The microRNA pathway controls germ cell proliferation and differentiation in C. elegans

Abstract

The discovery of the miRNA pathway revealed a new layer of molecular control of biological processes. To uncover new functions of this gene regulatory pathway, we undertook the characterization of the two miRNA-specific Argonaute proteins in Caenorhabditis elegans, ALG-1 and ALG-2. We first observed that the loss-of-function of alg-1 and alg-2 genes resulted in reduced progeny number. An extensive analysis of the germline of these mutants revealed a reduced mitotic region, indicating fewer proliferating germ cells. We also observed an early entry into meiosis in alg-1 and alg-2 mutant animals. We detected ALG-1 and ALG-2 protein expressions in the distal tip cell (DTC), a specialized cell located at the tip of both C. elegans gonadal arms that regulates mitosis-meiosis transition. Re-establishing the expression of alg-1 specifically in the DTC of mutant animals partially rescued the observed germline defects. Further analyses also support the implication of the miRNA pathway in gametogenesis. Interestingly, we observed that disruption of five miRNAs expressed in the DTC led to similar phenotypes. Finally, gene expression analysis of alg-1 mutant gonads suggests that the miRNA pathway is involved in the regulation of different pathways important for germline proliferation and differentiation. Collectively, our data indicate that the miRNA pathway plays a crucial role in the control of germ cell biogenesis in C. elegans.

Full text

Germ cells regulation by miRNAs
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Cell Research | Vol 22 No 6 | June 2012
ORIGINAL ARTICLE
The microRNA pathway controls germ cell proliferation
and differentiation in C. elegans
Syed Irfan Ahmad Bukhari1, Alejandro Vasquez-Rifo1, Dominic Gagné2, Eric R Paquet1, Monique Zetka3,
Claude Robert2, Jean-Yves Masson1, Martin J Simard1
1Laval University Cancer Research Centre, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec, Canada G1R 2J6; 2Laboratoire
de génomique fonctionnelle du développement embryonnaire, Centre de recherche en biologie de la reproduction, Pavillon Com-
tois, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, Québec, Canada G1V 0A6; 3Department of Bio-
logy, McGill University, 1205 avenue Doctor Peneld, Montréal, QC, Canada H3A 1B1
Correspondence: Jean-Yves Massona, Martin J Simardb
aE-mail: Jean-Yves.Masson@crhdq.ulaval.ca
bE-mail: Martin.Simard@crhdq.ulaval.ca
Received 16 August 2011; revised 16 November 2011; accepted 20 De-
cember 2011; published online 28 February 2012
The discovery of the miRNA pathway revealed a new layer of molecular control of biological processes. To uncover
new functions of this gene regulatory pathway, we undertook the characterization of the two miRNA-specic Argo-
naute proteins in Caenorhabditis elegans, ALG-1 and ALG-2. We rst observed that the loss-of-function of alg-1 and
alg-2 genes resulted in reduced progeny number. An extensive analysis of the germline of these mutants revealed a
re-
duced mitotic region, indicating fewer proliferating germ cells. We also observed an early entry into meiosis in alg-
1
and alg-2 mutant animals. We detected ALG-1 and ALG-2 protein expressions in the distal tip cell (DTC), a specia-
lized cell located at the tip of both C. elegans gonadal arms that regulates mitosis-meiosis transition. Re-establishing
the expression of alg-1 specically in the DTC of mutant animals partially rescued the observed germline defects.
Further analyses also support the implication of the miRNA pathway in gametogenesis. Interestingly, we observed
that disruption of ve miRNAs expressed in the DTC led to similar phenotypes. Finally, gene expression analysis of
alg-1 mutant gonads suggests that the miRNA pathway is involved in the regulation of different pathways important
for germline proliferation and differentiation. Collectively, our data indicate that the miRNA pathway plays a crucial
role in the control of germ cell biogenesis in C. elegans.
Keywords: argonaute; miRNA; germline
Cell Research (2012) 22:1034-1045. doi:10.1038/cr.2012.31; published online 28 February 2012
npg
Cell Research (2012) 22:1034-1045.
© 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00
www.nature.com/cr
Introduction
MiRNAs are 21-23 nucleotides long non-coding RNA
molecules processed from hairpin-structured RNAs by
Drosha and Dicer RNaseIII enzymes. These short RNA
molecules induce translational repression and gene si-
lencing of their target mRNAs via interaction with an
Argonaute protein. Members of this protein family are
classified into three groups: Argonaute-like proteins
(AGO); Piwi-like proteins and C. elegans-specic AGOs
(WAGOs) (reviewed in [1]). In C. elegans, AGO-clade
proteins ALG-1 and ALG-2 have been identied to be in-
volved exclusively in the miRNA pathway [2]. MiRNAs
regulate a plethora of biological processes including cell
proliferation, cell differentiation and apoptosis, processes
important to coordinate developmental timing (reviewed
in [3, 4]). Since the discovery of the prominent miRNA
families of lin-4 and let-7 in determining C. elegans de-
velopmental timing (reviewed in [4]), their mammalian
homologs have also been identied to control cell proli-
feration in human cell lines [5, 6]. Recent studies suggest
that mammalian let-7 miRNA could regulate develop-
mental processes via regulation of several cell cycle-
related genes [7, 8]. Since the deletion of a single family
of miRNA often fails to induce severe defects in vivo [9,
10], it has been suggested that most biological processes
are subjected to regulation of a cumulative effect by vari-
ous miRNAs.
Among these processes, the studies of animals carry-
ing mutations of important components of the miRNA

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pathway indicate the contribution of miRNAs in the ani-
mal germline. In Drosophila, mutations of dcr-1, ago-
1 and loquacious genes resulted in defects in germline
maintenance [11-14]. In C. elegans, alteration of the
dcr-1 gene rendered animals sterile despite retaining a
normal gonad due to the strong maternal rescue [15]. Ad-
ditionally, deletion of the Drosha-encoding gene drsh-1
also led to sterility in nematode [16].
Here, we report that Argonaute proteins ALG-1 and
ALG-2 are expressed in the distal tip cells (DTCs) of the
C. elegans germline. Mutations in alg-1 and alg-2 result
in drastically reduced number of progeny. We observed
that this reduction in fertility is caused by defects in
multiple processes in germline development, including
reduced germ cell proliferation and increase in apoptosis.
Our findings suggest that ALG-1/2 function, together
with a set of miRNAs expressed in the DTCs to regulate
diverse biological pathways important to maintain ani-
mal germline proliferation and differentiation.
Results
MicroRNA-specic Argonautes are required for germ cell
proliferation
Since defects in the development of the germline will
be reected in the progeny number, we rst investigated
the brood size of ALG-1 and ALG-2 loss-of-function
mutants, named as alg-1(gk214) and alg-2(ok304), re-
spectively. Compared to wild-type animals, we noticed
a significantly reduced brood size in alg-1(gk214) and
alg-2(ok304), though such reduction was more drastic in
alg-1(gk214) (Table 1). As any defects during germline
proliferation, meiosis or gamete formation could affect
progeny number, we first asked if these mutants have
defects in cell proliferation in the germline. We extruded
germline of young adults from wild-type, alg-1(gk214)
and alg-2(ok304) animals, followed by staining of DNA
with DAPI to monitor the spatio-temporal progression
of different meiotic phases. Cells with crescent-shaped
nuclear morphology mark the transition zone, which
represents the leptotene/zygotene stage of meiosis [17,
18]. Compared to the wild-type animals, the mitotic re-
gions of alg-1(gk214) and alg-2(ok304) are shorter as
determined by the morphology of the germ cell nuclei
(Figure 1A-1C) and by the number of cells in the mitotic
region (Table 1). To corroborate our findings from the
DAPI staining, we used an antibody specic to HIM-3, a
meiosis-specic axis component between sister chroma-
tids [19], as a bona de marker of entry into meiosis. In
agreement with our earlier ndings, anti-HIM-3 antibody
revealed an early entry into meiosis in alg-1(gk214) and
alg-2(ok304), compared to the wild type (Figure 1D-
1F). Similar defects were observed with another loss-of-
function allele of the alg-1 gene (alg-1(tm492)) as well
as in alg-1(RNAi) animals (Supplementary information,
Figure S1). These results suggest that ALG-1 and ALG-2
are involved in the regulation of germline proliferation.
Expression of ALG-1 in the DTC controls germ cell pro-
liferation
To better decipher how alg-1 and alg-2 control germ
cell proliferation and meiosis entry, we next decided to
observe the expression pattern of ALG-1 and ALG-2 pro-
teins in animal gonads. By immunostaining of extruded
gonads using a newly generated ALG-1-specic antibody
(Supplementary information, Figure S2), we observed
that ALG-1 is localized to the DTC of the wild-type go-
nads but not in alg-1(gk214) (Figure 2A). To overcome
the non-availability of ALG-2-specific antibody, we
generated a transgenic line somatically expressing GFP-
Table 1 Phenotypes observed in the germline of alg-1(gk214) and alg-2(ok304) animals
Strain Brood size (N=25) Nb of cells in Oocyte / arm Oocyte / arm Average corpses/
MR (N=5) day 1 (N=5) day 2 (N=5) arm (N=5)
Wild-type 266.4 ± 3.22 240.3 ± 0.63 9.06 ± 0.06 9.84 ± 0.12 2.98 ± 0.19
alg-1(gk214) 77.0 ± 5.64 203.9 ± 1.26 5.48 ± 0.16 6.08 ± 0.07 4.38 ± 0.18
*(5.68E−20) *(1.93E−05) *(6.51E−06) *(3.33E−05) *(0.01)
alg-2(ok304) 178.8 ± 4.27 213.7 ± 0.43 7.0 ± 0.07 7.18 ± 0.24 4.12 ± 0.05
*(4.24E−14) *(1.99E−06) *(1.03E−05) *(0.0015) *(0.001)
lag-2p::rfp::alg-1 142.12 ± 5.6 237.2 ± 1.4 6.7 ± 0.17 6.4 ± 0.2 3.28 ± 0.08
*(3.70E−17) *(0.134) *(9.61E−05) *(8.68E−05) *(0.184)
**(5.34E−08) **(0.0001) **(0.005) **(0.186) **(0.004)
N: number of animals (brood size) or number of replicates (10 animals/replicate); MR: Mitotic region. The brood sizes were dened as the num-
bers of viable larvae that developed to the L1 stage descended from a single hermaphrodite of its strain. In parenthesis, P-values were calculated
with a Student’s t-test to compare numbers with wild-type (*) or alg-1(gk214) (**) animals, and represented as ± SEM.

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Figure 1 alg-1 and alg-2 mutant gonads have shorter mitotic region and display early entry into meiosis. (A-C) DAPI-stained
germline depicting mitotic region (MR) and transition zone (TZ) in wild-type (WT), alg-1(gk214) and alg-2(ok304) mutant ani-
mals. (D-F) Staining with HIM-3 antibody (green) depicting entry into meiosis in wild-type (WT) gonads and alg-1(gk214) and
alg-2(ok304). Scale bar measures 20 µm.
tagged ALG-2 (MJS13) and found that ALG-2 is also lo-
calized in the DTC of the gonads (Figure 2B). DTC caps
the distal end of the germline, and provides the stem cell
niche. These specialized cells are also responsible for
maintaining proliferation in the distal part of the gonad
arm, which is the mitotic region [20-23]. When the con-
tact between the DTC and germ cells is breached, cells
enter into meiosis. To determine whether the presence
of ALG-1 in the DTC is crucial to control germ cell pro-
liferation and differentiation, we generated a transgenic
line where ALG-1 expression is under control of the pro-
moter of lag-2, a membrane-bound Delta/Serrate/LAG-2
ligand expressed exclusively in the DTC [24]. When re-
establishing ALG-1 expression in the DTC (Figure 2C),
we were able to partially rescue the brood size compared
to the alg-1 mutant (Table 1), as well as fully restore the

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Figure 2 ALG-1 and ALG-2 localize to the DTC. (A) Anti-ALG-1 antibody depicts ALG-1 localization to the DTC of wild-type (WT)
germline, but not in alg-1(gk214). (B) ALG-2 (green) expression in DTC of gfp::alg-2 (MJS13) transgenic line. Gonads were
counter stained with DAPI (blue) to visualize nuclei. (C) ALG-1 localized exclusively in DTC of transgenic animals expressing
RFP-tagged ALG-1 protein under control of the DTC-specic lag-2 promoter (lag-2p). Arrows indicate DTC. (D) Restoration of
mitotic region in lag-2p::rfp::alg-1-expressing animals shown by DAPI staining compared to wild-type gonad. Scale bar mea-
sures 20 µm.

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Figure 3 Effects of ALG-1 and ALG-2 on oocytes. (A) DAPI-stained germline. Arrowheads depict oocyte nuclei in diakinesis
stage in the proximal gonad arm of wild-type (WT), alg-1(gk214), alg-2(ok304) and plag-2::rfp::alg-1 animals. (B) Merged and
nomarski DIC micrographs of AO-stained germline in respective genetic backgrounds. Arrowheads mark apoptotic corpses.
(C) Brood size in alg-1 and alg-2 mutant animals as well as in plag-2::alg-1 and wild-type animals after mating with wild-type
males represented as average brood size ± SEM, P < 0.01.
normal length of the mitotic region (Figure 2D and Table
1, compared to wild type). To further decipher the role of
alg-1 beyond the DTC, we scored the brood size in trans-
genic animals expressing RFP-tagged ALG-1 under the
control of endogenous alg-1 promoter and 3′UTR. Apart
from the DTC, RFP::ALG-1 is also expressed in sheath

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cells besides other somatic tissues (AVR, unpublished
data). We observed that these transgenic animals showed
a signicant increase in brood size compared to animals
with DTC-expressed alg-1 (158 ± 2.7 vs 142.12 ± 5.6 for
plag-2::alg-1; P < 0.017). Taken together, our results in-
dicate that ALG-1 plays a role within the DTC as well as
in other tissues of the gonads.
ALG-1 and ALG-2 are important for gamete formation
and maintenance
Since germ cells undergo proliferation (mitosis),
gametogenesis (meiosis and differentiation) or apop-
tosis, we examined if the loss of the miRNA-specific
Argonautes alg-1 and alg-2 genes can affect the fates
of germ cells in animals. Although we did not observe
high incidence of males and the dead eggs phenotype
in alg-1(gk214) and alg-2(ok304) animals (two notable
consequences of meiotic defects in C. elegans, data not
shown), we found that both mutants have significantly
reduced number of oocytes than the wild-type animals
(Figure 3A and Table 1). Using the vital dye acridine or-
ange (AO), which has been used to stain apoptotic cells
in live animals [25], as well as differential interference
contrast (DIC) microscopy, we observed increased germ
cell corpses in the proximal region of the gonad arm in
alg-1(gk214) and alg-2(ok304) (Figure 3B and Table
1). The fact that both DIC microscopy and AO staining
detect the same number of germ cell corpses, suggests
that mutants have an increase in apoptosis rather than
defective engulfment in the germline. Re-establishing the
expression of ALG-1 in the DTC partially restores the
number of oocytes and reduces germ cell corpses (Fig-
ure 3B and Table 1). We next determined if the decrease
in brood size also results from defects in male gametes
formation. We observed that sperm nuclei number in alg-
1 and alg-2 mutant hermaphrodites is not significantly
different from the wild-type animals (data not shown).
However, when the two Argonaute mutants were mated
with wild-type males, we observed a signicant increase
in the brood size compared to the unmated mutant her-
maphrodites, but brood size is signicantly smaller than
that of the wild-type hermaphrodites mated with wild-
type males (Figure 3C). In addition, the brood size of
animals with DTC-expressed alg-1 is not enhanced upon
mating with wild-type males (Figure 3C). This observa-
tion supports that re-establishing ALG-1 expression in
the DTC mainly rescues spermiogenesis, and thus sus-
tains the fact that ALG-1 is required in other gonadal tis-
sues. Together, our data show that ALG-1 and ALG-2 are
involved in maintaining C. elegans fertility.
miRNAs expressed in the DTC are implicated in germ
cell proliferation and differentiation
Since ALG-1 and ALG-2 are imperative to miRNA-
induced gene silencing, we next decided to identify
candidate miRNAs involved in germline maintenance.
Recently, the expression pattern of several C. el-
egans miRNAs has been studied in vivo using miRNA
promoter::GFP fusion constructs [26]. Of the total of 70
transgenic C. elegans strains reported, 8 pmiRNA::gfp
strains (plet-7, plin-4, pmir-80, pmir-237, pmir-247-797,
pmir-359, pmir-53 and pmir-71) exhibited expression
in the DTC. We thus examined animals carrying mutant
alleles of the eight miRNA genes, to determine if they
Table 2 Different phenotypes observed in miRNA mutants
Strain Brood size (N=20) Nb of cells in MR (N=5) Oocytes / arm day 1 (N=5) Oocytes / arm day 2 (N=5)
Wild-type 266.4 ± 3.7 240.3 ± 0.63 9.06 ± 0.06 9.84 ± 0.12
let-7(n2853) 43.9 ± 3.1 192.02 ± 1.4 5.8 ± 0.07 *ND
(6.45E−22) (3.40E−06) (1.66E−06)
lin-4(e912) 30.0 ± 0.9 197.6 ± 1.2 5.6 ± 0.12 *ND
(1.07E−23) (4.11E−06) (1.81E−06)
mir-359(n4540) 235.6 ± 6.8 221.3 ± 0.9 7.7 ± 0.12 8.98 ± 0.09
(0.002) (5.39E−05) (0.001) (0.01)
mir-247(n4505) 207.7 ± 5.9 200.94 ± 2.6 7.6 ± 0.12 8.84 ± 0.08
(3.10E−07) (0.0002) (0.0003) (0.003)
mir-237(n4296) 222.1 ± 7.4 213.7 ± 1.9 8.82 ± 0.05 10.12 ± 0.32
(4.37E−05) (5.45E−05) **(0.70) **(0.40)
N: number of animals (brood size) or number of replicates (10 animals/replicate); MR: Mitotic region; ND: Not determined. In parenthesis, P-
values were calculated with a Student’s t-test to compare the signicance of the numbers with wild-type animals and represented as ± SEM.
*Most of the population died after the rst day of fertile adults.
**mir-237(n4296) animals have similar number of oocytes as the wild-type.

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Figure 4 A subset of miRNAs expressed in the DTC affects germline proliferation and oocytes. (A) DAPI and HIM-3 staining
depicting shorter mitotic region observed in let-7, lin-4, miR-237 and mir-247 in mutant animals. (B) DAPI-stained germline.
Arrowheads depict oocyte nuclei in diakinesis stage in the proximal gonad arm of miRNA mutant animals.

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Figure 5 Comparative microarray analyses of genes expressed in the germline. (A) Number of genes signicantly modulated
in extruded alg-1 mutant versus wild-type gonads. Selected signicant genes have a fold change > 2 and a P-value < 0.001
(N = 4; the list of can be found in Supplementary information, Table S1). (B) Gene ontology biological process enrichment
performed using DAVID. The upper part corresponds to enrichment obtained when using the list of downregulated genes and
the bottom part of the upregulated genes. Numbers to the right represent Benjamini-Hochberg P-values.
display phenotypes similar to alg-1(gk214) and alg-
2(ok304). Among them, we found that let-7(n2853), lin-
4(e912), miR-237(n4296), miR-359(n4540) and miR-
247(n4505) mutants displayed similar phenotypes to
alg-1(gk214) and alg-2(ok304). All five mutant strains
had signicantly smaller brood size and shorter mitotic
region with reduced number of cells within the mitotic
region compared to wild type (Figure 4 and Table 2).
While the let-7(n2853), lin-4(e912), miR-359(n4540) and
mir-247(n4505) mutant animals have shorter mitotic re-
gion as well as fewer number of oocytes, mir-237(n4296)
mutant animals have only shorter mitotic region but
normal oocyte number (Figure 4 and Table 2), and miR-
80(nDf53), mir-71(n4115) and mir-53(n4113) mutant
strains have no apparent germline defect (Supplementary
information, Figure S3). These observations suggest that
a variety of miRNAs regulate different processes at mul-
tiple steps in germline biogenesis.
Since the regulation by miRNAs often leads to a
decrease in target mRNAs [27-30], we next decided to
compare the level of mRNAs found in gonads of wild-
type and alg-1 animals to uncover putative targets of let-
7, lin-4, miR-237, miR-359 and miR-247 miRNAs in the
germline. When we compared microarray data from four
independent biological samples, we observed that the
level of 1 374 different mRNAs is signicantly altered in
the absence of alg-1 (with a threshold of > 2-fold change,
P ≤ 0.001; Figure 5 and Supplementary information,
Table S1). A clustering analysis of the gene expression
data revealed a signicant alteration in the expression of
genes associated with biological pathways important for
chromosome organization and segregation (Figure 5).
Although they are not signicantly enriched, we notably
found putative targets predicted by either TargetScan [31]
or miRWIP [32] algorithm for let-7, lin-4/miR-237 (since
they are similar in sequence, they are predicted to target
the same mRNAs [33]), miR-359 and miR-247 miRNAs
among mRNAs misregulated in the germline of alg-1
mutant (Supplementary information, Table S2). Thus,
our results implicate that these miRNAs contribute to
the regulation of the process of gamete formation and di-
fferentiation in C. elegans by affecting the expression of
multiple mRNA targets.
Discussion
Earlier studies performed in Drosophila have high-
lighted that components of the miRNA pathway are re-
quired for germline stem cell self-renewal, [11, 13, 14,
34] and that the maternally expressed bantam and miR-
184 miRNAs contribute to oogenesis [35, 36]. While
our data revealed that the role of the miRNA pathway
in germ line maintenance is conserved in C. elegans, our
results also support for the rst time that both miRNA-
specific Argonaute proteins ALG-1 and ALG-2 in the
stem cell niche are crucial for the proper control of germ
cell proliferation and gametes formation.
Previous studies of C. elegans strains carrying muta-
tions in Drosha (drsh-1) and Dicer (dcr-1) genes, two
important processors of miRNAs, showed that these mu-
tants were sterile [15, 16]. In our laboratory, we observed
that post-embryonic RNAi of ALG-1 on alg-2 back-
ground or vice versa could also render animals sterile
(unpublished data). These observations indicate that the
miRNA pathway is indispensible in animal reproduction.
Our current study showed that germline proliferation is

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reduced in both miRNA-specic Argonaute mutants. The
restoration of ALG-1 exclusively in the DTC, rescues
the mitotic cell number in the germline, which leads to a
partial but signicant increase in brood size, suggesting
that miRNAs play a role in germline cell division. Addi-
tionally, our observation of fewer oocytes and increased
apoptotic corpses in both alg-1 and alg-2 suggests that
miRNAs are also important regulators in gamete for-
mation. Consistent with our findings, mice with either
global deletion of dicer or with oocyte-specic deletion
of AGO2 display failures in oogenesis due to arrest at
meiosis I [37-40]. These animals also have severely re-
duced number of spermatogonia, which could be due to
proliferation defects and an increase in apoptosis [39, 41,
42]. However, it has yet to be determined in vertebrates
the contribution of miRNAs in gametogenesis, since the
loss of Dicer also affects the production of endogenous
siRNAs, a type of small RNAs that are also important in
this process [43-45].
In C. elegans, a single DTC in each gonad arm estab-
lishes germline stem cell niche. It controls germ cells
fate by employing in part the GLP-1/Notch signaling
through a network of RNA-binding regulatory proteins,
most notably, Pumilio and FBF, to maintain a balance
between proliferation and differentiation [46]. While the
RNA-binding proteins GLD-1, GLD-2/GLD-3 promote
the meiosis entry of germline stem cells, the activation of
the GLP-1/Notch signaling pathway inhibits these signals
and retains the cells in mitotic stage [47]. Interestingly,
gld-1 mRNA has been found in ALG-1-immunoprecipi-
tated complex [48], and other evidence also indicates that
gld-1 is subject to regulation by miRNAs [49]. These
studies suggest the possibility that miRNAs can regulate
the mitosis to meiosis decision by controlling key genes
involved in the process.
We demonstrate that mutants of ve different miRNAs
which are known to localize to the DTC display similar
phenotypes as the ones observed in alg-1 and alg-2 mu-
tants, suggesting that more than one miRNA participate
in stem cell fate regulation. Our extensive microarray
analysis of mRNAs expressed in alg-1 mutant gonads,
detected the mis-regulation of more than 1 300 genes.
Among them, we observed that the expression of the
major sperm proteins (MSPs), was upregulated in alg-
1 mutant gonads. MSP signaling is known to regulate
the oocyte production and development. The proximal
MSP signaling works coordinately with the distal GLP-
1 signaling to regulate the proper oocyte growth and
function [50, 51]. We thus envision that GLP-1 and MSP
signaling are both subject to the miRNA regulation, and
the loss of the miRNA-specific Argonaute genes leads
to alterations in both signaling pathways, likely due to
imbalance of these proteins, and thus affecting the fertil-
ity of the animals. Therefore, the phenotypes observed in
the germline of alg-1 and alg-2 mutants reect that dif-
ferent miRNAs are involved in germline biogenesis by
regulating different pathways. Hence, it may be difcult
to recapitulate the alg-1 and alg-2 mutant phenotypes by
inactivating specic miRNAs. We therefore propose that
the phenotypes of alg-1 and alg-2 mutants result from
the combinatorial effect of miRNAs and their targets.
Materials and Methods
Strains
All the strains were maintained according to standard proto-
cols [52]. The let-7(n2853) and lin-4(e912) mutant strains were
maintained at 15 °C and let-7(n2853) (shifted to 20 °C beyond
L4 stage). All other strains were maintained at 20 °C. The alg-
1(gk214) mutant carries an out-of-frame deletion of 200 bp after
the 28th amino acid and terminates by 2 additional amino acids.
The alg-2(ok304) allele is an out-of-frame deletion that removes
the nucleotides encoding amino acids 34-374 and terminates after
encoding 8 additional amino acids from another reading frame.
They are therefore likely to be null alleles of alg-1 and alg-2. Fur-
ther details can be found on the C. elegans Gene Knockout Con-
sortium website. All mutant strains have been outcrossed at least
three times before analyses.
Rescue experiments
Transgenic MJS13 line was generated by microinje cting a
mix of reporter plasmids (pRF4), MSp59 (alg-1p::RFP::alg-1)
and MSp72 (alg-2p::GFP::alg-2) and crossed into alg-1(gk214)
strain. Extrachromosomal arrays were UV integrated. The plag-
2::alg-1 line (MJS26) was generated by microinjecting a mix of
pRF4 and MSp151 plasmids and crossed into alg-1(gk214) strain.
A 3 kb promoter of lag-2 was excised by BamHI from pJK590
[53] and cloned into BamHI-digested pBluescriptII SK+ to gener-
ate MSp147. Using a primer set incorporating AII and NotI sites,
the promoter was amplified by PCR, digested and employed to
exchange the endogenous alg-1 promoter from AII/NotI-digested
MSp59 plasmid (alg-1p::RFP::alg-1) producing MSp150. Latter,
the NotI RFP cassette was reintroduced to generate MSp151 (lag-
2p::RFP::alg-1). Both alg-1 and alg-2 constructs contain their
respective 3′UTR regions.
Cytological methods
For antibody staining, gonads were dissected from young
adults (20-22 h post L4) in PBS. Extruded gonads were im-
mersed in fi xing solution (1% PFA+0.1% Tween-20) for 5 min
at room temperature, followed by freeze crack in liquid nitrogen
and transferred to −20 °C methanol for 1 min. The xed gonads
were washed three times in PBS with 0.1% Tween-20 for 15
min, followed by blocking in PBS-T + 1% BSA for 1 h at room
temperature. Gonads were then incubated with primary antibod-
ies (α-ALG-1 (1:500) or α-HIM-3 (1:200)) overnight at 4 °C, and
probed with Alexa Fluor 488 anti-rabbit as secondary antibody
(1:500). Gonads were counter-stained by 1 µg/ml DAPI in anti-
fading agent (Vectashield, Vector Laboratories). Images were
captured using Zeiss motorized Axioplan 2 microscope at 630×

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Syed Irfan Ahmad Bukhari
et al
.
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consisting of 15-20 serial Z sections of 0.5 µm thickness subsum-
ing entire nuclei. Fluorescence images were acquired with an Ax-
ioCam HRm camera and AxioVision acquisition software.
Brood size
Single L4 hermaphrodite from wild-type and mutant strains
were transferred to seeded NGM plates and maintained at 20
°C.
Animals were transferred to fresh plates each day until they
stopped laying eggs. The hatched larvae on each plate were count-
ed and total number of viable larvae that developed to the L1 stage
descended from a single hermaphrodite was calculated. The aver-
age number of viable larvae from 25 hermaphrodites of a strain
was plotted as brood size. Signicant differences were determined
by Student’s t-test (P < 0.05) and represented as ± SEM.
Scoring mitotic cell number counts and entry into meiosis
Mitotic region was established previously [23]. Cell numbers
within mitotic region were determined by counting from the row
immediately adjacent to the DTC to the row containing multiple
crescent-shaped nuclei, which is the early meiotic prophase I (lep-
totene/zygotene) or to the row where the nuclei stained positive
with α-HIM-3 antibody (marker for entry into meiosis). Entry into
meiosis was conrmed by looking at the gonad arm within each
category for nuclei that stained positive with α-HIM-3 antibody in
mitotic region/ transition zone.
Oocyte count and sperm defect
Gonads from L4 animals past 20 h and 40 h (day-1 and day-
2, respectively) were dissected and fixed with fixing solution
(1% PFA+0.1% Tween-20) for 30 min at room temperature. Go-
nads were washed stained by 1 µg/ml DAPI in anti-fading agent
(Vectashield, Vector Laboratories) and monitored under the micro-
scope at 630× magnication. Oocytes were counted from the loop
of the gonad arm in a linear fashion till the most proximal oocyte
also called as (−1). To check the sperm defect, L4 animals of mu-
tant background were mated with wild-type males. Progeny was
counted from animals, where cross progeny was monitored and
compared to progeny from unmated animals.
AO assay
To obtain the number of corpses in worms, 20-22 h past L4
adult animals were stained with AO. Adult animals were incubated
in dark for 1.5 h at room temperature on plates containing 1 ml of
M9 with 0.08 mg of AO. Stained adults were transferred to fresh
NGM plates to incubate for 45 min to clear the stained bacteria.
Worms were mounted on agarose pads and monitored under uo-
rescence microscope. Stained corpses as well as the ones which
were clearly visible under DIC as dark spots were counted.
Microarray analysis
Gonads from wild-type N2 and alg-1(gk214) animals from four
independent pools (around ve hundred gonads for each set) were
extruded and immediately placed in cold Tri-Reagent (Sigma) for
total RNA extraction. RNA purication was performed using the
PicoPure RNA isolation kit (Applied Biosystems), with DNase
(Qiagen) treatment on the purification column according to the
manufacturer’s instructions. Quantity and quality of RNA was
veried on a 2100 Bioanalyzer (Agilent Technologies). Samples
were stored at −80 °C.
Antisense RNA was produced using the Agilent LowInput
QuickAmp Labeling Kit Two Color (Agilent Technologies). A 100
ng of total RNA spiked in with Two-Color RNA Spike-In Kit from
Agilent was amplied and labeled as recommended by manufac-
turer; except for labelled aRNA purication, picopure RNA extrac-
tion kit was used. Quantity and labeling of aRNA was determined
using a Nanodrop ND-1000 (NanoDrop Technologies).
Samples from four biological replicates of each gonad strain
were hybridized on C. elegans Oligo Microarray (Agilent Tech-
nologies) using a dye-swap design (technical replicates) for a total
of four arrays. The manufacturer’s protocol provided with Agilent
Gene Expression oligo microarrays Version 6.5 (May 2010) was
followed adding the acetonitrile and Stabilization and Drying So-
lution (Agilent Technologies) to wash steps. Slide was scanned on
a G2505 B Agilent Microarray scanner and fluorescence values
extract using Feature extraction software (Agilent Technologies).
The raw expression data were imported in FlexArray (http://
www.genomequebec.mcgill.ca/FlexArray) and analyzed using
LIMMA [54]. First background was subtracted and the data were
normalized using loess normalization. The experimental design
was modeled using the function lmFit and the dye-swap arrays
were taken into account. Differential expression was assessed us-
ing an empirical Bayes’ statistics using the eBayes function. Gene
enrichment analysis was performed using DAVID on the gene on-
tology biological processes [55].
Acknowledgments
We thank members of the Zetka lab for their technical help as
well as members of the Simard’s group for their critical reading
of the manuscript. We also thank Judith Kimble (University of
Wisconsin-Madison) and Eric Miska (University of Cambridge)
for providing reagents and strains. Some nematode strains were
provided by the Caenorhabditis Genetics Center, which is funded
by the NIH National Center for Research Resources (NCRR) and
by the International C. elegans Gene Knockout Consortium. This
research was supported by NSERC grants (C R, J-Y M and M J S).
J-Y M is a Chercheur-Boursier Senior from Fonds de la Recherche
en Santé du Québec and M J S is a Canadian Institutes of Health
Research New Investigator.
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