2007 -2010)
Development and Longevity in C. elegans
HOW DO MIGRATING CELLS FIND THEIR WAY?How do migrating cells know which way to migrate and where to stop? Several graduate students in the lab are using genetics to identify and characterize genes whose products guide migrating cells to their targets; and are cloning interesting genes to learn how they function at the molecular level. We are focusing on the Q neuroblasts, QL and QR. These cells are located in symmetrical positions on the left and right sides of the animal, but they migrate in opposite directions. QR and its descendants migrate toward the head, whereas QL and its descendants migrate toward the tail. As they migrate, these cells divide; their descendants each stop at characteristic positions along the body axis, although there are no obvious landmarks at these positions.
The migratory behaviors of the Q descendants are influenced by the activities of Hox genes, which encode homeodomain proteins that specify cell fates along the anteroposterior axis of the worm (see below). The genes mab-5, specific for the posterior body region, and lin-39, specific for the central body region, play key roles in programming Q cell migrations. Steve Salser found that after QL migrates a short distance posteriorly into the mab-5 domain, it switches on the Hox gene mab-5. mab-5 functions within QL's descendants to program their migratory behaviors: it causes one of QL's daughters to stop migrating, and it causes the other daughter and its descendant to migrate posteriorly. In mab-5(-) animals, both cells migrate anteriorly. We and others have shown that lin-39 is required for the migrations of QR's descendants, which migrate anteriorly through the lin-39 domain. In lin-39 mutants, QR and its descendants still migrate anteriorly, but they stop prematurely.
The Q cells appear to use global rather than local cues to distinguish anterior from posterior. Steve showed that if mab-5 is expressed ubiquitously from a heat shock promoter, a Q descendant migrating anteriorly will turn around and migrate posteriorly. Lee Honigberg has now shown, by activating mab-5 specifically in the migrating Q descendant with a laser microbeam, that mab-5 is exerting its effect by reprogramming the Q cell itself. However, the fact that cells at any position can respond to mab-5 activity argues that the information cells use to distinguish anterior from posterior is distributed all along the body axis.
How are the different steps in Q cell migration specified? There must be a mechanism for establishing left-right asymmetry, for activating homeotic genes within migrating cells, for programming the Q cell descendants to respond differently to external signals, and for marking the destination points of the different Q descendants. To identify genes that regulate Q cell migration, we have analyzed mutations in which the descendants of QL are located the wrong places. Lee Honigberg is characterizing mutations that alter the left/right asymmetry of the Q migrations. Mutations in four other genes do not affect the direction of QL and QR migration, but affect the ability of the Hox gene mab-5 to be activated during QL's migration. It is possible that these mutations affect a positional signaling system that turns on mab-5 in cells that enter the posterior body region. Certain mutations shift the stopping-points of all the Q-cell descendants cells posteriorly along the anteroposterior body axis. These mutations, Jeanne Harris, Gregg Jongeward, Lee Honigberg and Jen Whangbo are studying, provide entry points for learning how positions along the anteroposterior body axis are selected as migratory targets.
The Hox genes, which regulate transcription, act within migrating cells to determine which direction, or how far, the Q descendants migrate. What downstream genes do they regulate? Naomi Robinson found that one gene, mig-13 behaves genetically like a downstream target of the Hox genes. Mary Sym is on the verge of sequencing mig-13 now. We are also studying genes that probably function in the process of migration itself. In collaboration with Lou Reichardt and Ed Hedgecock, Sonya Gettner found that Q cell migration requires the function of a beta integrin subunit encoded by the gene pat-3. Another gene, mig-2, which Ilan studies, may interact with the integrin in some way, since it has many of the same defects as pat-3 mutants. This aspect of the migration project is exciting because it may allow us to learn how the functions of these conserved adhesion receptors are integrated into the guidance system that directs all these cells to their final positions.
A CONSERVED HOMEOTIC GENE CLUSTER GENERATES PATTERN ALONG THE BODY AXIS OF C. elegansA highly conserved regulatory system generates anteroposterior body pattern across much of the animal kingdom. Clusters of genes encoding Antennapedia-class homeodomain proteins (Hox proteins) have been conserved between flies and vertebrates, where their patterns of expression and even their functions seem to be similar. Studies from our lab and other labs has shown that C. elegans also has a Hox cluster that patterns its anteroposterior body axis. These findings indicate that Hox-based pattern formation must have evolved before the many differences in morphology and embryology that now distinguish nematodes from other phyla. They also mean that one can study the regulation and function of this conserved patterning system at the single-cell level, using C. elegans.
Craig Hunter has shown that the Drosophila Hox genes Scr and Antp can partially compensate for the functions of their C. elegans homologs when expressed in C. elegans. The Drosophila genes can rescue mutant neurons and sensory structures, epidermal cells, and cell migrations. These findings indicate that the sequence specificity for DNA binding has been conserved over a great evolutionary distance.
TARGETING Hox GENE EXPRESSION TO THE CORRECT POSITIONA general feature of Hox genes is that they are expressed in cells that are related only by position. This is true in C elegans as well. The early embryogenesis of C. elegans seems at least superficially to be very different from that of other organisms, in that cell lineage and local cell-cell interactions rather than global positional information appear to determine many cell fates. If so, then how does C. elegans localize the expression of its conserved Hox genes? Is there a cryptic underlying similarity in the developmental mechanisms in all these organisms, or do they use different mechanisms to achieve the same end_localized Hox gene expression? Deborah Cowing has performed a simple experiment to ask whether localized positional information is required in order for the Hox gene mab-5 to be expressed in the posterior body region. The cell M migrates from the head region to the posterior body region_the mab-5 domain_and then, when it arrives, switches on mab-5 gene expression. Deborah managed to prevent the M cell from migrating using drugs that disrupt the cytoskeleton. To our surprise, the cell still expresses mab-5 even when it is nowhere near the posterior body region. This indicates that, at least for this cell, positional information is not required for proper mab-5 expression. Some other information_lineal information, or local cell-cell interactions_must instruct this cell to turn on mab-5 at a time that coincides with its arrival in the posterior domain. In a separate series of experiments, Deborah has asked whether certain stationary cells require positional information to express mab-5, and again, the answer is no.
One protein involved in Hox gene regulation is pal-1, a homolog of the Drosophila caudal gene. These homologs are required for posterior development of both worms and flies and probably vertebrates as well. Craig Hunter has examined the role of pal-1 in early C. elegans development. He finds that, as in Drosophila, early protein (but not RNA) expression is confined to posterior blastomeres. This is very interesting, because it suggests that pal-1 is localized by an ancient regulatory mechanism that operated in the common ancestor of worms and flies. We hypothesize that, later, zygotic expression of pal-1 is subject to lineage-specific regulators, which, in turn, bring Hox gene expression under lineal rather than positional control. But we don't know this for sure yet.
A C. elegans Hox gene switches ON, OFF, ON, and OFF again to regulate proliferation, differentiation, and morphogenesisHox genes pattern the A/P body axis throughout the animal kingdom, but it remains a mystery how these genes work at the cellular level to modify and shape particular body segments. It was once thought that Hox genes were expressed in every cell of a body region, and that other factors expressed in a cell-specific way determined the particular cell type that was formed. It was not known how many individual cell fate decisions a specific Hox gene can control within a single lineage. Steve Salser has taken advantage of the single-cell resolution possible with worms to ask exactly what the Antennapedia homolog mab-5 is doing to cells in its primary domain of funciton, and to what extent changes in its expression pattern are important in determining the final body pattern. He has found that the C. elegans Antennapedia homolog mab-5 acts at distinct times in individual ectodermal lineages to program first cell proliferation, then neuroblast formation, and then sense organ morphology. In one lineage, mab-5 is initially off, switches on, switches off, on again in part of the lineage, and finally off. Every regulatory phase is essential to direct the unique development of this lineage. Thus, much of the power of the Hox genes to generate body pattern may derive from fine control over their expression coupled with changing patterns of cellular response.
CONTROL OF HOX GENE EXPRESSION IN SPACE AND TIMESo how is this complex pattern of mab-5 expression set up? We have been examining this question in the V cells, which are lined up in rows along the sides of the animal. mab-5 is off in the most anterior four V cells, V1-V4. It suddenly comes on a little while after hatching in V5. In V6, expression begins in the embryo. Craig has found that pal-1, the caudal homolog mentioned above, turns mab-5 on in V6 in the embryo. We also know that lin-22, which Lisa has found is a homolog of Drosophila hairy, is required to keep mab-5 off in anterior V cells. In lin-22 mutants, anterior V cells exhibit a mab-5-expression pattern similar to that of V5.
How is the ON-OFF switching of mab-5 accomplished in V5? This is an important question, because dynamic patterns of Hox gene expression are probably responsible for a lot of the body pattern we see in the animal kingdom. Here is what we know so far: lin-22 plays a role in turning mab-5 OFF in one branch of the V5 lineage, and so does another gene Polyray, studied by Julin Maloof. Polyray also keeps mab-5 off just after the animal hatches. One thing that is extremely interesting about Polyray is the following: In polyray mutants, mab-5, and also the other Hox genes, are expressed normally early in development, but then the expression of all these genes switches on in body regions in which they are normally off. This is just what happens in the Polycomb mutants of Drosophila, in which maintenance of Hox gene repression is lost. It is interesting that C. elegans has a maintenance system. What is much more interersting is that this repression system is reversible, and that it is used to create the on-off switching pattern of expression seen in V5. This raises even more questions: for example, mab-5 expression in V1-V4 is first repressed by an unknown mechanism (in the embryo), then by polyray (right after hatching), then by lin-22 (later after hatching). Why does lin-22 suddenly become required for repression? Why can't lin-22 establish repression in a Polyray mutant? These are mysteries.
Cell contact and cell fate determinationIn some cells, the Antennapedia homolog mab-5 is also kept off by cell-cell interactions. If the neighbors of V5 are ablated, then V5 turns on mab-5 much sooner than normal and changes V5's fate. Judith Austin has shown that the signaling cells extend long processes that recognize the target cells and probably signal via direct cell contact. This is a very interesting signaling system, because if either of V5's neighbors are killed with a laser, V5's fate changes. This suggests that maybe it is the absense of cell contact that creates a change in cell fate. What signaling pathway is involved? No one knows, although we have identified several mutants in which this cell fate change does not occur following ablation. Jen Whangbo, a new graduate student, may look for signaling mutants.
To summarize: it appears that even in a single lineage, Hox gene expression can be subject to many levels of regulation. Examination of Hox gene expression patterns in higher organisms indicates that regulatory mechanisms will be just as complex. This makes an organism with single-cell resolution especially attractive as an experimental system for asking how Hox genes are deployed in single cells so that different body regions can acquire their different identities.
Genetic Analysis of C. elegans wnt-1, a Mysterious Signaling MoleculeWe have been trying to learn the functions of two C. elegans wnt homolog, wnt-1. wnt genes are especially interesting because they are intercellular signals that act in poorly understood signaling pathways to specify cell fates in many types of organisms. Supriya has generated a deletion mutant of wnt-1, which screws up embryonic development. She is now trying to learn what cell fate decisions are affected by the mutation using our 4D recording system. Gregg and she are in the process of making mutations in another C. elegans wnt gene homolog, wnt-2.
LIFESPAN CONTROL: Genetic Quest for the Fountain of YouthWe all age. There is clearly a genetic basis for determining the lifepspan of an animal; for example, mice live 2 years; canaries, 13; and bats, 30 or more. Yet practically nothing is known about how the rate of aging is controlled. We have found that mutations in the gene daf-2 can cause fertile, active, adult C. elegans hermaphrodites to live more than twice as long as wild type. Mutations in another gene, age-1, has previously been shown to extend lifespan as well. It's creepy. We have found that the lifespan extensions of both age-1 and daf-2 mutants require activity of another gene, daf-16. How can mutations in single genes have such large affects on lifespan? We are now trying to learn more about how these genes influence lifespan. Where do they act? When do they act? What do they do? How highly conserved are they? We are also looking for additional lifespan mutants using a couple of different mutant screening schemes.
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