In order to explore the temporal and spatial expression of SAC genes, we generated transcriptional reporter transgenic C. elegans strains for the five widely conserved checkpoint core components (mdf-1/MAD1, mdf-2/MAD2, san-1/MAD3, bub-1/BUB1 and bub-3/BUB3) and four SAC components only conserved in higher eukaryotes (hcp-1/CENP-F, hcp-2/CENP-F, czw-1/ZW10 and rod-1/ROD) (Table 1). 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 promoter constructs (Table 1, Additional file 1, Figure S1). The promoter::GFP fusions were generated using a "PCR stitching" technique 19 , rather than by cloning methods, to avoid potential interference from cloning vector backbones on transgene expressions, as reported recently by Etchberger and Hobert, 2008 20 . The putative "promoter" amplicons were "PCR-stitched" to the PCR products containing a gfp encoding sequence (S65C variant) that includes artificial introns and the unc-54 3'UTR from the pPD95.75 vector (developed by Dr. Andrew Fire, Carnegie Institution). The 5' regions examined in this study as putatively containing regulators of the SAC genes extended from the predicted ATG initiator site for a targeted gene to its adjacent upstream gene. The lengths of the upstream regions defined by these criteria range in size from 282 bp to 3,000 bp (Table 1) and are in accordance with previously described minimum and maximum promoter lengths used in large scale projects 21 22 23 24 25 . In total, 12 transcriptional fusions with gfp were constructed corresponding to the nine checkpoint genes of interest (Table 1). For each construct, we generated at least three independent lines that were compared for expression pattern consistencies. Due to the mosaicism issues associated with extrachromosomal concatameric arrays, we analyzed at least 30 replicates and recorded GFP-expressing cells and tissues that showed expression in at least 50% of the animals at any given developmental stage, as described previously 23 .

Our analysis of SAC gene regulatory activities revealed that all of the SAC constructs, except for pczw-1::GFP, confer GFP expression (Table 1). The 2,101 bp sequence upstream of czw-1 did not drive any detectable GFP expression at any developmental stage in any of the four independent transgenic lines analyzed. We also examined another construct that contained 3 kb upstream sequence of czw-1 and still did not observe any expression (Table 1). Importantly, our analysis of the other eight SAC genes revealed expression that was consistent between the independent lines for every given construct (Table 1). We have detected GFP at all developmental stages, except for very young embryos (younger than 12 cell-stage embryos), and have identified expressed GFP in all the major tissues, except for germline, likely due to germline silencing of concatameric arrays 26 .

GFP expression driven by the eight SAC gene upstream regions containing regulatory sequences (promoters) was commonly observed early in development, well before the comma stage of embryogenesis (Figures 1A-D and 2). In fact, we were able to detect GFP expression before embryos progressed to gastrulation (Figure 1A). Because we observed mosaicism due to mitotic loss of the concatamer arrays (Figures 1 and 2), we analyzed many embryos per construct. Our results show that SAC gene promoters drive GFP expression in the majority of the early embryonic cells (Figures 1A-D and 2). The only construct that did not drive ubiquitous GFP expression in early embryos is the putative promoter of mdf-1, which is in an operon, that extends upstream from the ATG initiator site in the first gene, his-35, of the operon to the adjacent upstream gene (his-41) (Additional file 1, Figure S1A). On the other hand, both transcriptional fusions that included an internal mdf-1 promoter revealed the same ubiquitous activities in early embryos (Additional file 1, Figure S1B, C). Considering the established role of the mdf-1 checkpoint gene in surveillance of the metaphase-to-anaphase transition, as well as the observed antibody localization in dividing cells in early embryos 27 , we conclude that the mdf-1 containing operon belongs to the "hybrid operons" class 28 , in which the internal promoter of mdf-1 is necessary to drive proper expression of this gene in embryonic cells.

The cell cycles of early embryonic cells in C. elegans are rapid, consist entirely of S phase and mitosis, and lack gap phases 29 . This rapid embryonic cell proliferation creates more than half of C. elegans' somatic cells, with the majority of cell divisions being completed in the first half of embryogenesis 30 . Thus, co-expression of SAC genes in the rapidly dividing early embryonic cells (Figures 1A-D and 2) is consistent with the well established role of these genes in cell division. In addition to the activities of SAC gene promoters in the early embryos, we also observed GFP expression in later embryos for all of the spindle-checkpoint promoters that we analyzed. The expression patterns in late embryos show GFP expression in the majority of the cells, although the majority of the promoter constructs tend to confer more localized GFP expression, as exemplified for mdf-2 (Figure 1G). Together, the expected promoter activities of SAC genes during embryogenesis, show that the promoters used for our analysis are appropriate.

Rapid cell proliferation occurs in all four larval stages especially in the second larval stage (L2) of development in C. elegans when many somatic cells are generated 30 . As expected, GFP expression conferred by SAC gene promoters was detected at all four larval stages (Table 1). Unlike embryonic expression, spatiotemporal analysis revealed that postembryonic expression of SAC genes is generally restricted to specific cells and tissues types (Table 1). For example, mdf-2 promoter drives GFP expression in seams cells (Figure 1I), gut cells (Figure 1K), and some additional tissue types (Table 1) at all larval stages. In contrast, mdf-1 ^internal and rod-1 promoters drive GFP expression specifically in gut cells after embryogenesis (Table 1). Unlike mdf-2, mdf-1 and rod-1 promoters, hcp-1 promoter was found to be active in the majority, but not all, tissues analyzed, including dorsal/ventral nerve cord, head/tail/body neurons and many other tissue types (Table 1). Thus, postembryonic spatial analysis revealed distinct, yet overlapping, tissue-specific expression of SAC genes during larval development.

Unexpectedly, we also observed tissue-specific expression of SAC genes at late larval (late L4) and adult stage (Figures 1M-T and 3). Since there are no cell divisions during late L4 and at adulthood except for the divisions in somatic gonads that lead to oocyte development 30 , our observations suggest that SAC genes are expressed in non-proliferating cells in C. elegans. Similar to larval expression profiles, tissue-specific expression is observed in adult animals as well. For example, as in larvae, mdf-2 promoter drives GFP expression in seam cells and hypodermis (Figure 1M), gut cells (Figure 1O), pharynx (Figure 1Q), and vulva (Figure 1S). The expression patterns detected in adult tissues further support the striking co-expression of the checkpoint genes in hypodermal seam cells (Figures 3H-L) and intestine (Figures 3A-G) that we observed in larval stages.

We were interested in testing whether absent or nonfunctional SAC would cause aberrant postembryonic seam cell development. For this analysis, we chose mdf-2. MDF-2 shares 40% sequence identity with budding yeast Mad2 and rescues benomyl sensitivity of the mad2 knockout strain in yeast, suggesting functional checkpoint conservation 9 . Like Δmdf-1, absence of MDF-2 leads to severe defects in larval and germ cell development, suggesting essential roles in postembryonic development 9 12 . Unlike Δmdf-1, knockout strain of mdf-2 is viable 12 .

Our spatiotemporal analysis using extra-chromosomal concatameric arrays revealed that the promoter of mdf-2 drives expression of the GFP reporter in hypodermis and seam cells (Figures 1I and 1M), and some other cell types. We also constructed two chromosomal integrant pmdf-2::GFP strains, a multi-copy stable line (putatively integrated into the genome), and a stable line generated using the recently developed Mos1-mediated Single-Copy Insertion (MosSCI) method 31 . Using the multi-copy stable line, we observed similar expression patterns in hypodermis and seam cells (Figure 4A), and other cell types. MosSCI method, on the other hand, allows integration of transgenes as single copies at a few specific loci in C. elegans' genome. Although the pmdf-2::GFP stable line generated using MosSCI had > 10 × lower intensity of the GFP expression than the multi-copy stable line (data not shown), it further confirmed the expression patterns that we observed using a pmdf-2::GFP extrachromosomal transgene in postembryonic hypodermis and seam cells (data not shown).

To determine the consequence of absence of MDF-2 on normal seam cell development, we examined and quantified the number of seam cell nuclei in transgenic strains expressing SCM::GFP 32 (seam cell marker fused to GFP) in the mdf-2(tm2190) knockout, Δmdf-2, background using fluorescence microscopy (Figures 4B, 5 and 6). The tm2910 deletion removes 864 nucleotides between intron 3 and exon 6 and is likely to be a null mutation. The SCM::GFP marker allows visualization of the number of seam cell nuclei and their morphology during development. Our analysis of young adult animals homozygous for Δmdf-2 revealed both qualitative and quantitative difference compared to wild-type animals (Figures 4-6; Table 2). While wild-type adult hermaphrodites usually contain 16 evenly spaced and aligned SCM::GFP nuclei on each side of the animals 32 (Figure 4B), Δmdf-2 adult hermaphrodites frequently have non-aligned seam cell nuclei clustered in one part of the body (Figures 5 and 6). Such clustering appears to be stochastic (Figure 5) and each cluster can contain two (Figures 5A,B), three (Figure 5C), four (Figure 5D) or even more seam cell nuclei (Figure 5E). More often, certain seam cells are missing (Figure 4C), resulting in fewer than 16 SCM::GFP nuclei observed in wild-type animals (Figure 4B). Collectively, in the absence of MDF-2, the number of SCM::GFP nuclei is significantly decreased in young adult worms from 16 (observed in wild-type animals) to 14 in Δmdf-2 homozygotes (unpaired t-test, p = 2.96E-12) (Table 2). Furthermore, using ajm-1::GFP apical junction marker 33 , we observed disruptions of seam syncytia in Δmdf-2 homozygote adult worms (data not shown), which further supports the importance of MDF-2 for proper seam cell development.

During normal development, 10 precursor seam cells, H0-2, V1-6 and T, are formed during embryogenesis and are present at L1 after hatching. During L2, six of the 10 precursors undergo symmetrical division to produce additional seam cells totaling 16 seam cells at the end of L2 and beyond 34 . Therefore, the seam cell defects observed in mdf-2(tm2190) young adult worms could be either due to defective embryonic cell divisions, or alternatively, defective postembryonic divisions. In order to address these two possibilities, we scored the number of seam cell nuclei in newly hatched wild-type and Δmdf-2 L1 larvae. The wild-type animals analyzed had an average number of 10.02 SCM::GFP nuclei per side (range 9-11) (Table 2). Similarly, the majority of the Δmdf-2 newly hatched larvae had 10 SCM::GFP positive nuclei with 9.75 average and 8-11 range (Table 2). Although, unpaired students t-test analysis revealed a significant difference (p = 0.012), both the quantitative and qualitative defects observed in Δmdf-2 newly hatched larvae were much less severe than defects observed in L4 (unpaired t-test, p = 3.35E-5) or adults (unpaired t-test, p = 2.96E-12) (Table 2). Therefore, we conclude that MDF-2 plays an important role in postembryonic seam cell development.

Recently, it was reported that MDF-1 plays an important role in nutrient-deprivation induced somatic cell arrest 35 . Namely, it was found that hemizygosity of mdf-1 causes an increase in seam cell numbers from 10, observed in wild-type L1 worms starved for four days, to between 12 and 17 in more than half of the mdf-1(gk2)/+ L1 worms. To analyze the ability of mdf-2(tm2190) hemizygotes to arrest the proliferation during L1 diapause, we starved wild-type and Δmdf-2/+ hatchlings for four days. Subsequent analysis of the seam cells revealed that neither wild-type (n = 25) nor Δmdf-2/+ (n = 25) larvae had more than 11 SCM::GFP-positive nuclei, indicating starvation-induced L1 larval arrest. Thus, unlike MDF-1, MDF-2 component of the SAC does not seem to be required for starvation-induced somatic cell cycle arrest.

The seam cells have stem cell-like properties and divide four times in developing larva for self-renewal maintenance, expansion, and to produce differentiated cells 30 . Six out of 10 embryonic seam cells, H1, V1-V4 and V6, undergo self-renewal expansion division at L2, resulting in an increase in the number of seam cells to 16 30 (Figure 4B). To determine if the seam cell defect observed in Δmdf-2 homozygotes is due to a defect in proliferative cell division, we determined the number of SCM::GFP positive nuclei at late L2 and L3. We observed a mean of 14.36 (n = 25) seam cell nuclei at late L2 in the Δmdf-2 homozygotes (wild-type 15.76) and a mean of 14.08 (n = 25) seam cell nuclei at L3 in the Δmdf-2 homozygotes (wild-type 15.96), which is not significantly different from the number of SCM::GFP nuclei observed in later stages of the Δmdf-2 homozygotes (unpaired t-test, p = 0.7 and p = 0.8, respectively) (Table 2). These data demonstrate that the seam cell defect observed in Δmdf-2 homozygotes is most likely due to cell division defects at L2.

We next examined whether reduction of seam cell number could be attributed to failure of cell cycle progression of specific seam cells (H0-2, V1-V6 or T). We counted how often the observed seam cell defect is a consequence of failure of cell cycle progression of one particular cell (Figure 4C). Our analysis only includes unambiguous instances, where the identity of the defective nuclei could be determined (Figures 5 and 6). The cases where the identity of the defective seam nucleus is ambiguous, as in Figure 6B, were excluded from the analysis. We observed defects in all of the seam cells, H0-2, V1-V6 and T (Figure 4C), suggesting that failure of cell division affects all the cells in the seam cell linage. However, the frequencies of defects are different between the seam cells. For example, H0 seam cell defect was observed only once in 251 animals scored (Figure 4C). The H0 cells are the only cells, from the seam cell lineage, that do not undergo postembryonic division, further confirming the previous findings that the seam cell defect observed in Δmdf-2 homozygotes is mainly due to postembryonic defects. Similarly, H2, V5 and T cell defects were rarely observed (Figure 4C). In contrast, frequent defects were observed in the six seam cells, H1, V1-V4 and V6 that undergo expansion division to generate an additional six seam cells at L2 and beyond. These data support the findings that seam cell defects likely arise in L2 Δmdf-2 homozygotes. Furthermore, we quantified extra seam cell nuclei (Figures 5 and 4C) versus missing seam cell nuclei (Figures 6 and 4C) and, as expected, we observed that reduction of the number of SCM::GFP positive nuclei is a much more common event (Figure 4C). Representative images of seam cell reduction due to a failure of cell cycle progression of a particular lineage are shown in Figure 6. Together, these data indicate that seam cell defects in the absence of MDF-2 are mainly attributed to cell proliferation failure at L2 which randomly affects H1, V1-V4 or V6 seam cells.

It is possible that the reduction of number of seam cells in Δmdf-2 worms is caused by cell damage followed by apoptotic cell death. CED-3 is a member of the caspase family of cystein proteases that is required for cell death in C. elegans 36 . To determine whether apoptotic cell death could account for loss of seam cells, we constructed ced-3(n717) unc-26(e205) mdf-2(tm2190) in which there is no cell death. We found that ced-3(n717) unc-26(e205) mutants do not affect seam cell development, as on average 15.92 seam cell nuclei were observed in young adults (unpaired t-test, p = 0.3, when compared to wild type young adults) (Table 3). Furthermore, we found that ced-3(n717) unc-26(e205) mdf-2(tm2190) animals had similar numbers of seam cell nuclei (on average 14.27; unpaired t-test, p = 0.98) as mdf-2(tm2190), suggesting that ced-3 dependent cell death is unlikely to be responsible for seam cell loss in the tm2190 background.

During postembryonic development, seam cell division is regulated at the G1 to S phase progression by a cascade of regulatory factors that include LIN-35/Rb, FZR-1/Cdh1, and CKI-1 37 38 39 40 41 42 . As LIN-35 and FZR-1 act redundantly to control the G1 to S phase progression, seam cell proliferation appears to be normal in lin-35 and fzr-1 single mutants, while extensive hyperproliferation is observed in lin-35; fzr-1 double mutants 39 . Furthermore, lin-35 and fzr-1 single mutants rescue postembryonic seam cell defects in bro-1 single mutants 42 . bro-1 is the C. elegans CBFβ homolog that is required for the normal proliferation and differentiation of seam cells 42 . To determine whether or not lin-35 and fzr-1 mutants play a role in the defective postembryonic cell proliferation in the mdf-2(tm2190) background, we examined genetic interactions by constructing lin-35(rr33); mdf-2(tm2190) and fzr-1(ok380); mdf-2(tm2190) double mutants. We found that 100% of the lin-35(rr33); mdf-2(tm2190) double mutants are sterile, making the analysis of seam cell development difficult. We also found synthetic enhanced interaction between fzr-1(ok380) and mdf-2(tm2190) mutants (Figure 7). The ok380 deletion removes 442 nucleotides between intron 3 and exon 3 and is predicted to result in truncated FZR-1, which may or may not be functional. fzr-1(ok380) homozygotes can be easily propagated and exhibit no major developmental abnormalities. As reported previously, mdf-2(tm2190) homozygotes can be maintained at 20°C indefinitely but display a severely reduced brood size of approximately 40 progeny/worm (Figure 7A) of which only 40% develop into adults 12 (Figure 7B). Once we constructed Δfzr-1; Δmdf-2 homozygotes, we immediately observed that these worms are extremely difficult to propagate due to the small number of progeny that reach adulthood. Our detailed analysis of Δfzr-1; Δmdf-2 double mutants revealed that they have significantly reduced brood sizes (~11 progeny per worm) (Figure 7A) and significantly reduced numbers of fertile adults (~50% of all adult progeny are fertile), resulting in only two or three fertile adult progeny per hermaphrodite compared to about 10 to 15 fertile adults produced by Δmdf-2 homozygotes (Figure 7). Furthermore, we observed that while Δmdf-2 homozygotes displayed CIN as determined by high incidence of males (Him) phenotype (~3% of the adult progeny are males; n = 252), Δfzr-1 increases this chromosome instability to ~6% (n = 107 adult progeny observed).

Even though Δfzr-1; Δmdf-2 double mutants are difficult to grow, we collected enough adult progeny for analysis of postembryonic seam cell proliferation. As expected, we found that Δfzr-1 homozygotes (n = 50) had on average 15.98 SCM::GFP nuclei (Table 3) not significantly different from wild-type (unpaired t-test, p = 0.8). However, we found that Δfzr-1 had no effect on seam cell proliferation in the mdf-2(tm2190) background as Δfzr-1; Δmdf-2 double mutants had on average 14.82 (Table 3) seam cell nuclei not significantly different from the Δmdf-2 animals (unpaired t-test, p = 0.6). Taken together, these data suggest that although mdf-2 displays synthetic lethality and enhanced phenotype with lin-35 and fzr-1, this pathway is unlikely explanation for postembryonic cell proliferation defect observed in the absence of MDF-2 spindle-checkpoint using the seam cell lineage.

The hypomorphic mutant allele of fzy-1,h1983, was isolated from the screen for suppressors of the mdf-1(gk2) lethal phenotype in search for additional components that function in the metaphase-to-anaphase transition 43 . The h1983 allele is a missense mutation and the resulting FZY-1D433N mutant protein cannot properly bind the APC/C substrate IFY-1 (securin) 43 . Subsequently, it has been shown that fzy-1(h1983) rescues mdf-1(gk2) lethality likely by delaying anaphase onset because the duration of mitosis in fzy-1(h1983) early-stage embryos is extended, presumably due to an increased level of securin 44 . While the main function of MDF-1 may be regulation of APC/C activity 43 44 , the precise role for MDF-2 is currently unknown.

fzy-1(h1983) homozygotes can be easily propagated and the strain exhibits a slight decrease in the brood size and an increase in incidence of males with no apparent abnormalities in growth or morphology 43 . To determine whether fzy-1(h1983) can rescue lethality of the mdf-2(tm2190), we constructed fzy-1(h1983); mdf-2(tm2190). We observed that fzy-1 has no significant effect on brood sizes of Δmdf-2 homozygotes (Figure 7A). However, fzy-1; Δmdf-2 worms produce on average 85% progeny that develop into adults, compared to ~40% observed for Δmdf-2 homozygotes (Figure 7B). Furthermore, the majority (~95%) of fzy-1; Δmdf-2 adult progeny are fertile (Figure 7C), suggesting that fzy-1(h1983) can suppress the sterility caused by the absence of MDF-2. Also, we observed that fzy-1 decreases incidence of males from ~3% observed in the Δmdf-2 homozygotes to ~0.8% observed in double mutants. Together, these data further confirm that like MDF-1, MDF-2 regulates APC/C^CDC20 activity during development.

Next, we examined if fzy-1(h1983) has an effect on seam cell development. Interestingly, we found that fzy-1(h1983) homozygotes had on average 16.04 (Table 3) seam nuclei not significantly different from wild-type animals (unpaired t-test, p = 0.8). Furthermore, seam cell development in fzy-1; Δmdf-2 double mutants appeared to be completely normal (Table 3). Namely, fzy-1; Δmdf-2 double mutants had on average 16.08 (Table 3) seam cell nuclei not significantly different from the wild-type or fzy-1(h1983) homozygous animals (unpaired t-test, p = 0.8). In addition, the majority of the analyzed fzy-1; Δmdf-2 young adults had 16 evenly spaced and aligned SCM::GFP nuclei. These results suggest that MDF-2 plays an important role in postembryonic seam cell proliferation by inhibiting the activity of the APC/C^CDC20.

The Bristol strain N2 was used as the standard wild-type strain 50 . The following mutant alleles were used in this work: dpy-5(e907), mdf-1(gk2), mdf-2(tm2190), ced-3(n717), unc-26(e205), lin-35(rr33); fzr-1(ok380) and fzy-1(h1983). The wls51 (SCM::GFP) strain JR667 was used to visualize the seam cell nuclei in wild-type worms and the mutant backgrounds. The strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN) unless otherwise stated. The following transgenic strains were generated: JNC104 [dpy-5(e907) I; dotEx104 (Y69A2AR.30::GFP + pCeh361)]; JNC105 [dpy-5(e907) I; dotEx105 (C50F4.13::GFP + pCeh361)]; JNC106 [dpy-5(e907) I; dotEx106 (ZC328.4::GFP + pCeh361)]; JNC107 [dpy-5(e907) I; dotEx107 (Y54G9A.6::GFP + pCeh361)]; JNC108 [dpy-5(e907) I; dotEx108 (R06C7.8::GFP + pCeh361)]; JNC109 [dpy-5(e907) I; dotEx109 (ZK1055.1::GFP + pCeh361)]; JNC110 [dpy-5(e907) I; dotEx110 (T06E4.1::GFP + pCeh361)]; JNC111 [dpy-5(e907) I; dotEx111 (C50F4.11::GFP + pCeh361)]; JNC112 [dpy-5(e907) I; dotEx112 (F20D12.4^{2101 bp}::GFP + pCeh361)]; JNC113 [dpy-5(e907) I; dotEx113 (F20D12.4^{3000 bp}::GFP + pCeh361)]; JNC114 [dpy-5(e907) I; dotEx114 (F55G1.4::GFP + pCeh361)]; JNC115 [dpy-5(e907) I; dotEx115 (C50F4.11^{1332 bp}::GFP + pCeh361)]; JNC116 [dpy-5(e907) I; dotIs104 (Y69A2AR.30::GFP + pCeh361)]; JNC117 [unc-119(ed3) I; dotSi104 II (Y69A2AR.30::GFP + unc-119(+)]. Animals were maintained using standard procedures 50 .

The promoter::GFP constructs were generated using the "PCR stitching" technique 19 . The PCR experiments were designed to amplify and fuse 5' sequence immediately upstream of the predicted ATG initiator site for a targeted gene to an adjacent upstream gene. All of the primers were designed semi-manually with the aid of primer3 51 and used in standard PCR procedures to amplify putative SAC gene promoters from C. elegans N2 (Bristol) single worm lysates. These amplicons were then fused to the PCR products containing gfp sequence and unc-54 3'UTR from pPD95.75 (developed by Dr. Andrew Fire http://www.addgene.org/pgvec1?f=c&identifier=1494&atqx=%C2%A0pPD95_75&cmd=findpl). For fusion PCR reactions we used Phusion (NEB) high-fidelity DNA polymerase. All of the promoter::GFP fusion PCR products were confirmed by sequencing before injection. We injected fusion PCR products, without purification, into the gonad of young adult hermaphrodites of CB00907 at a concentration of 10 ng/μL together with 100 ng/μL dpy-5(+) plasmid (pCeh361) in 1XTE buffer to generate extrachromosomal arrays. On average, 25-30 P₀ dpy-5(e907) hermaphrodites were injected with each pSAC::GFP construct. Rescued Dpy-5 mutant phenotype was indicative of transformants. These wild-type looking F₁ progeny were plated individually and screened for the presence of wild-type F₂ progeny. On average, we obtained three to five lines yielding at least 30% wild-type progeny. Aware of the mosaicism issues associated with extrachromosomal concatamer arrays, we analyzed at least 30 replicates for each developmental stage. Once these lines were genotyped and confirmed to have similar expression patterns, one line for each construct was frozen and kept as a transformed stock. Genotyping was performed using promoter specific primer and GFP specific primer.

For each transgenic line, we prepared mixed-staged population of worms and immobilized them in 100 mM sodium azide (in water) immediately before imaging. Initially, worms were analyzed using a Zeiss Axioskop equipped with QImaging camera to confirm the consistency of expression patterns between the transgenic lines. Then more detailed analysis, which involved taking stacks of confocal images with 0.2-0.5 μm between focal planes, was performed using Quorum WaveFX Spinning Disk system mounted on a Zeiss Axioplan microscope. All images were taken at 400X, image acquisition and analysis was performed using a Volocity software package (Improvision, Coventry, England).

For all the double and single mutants, five L4 wild-type looking worms were individually plated at 20°C. The worms were transferred to fresh plates every 12 hours and the plates were scored. Total numbers of eggs laid defined the brood sizes. The eggs that did not hatch in 24 hours were scored as embryonic arrest. The eggs that hatched but did not reach adulthood were scored as larval arrest. The progeny that developed to adulthood were scored for incidence of males. The percent fertility was determined by individually plating all progeny that developed to adulthood. All of the single and double mutants were then analyzed in a SCM::GFP background for number of seam cells by using Zeiss Axioskop equipped with QImaging.