DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 4375
Development 138, 4375-4385 (2011) doi:10.1242/dev.066431
© 2011. Published by The Company of Biologists Ltd
Cell shape and Wnt signaling redundantly control the
division axis of C. elegans epithelial stem cells
Marjolein Wildwater1,*, Nicholas Sander2, Geert de Vreede1 and Sander van den Heuvel1,*
SUMMARY
Tissue-specific stem cells combine proliferative and asymmetric divisions to balance self-renewal with differentiation. Tight
regulation of the orientation and plane of cell division is crucial in this process. Here, we study the reproducible pattern of
anterior-posterior-oriented stem cell-like divisions in the Caenorhabditis elegans seam epithelium. In a genetic screen, we
identified an alg-1 Argonaute mutant with additional and abnormally oriented seam cell divisions. ALG-1 is the main subunit of
the microRNA-induced silencing complex (miRISC) and was previously shown to regulate the timing of postembryonic
development. Time-lapse fluorescence microscopy of developing larvae revealed that reduced alg-1 function successively
interferes with Wnt signaling, cell adhesion, cell shape and the orientation and timing of seam cell division. We found that Wnt
inactivation, through mig-14 Wntless mutation, disrupts tissue polarity but not anterior-posterior division. However, combined
Wnt inhibition and cell shape alteration resulted in disordered orientation of seam cell division, similar to the alg-1 mutant. Our
findings reveal additional alg-1-regulated processes, uncover a previously unknown function of Wnt ligands in seam tissue
polarity, and show that Wnt signaling and geometric cues redundantly control the seam cell division axis.
INTRODUCTION
The development of complex organisms with specialized cell
types, tissues and organs requires tight coordination between cell
division and differentiation. This is achieved in part through
asymmetric cell divisions that segregate the potential to
proliferate and the commitment to differentiate to different
daughter cells (Galli and van den Heuvel, 2008; Gonczy, 2008;
Knoblich, 2008). For example, asymmetric divisions allow adult
stem and precursor cells to self-renew and simultaneously
produce daughter cells that will go on to differentiate. By
contrast, proliferative (or symmetric) cell divisions generate two
essentially identical daughter cells and promote exponential
increases in cell number. The proper balance between
proliferative and asymmetric cell divisions is crucial in stem cell
maintenance and tissue homeostasis.
Differences between daughter cells may arise during division
or after completion of division (Horvitz and Herskowitz, 1992).
The first mode of division is considered intrinsically asymmetric
and applies, for instance, to the C. elegans zygote and to
Drosophila neuroblasts (Galli and van den Heuvel, 2008;
Gonczy, 2008; Knoblich, 2008). During intrinsically asymmetric
division, cell-fate determinants become unequally localized
according to the polarity of the cell or tissue, and the mitotic
spindle aligns along the polarity axis. Consequently, cell
cleavage, which takes place perpendicular to the spindle,
generates two daughter cells with different constituents. As an
alternative mode of asymmetric division, external signals (e.g.
from a stem cell niche) may instruct the fate of daughter cells
1
Department of Developmental Biology, Utrecht University, Padualaan 8, 3584 CH
Utrecht, The Netherlands. 2Department of Genetics, University of Minnesota, 321
Church St SE, Minneapolis, MN 55455, USA.
*Authors for correspondence ([email protected]; [email protected])
Accepted 4 August 2011
that are initially identical after division. Importantly, the axis and
plane of cell cleavage are critical in both forms of asymmetric
division, as they determine the distribution of determinants as
well as the size and position of daughter cells.
The mechanisms that determine the axis of division in the
context of a tissue are poorly understood. Seam cells in the
C. elegans epidermis provide a powerful model for studies of
stem cell-like divisions within a polarized epithelium. These
cells undergo symmetric and asymmetric divisions with a
reproducible anterior-posterior orientation at stereotypical times
during postembryonic development (Fig. 1A) (Sulston and
Horvitz, 1977). The left and right lateral sides of the first stage
larva (L1) each contain a row of ten seam cells, which continue
to divide during each larval stage (Fig. 1A,B). Usually, the
posterior daughter cell maintains the seam cell fate, while the
anterior daughter terminally differentiates and fuses with the
epidermal syncytium hyp7 (in the V1-V4 and V6 lineages) or
becomes a neuron or neuronal support cell (in the H2, V5 and T
lineages). In the second larval stage (L2), six seam cells (V1p4p, V6p and H1aa) also go through a proliferative division,
which expands the seam cell number to sixteen. Seam cells are
polarized in the apical-basal as well as anterior-posterior
direction. Anterior-posterior polarity involves asymmetric
localization of Wnt/-catenin asymmetry pathway components,
such as APR-1 APC, WRM-1 -catenin and POP-1 TCF/LEF
(Mizumoto and Sawa, 2007b). Apical junctions that contain
cadherin-catenin complexes separate the apical and basolateral
domains and connect neighboring seam cells (Labouesse, 2006;
Lynch and Hardin, 2009).
Previous studies revealed that a ‘heterochronic’ pathway controls
the stage-dependent timing of seam cell divisions (Ambros and
Horvitz, 1984; Chalfie et al., 1981). Several heterochronic genes
encode microRNAs (miRNAs), which promote the transition from
the L1 to L2 (lin-4), the L2 to L3 (let-7 sister miRNAs) and the L4
to adult (let-7) stages (Abbott et al., 2005; Lee et al., 1993;
Reinhart et al., 2000; Wightman et al., 1993). lin-4 mutant larvae
DEVELOPMENT
KEY WORDS: C. elegans, microRNA, Argonaute, Wnt signaling, Cleavage plane, Asymmetric division
4376 RESEARCH ARTICLE
skip the L2 stage-specific proliferative divisions and show a
reduced number of seam cells, whereas mutants that lack the let-7
sister (let-7s) miRNAs reiterate these divisions and contain extra
seam cells (Resnick et al., 2010). Although this heterochronic
pathway provides stage-specific timing, what distinguishes
proliferative from asymmetric divisions and what controls the seam
cell division axis remain unknown.
In this study, we investigate the reproducible anterior-posterior
orientation of the stem cell-like seam cell divisions. For advanced
analysis, we created transgenic reporter strains that allow timelapse fluorescence microscopy of seam cell divisions. We identified
a mutant with large clusters of abnormally positioned seam cells
and traced the mutation to the alg-1 Argonaute gene. We show that
alg-1 not only contributes to the heterochronic developmental
timing pathway, but also to the orientation of seam cell division
from the L3 stage onward. We discovered that alg-1 inhibition also
creates defects in the Wnt/-catenin asymmetry pathway and in
seam cell contacts and shape, which could potentially underlie the
abnormal orientation of seam cell divisions. However, interfering
with Wnt signaling, seam cell contacts or cell shape individually
did not alter the division axis. Loss of Wnt secretion resulted in a
random pattern of seam versus non-seam fates, indicating that Wnt
ligands instruct tissue polarity in this system. However, when
combined with seam cell rounding, Wnt inactivation also caused
abnormally oriented seam cell divisions. These findings point to
robust control of the cell division axis through a combination of
Wnt signaling and geometric constraints.
MATERIALS AND METHODS
Nematode strains
We used the wild-type Caenorhabditis elegans strain N2 and the
derivatives listed in Table 1.
EMS screening and mapping
Mutants with increased numbers or abnormally positioned seam nuclei
were selected in a clonal EMS mutagenesis screen of ~3500 haploid
genomes (Brenner, 1974) with scm::GFP transgenic animals (Terns et al.,
1997). Our strategy aimed to evade maternal contribution and larval
lethality but allow identification of temperature-sensitive alleles. Adult F1
animals were washed two to three times to remove bacteria, and single
animals allowed to lay eggs in 10 l M9 containing 0.05% Tween 20
overnight at 15°C. Synchronized F2 progeny were fed, placed at 25°C and
examined after 24 hours. At this time, wild-type animals finished the L3
Development 138 (20)
seam divisions and nuclei could be scored prior to hyp7 fusion of anterior
daughter cells. Mutants were backcrossed multiple times. SNP mapping
was performed according to Davis et al. (Davis et al., 2005).
Immunofluorescence
Synchronized animals on poly-L-lysine-coated slides were freeze cracked
and fixed with methanol (5 minutes at –20°C) and acetone (20 minutes at
–20°C) (Duerr, 2006). Primary antibodies used were MH27 (1:20
supernatant or 1:200 concentrate; mouse, Developmental Studies
Hybridoma Bank) and rabbit anti-GFP (1:100; Molecular Probes).
Secondary antibodies used were Alexa 568 goat anti-mouse and Alexa 488
goat anti-rabbit (both 1:500; Invitrogen). Worms were mounted in Prolong
Anti-Fade Gold (Invitrogen) supplemented with 2 g/ml 4⬘,6-diamidino2-phenylindole (DAPI; Sigma).
RNAi-mediated interference
Starved L1s were transferred onto alg-1 RNAi feeding plates and grown at
20°C. For strong knockdown by RNAi, we either injected double-stranded
RNA into young adults or transferred synchronized L3 stage larvae to
RNAi feeding plates and analyzed the next generation. For the dpy-11
RNAi experiments, several generations of animals were grown on RNAi
food to ensure the most severe dumpy phenotype.
Molecular cloning
A 1.6 kb wrt-2 promoter fragment was amplified by PCR (primers 5⬘CTACAAGCTTCAGGTCGACTCCAGGTAATT-3⬘ and 5⬘-TAGGATCCCCGAGAAACAATTGCCAGGTTG-3⬘) from plasmid pPD95.69
containing Pwrt-2::gfp [a kind gift from Thomas Bürglin (Aspöck et al.,
1999)], and cloned into the PCGSI expression vector with HindIII and
BamHI. Expression constructs for GFP::H2B and GFP::PH were recloned
from pAZ132 (Ppie-1::GFP::H2B) and pAA1 into the BamHI site of the
Pwrt-2-containing PCGS1 vector. Constructs were injected into N2 animals
at 5 ng/l together with Plin-48::mCherry (15 ng/l) and digested  DNA
(60 ng/l). The alg-1 rescue construct was created by combining
independent PCR reactions of an 8.3 kb genomic alg-1 fragment containing
the alg-1 promoter, intron/exon sequences and 3⬘UTR. The PCR mix was
injected into he210 animals (3-5 ng/l) together with Plin-48::tdTomato
(15 ng/l) and digested  DNA (60 ng/l).
Microscopy
Time-lapse movies of larval development were recorded at 30-second
intervals for 2-7 hours at 20°C using a 63⫻ 1.4 N/A PlanApochromat
objective and a motorized microscope (Zeiss Axioplan). Animals were
sedated with 10 mM muscimol (M1523 Sigma) and mounted on 5%
agarose. Coverslips were sealed with Zeiss Immersol 518N oil to prevent
liquid evaporation. GFP excitation light from the X-Cite source was filtered
to 10% transmission, and 50-msecond exposure times were used.
Table 1. Strains used
JR667
SV875
SV1009
SV1050
SV1045
CB185
VT1145
VT581
VT1101
MT1524
MT2306
HS1486
WM53
JJ1136
OLB19
JK3437
SV1176
SV1124
SV1245
Genotype
unc-119(e2498::Tc1) III; wIs51[scm::gfp; unc-119(+)]
unc-119(e2498::Tc1) III; alg-1(he210) X; wIs51s
heIs63[Pwrt-2::GFP::PH; Pwrt-2::GFP::H2B; Plin-48::mCherry]
unc-119(e2498::Tc1) III; alg-1(he210) X; wIs51; heEx357[alg-1(+); Plin-48::mcherry]
alg-1(he210) X; heIs63(may contain (unc-119(e2498::Tc1) III; wIs51)
lon-1(e185) III
lin-46 (ma164) lin-58 & mir-241 (nDf51) V; mir-84(n4037) X
dpy-5(e61) lin-28(n719) I; lin-46(ma164) unc-76(e911) V
lin-28(n719) I; lin-46(ma164) lin-58 &mir-241(nDf51) V; mir-84(n4037) X
lin-28(n719) I
lin-28(n1119) I
unc-76(e911) V; osIs13[Papr-1::apr-1::venus; unc-76 (+)]
alg-2(ok304) II
unc-119(e2498) III; zuEx2[unc-119 (+) hmp-1::GFP]
dusIs[let-413::gfp; rol-6(su1006)]
him-5(e1490) V; qIs74[Ppop-1::pop-1::gfp]
rol-1(e91) mig-14(or78) II; muIs35[Pmec-7::gfp; lin-15(+)]; heIs63; huEx119 [myo-2::GFP; R06B9]
dpy-11(e224); heIs63
lin-46(ma164) lin-58 & mir-241(nDf51) V; mir-84(n4037) X; heEx421([Pwrt-2-GFP-PH; Pwrt-2::GFP::H2B; Plin-48::mCherry]
DEVELOPMENT
Strain
RESULTS
The alg-1 Argonaute gene is required for normal
epithelial stem cell-like division
To identify regulators of the cell division axis, we performed a
genetic screen for mutants with abnormally positioned seam cell
nuclei, based on seam-specific expression of the green fluorescent
protein (scm::GFP; see Materials and methods). Of several
identified mutants, he210 larvae displayed the most dramatically
disorganized seam cell phenotype (Fig. 1). Starting at the L3
developmental stage, he210 mutant larvae showed an increased
number of seam nuclei [average 22±4.6 after fusion in L3 versus
16±0.3 in wild type (±s.d.)] and an accumulation of nuclei outside
the normally linear row (Fig. 1D). Some of the nuclei outside the
row lost expression of the seam cell marker (data not shown),
which indicates fusion with the hyp7 syncytium (Fig. 1B). Thus,
seam cells in he210 mutants may still divide asymmetrically, but
appear to lose the proper timing and orientation of cell division.
Immunostaining of the cell junction-associated protein AJM-1
confirmed that the scm::GFP-positive nuclei represent individual
seam cells (Fig. 1E,F). Moreover, gaps between seam cells were
frequently observed in he210 mutants (see below, Fig. 2D,H, Fig.
3H). These abnormalities, in particular the accumulation of seam
nuclei outside the row, became more severe during the L4 stage
(see below). Adult he210 animals displayed a normal overall
morphology, but showed defects in the formation of adult alae,
frequently contained a protruding vulva or had burst at the vulva,
and produced few progeny. Thus, the he210 mutation severely
affects the seam lineage and a limited number of other cells.
We located the he210 mutation to a single base pair alteration in
the alg-1 Argonaute gene. This mutation is predicted to change
glycine 716 to arginine at the beginning of the highly conserved
PIWI domain (Fig. 1G). Introduction of PCR-amplified genomic
RESEARCH ARTICLE 4377
alg-1 DNA rescued the he210 mutant phenotype to wild type
(20/28 animals). Moreover, feeding RNAi of alg-1 during larval
development phenocopied he210 (see below; Fig. 1H). Thus,
downregulation of ALG-1 leads to the overproliferation and
mislocalization of seam cells.
ALG-1 controls seam organization independently
of the developmental timing pathway
ALG-1 is the core component of an RNA-induced silencing
complex (RISC), which, in association with miRNAs, represses
specific mRNAs (Grishok et al., 2001). The best-characterized
miRNAs are key regulators of the heterochronic developmental
timing pathway, including the pioneering lin-4 and let-7 miRNAs
and the let-7s miRNAs mir-48, mir-84 and mir-241 (Abbott et al.,
2005; Lee et al., 1993; Reinhart et al., 2000). These miRNAs
require ALG-1, or the closely related ALG-2, for their function
(Grishok et al., 2001; Tops et al., 2006). In agreement, alg-1 has
also been associated with a heterochronic phenotype and increased
seam cell numbers (Cai et al., 2008; Grishok et al., 2001).
We observed that alg-1 feeding RNAi in the alg-2(ok304)
mutant reduced the seam cell number to ~10 (Table 2). This
indicates that ALG-1/ALG-2 double inhibition blocks lin-4
miRNA function and prevents the L2-specific proliferative
divisions. By contrast, homozygous alg-1(he210) mutants and
larvae exposed to alg-1 RNAi by feeding from the L1 stage onward
invariably formed more than 16 seam cells. In such alg-1 feeding
RNAi larvae, most seam cells in the V1-V4 and V6 lineages
completed two rounds of division during the L3 stage, which
increased the seam cell number in late L3 larvae from 16 to 24±4.9
(±s.d.). Thus, alg-1 feeding RNAi and alg-1(he210) mutation cause
a heterochronic phenotype that resembles let-7s miRNA inhibition,
with repeated proliferative divisions and extra seam nuclei.
Fig. 1. Identification of ALG-1 Argonaute as a regulator of seam epithelium organization. (A)Seam cells (green) are aligned in a linear row
on each lateral side of C. elegans larvae. (B)Postembryonic seam cell lineages. y-axis indicates time (hours) of development. Circle color indicates
fate: green, seam cells; blue, hyp7 fusion fate; red, neuronal fate; cross, apoptosis. V1-V4 and V6 undergo a proliferative division in early L2.
(C,D)Wild-type (WT) and he210 larvae after seam cell divisions in L4. Expression of scm::GFP (green) marks seam cell nuclei. The he210 mutant
shows additional and abnormally positioned seam nuclei (D, arrowhead). (E,F)Seam cells visualized by apical junction staining (red, AJM-1) and
DNA (blue, DAPI) of wild-type (E) and he210 (F) animals prior to L4 stage divisions. Arrowhead in F marks the disorganized seam. (G)he210
contains a G-to-A transition in alg-1, which is predicted to change glycine 716 to arginine in the conserved PIWI domain. (H)alg-1 RNAi results in
seam cell defects similar to he210. L4 stage animal with disorganized seam cells (arrowhead). Seam-specific GFP::H2B and GFP::PH mark the DNA
and cell membranes, respectively. Scale bars: 10m.
DEVELOPMENT
Control of epithelial stem cell division
4378 RESEARCH ARTICLE
Development 138 (20)
We examined whether the abnormal positions and gaps between
seam cells are also normal aspects of the retarded heterochronic
phenotype. Retarded daf-12(rh285) larvae showed a moderately
increased number of seam cells, but these cells invariably remained
present in a single linear row (Table 2). To obtain stronger
reiterative L2 divisions, we combined mutation of the three let-7s
miRNAs and lin-46, which act in parallel to promote the L2-to-L3
transition (Abbott et al., 2005). These quadruple mutants showed
substantially increased numbers of seam cells [average 26±0.8
(±s.d.); Table 2], which again invariably remained aligned in a
single row (Fig. 2C; 100%, n160 cells observed in 16 animals).
However, when these mutants were exposed to alg-1 feeding
RNAi, the seam cell pattern became disorganized, with gaps and
cells located outside the row, similar to the alg-1 RNAi phenotype
(Fig. 2D; 100%, n6 animals). Thus, disorganization of the seam
epithelium does not automatically result from increased cell
numbers, but indicates an alg-1 function that is independent of the
let-7s miRNAs.
Notably, alg-1 RNAi induced very few abnormally positioned
seam cells in lin-28 mutants (Fig. 2F; 2 of 11 animals showed a
single mispositioned seam cell). As lin-28 is a target for inhibition
by lin-4 miRNA, we examined whether enhanced lin-28 activity
contributes to the alg-1 disorganized seam phenotype. Mammalian
LIN28 is highly expressed in stem cells and can contribute to the
conversion of differentiated somatic cells to induced pluripotent
stem (iPS) cells (Yu et al., 2007). C. elegans lin-28 mutants skip
the L2 stage proliferative seam cell divisions and contained only
~10 seam cells (Table 2). However, larvae with combined lin-28,
lin-46 and let-7s mutations reiterated L2 divisions during the L3
and subsequent stages (Abbott et al., 2005) (Table 2). These
quintuple mutants showed a substantial increase in the number of
seam cell nuclei [average 29±0.7 after fusion (±s.d.)], which
remained localized in a continuous linear row (Fig. 2G; 100%,
n609 cells in 21 animals), unless alg-1 was also inhibited (Fig.
2H; 100%, n27 animals). Thus, alg-1 RNAi causes
disorganization of the seam epithelium independently of lin-28. In
summary, ALG-1 promotes the developmental timing of seam cell
divisions through the heterochronic pathway and has an additional
role in organizing the seam epithelium.
ALG-1 controls the orientation of the cell division
axis and motility of the cell membranes
To examine whether alg-1 inhibition causes an abnormal seam cell
division axis we generated seam cell-specific GFP reporters that
mark the cell membrane (Pwrt-2::GFP::PHPLC1) and DNA (Pwrt2::GFP::H2B), and established a protocol for time-lapse recording
DEVELOPMENT
Fig. 2. ALG-1 regulates seam cell localization
independently of the developmental timing pathway.
Seam cells visualized by immunostaining of apical junctions
(red, AJM-1) and DNA (blue, DAPI) in L3 or L4 C. elegans
larvae of various genotypes. Stars mark fusion of anterior
daughter cells. (A)Normal L4 larva. (B)alg-1(RNAi) L4 larva
with seam cells outside the row (arrowhead) and lost seam
cell connections (arrow). (C)L4 stage quadruple
heterochronic mutant [lin-46(ma164) mir-48 & mir241(nDf51); mir-84(n4037)] with additional seam cells
located within a single row. (D)L4 stage quadruple mutant
as in C treated with alg-1 RNAi. Arrow marks lost seam cell
contact. (E)L3 stage lin-28(n719) mutant with a reduced
seam cell number (arrow). (F)L3 stage lin-28(n719) mutant,
illustrating suppression of the alg-1 RNAi phenotype. (G)L4
stage heterochronic lin-28(n719); lin-46(ma164) let-7s larva
with additional seam cells that remained properly aligned.
(H)Animals as in G after alg-1 RNAi show the typical alg-1
RNAi phenotype. Seam cells are disorganized (arrowhead)
and cell-cell contacts are often lost (arrow). Scale bar: 10m.
Control of epithelial stem cell division
RESEARCH ARTICLE 4379
Fig. 3. ALG-1 regulates the seam cell division axis and cell motility. (A-B⬙) Still images from time-lapse recordings of asymmetric divisions in
wild-type and alg-1(RNAi) L3 C. elegans larvae. (A-A⬙) Normal seam cells divide along the anterior-posterior axis. (B-B⬙) alg-1 inhibition changes the
seam cell division axis. Metaphase (A, arrowhead B), anaphase (A⬘, arrowhead B⬘) and telophase (A⬙, arrowhead B⬙). Time is indicated from
metaphase onward. (C-F)Quantification of cell division angles (C) in wild-type (D) and alg-1(RNAi) (E,F) L3 larvae. (G-G⬙) A sequential series of timelapse microscopy images of seam cells migrating after division (arrowheads) in an alg-1(RNAi) L3 larva. The migrating cells end up outside the row.
Cell migration time is indicated. (H,I)Late alg-1(RNAi) larvae show disconnected seam cell clusters (H) and occasional networks of aberrantly
connected seam cells (I). Seam-specific GFP::H2B and GFP::PH mark DNA and cell membrane, respectively. Scale bars: 10m.
of the developing larvae. Animals with integrated reporter
transgenes were synchronized at the early L1 stage, transferred to
control bacteria or exposed to alg-1 feeding RNAi, and followed
for various time periods of larval development by time-lapse
fluorescence microscopy (see Materials and methods and Movies
1-4 in the supplementary material).
This approach revealed abnormally oriented seam cell divisions
in alg-1(RNAi) larvae from the early L3 stage onward. In the wild
type, chromosomes invariably segregated along the anteriorposterior axis, with a maximal deviation of 10 degrees (Fig. 3AA⬙,C,D). After alg-1 RNAi, the division axis was less stringent
during the reiterated proliferative divisions in L3 (Fig. 3E), and
even more abnormal during the subsequent second round of
division in the L3 stage (Fig. 3B-B⬙,F). We even observed cell
divisions perpendicular to the normal anterior-posterior axis (a
deviation of 90 degrees) (see Movie 2 in the supplementary
material). Thus, ALG-1 is required for normal orientation of seam
cell division.
Our time-lapse recordings also revealed an additional
mechanism for abnormal seam organization. In contrast to wildtype animals, alg-1 RNAi-treated L3 larvae showed highly motile
seam cell membranes with apparent ruffles and extensions (11 out
of 12 animals; see Fig. S2 in the supplementary material). As a
potential consequence of this high motility, we observed two
initially neighboring cells extending next to each other (Fig. 3GG⬙; see Movie 3 in the supplementary material). Thus, seam cells
Table 2. Summary of seam cell phenotypes in several genetic backgrounds
Genetic background
Seam number (±s.d.)
Orientation defects
Loss of contacts
16±0.3
22±4.6
24±4.9
26±0.8
25±2.1
ND
10.4±0.5
ND
29±0.7
29±1.3
16±0.7
22±3.4
16±0.3
16±0.7
17±1.3
16±1.3
16±0.9
–
+
+
–*
+
+
–
+/–
–
+
–
–
–
–
+
–
+/–
–
+
+
–
+
–
–
+
–
+
–
+
–
–
–
+/–
+
15.9±0.74
16.1±0.75
10.8±1.9
–
–
–
–
+
++
WT (N2)
alg-1(he210)
alg-1(RNAi)
let-7s + lin-46(ma164)
let-7s + lin-46(ma164) + alg-1(RNAi)
let-7s + lin-46(ma164) + mig-14(RNAi)
lin-28(n719)
lin-28(n719) or (n947) + alg-1(RNAi)
lin-28(n719) + let-7s + lin-46(ma164)
lin-28(n719) + let-7s + lin-46(ma164) + alg-1(RNAi)
lon-1(e185)
lon-1(e185) + alg-1(RNAi)
dpy-11(RNAi)
mig-14(or87)
mig-14(or87) + dpy-11(RNAi)
hmr-1(RNAi)
hmr-1(RNAi) + dpy-11(e224)
B. alg-1 and alg-2 are partially redundant
alg-2(ok304)
alg-1(he210) + alg-1(RNAi)
alg-2(ok304) + alg-1(RNAi)
Seam cells were counted after L3 divisions, fusion with hyp7 and reconnection of seam cells (column 2 in A). Division orientation defects were scored during L3 and L4
divisions. The plus sign indicates defects, whereas minus indicates normal. Note that let-7s indicates triple inactivation of let-7 sister miRNAs through mir-48 mir-241(nDf51);
mir-84(n4037) mutation.
*Defects were seen in late L4 division: 8 of 23 cells showed a division orientation defect.
ND, not determined.
DEVELOPMENT
A. Changes in cell shape and Wnt signaling affect the division axis
in alg-1(RNAi) larvae show aberrant cell division axes and
hypermotile cell membranes, which lead to a highly disorganized
seam epithelium.
ALG-1 affects seam cell fate and localization of
Wnt/-catenin asymmetry pathway components
To determine how alg-1 controls the axis of seam cell division, we
searched for abnormalities that precede the abnormal division
orientation. Using time-lapse fluorescence microscopy, we detected
abnormalities as early as the L2 division program. At the L2 stage,
the V1-4 and V6 seam cells undergo a proliferative symmetric
division followed by an asymmetric division in which the anterior
daughter fuses with hyp7 (Fig. 1B, Fig. 4A-A⬙) (Sulston and
Horvitz, 1977). In alg-1(RNAi) larvae, we occasionally observed
pairs of cells of which both the posterior and anterior daughter
fused with the hyp7 syncytium (Fig. 4B-B⬙, 2 of 30 pairs in three
animals). Moreover, we observed that some anterior daughters
failed to fuse, and that some seam-derived hyp7 nuclei were
missing (4 of 49 cells; data not shown). All seam cells reconnect
after fusion to form a continuous row in wild-type animals,
whereas some gaps remained in alg-1(RNAi) larvae (data not
shown). A few other seam cells appeared hyperextended,
suggesting that they closed gaps generated by aberrant hyp7 fusion
Development 138 (20)
events (data not shown). These results illustrate that alg-1 RNAi
leads to occasional defects in the segregation of the seam versus
non-seam (hyp7) fate.
The defects in daughter cell fate could reflect defects in the
Wnt/-catenin asymmetry pathway (Herman, 2001; Mizumoto and
Sawa, 2007a; Takeshita and Sawa, 2005a). This pathway promotes
the seam fate through export of the POP-1 TCF transcriptional
repressor and activation of POP-1–SYS-1 -catenin in the posterior
nucleus (Kidd et al., 2005; Shetty et al., 2005). After asymmetric
division, the anterior non-seam daughter cell contains high levels
of nuclear POP-1 and the posterior seam daughter cell contains low
levels of nuclear POP-1 (Fig. 4D) (Phillips and Kimble, 2009). The
upstream component APR-1 APC localizes asymmetrically to the
anterior cortex of seam cells before division, and becomes enriched
in the anterior daughter cell after division (Fig. 4G) (Mizumoto and
Sawa, 2007b). Surprisingly, we observed that POP-1 and APR-1
localize asymmetrically not only during the asymmetric L2
divisions, but also during the proliferative symmetric divisions in
wild-type L2 stage larvae (Fig. 4C,F). Thus, the symmetric L2
divisions seem to uncouple high nuclear POP-1 levels from the
hyp7 fate. Immunostaining of POP-1 in alg-1(RNAi) larvae showed
occasionally reversed POP-1 asymmetry after asymmetric L2
divisions [2 of 60 cell pairs in nine alg-1(RNAi) L2 larvae versus
Fig. 4. ALG-1 regulates the localization of Wnt pathway components and cell fate. (A-B⬙) Time-lapse microscopy shows fusing seam
daughter cells after division in wild-type (A-A⬙) and alg-1(RNAi) (B-B⬙) L2 stage C. elegans larvae. alg-1 RNAi occasionally leads to the fusion of
neighboring daughter cells (arrowheads in B-B⬙). Time is measured from completion of cytokinesis onward. Seam-specific GFP::H2B and GFP::PH
mark the DNA and cell membrane, respectively. (C-E)POP-1 TCF/LEF asymmetry. In wild-type L2 larvae, POP-1 levels are high (arrowheads) in
anterior daughter nuclei after symmetric (C) and asymmetric (D) cell divisions. After alg-1 RNAi, localization of POP-1 is occasionally reversed (E,
arrowheads point to high-level nuclear POP-1). Green, POP-1; red, AJM-1. (F-L)APR-1::Venus localization in wild-type and alg-1(RNAi) larvae. APR-1
APC (green) is enriched at the anterior cortex during symmetric (F) and asymmetric (G) cell division in wild-type (arrowheads) and remains enriched
at the cortex of the anterior daughter cell after division (the left-hand cells within the brackets in F,G,I). After alg-1 RNAi, APR-1 can be posteriorly
enriched or fails to localize (arrowhead, H), and an abnormally localized APR-1 crescent has been observed (compare K with L; dotted lines indicate
the metaphase plate). pd, neuronal postdereid cells. Scale bars: 10m.
DEVELOPMENT
4380 RESEARCH ARTICLE
0 of 62 cell pairs in 13 wild-type animals] (Fig. 4E). By the L3
stage, abnormal POP-1 localization was much more prominent [34
of 183 cell pairs (19%) in 14 animals]. Live imaging of APR1::Venus localization showed occasional enrichment of APR-1 at
the posterior cortex prior to L3 division in alg-1(RNAi) larvae (3
of 240 cells in 30 animals) (Fig. 4H), or high APR-1 localization
all around the cell membrane (6 of 240 cells) as compared with no
abnormal APR-1 localization in wild-type animals (n300 cells in
30 animals). By the L3 divisions, abnormal APR-1 localization was
detected in 38 of 168 (23%) seam cells (or cell pairs after division)
(Fig. 4J,L). These observations indicate that ALG-1 contributes to
the proper localization of Wnt/-catenin asymmetry pathway
components and to cell fate acquisition of the seam cell daughters.
The upstream components of the Wnt/-catenin asymmetry
pathway not only control cell fate, but also the cell division axis of
specific embryonic blastomeres (Cabello et al., 2010). As a
possible explanation for the abnormally oriented divisions in alg-1
larvae, we examined whether Wnt signaling controls the seam cell
division axis in wild-type animals. As C. elegans expresses five
different Wnt ligands, we focused on the transmembrane protein
MIG-14 Wntless, which is thought to be generally required for Wnt
secretion (Bänziger et al., 2006; Pan et al., 2008; Yang et al., 2008).
We used the lethal mutation mig-14(or87), rescued with an
extrachromosomal array with wild-type mig-14 (Bänziger et al.,
2006). Larvae that do not inherit the extrachromosomal array from
the mother complete embryogenesis but lack zygotic mig-14
function. To reduce the risk of residual maternal function, we
studied seam cell divisions at the L3 and L4 larval stages.
Interestingly, 48% of the seam cell divisions in mig-14 larvae
(n35 cell divisions in five animals) showed a reversed fate, such
that the anterior daughter remained present as a seam cell and the
posterior daughter cell fused with the hyp7 syncytium (Fig. 6E).
However, we never observed cells outside the seam cell row and
seam-seam cell contacts were invariably retained. Thus, whereas
RESEARCH ARTICLE 4381
alg-1 affects the Wnt/-catenin pathway and seam cell fate
determination, loss of Wnt signaling alone does not lead to seam
cell division with an abnormal axis.
The elongated seam cell shape is an important
cleavage plane determinant
We considered other factors that might contribute to the abnormally
oriented divisions in alg-1 larvae. The disrupted contacts between
seam cells could be important, as cell junctional contacts contribute
to cell polarity, proliferation control, tissue organization and cell
division orientation (Lu et al., 2001). However, immunostaining of
AJM-1 showed that the localization of apical junctions is
maintained in seam cells that are detached (see Fig. S1 in the
supplementary material). Based on the localization of LET-413
Scribble and HMP-1 -catenin, alg-1 RNAi did not prevent
the establishment of apical-basal polarity (see Fig. S1 in
the supplementary material). Moreover, most seam cells in
alg-1(RNAi) larvae remained connected to at least one neighbor. A
comparison of seam cells connected on both sides with seam cells
neighboring a gap did not reveal a significant difference in the
frequency or angle of aberrant divisions (see Fig. S1E,F in the
supplementary material). Finally, partial reduction of hmr-1
E-cadherin in wild-type animals with hmr-1 feeding RNAi
occasionally disrupted seam cell contacts [Fig. 6C; 13 of 400 cells
(3.25%) in 40 animals], but these cells did not change their
orientation of division. We conclude that partial loss of seam cell
contacts does not lead to abnormally oriented seam cell divisions.
We noticed that seam cells that were relatively round after
alg-1 RNAi frequently showed an aberrant division axis. Indeed,
measurements of the seam cell length (X) and width (Y) showed a
significant correlation between increased roundness (X/Y closer to
1) and abnormal axis of division (P0.009) (Fig. 5C). Examination
of z-stack images obtained by confocal microscopy showed that
cells round up in all three dimensions. The normally relatively flat
Fig. 5. The seam cell division axis follows geometric constraints. (A,B)z-stacks of seam cells in wild-type and alg-1(RNAi) L4 C. elegans larvae.
Normal seam cells are flat and elongated (A); alg-1 RNAi results in rounded seam cells (B). Red and green lines indicate the position of the crosssections shown at the top and left, respectively. The star marks a cell fusing with hyp7. GFP::H2B and GFP::PH mark the DNA and cell membrane,
respectively. (C)Elongation factor [cell length (X) divided by width (Y)] comparison between cells with normal versus abnormal division axes in alg1(RNAi) larvae (second division in L3). Division angles were measured at anaphase onset. Bars indicate the average X/Y ratio±s.d. (D) Cell elongation
factor during interphase in several mutant/RNAi backgrounds. Dev timing refers to lin-28(n719); lin-46(ma164) triple let-7s mutant. Error bars
indicate s.d. (E-G) Seam cell elongation suppresses aberrant cell division orientation. Seam cells are disorganized in alg-1(RNAi) larvae (E, arrow),
whereas they are stretched in lon-1 larvae (F). alg-1 RNAi-treated lon-1(e185) mutant shows disconnected seam cell-cell contacts (arrow, G), but no
seam cells outside the row (G). Red, AJM-1; blue, DNA (DAPI). Scale bars: 10m.
DEVELOPMENT
Control of epithelial stem cell division
seam cells became progressively more spherical during the L3 to
L4 stage of development in alg-1(RNAi) larvae (compare Fig. 5A
with 5B).
To examine whether the rounded shape influences the division
axis, we used the abnormally long (Lon) mutant lon-1(e185). These
mutants display, on average, a 25% increased body length
(Maduzia et al., 2002). Examination of lon-1 mutants showed that
seam cells are present in normal numbers, form a single row, and
are more stretched compared with those in the wild type (Fig. 5F).
Inhibition of alg-1 in lon-1(e185) mutants resulted in gaps between
the seam cells, but the seam cells almost invariably remained
present in a single linear row (Fig. 5G) [only 1 of 16 lon-1(e185),
alg-1(RNAi) animals formed a single small seam cell cluster; 7 of
12 control alg-1(RNAi) animals formed one or more groups of
misplaced seam cells]. These data indicate that the elongated cell
shape contributes an important cue for the orientation of seam cell
divisions. The rounding of seam cells after alg-1 loss of function
eliminates this cue.
Redundant control of the anterior-posterior
division axis
To further investigate the influence of cell shape on the seam cell
division plane, we examined whether Dpy (short and fat) animals
have fewer elongated seam cells. As a severe Dpy mutant, we used
dpy-11, which encodes a thioredoxin-like enzyme that probably
modifies collagens. Feeding RNAi of dpy-11 in Pwrt-2::GFP::PH,
Pwrt-2::GFP::H2B animals caused a severe Dumpy phenotype.
Such animals formed a normal number of seam cells, although
these were shorter and wider than normal (Fig. 6B). Cell divisions
almost invariably took place along the proper anterior-posterior
division axis (one abnormal division, n140 cells in 14 animals
examined). Interestingly, the cell elongation factor (X/Y) was
similar for seam cells in dpy-11 and alg-1 RNAi larvae (Fig. 5D).
The seam cells in the retarded heterochronic lin-28; lin-46 let-7s
Development 138 (20)
mutants used in our study also showed a similar elongation factor
(Fig. 5D). The near normal division orientations in dpy-11 and
retarded heterochronic larvae indicate that cell shape is not the sole
factor determining the cell division axis.
We concluded that alg-1 mutants contain multiple seam cell
defects, several of which might affect the division axis: defects in
the Wnt/-catenin asymmetry pathway, reduced cell adhesion,
more rounded seam cell shape, and extra proliferative divisions that
reduce the cell elongation factor. As none of the single disruptions
appeared to alter the division axis in wild-type animals, we
examined whether reduced seam cell elongation in combination
with reduced Wnt signaling or cell adhesion could disrupt the cell
division axis. To test this, we transferred either mig-14(or87)
segregating animals to dpy-11 RNAi or dpy-11(e224) mutants to
hmr-1 RNAi. Partial hmr-1 RNAi did not affect the seam cell
division axis (see above), and treatment of dpy-11 mutants with
hmr-1 RNAi only occasionally resulted in a misplaced seam cell
(Fig. 6D; in 6 of 39 animals). However, exposure of mig-14(or87)
animals to dpy-11 RNAi caused severe disorganization of the seam
epithelium, with multiple seam cell clusters (Fig. 6F; in 7 of 13
animals), whereas the mig-14(or87) single mutant formed a linear
row of seam cells (see above, Fig. 6E).
As an alternative means to alter seam cell shape, we made use
of the extra proliferative divisions in heterochronic lin-46 let-7s
mutants (see above, Fig. 2C, Fig. 5D, Fig. 6G). We exposed this
mutant to mig-14 feeding RNAi, starting in the L1 stage, which
caused a weak seam cell fate phenotype in L4 larvae (only 1 of 56
cells showed a reversed fate). Nevertheless, the mig-14(RNAi); lin46 let-7s mutant combination resulted in abnormally oriented seam
cell divisions and a highly disorganized seam epithelium (15 of 23
cells showed abnormal division orientation) (Fig. 6H; see Movie 4
in the supplementary material). We conclude that multiple factors
promote the anterior-posterior orientation of seam cell divisions,
including the elongated cell shape, the Wnt/-catenin asymmetry
Fig. 6. Cell shape and Wnt signaling redundantly control the cell division axis. (A-F)Combined effects of Wnt signaling, cell adherence and
cell shape on division orientation. (A,B)Seam organization is abnormal in alg-1(RNAi) (A) but normal in dpy-11(RNAi) (B) L4 C. elegans larvae.
(C)hmr-1 E-cadherin weak RNAi partly disrupts seam cell contacts (arrow). (D)In combination with dpy-11(e224), abnormally oriented divisions
were also observed at low frequency (arrowhead, 6/39 animals). (E)mig-14(or87) Wntless mutants show a random anterior-posterior orientation of
cell fate, but seam cells continue to divide along the anterior-posterior body axis. Similar numbers of anterior (arrow) and posterior daughter cells
fuse with hyp7 (stars). (F)dpy-11 RNAi in mig-14(or87) leads to highly disorganized seam epithelium (arrowhead). (G,H)A heterochronic L4 larva
shows a largely normal seam epithelium (G). However, weak mig-14 RNAi causes dramatic disorganization of the seam in these mutants (H) (see
Movie 4 in the supplementary material). Seam-specific GFP::H2B and GFP::PH mark DNA and cell membranes, respectively. Note that nuclei without
membranes are part of hyp7. Scale bar: 10m.
DEVELOPMENT
4382 RESEARCH ARTICLE
pathway and cell junctional contacts. Because of redundancy
between these contributions, the orientation of seam cell division
is robust and only becomes abnormal when two different levels of
control are lost.
DISCUSSION
In this study, we examined the regulation of the cell division axis
in a C. elegans epithelial stem cell-like lineage. Mutants with
abnormally orientated seam cell divisions appeared relatively rare;
in a moderately sized screen, only the alg-1(he210) mutant stood
out for its remarkably disorganized seam epithelium. Detailed
analysis of this phenotype uncovered novel roles for ALG-1 in
contributing to proper asymmetry in the Wnt/-catenin pathway,
cell adhesion, cell shape, cortical dynamics and division
orientation, in addition to its previously reported role in the
heterochronic pathway. Cell rounding in alg-1(RNAi) larvae
correlated with abnormally oriented divisions, and introduction of
a lon-1 mutation suppressed both defects. Unexpectedly, we found
that mig-14 Wntless, a transmembrane protein dedicated to Wnt
ligand secretion (Bänziger et al., 2006; Pan et al., 2008; Yang et al.,
2008), controls the anterior-posterior pattern of seam and non-seam
cell fates. Moreover, when combined with reduced seam cell
elongation, inactivation of mig-14 resulted in a highly disorganized
seam epithelium. These data support redundant roles for Wnt
signaling and cell shape in determining the orientation of seam cell
division (see Fig. S3 in the supplementary material).
alg-1 provides multiple developmental functions
C. elegans alg-1 was known to contribute to the developmental
timing of larval development (Cai et al., 2008; Grishok et al., 2001;
Morita and Han, 2006), but not to the control of the cell division
axis. Although ALG-1 probably acts with miRNAs to control the
division axis, it remains unclear which miRNAs are involved.
Recent studies identified large numbers of high-confidence
miRNA-mRNA interactions, which are associated with various
biological processes (Chan et al., 2008; Zhang et al., 2007; Zisoulis
et al., 2010). Genetic confirmation of such interactions is hampered
by redundancies between miRNAs and, in general, by the modest
inhibition of target genes by individual miRNAs (Baek et al., 2008;
Inui et al., 2010). Key targets should be upregulated after alg-1
inhibition and thereby contribute to disrupted Wnt signaling,
increased cortical dynamics, loss of adherence or cell rounding.
The effects could be indirect, as indicated by ipla-1, which is the
only gene previously found to act in seam division orientation
(Kanamori et al., 2008). IPLA-1 has phospholipase A(1) activity
and is needed for proper localization of WRM-1 -catenin and
membrane lipid composition (Imae et al., 2010; Kanamori et al.,
2008). Future studies might focus on candidate genes that are
present in the target lists, including Wnt pathway components and
regulators of cell adhesion, membrane transport and cortical
contractility.
Wnt signaling depends on alg-1 and promotes
tissue polarity
Several observations implicate alg-1 in the Wnt/-catenin
asymmetry pathway. During seam cell division, several Wnt
signaling components become asymmetrically localized to the
anterior (e.g. APR-1 APC) or the posterior (e.g. LIN-17 Frizzled)
cortex (Mizumoto and Sawa, 2007a; Mizumoto and Sawa, 2007b;
Sawa et al., 1996; Takeshita and Sawa, 2005b). This leads to
activation of the POP-1–SYS-1 -catenin transcriptional complex
in the posterior nucleus (Kidd et al., 2005; Shetty et al., 2005), and
RESEARCH ARTICLE 4383
establishment of the posterior seam fate. Interestingly, we observed
that the L2-specific symmetric divisions also showed asymmetric
APR-1 and POP-1 localizations. Hence, the seam cell fate might
be established independently of Wnt/-catenin signaling during the
proliferative L2 divisions.
A recent study suggested a role for Wnt/-catenin asymmetry
signaling upstream of the alg-1/miRNA heterochronic pathway (Ren
and Zhang, 2010). Our results indicate that alg-1, independently of
the heterochronic pathway, also affects Wnt signaling. The observed
contribution of Wnt ligands was remarkable, as only the V5 and T
seam cell lineages were previously shown to depend on Wnt ligands
(Herman et al., 1995; Whangbo et al., 2000). Mutation of mig-14
Wntless was previously reported to have no effect on the seam cell
number (Gleason and Eisenmann, 2010). However, we found that
mig-14 mutation causes a randomized order of seam and non-seam
cell fates. This indicates that seam cells can polarize independently
of Wnt ligands, but coordinating this polarity with respect to the
anterior-posterior body axis requires Wnt. The mig-14 phenotype
thus resembles a tissue polarity or planar cell polarity (PCP)
phenotype, in that cells fail to coordinate their polarity with the plane
of the epithelium. Similar observations have been made for a
quintuple Wnt mutant (Yamamoto et al., 2011).
The elongated seam cell shape depends on alg-1
and promotes anterior-posterior division
Although alg-1 RNAi caused seam cells to lose adhesive
connections, this did not appear to cause abnormally oriented
divisions. By contrast, reduced elongation of the seam cells clearly
contributes to the aberrant anterior-posterior orientation of seam
cell divisions. What causes the cell-shape change in alg-1 mutants?
Reiterated L2 proliferative divisions probably provide one
mechanism; extra proliferative divisions add extra cells to the row
and reduce elongation. Indeed, alg-1 RNAi did not cause abnormal
division orientation in lin-28 mutants that skip L2 proliferative
divisions. However, some of the seam cells in alg-1(RNAi) larvae
were more rounded even before extra cells were formed, and cells
rounded off in three dimensions (Fig. 5). This suggests that alg-1
promotes seam cell elongation independently of cell number,
possibly by affecting the actin cytoskeleton or adherence to the
extracellular matrix.
It has long been recognized that cell shape affects the division
axis through preferential orientation of the mitotic spindle along the
longest axis of the cell (‘Hertwig’s rule’) (Théry and Bornens,
2006). However, in a developmental context, cortical cues
generally determine spindle orientation, as is well described for C.
elegans early embryos and Drosophila neuroblasts and sensory
organ precursor cells (Galli and van den Heuvel, 2008; Gonczy,
2008; Knoblich, 2008). Such cues can overrule the effect of
geometric constraints. For example, PCP instructs a spindle
position perpendicular to the long cell axis in dorsal tissue during
zebrafish gastrulation (Gong et al., 2004).
Cells in culture round up during mitosis, yet orient the division
axis according to their shape in interphase (Théry et al., 2007).
During cell rounding, adhesive contacts with the extracellular
matrix lead to the formation of retraction fibers (Théry et al., 2007;
Toyoshima and Nishida, 2007). This creates cortical tension and
provides anchor points in mitosis that presumably promote the
stabilization of microtubule attachments and localization of force
generators (Théry et al., 2007; Toyoshima and Nishida, 2007; Wen
et al., 2004). Interestingly, although seam cells round off in mitosis,
they remain attached at apical junctions through long thin
extensions. Thus, tension and shape are partly maintained in
DEVELOPMENT
Control of epithelial stem cell division
mitosis, which probably promotes spindle orientation. The cell
rounding in alg-1 and dpy-11 larvae is likely to reduce the tension
in the anterior-posterior direction and thereby the bias in spindle
orientation.
Other factors, such as the stiffness and resistance of the
surrounding tissue, are also important in creating tension (Erler
and Weaver, 2009). The composition of the extracellular matrix
and collagen cuticle contribute to these factors (Clark and
Brugge, 1995; Erler and Weaver, 2009; Miranti and Brugge,
2002). As such, mutation of dpy-11 might affect seam cell
division through reduced collagen rigidity. However, division
orientation was affected by alg-1, lon-1, lin-28, dpy-11 and
retarded heterochronic mutations, which all change the seam cell
elongation factor. Hence, cell shape is likely to be a major
determinant.
Redundant control of the cell division axis
The redundant control of cell division orientation explains the low
number of mutants identified in our screen. alg-1 is exceptional in
that it affects cell shape as well as Wnt signaling. Redundant
geometric and polarity contributions are probably used broadly to
provide robust control over the cell division axis. Indeed, the
elliptical egg shape contributes to longitudinal division of the
C. elegans zygote even when PAR polarity is lost (Tsou et al.,
2002). This insight can be used in future studies aimed at
determining how each of the individual cues contributes to cell
division orientation and how ALG-1 promotes seam cell elongation
and Wnt signaling. Ultimately, these studies will improve our
understanding of the formation and maintenance of normal
epithelia and of the defects that contribute to carcinogenesis.
Acknowledgements
We thank Atilla Stetak for help with SNP mapping; Hitoshi Sawa, Rik
Korswagen, Fried Zwartkruis and Olaf Bossinger for strains; Karen Oegema
and Thomas Bürglin for reagents; Mike Boxem and Inge The for critically
reading the manuscript; and Frits Kindt for artwork. We acknowledge the
Developmental Studies Hybridoma Bank for antibodies and the Caenorhabditis
Genetics Center, supported by the National Institutes of Health National
Center for Research Resources, for several strains used in this study.
Funding
This work was sponsored by NWO-VENI grant 016.071.080 to M.W.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.066431/-/DC1
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