Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans.

Abstract

The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.
We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.
Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.

Full text

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RESEARCH ARTICLEOpen Access
Spindle assembly checkpoint genes reveal
distinct as well as overlapping expression that
implicates MDF-2/Mad2 in postembryonic seam
cell proliferation in Caenorhabditis elegans
Maja Tarailo-Graovac, Jun Wang, Jeffrey SC Chu, Domena Tu, David L Baillie, Nansheng Chen*
Abstract
Background: The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the
anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the
spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play,
during postembryonic development of a multicellular organism, remain largely unexplored.
Results: We have used GFP fusions of 5’ upstream intergenic regulatory sequences to assay spatiotemporal
expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in
Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP
expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-
specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we
show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of
seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show
that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20by
the SAC component MDF-2 is important for proper postembryonic cell proliferation.
Conclusion: Our results indicate that SAC genes display different tissue-specific expression patterns during
postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells,
suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or
may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam
cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly,
our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular
MDF-2 loss, than embryonic cell division.
Background
The spindle assembly checkpoint (SAC) acts as a sur-
veillance mechanism by delaying the metaphase-to-ana-
phase transition until all the chromosomes have
properly aligned and attached to the mitotic spindle;
thus, preventing chromosome instability (CIN). In the
presence of even a single improperly attached kineto-
chore, SAC is activated to inhibit a large multisubunit
E3 ubiquitin ligase complex, the anaphase promoting
complex/cyclosome (APC/C), and prevents anaphase
onset [1]. APC/C activity requires the association of
Cdc20 in early mitosis, while Cdh1 (encoded by Fzr1 in
mammals) is required to activate APC/C in late mitosis
and during G1 [2,3]. The primary target of SAC is the
Cdc20 activator that, when inhibited, cannot activate
APC/C to degrade securin [1]. Degradation of securin is
required for activation of separase and cleavage of cohe-
sion between sister chromatids which in turn triggers
anaphase onset in mitotic cells [1].
* Correspondence: chenn@sfu.ca
Department of Molecular Biology and Biochemistry, Simon Fraser University,
Burnaby, British Columbia, V5A 1S6, Canada
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© 2010 Tarailo-Graovac et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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The first identified components of SAC were isolated
in two independent genetic screens in Saccharomyces
cerevisiae and include MAD1, MAD2, MAD3, BUB1,
and BUB3 [4,5]. These proteins are widely conserved,
both structurally and functionally, throughout the eukar-
yotic kingdoms [1]. However, additional proteins essen-
tial for the checkpoint activity have continued to be
discovered in higher eukaryotes. These include Rod
(ROugh-Deal), Zw10 (Zeste-White 10) and CENP-F pro-
teins, among others [6-8]. These components lack clear
yeast orthologs, suggesting that, in higher eukaryotes,
checkpoint signaling is more elaborate.
The SAC components and the checkpoint signalling
pathway are highly conserved in C. elegans. The C. ele-
gans homologues of the SAC components, originally dis-
covered in yeast, have been identified and named mdf-1,
mdf-2, san-1, bub-1 and bub-3, respectively [9-13].
Recent availability of knockout alleles of these checkpoint
components, in addition to RNA interference (RNAi)
experiments, allowed assessment of the phenotypic con-
sequences in the absence of the SAC gene products
[9,12,14]. All of these genes are important for genome
stability and viability in the presence of spindle damage
[9,11,12,15]. However, while mdf-2, san-1 (known as
MAD3 in other systems) and bub-3 become essential
only in the presence of chemical or mutational disrup-
tions of the mitotic spindle [9,11,12,15], bub-1 and mdf-1
are essential for embryonic viability, long-term survival
and fertility under normal laboratory conditions in C. ele-
gans [9,16]. In fact, analysis of an mdf-1 deletion mutant,
mdf-1(gk2), gave the first demonstration of what affect a
defective checkpoint has on animal development [9]. In
the absence of MDF-1, severe developmental defects are
observed, including embryonic lethality, larval arrests,
abnormal vulva development, and sterility, which lead to
lethality of the homozygous strain after three generations
[9]. Similar developmental defects have also been
observed in the absence of MDF-2 [9,12]; however, unlike
Δmdf-1 animals, Δmdf-2 homozygotes can be propagated
indefinitely [12]. The fact that absence of different SAC
components leads to different developmental conse-
quences in C. elegans, as well as other organisms [17,18],
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(CDC20) fully restored proper
seam cell proliferation; fzr-1/CDH1 mutant had no effect
on seam cell development in a Δmdf-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 (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 promo-
ter 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 puta-
tive “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 regula-
tors 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 pre-
viously described minimum and maximum promoter
lengths used in large scale projects [21-25]. 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 expres-
sion 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].
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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 exam-
ined another construct that contained 3 kb upstream
sequence of czw-1 and still did not observe any expres-
sion (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].
Promoters of spindle assembly checkpoint genes drive
similar early embryonic expression
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 major-
ity 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 sur-
veillance 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 neces-
sary to drive proper expression of this gene in embryo-
nic 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 prolif-
eration 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-expres-
sion of SAC genes in the rapidly dividing early embryo-
nic 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
Table 1 Summary of postembryonic spatial GFP expression observed for SAC gene transcriptional reporters
Gene5’ Regulatory Region Size
(bp)System
2951
C50F4.11 (mdf-1)5432
C50F4.11 (mdf-1)13323
Y69A2AR.30 (mdf-2) 301
ZC328.4 (san-1) 597
R06C7.8 (bub-1)1130h
Y54G9A.6 (bub-3)282
ZK1055.1 (hcp-1) 591 d,v,h,t,b
T06E4.1 (hcp-2) 427t
F20D12.4 (czw-1)2101
F20D12.4 (czw-1)3000
F55G1.4 (rod-1)1163
Nervous Intestine Pharynx Gonad HypodermisSeam
cells
Vulva Coelomocytes
C50F4.11 (mdf-1)
X
X
X
X
X
X
X
X
X
X
XX
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X s,u
dtc
X
X
5’ Regulatory Region column lists the corresponding sizes of the putative promoters used to drive GFP expression.
X = expression observed; h = head neurons; d = dorsal nerve cord; v = ventral nerve cord; t = tail neurons; b = mid-body neurons; s = spermatheca; u = uterus;
dtc = distal tip cells.
1Upstream region, from the ATG initiator site in the first gene, his-35, of the mdf-1 containing operon to an adjacent upstream gene his-41.
2Upstream region from the ATG initiator site in mdf-1 to an adjacent upstream gene his-35.
3Upstream region from the ATG initiator site in mdf-1 to the operon upstream gene his-41.
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tend to confer more localized GFP expression, as exem-
plified 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.
SAC promoters drive tissue-specific gene expression later
in development
Rapid cell proliferation occurs in all four larval stages
especially in the second larval stage (L2) of develop-
ment in C. elegans when many somatic cells are
Figure 1 Expression patterns driven by the mdf-2 promoter. pmdf-2::GFP is expressed at all developmental stages of C. elegans.
Representative images of the pre-comma (A-D), mid (E and F) and late (G and H) embryonic stages with the majority of cells expressing GFP.
(I-L) Representative images of the GFP signal in seam cells at L3 (I and J) and gut cells at L2 (K and L). (M-T) Representative images of the adult
tissues that contain GFP signal including seam (M and N), gut (O and P), pharyngeal (Q and R) and vulval cells (S and T). The scale bar
represents 25μm.
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generated [30]. As expected, GFP expression conferred
by SAC gene promoters was detected at all four larval
stages (Table 1). Unlike embryonic expression, spatio-
temporal analysis revealed that postembryonic expres-
sion 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-
1internaland rod-1 promoters drive GFP expression spe-
cifically in gut cells after embryogenesis (Table 1).
Unlike mdf-2, mdf-1 and rod-1 promoters, hcp-1 pro-
moter 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 expres-
sion 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 pat-
terns 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.
Absence of MDF-2 results in aberrant number and
alignment of seam cell nuclei
We were interested in testing whether absent or non-
functional 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 check-
point conservation [9]. Like Δmdf-1, absence of MDF-2
leads to severe defects in larval and germ cell develop-
ment, suggesting essential roles in postembryonic devel-
opment [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
Figure 2 Embryonic expression of seven spindle assembly
checkpoint genes. (A-G) Representative images of early embryonic
(pre-comma stage) SAC promoter activities; GFP and DIC images are
shown. (A) For mdf-1, the 1332 bp 5’ regulatory region that contains
the internal promoter is depicted. (F) For the hcp-2 promoter, bean
stage embryo is shown. Notice that in some images, like (E), clear
mosaicism is observed. Some nuclei have weak GFP signal, while
some have no detectable signal. This is likely due to loss of the
array. The scale bar represents 10μm.
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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 speci-
fic 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 ani-
mals homozygous for Δmdf-2 revealed both qualitative
and quantitative difference compared to wild-type ani-
mals (Figures 4-6; Table 2). While wild-type adult her-
maphrodites usually contain 16 evenly spaced and
aligned SCM::GFP nuclei on each side of the animals
[32] (Figure 4B), Δmdf-2 adult hermaphrodites fre-
quently 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 (Fig-
ure 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 homo-
zygotes (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
Figure 3 Co-expression of the SAC genes in gut and seam cells
of the adult animals. (A-G) All of the SAC promoters drive GFP
expression in gut cells, depicted by arrows. (H-L) The majority of the
SAC gene promoters drive GFP expression in seam cells, depicted
by arrowheads. The scale bar represents 25μm.
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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
Figure 4 mdf-2/MAD2 (a spindle-checkpoint gene) is expressed in hypodermal seam cells and is important for their proper
development. (A) Expression driven by mdf-2 promoter in hypodermis (long arrow) and seam cells (short arrow) using the multi-copy stable line
JNC116. The scale bar represents 25μm. (B) An adult wild-type worm containing 16 SCM::GFP nuclei, originating from H0-H2, V1-V6, and from the
T seam cell. (C) Quantitative analysis of 251 animals with the seam cell defect. Black bars represent percentage of extra cells observed in
individual seam cells, while the grey bars represent percent of the individual seam cells that are missing in the defective animals.
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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 post-
embryonic seam cell development.
Recently, it was reported that MDF-1 plays an impor-
tant 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/+ hatchl-
ings 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.
Figure 5 Representative images of extra SCM::GFP positive nuclei observed in the mdf-2(tm2190) animals. (A) An animal has 17 positive
SCM::GFP nuclei. It appears to have one extra V3 seam cell with two fused together. (B) An animal has 17 positive SCM::GFP nuclei. It appears to
have one extra V6 seam cell. (C) An animal has 18 positive SCM::GFP nuclei. It appears to have two extra V6 seam cells. (D) An animal has 19
positive SCM::GFP nuclei. It appears to have three extra V3 seam cells. (E) An example of five SCM::GFP positive nuclei in the region where only a
single seam cell should reside. The animals depicted are in either L4 or young adults.
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The seam cell defect of mdf-2(tm2190) is due to defects in
the proliferative seam cell division
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
Figure 6 Representative images of mdf-2(tm2190) homozygotes with only 15 SCM::GFP positive nuclei. (A) H1 seam cell appears to be
missing. (B) An example of ambiguous case where either V1 or V2 seam nucleus is missing. (C) V2 seam cell appears to be missing. (D) V3 seam
cell appears to be missing. (E) V4 seam cell appears to be missing. (F) V6 seam cell appears to be missing. Arrowheads depict a single seam
nucleus in the lineages where two seam nuclei are expected to be observed.
Table 2 The average number of SCM::GFP nuclei is altered in mdf-2(tm2190) mutants
Genotype Hypodermal seam-cell nuclei (n)
L1 L2L3 L4 Adult
Wild type
mdf-2(tm2190)
10.02 ± 0.08 (48)
9.75 ± 0.09* (59)
15.76 ± 0.12 (25)
14.36 ± 0.30* (25)
15.96 ± 0.04 (25)
14.08 ± 0.25* (25)
16.04 ± 0.09 (25)
14.20 ± 0.39* (25)
16.00 ± 0.08 (49)
14.28 ± 0.17* (64)
All of the strains are homozygous for the seam cell marker wIs51 (SCM::GFP). The number of SCM::GFP positive nuclei was scored 0 h after hatching (L1), two
days after hatching (L4) and 12 h after L4 stage (young adults). Asterisk denotes significant difference when compared to wild type using unpaired students
t-test analysis (p = 0.012 for L1 stage; p = 7.21E-5 for L2 stage; p = 2.57E-10 for L3 stage; p = 3.35E-5 for L4 stage and p = 2.96E-12 for adult stage.
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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 homozy-
gotes (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 pro-
gression 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 defec-
tive 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 divi-
sion 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. Further-
more, 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 pro-
gression 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 pro-
liferation failure at L2 which randomly affects H1, V1-
V4 or V6 seam cells.
The seam cell reduction in mdf-2(tm2190) is not likely to
be caused by ced-3 dependent cell death
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 con-
structed 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 develop-
ment, 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). Further-
more, 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.
Absence of FZR-1 enhances sterility of mdf-2 mutants
without causing any effect on seam cell development
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 [37-42]. 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 hyperprolifera-
tion is observed in lin-35; fzr-1 double mutants [39].
Furthermore, lin-35 and fzr-1 single mutants rescue post-
embryonic seam cell defects in bro-1 single mutants [42].
bro-1 is the C. elegans CBFb 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) homo-
zygotes 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
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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 immedi-
ately 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 sig-
nificantly 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 homo-
zygotes (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 diffi-
cult 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) back-
ground as Δfzr-1; Δmdf-2 double mutants had on aver-
age 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 pheno-
type 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.
Hypomorphic mutant fzy-1(h1983) partially suppresses
lethality of mdf-2 mutants and completely rescues seam
cell defects
The hypomorphic mutant allele of fzy-1,h1983, was iso-
lated 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].
Table 3 The average number of SCM::GFP positive nuclei observed in different genetic backgrounds
GenotypeHypodermal seam-cell nucleiRangen (sides)
Wild type
mdf-2(tm2190)
ced-3(n717) unc-26(e205)
ced-3(n717) unc-26(e205) mdf-2(tm2190)
fzr-1(ok380)
fzr-1(ok380); mdf-2(tm2190)
fzy-1(h1983)
fzy-1(h1983); mdf-2(tm2190)
16.00 ± 0.08
14.28 ± 0.17
15.92 ± 0.08
14.27 ± 0.25
15.98 ± 0.07
14.82 ± 0.26
16.04 ± 0.12
16.08 ± 0.11
15-17
8-19
15-17
8-17
15-17
10-20
14-18
14-18
49
64
48
55
50
50
50
50
All of the strains are homozygous for the seam cell marker wIs51 (SCM::GFP). The number of SCM::GFP positive nuclei was scored in young adult hermaphrodites.
Note that data for wild-type and mdf-2(tm2190) young adults are duplicates of the data shown in Table 2.
0
10
20
30
40
50
60
*
Progeny/worm
mdf-2(tm2190)
mdf-2(tm2190);
fzy-1(h1983)
mdf-2(tm2190);
fzr-1(ok380)
0
10
20
30
40
50
60
70
80
90
100
*
mdf-2(tm2190)
mdf-2(tm2190);
fzy-1(h1983)
mdf-2(tm2190);
fzr-1(ok380)
Percent Adult Progeny
0
10
20
30
40
50
60
70
80
90
100
mdf-2(tm2190)
mdf-2(tm2190);
fzy-1(h1983)
mdf-2(tm2190);
fzr-1(ok380)
*
*
Percent Fertile Adult Progeny
A
BC
Figure 7 Genetic interactions between mdf-2(tm2190)/MAD2, fzr-1(ok380)/CDH1 and fzy-1(h1983)/CDC20. (A) Brood size - scored as total
number of eggs laid by a worm. (B) Viability - scored as percent of the progeny that developed to adulthood. (C) Fertility - percent of adult
progeny that are fertile.
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Subsequently, it has been shown that fzy-1(h1983) res-
cues 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 deter-
mine 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). Further-
more, 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/CCDC20
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 sug-
gest that MDF-2 plays an important role in postembryo-
nic seam cell proliferation by inhibiting the activity of
the APC/CCDC20.
Discussion
In this work we have examined for the first time in vivo
spatiotemporal expression profiles of eight spindle-
checkpoint genes in C. elegans. Among these eight
genes, five are conserved from yeast to human (mdf-1,
mdf-2, san-1, bub-1 and bub-3) [9-13], while three are
conserved in higher eukaryotes (hcp-1, hcp-2 and rod-1),
including C. elegans [12,16,45]. Our study focused on
analysis of the expression patterns by using extra-chro-
mosomal arrays. To maximally reduce the effect of
mosaicism, the known caveat of this approach, we
analyzed a large number of animals for each develop-
mental stage, and recorded the tissues and cells where
GFP expression was consistently observed. On the other
hand, we found the mosaicism to be beneficial for a bet-
ter identification of tissues where GFP is expressed.
When promoters drive GFP expression in more than
one tissue types, then expression restricted to only small
groups of cells, due to loss of the array, offers more con-
fident identification of these tissues. Also, GFP expres-
sion is a sensitive technique which is important for SAC
gene expression analysis because generally SAC genes
do not produce an abundant number of transcripts.
Concatamer arrays were previously suggested to be a
sensitive tool for detecting gene expression for genes
with low levels of transcription [23]. We confirmed the
sensitivity of this approach when we generated a pmdf-
2::GFP stable line using MosSCI [31]. This stable line
had very low GFP signal intensity and required long
exposure times for the expression to be observed.
The 5’ DNA sequences selected as containing putative
promoters of the SAC genes displayed common early
embryonic activities in the majority, if not all, of the
rapidly dividing embryonic cells. This finding is consis-
tent with the known roles of the checkpoint genes in
cell division. We expected this result because of the fact
that 556 of the 959 somatic cells present in adult her-
maphrodite are generated during embryogenesis [30].
Furthermore, our observations of early embryonic
expression is supported by published analyses which
used antibodies against some of the SAC gene products
[9,11,15,27,45]. Thus, it is likely that these transcrip-
tional fusions recapitulate endogenous SAC gene pro-
moter activities. Importantly, this common “ubiquitous”
expression of SAC genes (including mdf-1) during early
embryogenesis, suggests that expression of mdf-1, the
only one located within an operon, has to be driven by
the internal promoter (Additional file 1, Figure S1).
Thus, the mdf-1 containing operon is likely a “hybrid
operon” [28].
czw-1 (known as ZW10 in other organisms) was also
included in our study; however, analysis of two different
constructs did not reveal any detectable GFP expression.
It is possible that expression of the analyzed transgenes
was either too low for visible detection, germline speci-
fic, conditional, or that regulatory elements of this gene
are located in regions not included by our putative pro-
moter selection criteria.
In contrast to expression in embryos, postembryonic
expression of SAC genes in C. elegans is more localized.
During the four larval stages in a hermaphrodite, the 53
undifferentiated somatic blast cells generate an addi-
tional 403 somatic nuclei [30]. The somatic blast cell
divisions generate somatic gonad, muscle, coelomocytes,
nerves, hypodermis and intestine [30,46,47]. If all of the
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checkpoint genes played the same role in postembryonic
development, one would expect to observe the same
expression patterns for the SAC genes. However, our
analysis revealed that checkpoint promoters generally
dictate differential postembryonic expression patterns.
For example, it is very interesting that mdf-1internaland
the rod-1 promoters drive GFP expression exclusively in
intestine after embryogenesis, while the hcp-1 promoter
drives GFP expression in multiple tissues (Table 1).
These findings suggest distinct, yet overlapping, roles of
the checkpoint genes in postembryonic development
and provide an excellent resource for further research.
Recently, staining of newly hatched L1 larva with anti-
MDF-1 antibody revealed specific localization of MDF-1
to intestinal cells and germ cell precursors [35], which
further supports our findings from using the transcrip-
tional reporter system. We did not observe expression
in germ cell precursors or any other germ cells possibly
due to silencing of concatamer transgenes in the germ-
inal gonad.
An unexpected finding from our analysis was tissue-
specific expression of SAC genes in late L4 and adults
that contain no somatic cells destined to divide. Consid-
ering that tissue-specificity observed in these stages was
similar to the tissue-specificity observed in larval stages,
it is possible that the observed patterns reflect longer
turnover times for the GFP carried over from earlier lar-
val stages [23]. On the other hand, it is possible that 5’
upstream sequences used in our analysis do not include
important “repressor” elements that are required for
proper expression of SAC genes. Alternatively, it may be
that SAC genes have roles in these adult tissues that
remain to be uncovered.
We have found that spindle-checkpoint genes reveal
an intriguing co-expression in hypodermal seam cells.
This finding prompted us to use the seam cell lineage to
test the functional importance of the checkpoint for
proper postembryonic cell proliferation. Here, we
demonstrated that the knockout allele, tm2190, of mdf-2
results in defective seam cell development that is mainly
attributed to seam cell proliferation failure at L2. In the
absence of MDF-2, on average 14 seam cell nuclei were
observed instead of expected 16. The number of SCM::
GFP nuclei per side of an animal ranges from 8 to 19 in
the absence of MDF-2 (Table 3). While the majority of
the Δmdf-2 homozygotes contains less than expected 16
seam cell nuclei per side in young adults, we also
observed animals that had more than 16 seam cell
nuclei (Figure 5), which could be attributed to defective
cell division. The results presented in this paper provide
the first evidence that embryonic cell divisions are more
tolerant to the loss of SAC, in particular MDF-2, than
postembryonic cell divisions, as determined using the
seam cell lineage. Furthermore, we show that the impor-
tance of MDF-2 for proper seam cell proliferation
depends on its regulation of APC/CCDC20. The seam cell
defect in Δmdf-2 homozygotes cannot be explained
by cell damage followed by caspase-dependent apoptotic
cell death, since ced-3 mutant had no effect on seam
cell defect in Δmdf-2 worms. Furthermore, fzy-1(h1983)
rescued all of the Δmdf-2 phenotypes, except for the
brood size. On the other hand, G1 phase regulators,
LIN-35 and FZR-1, when defective affect only brood
size in the absence of MDF-2. The analysis presented
here, using the Δmdf-2, serves as an excellent model for
further studies on effects of a defective SAC on develop-
ment of different tissues in a multicellular organism.
A striking emerging pattern is that essentially all SAC
genes are expressed in intestine and hypodermis. SAC
components MDF-2 [9] and MDF-1 [35] have previously
been observed to be localized to gut cells by using anti-
body staining. Endoreduplication, also known as endore-
plication, is a process in which S phases are not
followed by mitosis. This process gives rise to cells with
extra copies of chromosomes, permitting amplification
of the genome in specialized cells. In humans, these
include hepatocytes, cardiomyocytes and megakaryocytes
[48]. In C. elegans, two tissues are polyploid: the hypo-
dermis and the intestine [49]. Our finding of co-expres-
sion of SAC genes in these tissues may suggest a
possible role of these genes in the process of endoredu-
plication in C. elegans. Furthermore, our findings clearly
suggest that SAC genes are differentially regulated at
the transcription level at different developmental stages.
Conclusion
We have examined for the first time in vivo spatiotem-
poral expression profiles of eight conserved spindle
assembly checkpoint genes in C. elegans. Our compre-
hensive analysis revealed that all of the SAC gene pro-
moters displayed common early embryonic activities in
the majority, if not all, of the rapidly dividing embryonic
cells. Furthermore, we found that all of the SAC gene
promoters drive tissue specific postembryonic expres-
sion. The expression patterns differ between the SAC
genes; the majority of the SAC genes co-express in
hypodermal seam cells and gut cells. These findings sug-
gest that the SAC components may have distinct roles
in postembryonic development which could be different
from their role in mitosis. Furthermore, our analysis
provides an important starting point for analysis of the
checkpoint roles in development of a multicellular
eukaryote that may offer explanation for distinct pheno-
typic consequence upon inactivation of different SAC
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genes. It is extremely important to determine how
defects in different SAC components affect cell prolif-
eration, cell fate determination and cell differentiation in
a multicellular organism.
Methods
C. elegans strains, alleles and culturing
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
(F20D12.42101 bp::GFP + pCeh361)]; JNC113 [dpy-5
(e907) I; dotEx113 (F20D12.43000 bp::GFP + pCeh361)];
JNC114 [dpy-5(e907) I; dotEx114 (F55G1.4::GFP +
pCeh361)]; JNC115[dpy-5(e907)
(C50F4.111332 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].
I;dotEx115
Generation of pSAC::GFP transgenic animals
The promoter::GFP constructs were generated using
the “PCR stitching” technique [19]. The PCR experi-
ments 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 pro-
cedures to amplify putative SAC gene promoters from
C. elegans N2 (Bristol) single worm lysates. These
amplicons were then fused to the PCR products con-
taining 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 gener-
ate extrachromosomal arrays. On average, 25-30 P0
dpy-5(e907) hermaphrodites were injected with each
pSAC::GFP construct. Rescued Dpy-5 mutant pheno-
type was indicative of transformants. These wild-type
looking F1 progeny were plated individually and
screened for the presence of wild-type F2progeny. 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 developmen-
tal stage. Once these lines were genotyped and con-
firmed to have similar expression patterns, one line for
each construct was frozen and kept as a transformed
stock. Genotyping was performed using promoter spe-
cific primer and GFP specific primer.
In vivo analysis and imaging of pSAC::GFP transgenic
lines
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 consis-
tency of expression patterns between the transgenic
lines. Then more detailed analysis, which involved tak-
ing 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).
Viability measurement
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 lar-
val 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.
Tarailo-Graovac et al. BMC Cell Biology 2010, 11:71
http://www.biomedcentral.com/1471-2121/11/71
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Page 15

Additional material
Additional file 1: Figure S1: Putative mdf-1/MAD1 promoter
activities. (A) 295 bp of the 5’ regulatory region immediately upstream
of his-35, the first gene in the mdf-1 containing operon, drives localized
GFP expression from the embryonic stage. (Left) GFP images; (Right) DIC
images. (B) The internal promoter is the 543 bp sequence between his-
35 and mdf-1. This promoter drives ubiquitous GFP expression in the
embryo as expected. (C) 1332 bp of the 5’ regulatory region upstream
from the ATG initiator site in mdf-1 - extending to the operon adjacent
upstream gene (his-41) - results in ubiquitous GFP expression in embryos,
similar to the internal mdf-1 promoter expression pattern.
Abbreviations
APC/C: anaphase promoting complex or cyclosome; CIN: chromosome
instability; DIC: differential interference contrast; GFP: green fluorescent
protein; Him: high incidence of males; MosSCI: Mos1-mediated single-copy
insertion; RNAi: RNA interference; SAC: spindle assembly checkpoint.
Acknowledgements
This project was supported by funding from the Natural Science and
Engineering Research Council (NSERC) of Canada Discovery Grants to NC
and DLB. We thank the Caenorhabditis Genetics Center and Ann M. Rose for
strains, as well as Harald Hutter for sharing experimental equipment. We also
thank Robert Johnsen for critical reading for the manuscript. NC is a Michael
Smith Foundation for Health Research (MSFHR) Scholar and a Canadian
Institutes of Health Research (CIHR) New Investigator.
Authors’ contributions
NC and MTG conceived of the study. MTG designed the experiments,
performed the expression analysis, seam cell analysis, genetic interaction
analysis and wrote the manuscript. JW participated in seam cell analysis.
JSSC participated in spatiotemporal expression analysis. DT generated
transgenic strains. NC and DLB helped to draft the manuscript. All authors
read and approved the final manuscript.
Received: 17 February 2010 Accepted: 21 September 2010
Published: 21 September 2010
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