1 Department of Genetics, Yale University School of Medicine, New Haven, CT
06520, USA
2 Department of Molecular and Cellular Biology, University of Arizona, Tuscon,
AZ 85721, USA
* Author for correspondence (e-mail: valerie.reinke{at}yale.edu)
Accepted 9 October 2003
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SUMMARY |
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Key words: C. elegans, Germline, Sex determination, Microarrays, Gene expression, X chromosome
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Introduction |
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In the bi-lobed gonad of the hermaphrodite nematode C. elegans, the cells destined to become gametes originate from a common pool of germline stem cells that exist within a niche at the distal end of the gonad. Upon leaving the niche, germ cells enter meiosis I and undergo chromosomal synapsis and recombination. During the fourth larval stage (L4), meiotic germ cells within the proximal region of the gonad differentiate into spermatocytes. After the L4-to-adult molt, the germ cells in the proximal gonad instead differentiate into oocytes.
Regulation of gene expression at transcriptional and post-transcriptional
levels is crucial for the proper specification, proliferation and
differentiation of germ cells (reviewed by
Seydoux and Strome, 1999;
Goodwin and Evans, 1997
;
Kuwabara and Perry, 2001
).
However, the regulatory networks controlling genes involved in either germ
cell fate decisions or terminal differentiation of gametes are only
superficially understood. Additionally, global silencing mechanisms that
control the expression of large regions of the genome overlay gene-specific
regulation, as demonstrated by the selective silencing of repetitive
transgenes in the germline (Kelly et al., 1997). The largest endogenous target
of silencing identified to date is the X chromosome, which is silenced to
differing degrees in the germline of males and hermaphrodites
(Kelly et al., 2002
;
Fong et al., 2002
). A thorough
understanding of how both global and gene-specific regulation of gene
expression contributes to the proper functioning of the germline requires a
comprehensive knowledge of the genes expressed in that tissue.
Males appear among the hermaphrodite population at a low frequency, when X
chromosome nondisjunction occurs during meiosis. Somatic tissues of males
differ from hermaphrodites in several ways: males lack a vulva and uterus,
have a single-lobed gonad, and do not produce vitellogenin in the intestine.
Male-specific structures include both a broad, fan-shaped tail as well as
sex-specific neurons that direct physical and behavioral aspects of mating
(Emmons and Sternberg, 1997).
In the male germline, many of the initial steps in germ cell development are
morphologically similar to hermaphrodites, although they make only sperm. Male
spermatozoa are larger than hermaphrodite spermatozoa, and this size
difference is one factor that promotes the preferential use of male sperm over
self-sperm by a mated hermaphrodite
(LaMunyon and Ward, 1998
).
Genome-wide gene expression studies that identify molecular similarities and
differences between the two sexes for both somatic and germline tissues are
crucial for investigations of the underlying genetic pathways that generate
sex-specific structures.
In the past few years, many microarray-based expression studies have been
performed on diverse aspects of C. elegans development, such as
embryogenesis, aging, pharyngeal development, muscle development and dauer
formation (Baugh et al., 2003;
Lund et al., 2002
;
Gaudet and Mango, 2002
;
Roy et al., 2002
;
Wang and Kim, 2003
).
Additionally, several large-scale expression and functional analyses have been
performed in C. elegans, including in situ hybridization of ESTs and
genome-wide RNAi (Kohara,
2001
; Kamath et al.,
2003
). Integration of the diverse data types from these global
investigations can result in increased strength and specificity of functional
predictions. For example, in situ data provides spatial patterns that are only
inferred in whole-animal microarray studies, while RNAi studies provide
valuable information about reduction-of-function phenotypes. Beyond compiling
evidence for single genes, genome-wide functional approaches also facilitate
observations on a different scale from single-gene studies. In particular,
several of these global studies have demonstrated that genes are non-randomly
arranged in the C. elegans genome with respect to both gene
expression and function (reviewed by
Reinke, 2002
;
Piano et al., 2002
;
Kamath et al., 2003
).
We use DNA microarrays corresponding to 92% of the currently predicted genes in the C. elegans genome to examine expression profiles among mutant strains with defects in germline proliferation or gamete production, as well as between males and hermaphrodites. Together these experiments identify 5629 genes that show distinct germline- or sex-dependent expression profiles. In addition, we have pinpointed a small set of genes with expression likely to be specific in the male germline. Investigation of the kinetics of gene expression during wild-type hermaphrodite larval and adult development demonstrates that sets of genes with germline-enriched expression are highly temporally co-regulated, but that genes with sex-biased expression in the soma are less so. We also extend the previous observation that the chromosomal location of germline-enriched transcripts is non-random in the genome. Our studies show that genes expressed in the germline are under-represented on the X chromosome and, conversely, that genes with hermaphrodite soma-biased expression are enriched on the X chromosome. We also discuss previously unreported biases on autosomes. Comparison between germline- and sex-regulated genes and large-scale RNAi screens demonstrates that genes with expression in oogenic germlines are enriched for visible phenotypes relative to other gene expression sets.
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Materials and methods |
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The wild-type reference RNA used is identical to that of Reinke et al.
(Reinke et al., 2000), and by
mass is approximately composed of 40% gravid adults, 30% larvae, 15% embryos
and 15% post-reproductive adults. The reference sample was used solely to
allow comparison between genotypes; we did not infer any biological meaning
for the sample/reference ratios. The L4 and adult N2 and glp-4
hermaphrodite samples and the fem-1 and fem-3 hermaphrodite
samples are also identical to those described by Reinke et al.
(Reinke et al., 2000
). The
him-5 samples and glp-4;him-5 samples were prepared by
growing worms on 15-cm2 plates and synchronizing as described
previously (Reinke et al.,
2000
). Starved L1 larvae were plated on 15 cm2 plates,
raised at 25°C until the young adult stage, and then harvested and washed
in S basal buffer. The worms were centrifuged on a sucrose cushion to remove
debris and recovered in several washes of S-basal. The worms were then
filtered through a 30 µm nylon mesh (Spectrum Laboratories, Rancho
Dominguez, CA) into a petri dish containing S-basal for 10 minutes. Males
passed through the mesh, while hermaphrodites were retained on top. The
filtration was repeated a second time, and male purity was determined by
examining the sex of 100 animals. A population had to consist of 90-95% males
before it was used in a microarray experiment.
Timecourse samples were grown by taking synchronized L1 larvae, plating them on 15 cm plates and growing them at 25°C until the middle of L3. Samples were taken every three hours for 36 hours by harvesting approx. five plates and washing the worm pellet several times in S-basal until most bacteria was removed, with a final resuspension of the worm pellet in four volumes of Trizol (Gibco/BRL). At each collection, the developmental stage of the animal was verified by inspection of vulval and germline formation using Nomarski optics.
Total RNA and polyA purification was performed as described elsewhere
(Reinke et al., 2000).
Microarrays
Microarrays were constructed essentially as described elsewhere
(Reinke et al., 2000;
Jiang et al., 2001
). The set
of 19,213 primer pairs corresponding to
94% of the genes in the genome
was used to PCR amplify gene fragments as described previously
(Jiang et al., 2001
). Out of
these, 18,010 produced a single band of the correct size. Reverse
transcription, labelling and hybridization to the arrays were performed as
described elsewhere (Reinke et al.,
2000
).
Data analysis
The average mean log2 ratio was calculated for each set of
replicates comparing a staged sample of specific genotype to the common
reference. Comparisons were made between genotypes by subtracting the mean log
value of one ratio from another, and the significance of the difference was
evaluated using Student's t-test for two populations. For the
fem-3(gf) versus fem-1(lf) direct comparison, we performed
the same analysis, except we used a Student's t-test for one
population. We chose a combination of a twofold difference with a t
value exceeding 99% confidence (P<0.01), because these criteria
allowed the inclusion of essentially all genes that had previously been
identified as germline-enriched in a wt/glp-4 hermaphrodite
comparison (Reinke et al.,
2000). Additionally, requiring a twofold difference reduced false
positives, as the number of genes with two-fold difference and a
P<0.01 only included
100 genes more than with
P<0.001, and almost all genes showed germline expression by in
situ hybridization (Table
1).
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Results |
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We collected poly A+ RNA from populations of wild type (N2) and
glp-4(bn2) hermaphrodites that were synchronized to either the fourth
larval stage (L4) or young adult stage. We also collected poly A+ RNA from
him-5(e1490) and glp-4(bn2);him-5(e1490) purified adult male
populations. Each sample was independently grown and harvested four times.
Using DNA microarrays representing 18,010 of the 19,546 protein-encoding genes
currently annotated in the C. elegans genome, we compared each of
these samples with a common reference sample. The reference sample comprises
hermaphrodites at all life stages: embryos, L1-L4 larvae, young adults and
post-reproductive adults. For each gene in each microarray experiment, we
calculated a log2 ratio of the staged sample relative to the
reference sample. We averaged the log2 ratios for each set of four
replicates. The common reference used in all hybridizations allows us to
compare the average expression ratios among these four genotypes and define
sex- and germline-regulated genes (Fig.
1). In each comparison, we required the fold difference in
expression to exceed twofold and a confidence level of 99%
(P<0.01, Student's t-test) for inclusion in the defined
gene set (see Materials and methods). These criteria were selected based on
the expression profiles of genes that had been previously characterized as
germline-enriched by microarray analysis
(Reinke et al., 2000).
To identify genes with germline-enriched expression relative to somatic tissues, we compared either L4 or young adult wild-type hermaphrodites to glp-4(bn2) hermaphrodites of the corresponding stage. Hermaphrodites produce sperm as L4 larvae and switch to oogenesis as young adults, so we combined the L4 and adult gene sets to include genes expressed during both sperm and oocyte production. This set of comparisons defined 3144 genes with germline-enriched gene expression in hermaphrodites (set I, Fig. 1). Comparison of adult him-5(e1490) males to adult glp-4(bn2);him-5(e1490) males identified a total of 1092 genes with germline-enriched expression in males (set II, Fig. 1).
To define genes with differential expression between the two sexes, we compared wild-type (N2) hermaphrodite gene expression with that of him-5(e1490) males. This comparison identified 1935 genes with hermaphrodite-biased expression, and 1269 genes with male-biased expression (sets IIIa and IIIb). Finally, we compared glp-4(bn2) hermaphrodites and glp-4(bn2);him-5(e1490) males, both of which lack a germline, to define 460 genes with enriched expression in the hermaphrodite adult soma and conversely, 430 genes with enriched expression in male adult soma (sets IVa and IVb).
In an independent set of microarray experiments, we further characterized
germline-enriched gene expression by determining which genes were
differentially expressed during spermatogenesis and oogenesis in
hermaphrodites. In three independent replicates, we directly compared gene
expression levels in adult fem-1(lf) hermaphrodites, which make only
oocytes, with adult fem-3(gf) hermaphrodites, which make only sperm
(Nelson et al., 1978;
Barton et al., 1987
). This
comparison identified 1652 genes with high levels of expression during
oogenesis [high in fem-1(lf) relative to fem-3(gf); set Va],
and 1343 genes with high levels of expression during spermatogenesis [high in
fem-3(gf) relative to fem-1(lf); set Vb]. Genes with high
expression during oogenesis would probably encode proteins required for oocyte
differentiation as well as maternally provided factors necessary for proper
development of the early embryo. Genes with high expression during
spermatogenesis are likely to be involved in spermatocyte specification and
differentiation.
Together the above experiments identify 5629 genes, 29% of the
protein-encoding genome, the expression of which is regulated by sexual
identity and/or the presence of a germline. All of these data are available
for either single or multiple gene queries at
http://wormgermline.yale.edu,
and the raw data are available at the Gene Expression Omnibus at NCBI and Yale
Microarray Database (see Materials and methods).
Classification of hermaphrodite germline-enriched gene expression
We consider all genes with increased expression in wild type relative to
glp-4 (set I) and/or significantly different expression between
fem-1(lf) and fem-3(gf) (set V) as the entire set of genes
with hermaphrodite germline-enriched expression. To examine the relationship
among these sets of genes, we performed a Venn diagram analysis
(Fig. 2A). The combination of
sets Va and Vb with set I define the total set of 4245 genes with
germline-enriched expression. The intersection of Va or Vb with set I
identifies those germline-enriched genes with increased expression in
hermaphrodites producing only sperm or only oocytes. These gene sets are
termed spermatogenesis-enriched and oogenesis-enriched, respectively. In our
prior experiments, we categorized germline-enriched genes with no significant
difference between spermatogenesis or oogenesis (i.e. genes in set I that do
not overlap Va or Vb) as `intrinsic'
(Reinke et al., 2000). Genes
with germline-intrinsic expression are predicted to function in mitotic
proliferation and early meiosis I in the distal germline, rather than in
gametogenesis or embryogenesis. However, as the data presented below show, we
cannot distinguish between the intrinsic and oogenesis sets by their temporal
regulation, their in situ hybridization patterns, or the predicted functions
of the encoded proteins. We therefore consider these two groups to have
significant functional overlap, and thus some genes found in the oogenesis set
are likely to have intrinsic functions, and vice versa.
A fraction of the fem-3(gf)-enriched or
fem-1(lf)-enriched transcripts, encoded by 479 and 622 genes,
respectively, did not meet the criteria for germline-enrichment
(Fig. 2A). The
fem-3(gf) and fem-1(lf) mutants make larger numbers of
gametes than wild-type hermaphrodites; thus, many differentially expressed
transcripts of low abundance are reliably detected in the
fem-3(gf)/fem-1(lf) comparison, but not in the
wild-type/glp-4 comparison
(Reinke et al., 2000).
Additionally, fem-1 is expressed in somatic tissues as well as the
germline (Gaudet et al.,
1996
). Even though the fem-1 mutation we used does not
have a phenotypic effect in the soma of hermaphrodites, the expression of a
subset of somatically expressed genes could still be affected in this
background. Because some of the genes that appear differentially regulated in
the fem-3(gf)/fem-1(lf) comparison may actually be somatically
expressed, we term these subsets `mixed spermatogenesis/somatic' and `mixed
oogenesis/somatic' (Fig. 2A).
Additional evidence supporting this possibility is discussed below.
We examined the predicted molecular functions of the genes within the
intrinsic, oogenesis and spermatogenesis gene sets using the existing gene
ontology (GO) annotation for C. elegans because it is a structured
annotation system and allows objective classification
(Ashburner et al., 2000).
Currently,
20% of the genes in the nematode have an entry in the
`molecular function' category of GO
(www.geneontology.org).
We cross-referenced these molecular annotations with the intrinsic, oogenesis
and spermatogenesis subsets to provide a preliminary annotation of these
genes. To simplify inspection of the annotations, we combined related
annotations into broader categories based on the `GO slim' ontology for
Drosophila
(www.flybase.org).
The results are presented as pie charts in
Fig. 3A. Most notably, the
oogenesis and intrinsic gene sets have genes in the same categories at
approximately the same ratios. For example, genes encoding predicted nucleic
acid-binding proteins comprise 31% and 32% of the intrinsic and oogenesis gene
sets, respectively. In both sets, 27% of the nucleic acid binding proteins are
predicted to bind RNA specifically (aqua box in bar). By contrast, only 4% of
genes in the spermatogenesis set encode predicted nucleic acid-binding
proteins. Of these, fewer than 1% encode RNA-binding proteins, probably
because mature sperm have extremely low levels of mRNA
(Roberts et al., 1986
).
Instead, the spermatogenesis set has an enrichment of cytoplasmic signaling
molecules such as protein kinases and protein phosphatases, as noted
previously (Reinke et al.,
2000
).
Identification of genes expressed in the male germline
Most events in male germ cell development also occur in hermaphrodite germ
cells, such as mitotic proliferation, recombination, chromosome segregation
and spermatogenesis. We therefore expected a large overlap between male and
hermaphrodite germline-enriched gene sets. To determine which of the male
germline-enriched genes (set II) were also enriched in the hermaphrodite
germline, we performed a Venn diagram analysis with three datasets: the male
and hermaphrodite germline-enriched gene sets and the spermatogenesis gene set
(sets I, II and Vb; Fig. 2B).
We found that 87% of genes with male germline-enriched expression are also
enriched in the hermaphrodite germline. Of these 956 shared genes, 702 show
significantly enriched expression during spermatogenesis, while 254 do not
show spermatogenesis-enrichment and are therefore likely to be involved in
other aspects of germline development shared between the two sexes, such as
mitotic proliferation. Another 105 male germline-enriched genes overlap with
the mixed spermatogenesis/somatic subset, and thus likely also represent
shared spermatogenesis-enriched genes.
The remaining 31 genes with germline-enriched expression in males, but not
hermaphrodites, are candidates for the molecular basis of two known male
germline-specific characteristics: heterochromatization of the X chromosome
(Kelly et al., 2002), and the
ability of male sperm to out-compete hermaphrodite sperm for fertilization of
oocytes (LaMunyon and Ward,
1998
). Under the strictest definition, a truly male-specific
germline gene should show no evidence of expression in hermaphrodites. We
excluded any genes with hermaphrodite expression by any of several criteria:
(1) a high fem-3(gf)/fem-1(lf) ratio, (2) significant fluctuation in
either of two hermaphrodite timecourses in larvae or embryos (see below)
(Baugh et al., 2003
), (3)
hermaphrodite expression by in situ hybridization
(Kohara, 2001
), or (4) an RNAi
phenotype in hermaphrodites (e.g. Kamath
et al., 2003
). The remaining eight genes include several that
encode novel proteins as well as one that contains a predicted MSP (major
sperm protein) domain found in structural and signaling sperm proteins, and
two that potentially bind DNA (Fig.
4).
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Comparison with independent large-scale gene expression datasets
To validate our gene subsets for the hermaphrodite germline, we compared
our results with other large-scale gene expression datasets. Our experiments
measure the abundance of transcripts in one sample relative to another.
Therefore, we will not identify germline-expressed genes with only mild or no
enrichment in the germline, relative to somatic tissues. Additionally, the
criteria we set for inclusion in a gene set are stringent, so genes with mild
germline-enriched expression in our experiments are excluded. Comparisons with
gene expression data generated by in situ hybridization or Affymetrix
microarray analysis allow us to estimate both the number of genes we are
missing, and the number of genes that are incorrectly included in our germline
datasets.
First, we examined whole-mount in situ expression patterns in
hermaphrodites in NextDB
(http://nematode.lab.nig.ac.jp)
(Kohara, 2001) for randomly
chosen genes in each subset (Table
1). Considering only genes for which expression was detected by in
situ hybridization, this comparison showed that 98% of the genes in the
intrinsic and oogenesis-enriched groups had detectable in situ staining solely
or primarily in the germline. Additionally, 88% of spermatogenesis-enriched
transcripts were detected either in the germline or spermatheca of the animal.
Overall, the in situ data indicates that most of our germline datasets have a
low false positive rate, as few genes that had germline-enriched expression by
microarray were found solely in the soma by in situ hybridization. However, we
note that the sensitivity of the large-scale in situ hybridization is unknown,
so it is possible that some genes with detectable expression only in the
germline are also expressed in the soma.
In contrast to all other germline-enriched gene sets, 34% of the genes in the mixed spermatogenesis/somatic gene subset (see Fig. 2A) show only somatic expression by in situ hybridization. This finding is consistent with the possibility discussed above that some of the genes in this subset might be those whose expression is affected by the fem-1 mutation in somatic tissues.
In this analysis, we found that our gene expression subsets had an unequal distribution of associated ESTs and detectable in situ staining. Approximately 60% of all annotated genes have a corresponding EST. Within most of our hermaphrodite gene expression subsets, an average of 74% of the genes were represented by ESTs; of these, 73% had visible staining (54% of subset). However, only 40% of genes with spermatogenesis-enriched or mixed spermatogenesis/somatic expression had associated ESTs; of these, 60% displayed visible staining (24% of subset). Thus, genes expressed during spermatogenesis are less likely to be represented by ESTs than are genes expressed in the oogenic germline. We note that the low numbers seen for genes with male-biased expression are not surprising, because the EST and in situ projects focused on hermaphrodites.
The majority of genes with hermaphrodite soma-biased expression (78%) had staining in specific somatic tissues, such as the intestine, vulva and body wall muscle, as well as broad staining that was difficult to attribute to a specific tissue(s). About 20% of the examined transcripts from the hermaphrodite-biased soma-enriched subset stained the germline, indicating that we missed about 20% of genes with detectable expression in the germline with our experimental design and statistical criteria.
We also compared our gene subsets to gene expression profiles of early
embryogenesis, collected using an Affymetrix microarray platform
(Baugh et al., 2003). By
examining embryos prior to the onset of zygotic transcription, this study
identified a large set of transcripts that are maternally provided, which (by
definition) must be expressed in the adult germline. As expected, genes with
intrinsic or oogenesis-enriched expression had very high overlap with the
maternally provided gene set, ranging from 71-88%
(Table 1). Genes enriched
during spermatogenesis, or in somatic tissues, had a lower overlap of 9-19%.
Our comparisons to both the in situ dataset and the maternally provided
dataset allows us to estimate that we are missing
20% of genes expressed
in the germline.
Temporal regulation during hermaphrodite development
In a second set of microarray experiments, we performed a temporal analysis
of gene expression during wild-type hermaphrodite development. This analysis
allows us to examine the normal kinetics of gene expression of many of the
genes defined in our mutant sets. We collected 12 samples at 3-hour intervals,
beginning in the middle of the third larval stage (L3) and extending through
adulthood (Fig. 5A). During
this time, germ cells initiate several events, including exiting mitosis and
initiating meiosis, differentiating into sperm then oocytes, and launching
embryogenesis. Formation of several somatic gonad structures such as the
vulva, the spermatheca and the uterus also occurs during this time. We
collected three series of staged hermaphrodites and performed 36
hybridizations against the same mixed stage reference sample used in the
mutant hybridizations. For each gene, we averaged the three replicates at each
time point, and used ANOVA to identify 5083 genes that showed a significant
alteration in gene expression levels between two or more time points
(P<0.01). Of these, 2925 are germline- or sex-regulated genes.
We used hierarchical clustering to group all 5083 genes solely by the
similarity in their temporal expression profiles, and defined six large
clusters (A-F) with distinct patterns of expression over time, as defined by a
correlation coefficient exceeding 0.80 within a cluster
(Fig. 5B)
(Eisen et al., 1998). So that
these genes would be grouped only by their temporal expression profiles, we
`carried' the mutant expression data in the analysis and did not give it any
weight in the clustering (Fig.
5C). We then asked whether specific sets of germline- or
sex-regulated genes were over-represented in these clusters that were defined
solely by temporal regulation. We found that 98% of the genes in the intrinsic
and oogenesis-enriched subsets included in the analysis comprise
84% of,
and are evenly distributed among, clusters E and F. The remaining 16% are
likely also expressed in the germline, based on the striking similarity in
temporal expression. Clusters E and F have largely similar expression profiles
(correlation coefficient of 0.74), with a few subtle differences. Genes in
Cluster E first display very low levels of expression from mid-L3 to the end
of L4, and then show an abrupt increase at the transition to young adulthood
(time points 6 and 7), with high levels persisting through the rest of the
time course, while genes in Cluster F have higher levels of expression at the
earlier time points and show a gradual increase starting slightly earlier
(timepoints 5 and 6).
In contrast to the intrinsic and oogenesis gene sets, 99% of spermatogenesis-enriched genes (as defined in Fig. 2A) are found in cluster D, which is characterized by sharp induction at the beginning of L4 (time point 3) and a sharp decline at the end of L4 (time points 6 and 7). The reciprocal relationship of the expression of the spermatogenesis-enriched and oogenesis-enriched/intrinsic transcripts at the onset of adulthood reflects the switch from spermatogenesis to oogenesis that occurs at this time. Strikingly, only 26% of genes in the mixed spermatogenesis/somatic gene set (see Fig. 2A) are present in cluster D, with the remaining 74% distributed among the genes in clusters A-C (Fig. 5B). The fact that most genes in the mixed spermatogenesis/somatic set differ in temporal regulation from the spermatogenesis-enriched set supports the possibility that the mixed spermatogenesis/somatic set contains many genes that are likely to be differentially expressed in somatic tissues in response to the effects of the fem-1(lf) mutation. By contrast, 86% of the genes in the mixed oogenesis/somatic subset are found in the E and F clusters with the intrinsic and oogenesis-enriched gene subsets, and thus are largely co-regulated with those sets.
In general, genes with sex-biased somatic expression were evenly distributed among clusters A-C, along with most remaining temporally regulated genes that showed no sex- or germline-regulated expression. Cluster A includes genes with a moderate level of expression at the L3 stage, which decreases at later stages, while Clusters B and C contain genes with a high level of expression in L3 and L4 larvae that decreases at the onset of adulthood to varying extents. Similarity in temporal regulation of somatic genes will help to determine which genes are likely to be co-expressed and possibly function together.
Biased chromosomal distribution of germline- and sex-regulated genes
Our previous study defining the partial set of genes with germline-enriched
expression demonstrated that many spermatogenesis-enriched and intrinsic genes
are not present on the X chromosome at the numbers expected given a random
distribution (Reinke et al.,
2000). Subsequent studies have identified transcriptional
silencing of the X chromosome in the male and hermaphrodite germline as a
potential force prohibiting X-linkage of germline-expressed genes
(Kelly et al., 2002
). In the
male, the single X chromosome remains silent at all stages of male germ cell
development, and displays a hallmark of heterochromatin formation, extensive
methylation of lysine 9 on histone H3
(Nakayama et al., 2001
;
Rea et al., 2000
). However, in
oogenic hermaphrodites, the pair of X chromosomes display only transient and
partial H3 lysine 9 methylation and is silenced only in mitotically dividing
and meiotic germ cells through the pachytene stage of meiosis I before
becoming transcriptionally active late in pachytene, just prior to diplotene
and diakinesis (Kelly et al.,
2002
). The entire genome is apparently silenced in meiotic
metaphase I just prior to fertilization.
We examined the chromosomal distribution of our genome-wide set of
germline-enriched and sex-biased somatic genes and, as before, found that
genes in the spermatogenesis and intrinsic sets are greatly under-represented
on the X chromosome (Fig. 6). Approximately 196 genes in the spermatogenesis set were expected on the X
chromosome given the number of X-linked genes on the microarray, and we only
found 25 (P<0.001). Similarly, 180 genes with intrinsic expression
were expected on the X chromosome, but only 47 are present
(P<0.001). The increased numbers in the genome-wide data set
allowed us to determine that genes with oogenesis-enriched expression are also
significantly under-represented on the X chromosome, although to a lesser
extent than the spermatogenesis-enriched and intrinsic genes; we expected 241
genes in the oogenesis subset to be X-linked, and found 138
(P<0.001; see Discussion). Conversely, genes encoding
hermaphrodite-biased somatically expressed transcripts are significantly
enriched on the X chromosome. A recent report on X chromosome distribution of
genes with male-biased expression in Drosophila demonstrated that
genes expressed in both the soma and germline of the male were depleted from
the X chromosome (Parisi et al.,
2003). In contrast to these observations in Drosophila,
male-biased somatically expressed genes in C. elegans are present at
close to expected numbers on the X chromosome, and only genes expressed in the
male germline of C. elegans are strongly depleted from the X
chromosome, as described above (see Discussion).
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RNA-mediated interference phenotypes of germline- and sex-regulated genes
The vast majority of the genes in the C. elegans genome have been
assayed for reduction-of-function phenotypes in hermaphrodites by large-scale
RNA-mediated interference screens (Piano
et al., 2002; Maeda et al.,
2001
; Kamath et al.,
2003
; Gonczy et al.,
2000
). The combined results of these large-scale screens have
identified visible phenotypes for
13% of assayed genes, with almost 10%
showing either an embryonic lethal or sterile phenotype. One study focusing on
a set of 751 genes expressed primarily in oogenic germlines found that 322
(42%) produced either an embryonic lethal or sterile phenotype upon functional
depletion by RNAi, demonstrating that these phenotypes are enriched among
germline-expressed genes (Piano et al.,
2002
).
We compared the genes with germline-enriched expression that we identified
in our microarray analysis to the combined results from these large-scale RNAi
screens, focusing on embryonic lethality and sterility
(Fig. 7). Of the genes within
the intrinsic and oogenesis gene sets that had been tested by RNAi, functional
depletion of 28% of the genes results in either or both of these
phenotypes. This number is lower than the 42% mentioned above, probably
because of differing false negative rates among the various studies
(Piano et al., 2002
).
Functional disruption of <3% of the genes in the spermatogenesis set causes
embryonic lethality or sterility. This number is a significant underestimate
of the genes whose function is required for spermatogenesis, as RNAi is
inefficient at reproducing phenotypes of known mutants in
spermatogenesis-expressed genes (S.W., unpublished).
|
We also examined a diverse group of phenotypes that affect post-embryonic
development without affecting fecundity [i.e. Vpep + Gro, as defined by Kamath
et al. (Kamath et al., 2003)].
We found that genes in the intrinsic and oogenesis data sets display
post-embryonic phenotypes more frequently than other gene sets, including the
hermaphrodite-biased somatic set. Genes in the intrinsic and oogenesis sets
that display post-embryonic phenotypes upon RNAi include multiple genes with
known somatic functions, such as egl-27, nhr-23, daf-18 PTEN,
lin-35 Rb and ptp-2
(Ch'ng and Kenyon, 1999
;
Kostrouchova et al., 2001
;
Mihaylova et al., 1999
;
Lu and Horvitz, 1998
;
Gutch et al., 1998
). This
observation suggests that a subset of the genes expressed primarily in the
germline also function in other tissues at other places and times. Because
incomplete RNAi can result in partial depletion of a gene product, many of
these post-embryonic phenotypes might be caused by decreased levels of a gene
product that causes embryonic lethality or sterility when completely
depleted.
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Discussion |
---|
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---|
Characterization of germline-enriched genes
The germline-enriched gene set has been resolved into three subsets, which
roughly correspond to different germline functions: spermatogenesis, oogenesis
and intrinsic. This last group is defined by a lack of significant regulation
during gametogenesis, and was originally thought to correspond to distal
germline functions, such as stem cell proliferation and early meiosis I
(Reinke et al., 2000).
However, several observations made in this study point to the conclusion that
genes in the intrinsic and oogenesis-enriched subsets are highly similar and
not easily functionally distinguishable. First, these genes encode similar
types and proportions of proteins with predicted or known functions
(Fig. 3A). Second, the temporal
regulation of these genes is essentially identical, with the vast majority of
the genes showing an abrupt induction at the onset of young adulthood
(clusters E and F; Fig. 5B).
Third, the range and percentage of RNAi phenotypes is comparable between these
two categories (Fig. 7).
Fourth, many genes among the oogenesis gene set show detectable expression in
the distal germline by in situ hybridization. Why then can we distinguish
these two gene sets by microarray analysis? One possibility is based on our
observation that the fem-3 mutant makes excess sperm at the expense
of more distal germ cells, reducing the number of cells in pachytene of
meiosis I relative to fem-1 mutants. Thus, many genes that are
expressed in distal germ cells (`intrinsic') actually appear enriched in
fem-1 relative to fem-3 and are classified as
`oogenesis-enriched'. From the above set of observations, we conclude that
many genes with `intrinsic' function are likely to be found in the oogenesis
category, and vice versa.
The single distinguishing characteristic we have observed between the
intrinsic and oogenesis sets is that genes in the oogenesis set are found on
the X chromosome at a slightly higher frequency than genes in the intrinsic
set, although they are still present at much lower levels than expected
(Fig. 6). Genes expressed
during early oogenesis are probably not as strongly excluded from the X
chromosome because the X chromosome becomes re-activated as germ cells enter
oogenesis (Kelly et al.,
2002). This observation suggests that although the oogenesis
category contains many genes with intrinsic function, it also contains genes
that are specifically expressed at a later point in the germline, relative to
the intrinsic category. Indeed, some autosomal and X-linked genes in the
oogenesis category have detectable in situ hybridization signals only in the
proximal germline
(http://nematode.lab.nig.ac.jp)
(Kelly et al., 2002
).
In our analysis of in situ hybridization patterns presented in NextDB for the germline- and sex-regulated genes in hermaphrodites, we found that more genes in the spermatogenesis set fail to have a corresponding EST than do hermaphrodite-biased somatic genes or intrinsic/oogenesis genes. One possible explanation for this observation is that genes required for spermatogenesis are expressed only briefly during the development of the animal and are therefore likely to be under-represented in a cDNA library. However, hermaphrodite-biased, somatically expressed genes, many of which are also expressed in a restricted area and for a brief period of time (e.g. during vulval induction), have a corresponding EST as frequently as genes in the intrinsic or oogenesis sets. Interestingly, the fraction of ESTs that give visible in situ expression patterns is roughly equivalent between hermaphrodite-biased somatic and spermatogenesis gene sets (50-56%), but is considerably lower than for the intrinsic/oogenesis gene sets (71-79%). This observation suggests that genes with highly restricted spatial or temporal expression tend not to be detected by in situ hybridization, or that genes with expression in the oogenic germline are more easily detectable than other tissues.
Comparison between male-biased and hermaphrodite-biased germline-enriched gene sets defined a small subset of genes whose expression appears largely restricted to the male germline. These genes are candidates for male germline-specific functions, such as heterochromatization of the X chromosome and effective sperm competition. Interestingly, among the eight genes that show no apparent expression in hermaphrodites, one contains an MSP domain commonly found among structural sperm proteins, as well as additional domains of unknown function. Future functional studies using RNAi or deletion mutant analysis to investigate the role of these proteins in male germline development and function will shed light on how the male germline performs its unique roles.
Temporal analysis of germline- and sex-regulated gene expression
We analyzed high-resolution temporal gene expression profiles during the
stages that encompass most of germline development: in L3 and L4 larvae, and
in pre- and post-reproductively mature adults. When we clustered genes based
solely on their temporal expression profiles, we found that subsets of the
germline genes showed very similar co-regulation. Genes in the spermatogenesis
set show expression profiles that correspond with spermatogenesis in the
fourth larval stage, as expected (Reinke
et al., 2000). Surprisingly, even with a fairly high-resolution
sampling, we were able to distinguish only two other germline-enriched
clusters (clusters E and F), each containing a mixture of genes in the
intrinsic and oogenesis sets. Not only do the genes within each cluster show a
strong degree of correlated expression (>0.85), but the correlation between
clusters E and F is also high (0.74). These temporal gene expression profiles
indicate that essentially all gene expression in the germline is tightly
temporally controlled, not just the genes with spermatogenesis-enriched
expression. In addition to providing profiles of germline-enriched gene
expression, these data also provide temporal profiles for genes expressed
during somatic events that occur during late larval and early adult stages,
including formation of several structures of the somatic gonad.
The timecourse data also provide supporting evidence that some genes
differentially regulated in the fem-3(gf)/fem-1(lf)
comparison are actually somatically expressed, because a subset of genes with
enrichment only in fem-3(gf) animals (`mixed spermatogenesis/somatic'
subset, see Fig. 2B) do not
cluster with all the other spermatogenesis genes in cluster D. For example,
among this subset is the family of Mariner transposases that have been found
to have sperm-enriched expression (Reinke
et al., 2000; Kim et al.,
2001
). However, transposons are known to be silenced in the
germline (Emmons and Yesner,
1984
), so the expression of transposases, which excise and
mobilize transposons, during spermatogenesis was surprising. The data
presented in this report demonstrate that although these transposases show
fem-3-enriched expression, their transcripts are not
germline-enriched in wild type relative to glp-4(lf) animals and they
do not cluster with the other spermatogenesis-enriched transcripts. Instead,
these transposases either show no temporal regulation or are found in cluster
A. Thus, it is very likely that these transposases are not expressed during
spermatogenesis, but show increased activity in somatic tissue upon loss of
the activity of the fem-1 gene product.
Chromosomal biases of germline- and sex-regulated genes
Our data show that the chromosomal locations of the genes with enriched
expression in the germline are non-random. We found a very strong bias against
genes in the spermatogenesis and intrinsic sets residing on the X chromosome,
as seen before (Reinke et al.,
2000). Additionally, with our expanded data set, we were also able
to detect a significant reduction in the number of observed X-linked genes in
the oogenesis category compared with the expected number. Our data also
indicate that the location of hermaphrodite-biased, somatically expressed
genes is reciprocal with the germline-enriched gene sets: whereas genes with
germline-enriched expression are enriched on chromosome I and lacking on the X
chromosome, hermaphrodite-biased, somatically expressed genes are enriched on
the X chromosome and lacking on chromosome I. Notably, this reciprocal
relationship is not limited to chromosomes I and X; a similar trend is also
seen for chromosomes II, III and V.
Recently, several investigations of gene expression in different organisms
have revealed sex chromosome biases for genes expressed in the germline. These
biases differ between organisms: in mice, genes expressed in male
spermatogonia are concentrated on the X chromosome, while in both
Drosophila and C. elegans, genes expressed during
spermatogenesis are found on the X chromosome much less frequently than
expected (Wang et al., 2001;
Parisi et al., 2003
;
Reinke et al., 2000
) (this
work). However, genes with male-biased expression in somatic tissues in
Drosophila are also under-represented on the X chromosome, whereas
male-biased somatically expressed genes in C. elegans are not. In
C. elegans, the silencing of the X chromosome in the germline
provides an excellent candidate for the mechanism behind under-representation
of germline genes on the X chromosome
(Kelly et al., 2002
). By
contrast, in Drosophila, several lines of evidence indicate that the
X chromosome remains transcriptionally active during spermatogenesis (reviewed
by McKee and Handel, 1993
);
therefore the forces preventing genes with male-biased expression from staying
on the X chromosome must differ from those in C. elegans. Genes with
male-biased expression in Drosophila show a strong tendency to move
off the X chromosome, based on comparisons to the distantly related mosquito,
Anopheles gambiae (Parisi et al.,
2003
). It will be interesting to perform a similar analysis of
genes with spermatogenesis-enriched expression in C. elegans, once
the genome sequence of the related nematode C. briggsae is fully
assembled and chromosomes are assigned.
When we compared our gene sets with existing RNAi phenotypic data, we found
that the intrinsic and oogenesis gene sets contain a high percentage of genes
that result in either embryonic lethality or sterility when functionally
depleted, as observed previously (Piano et
al., 2002). Large-scale functional studies have demonstrated that
RNAi of X-linked genes results in embryonic lethal or sterile phenotypes much
less frequently than expected (Piano et
al., 2002
; Kamath et al.,
2003
). Much of this observation can therefore be attributed to the
fact that genes with germline-enriched expression are excluded from the X
chromosome and that the X chromosome is silenced throughout much of the
germline, as discussed above. However, even taking into account the reduced
number of germline-enriched genes on the X chromosome, embryonic lethal and
sterile phenotypes are still much rarer than expected
(Piano et al., 2002
). One
possible explanation for this observation is that the brief window of
expression of X-linked genes in the germline reduces the ability of RNAi to
effectively functionally deplete them. Another possibility is that genes on
the X chromosome are simply less likely to encode proteins required for
viability and fecundity (Piano et al.,
2002
). This second possibility is supported by the fact that
post-embryonic phenotypes are found more frequently among X-linked genes
(Kamath et al., 2003
).
However, we did not see an enrichment of post-embryonic phenotypes among the
hermaphrodite-biased, somatically expressed genes, which are enriched on the X
chromosome (Figs 6 and
7).
![]() |
Conclusion |
---|
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---|
Our work has identified the vast majority of genes with expression in the germline of C. elegans. The identification and characterization of cis-regulatory elements in the noncoding regions surrounding these genes will allow us to better understand the gene-specific and global regulatory mechanisms that govern gene expression in the germline. In the future, computational analysis, in conjunction with experiments using genomic DNA microarrays to investigate the interactions of regulatory proteins with cis-regulatory elements, should shed light on these still-mysterious mechanisms.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
---|
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---|
Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M., Davis, A. P., Dolinski, K., Dwight, S. S., Eppig, J. T. et al. (2000). Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25-29.[CrossRef][Medline]
Barton, M. K., Schedl, T. and Kimble, J.
(1987). Gain-of-function mutations of fem-3, a
sex-determination gene in Caenorhabditis elegans.Genetics 115,107
-119.
Baugh, L. R., Hill, A. A., Slonim, D. K., Brown, E. L. and
Hunter, C. P. (2003). Composition and dynamics of the
Caenorhabditis elegans early embryonic transcriptome.
Development 130,889
-900.
Beanan, M. and Strome, S. (1992).
Characterization of a germline proliferation mutation in C. elegans.Development 116,755
-766.
Broverman, S. A. and Meneely, P. M. (1994).
Meiotic mutants that cause a polar decrease in recombination on the X
chromosome in Caenorhabditis elegans. Genetics
136,119
-127.
Ch'ng, Q. and Kenyon, C. (1999).
egl-27 generates anteroposterior patterns of cell fusion in C.
elegans by regulating Hox gene expression and Hox protein function.
Development 126,3303
-3312
Colaiácovo, M. P., Stanfield, G. M., Reddy, K. C.,
Reinke, V., Kim, S. K. and Villeneuve, A. M. (2002). A
targeted RNAi screen for genes involved in chromosome morphogenesis and
nuclear organization in the Caenorhabditis elegans germline.
Genetics 162,113
-128.
Eisen, M. B., Spellman, P. T., Brown, P. O. and Botstein, D.
(1998). Cluster analysis and display of genome-wide expression
patterns. Proc. Natl. Acad. Sci. USA
95,14863
-14868.
Emmons, S. W. and Sternberg, P. S. (1997). Male development and mating behavior. In C. elegans II (ed. D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess), pp.295 -334. Plainview, NY, Cold Spring Harbor Laboratory Press.
Emmons, S. W. and Yesner, L. (1984). High-frequency excision of transposable element Tc1 in the nematode Caenorhabditis elegans is limited to somatic cells. Cell 36,599 -605.[Medline]
Fong, Y., Bender, L., Wang, W. and Strome, S.
(2002). Regulation of the different chromatin states of autosomes
and X chromosomes in the germ line of C. elegans.Science 296,2235
-2238.
Gaudet, J. and Mango, S. E. (2002). Regulation
of organogenesis by the Caenorhabditis elegans FoxA protein PHA-4.
Science 295,821
-825.
Gaudet, J., VanderElst, I. and Spence, A. M. (1996). Post-transcriptional regulation of sex determination in Caenorhabditis elegans: widespread expression of the sex-determining gene fem-1 in both sexes. Mol. Biol. Cell 7,1107 -1121[Abstract]
Giese, A., Jude, R., Kuiper, H., Raudsepp, T., Piumi, F., Schambony, A., Guerin, G., Chowdhary, B. P., Distl, O., Topfer-Petersen, E. and Leeb, T. (2002). Molecular characterization of the equine testis-specific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family. Gene 299,101 -109.[CrossRef][Medline]
Gonczy, P., Echeverri, C., Oegema, K., Coulson, A., Jones, S. J., Copley, R. R., Duperon, J., Oegema, J., Brehm, M., Cassin, E. et al. (2000). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408,331 -336.[CrossRef][Medline]
Goodwin, E. B. and Evans, T. C. (1997). Translational control of development in C. elegans. Semin. Cell Dev. Biol. 8,551 -559.[CrossRef][Medline]
Gutch, M. J., Flint, A. J., Keller, J., Tonks, N. K. and
Hengartner, M. O. (1998). The Caenorhabditis elegans
SH2 domain-containing protein tyrosine phosphatase PTP-2 participates in
signal transduction during oogenesis and vulval development. Genes
Dev. 12,571
-585.
Hodgkin, J., Horvitz, H. R. and Brenner, S.
(1979). Nondisjunction mutants of the nematode Caenorhabditis
elegans. Genetics 91,67
-94.
Jiang, M., Ryu, J., Kiraly, M., Duke, K., Reinke, V. and Kim, S.
K. (2001). Genome-wide analysis of developmental and
sex-regulated gene expression profiles in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA
98,218
-223.
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., le Bot, N., Moreno, S., Sohrmann, M. et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421,231 -237.[CrossRef][Medline]
Kelly, W. G., Schaner, C. E., Dernburg, A. F., Lee, M.-H., Kim, S. K., Villeneuve, A. M. and Reinke, V. (2002). X-chromosome silencing in the germline of C. elegans. Development 129,479 -492.[Medline]
Kim, S. K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart,
J. M., Eizinger, A., Wylie, B. N. and Davidson, G. S. (2001).
A gene expression map for Caenorhabditis elegans.Science 293,2087
-2092.
Kohara, Y. (2001). Systematic analysis of gene expression of the C. elegans genome. Tanpakushitsu Kakusan Koso 46,2425 -2431.[Medline]
Kostrouchova, M., Krause, M., Kostrouch, Z. and Rall, J. E.
(2001). Nuclear hormone receptor CHR3 is a critical regulator of
all four larval molts of the nematode Caenorhabditis elegans. Proc.
Natl. Acad. Sci. USA 98,7360
-7365.
Kuwabara, P. E. and Perry, M. D. (2001). It ain't over till it's ova: germline sex determination in C. elegans.BioEssays 23,596 -604.[CrossRef][Medline]
LaMunyon, C. W. and Ward, S. (1998). Larger sperm outcompete smaller sperm in the nematode Caenorhabditis elegans.Proc. R. Soc. Lond. B. Biol. Sci. 265,1997 -2002.[CrossRef][Medline]
Lu, X. and Horvitz, H. R. (1998). lin-35 and lin-53, two genes that antagonize a C. elegans Ras pathway, encode proteins similar to Rb and its binding protein RbAp48. Cell 95,981 -991.[Medline]
Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K. and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr. Biol. 12,1566 -1573.[Medline]
MacQueen, A. J. and Villeneuve, A. M. (2001).
Nuclear reorganization and homologous chromosome pairing during meiotic
prophase require C. elegans chk-2. Genes Dev.
15,1674
-1687.
Maeda, I., Kohara, Y., Yamamoto, M. and Sugimoto, A. (2001). Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr. Biol. 11,171 -176.[CrossRef][Medline]
McKee, B. D. and Handel, M. A. (1993). Sex chromosomes, recombination, and chromatin conformation. Chromosoma 102,71 -80.[Medline]
Mihaylova, V. T., Borland, C. Z., Manjarrez, L., Stern, M. J.
and Sun, H. (1999). The PTEN tumor suppressor homolog in
Caenorhabditis elegans regulates longevity and dauer formation in an
insulin receptor-like signaling pathway. Proc. Natl. Acad. Sci.
USA 96,7427
-7432.
Miller, M. A., Ruest, P. J., Kosinski, M., Hanks, S. K. and
Greenstein, D. (2003). An Eph receptor sperm-sensing control
mechanism for oocyte meiotic maturation in Caenorhabditis elegans.Genes Dev. 17,187
-200.
Nakayama, J.-L., Rice, J. C., Strahl, B. E., Allis, C. D. and
Grewal, S. I. S. (2001). Role of histone H3 lysine 9
methylation in epigenetic control of heterochromatin assembly.
Science 292,110
-113.
Nelson, G. A., Lew, K. K. and Ward, S. (1978). Intersex, a temperature-sensitive mutant of the nematode C. elegans.Dev. Biol. 66,386 -409.[Medline]
Parisi, M., Nuttall, R., Naiman, D., Bouffard, G., Malley, J.,
Andrews, J., Eastman, S. and Oliver, B. (2003). Paucity of
genes on the Drosophila X chromosome showing male-biased expression.
Science 299,697
-700.
Pellettieri, J., Reinke, V., Kim, S. K. and Seydoux, G. (2003). Coordinate activation of maternal protein degradation during the egg-to-embryo transition in C. elegans. Dev. Cell 5,451 -462.[Medline]
Piano, F., Schetter, A. J., Morton, D. G., Gunsalus, K. C., Reinke, V., Kim, S. K. and Kemphues, K. J. (2002). Gene clustering based on RNAi phenotypes of ovary-enriched genes in C. elegans.Curr. Biol. 12,1959 -1964.[CrossRef][Medline]
Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C. P., Allis, C. D. and Jenuwein, T. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406,593 -599.[CrossRef][Medline]
Reinke, V. (2002). Functional exploration of the C. elegans genome using DNA microarrays. Nat. Genet. 32,541 -546.[CrossRef][Medline]
Reinke, V., Smith, H. E., Nance, J., Wang, J., van Doren, C., Begley, R., Jones, S. J. M., Davis, E. B., Scherer, S., Ward, S. and Kim, S. K. (2000). A global profile of germline gene expression in C. elegans. Mol. Cell 6,605 -616.[Medline]
Roberts, T. M., Pavalko, F. M. and Ward, S. (1986). Membrane and cytoplasmic proteins are transported in the same organelle complex during nematode spermatogenesis. J. Cell Biol. 102,1787 -1796.[Abstract]
Roy, P. J., Stuart, J. M., Lund, J. and Kim, S. K. (2002). Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature 418,975 -979.[CrossRef][Medline]
Seydoux, G. and Strome, S. (1999). Launching
the germline in Caenorhabditis elegans: regulation of gene expression
in early germ cells. Development
126,3275
-3283.
Walhout, A. J. M., Reboul, J., Shtanko, O., Bertin, N., Vaglio, P., Ge, H., Lee, H., Doucette-Stamm, L., Gunsalus, K. C., Schetter, A. J. et al. (2002). Integrating interactome, phenome, and transcriptome mapping data for the C. elegans germline. Curr. Biol. 12,1952 -1958.[CrossRef][Medline]
Wang, J. and Kim, S. K. (2003). Global analysis
of dauer gene expression in Caenorhabditis elegans.Development 130,1621
-1634.
Wang, P. J., McCarrey, J. R., Yang, F. and Page, D. C. (2001). An abundance of X-linked genes expressed in spermatogonia. Nat. Genet. 27,422 -426.[CrossRef][Medline]
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