In the absence of MDF 1, severe developmental defects are observed, including Crenolanib IC50 embryonic lethality, larval arrests, abnormal vulva development, and sterility, which lead to lethality of the homozygous strain after three generations. Similar developmental defects have also been observed in the absence of MDF 2, however, unlike mdf 1 animals, mdf 2 homozygotes can be propagated indefinitely. The fact that absence of different SAC components leads to different developmental conse quences in C. elegans, as well as other organisms, suggests differential requirement of these genes in devel opment and fertility that may or may not be distinct from their function in SAC. To investigate roles SAC genes have during postem bryonic development of a multicellular organism, we studied spatiotemporal expression patterns of the check point genes.
As expected, SAC promoters drive mainly ubiquitous GFP expression during early embryonic development. However, all SAC promoters drive tissue specific expression in later developmental stages. Further analysis revealed that the MDF 2 checkpoint component is required for proper postembryonic proliferation of seam cells by regulating APC CCDC20. In fact, seam cell proliferation was abrogated at a higher frequency during the proliferative L2 stage than in the embryo, suggesting that postembryonic cell divisions may be more sensitive to loss of the checkpoint than the embryonic cell divi sions. Furthermore, we showed that while the hypo morphic mutant fzy 1 fully restored proper seam cell proliferation, fzr 1 CDH1 mutant had no effect on seam cell development in a mdf 2 background.
Results Generation of pSAC,GFP C. elegans strains and characterization of SAC expression patterns In order to explore the temporal and spatial expression of SAC genes, we generated transcriptional reporter transgenic C. elegans strains for the five widely con served checkpoint core components and four SAC components only conserved in higher eukaryotes. All of the selected genes, except for mdf 1, are not in operons, and thus sequences immediately upstream were used for their promoter analysis. mdf 1, on the other hand, is part of an operon and was probed using three different promo ter constructs. The promoter,GFP fusions were generated using a PCR stitching technique, rather than by cloning methods, to avoid potential interference from cloning vector backbones on transgene expressions, as reported recently by Etchberger and Hobert, 2008.
The puta tive promoter GSK-3 amplicons were PCR stitched to the PCR products containing a gfp encoding sequence that includes artificial introns and the unc 54 3UTR from the pPD95. 75 vector. The 5 regions examined in this study as putatively containing regula tors of the SAC genes extended from the predicted ATG initiator site for a targeted gene to its adjacent upstream gene.