C. elegans journal club:
Where and how does a transcriptional repression complex functions?
Background:
The C. elegans vulva is the mating and egg-laying organ. Developmental genetic studies have focused on vulva development because it is a simple organ, comprised of only 22 cells, and it is easy to isolate mutants that affect its development, such as vulvaless (vul) or multivulva (muv) mutants.
Six vulval precursor cells (VPCs), consecutively named P3.p through P8.p, have the potential to adopt one of three fates. In wildtype, they always adopt the same pattern of fates, denoted as: 3°-3°-2°-1°-2°-3°. The 1° (P6.p in wildtype) and 2° (P5.p and P7.p) cells are vulval, and their descendants comprise the mature organ. The 3° cells (P3.p,
P4.p and P8.p in wildtype) divide once and their daughters fuse to the hypodermal
(skin) cell called hyp7. Thus, even though P3.p, P4.p and P8.p have the potential to become vulval, they typically do not.
This pattern of cell fates is regulated by at least three signals. Fir st, an “inductive” signal, LIN-3/EGF, is produced by the gonadal Anchor Cell (AC). This signal is received in VPCs by the receptor LET-23/EGFR and transduced by a canonical LET-60/Ras,
MPK-1/MAPK pathway. Low levels of LIN-3/EGF cause the three central VPCs to adopt vulval fates, while the highest level seen by P6.p promotes the 1° fate. The presumptive
1° cell is the source of a second, ”lateral”, signal that is received by the neighboring cells (P5.p/P7.p) via the receptor LIN-12/Notch. This signal induce s the 2° fate, and/or inhibits the 1° fate. Finally, there is a third signal, classically known as “inhibitory”, which prevents P3.p, P4.p and P8.p from adopting vulval fates. This last signal is the focus of our journal club.
QuickTime™ and a
decompressor are needed to see this picture.
Figure 1A from Myers and Greenwald, 2005.
The “inhibitory” signal was initially defined by studying mutants known as “synthetic multivulva”, or SynMuv. The SynMuv genes fall into two groups: A and B. Single mutants in each class, or animals carrying two mutations of the same class, do not exhibit a phenotype. However, when a SynMuvA and a SynMuvB mutation are combined a strong multivulva phenotype is obtained. In such double mutants P3.p, P4.p and P8.p inappropriately adopt vulval fates (1° or 2°). Thus, SynMuvA and SynMuvB genes define two pathways that redundantly inhibit vulval fates. Genetic analyses suggest that the SynMuv genes function by antagonizing the “inductive”
EGF/EGFR/Ras/MAPK pathway. Therefore, when SynMuv function is removed, all
VPCs adopt vulval fates because the inductive pathway becomes hyper-activated

Molecular characterization of several SynMuv genes revealed that they encode components of transcriptional repressor complexes. Of particular importance was the discovery that the SynMuvB gene lin-35 encodes the C. elegans homologue of Rb, an important tumor suppressor. Additionally, C. elegans homologues of proteins known to work with Rb in mammalian cells (such as E2F and Dp) also have SynMuv function.
This finding was exciting, as it hinted that a well-known tumor suppressor (Rb) might have an important role in negatively regulating the activity of an established oncogenic pathway (the Ras/MAPK cascade).
The controversy:
Two related questions are addressed in the four papers up for discussion.
1.- Where (in wh at tissue) do SynMuv genes function to “inhibit” VPCs from adopting vulval fates?
2.- How do the SynMuv genes intersect with the EGF/EGFR/Ras/MAPK pathway to reduce its function?
The two papers from the Horvitz Lab (Lu and Horvitz, 1998. Thomas and Horvitz, 1999) present evidence that SynMuv genes function cell autonomously (i.e. in the cells that exhibit the loss-of-function phenotype. In this case, the VPCs). They also suggest that the SynMuv genes function downstream of the receptor LET-23/EGFR to antagonize the function of this pathway.
The two other papers (Myers and Greenwald, 2005. Cui, et. al., 2006) present evidence that the SynMuv genes function cell non-autonomously , in hyp7, the hypodermal (skin) cell that surrounds the VPCs. They also show that SynMuv genes function to prevent inappropriate expression of LIN-3/EGF in hyp7. Therefore, they suggest that SynMuv genes function upstream of the LET-23/EGFR receptor.
For journal club I suggest we divide into two groups. Group 1 should defend the Horvitz
Lab papers. Group 2 should defend the other two papers. Each group should pay particular attention to experiments addressing the site of action of the SynMuv genes.
These include antibody staining, tissue-specific rescue, tissue-specific loss-of-function and mosaic analyses (i.e., the analysis of animals in which some cells are genetically wildtype while others are genetically mutant). How well controlled were these experiments? How were these data analyzed and results interpreted?
I know that the Horvitz Lab papers are longer. However, much of the work in these concerns the molecular cloning and characterization of several SynMuv genes. This is not what I want you to focus on (although by all means you should read it). Furthermore, everyone should have at least some idea of the papers being presented by the other group. Don’t ignore your competitors!