|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 25 July 2007
doi: 10.1242/dev.008375
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Max-Planck Institute for Developmental Biology, Department for Evolutionary Biology, Spemannstrasse 37, D-72076 Tübingen, Germany.
* Author for correspondence (e-mail: ralf.sommer{at}tuebingen.mpg.de)
Accepted 22 June 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: P. pacificus, Vulva development, C. elegans, Pax genes, lin-39
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In
Caenorhabditis elegans, detailed genetic and molecular studies of
vulva formation present a framework for studying pattern formation, cell fate
specification, developmental competence and induction
(Sternberg, 2005). At the same
time, comparative studies in other nematodes, in particular the satellite
organism Pristionchus pacificus, provide insight into the
evolutionary changes of developmental processes and mechanisms
(Sommer, 2005). Nearly all
aspects of vulva formation are subject to evolutionary change. P.
pacificus is amenable to forward and reverse genetic analysis, and
provides a platform to identify genetic and molecular alterations that control
evolutionary changes of developmental processes. P. pacificus
propagates as a self-fertilizing hermaphrodite and has an integrated genome
map that contains a genetic linkage map of more than 600 molecular markers and
a physical map of nearly 10,000 fingerprinted BAC clones
(Srinivasan et al., 2002;
Srinivasan et al., 2003). A
whole-genome sequencing project has recently been finished
(Dieterich et al., 2007).
The nematode vulva is a derivative of the ventral epidermis, which consists
of 12 precursor cells, called P(1-12).p, in all nematodes studied to date
(with `Pn.p' denoting the complete group of ventral epidermal precursor cells)
(Fig. 1A). These 12 Pn.p cells
adopt different cell fates according to positional information provided by
homeotic control genes. In the C. elegans hermaphrodite, the first
cell fate decision in the ventral epidermis is between potential vulval
precursor cells (VPCs) and `non-vulval' cells. Cells in the anterior and
posterior body region, P(1,2,9-11).p, fuse with the hypodermal syncytium hyp7
early in development. By contrast, P(3-8).p in the central body region remain
non-fused, become VPCs and form a so-called vulva equivalence group (VEG)
(Fig. 1A). Genetically, the Hox
gene lin-39 defines the developmental competence of P(3-8).p and
establishes this group of cells as VPCs. In C. elegans lin-39
(Cel-lin-39) mutants, P(3-8).p fuse with the hypodermis, resulting in
a phenotype that has been designated as `generation vulvaless' (Gev;
Fig. 1B)
(Clark et al., 1993;
Wang et al., 1993). It has
been suggested that the early role of Cel-LIN-39 is the indirect
regulation of the cell fusion effector eff-1. Cel-LIN-39 regulates
the GATA transcription factors egl-18 and elt-6, which in
turn might regulate eff-1 (Fig.
2A) (Shemer and Podbilewicz,
2002; Koh et al.,
2002; Cassata et al.,
2005).
In C. elegans, P(3-8).p adopt one of three alternative fates later
in development (Sternberg,
2005). P6.p generates eight progeny and forms the central part of
the vulva, a fate that is designated as 1° fate. P(5,7).p have a 2°
fate, generate seven progeny each and form the anterior and posterior part of
the vulva, respectively. P(3,4,8).p do not participate in vulva formation in
wild-type animals, remain epidermal and have the so-called 3° fate. After
cell ablation of P(5-7).p, P(3,4,8).p can substitute for the other VPCs,
indicating that all VPCs have the competence to form part of the vulva.
Detailed genetic and molecular studies revealed a complex regulatory network
involved in C. elegans vulva formation. An epidermal growth factor
(EGF)-like molecule encoded by the gene lin-3 is secreted from the
gonadal anchor cell (AC) and induces P(5-7).p to adopt 1° and 2°
vulval fates. The LIN-3 signal is transmitted within the VPCs by an EGFR/RAS
pathway. Wnt signaling and DSL/Notch signaling have been shown to participate
in vulva development, and various specification events involve redundant
functions of different signaling cascades. The Hox gene lin-39 is a
downstream target of EGF and Wnt signaling during vulva induction
(Maloof and Kenyon, 1998;
Wagmaister et al., 2006).
Thus, Cel-LIN-39 is used twice during vulva formation: first, to
establish the VEG and, second, in response to vulva induction.
|
|
). Four important differences in cell fate specification are
apparent between P. pacificus and C. elegans
(Fig. 1C). First, in P.
pacificus, non-vulval Pn.p cells do not fuse with hyp7 as they do in
C. elegans; instead, they die from programmed cell death (PCD)
(Sommer and Sternberg, 1996).
Second, this PCD involves P(1-4).p in the anterior body region and P(9-11).p
in the posterior body region, and has an influence on the size of the VEG;
therefore, P(3,4).p are members of the VEG in C. elegans but die in
P. pacificus. Thus, PCD limits the size of the VEG in P.
pacificus (Eizinger and Sommer,
1997). Third, vulva induction in P. pacificus involves
multiple cells of the somatic gonad and takes more than 10 hours, whereas the
AC induces the vulva in a one-step interaction in C. elegans
(Sigrist and Sommer, 1999).
Fourth, the epidermal cell P8.p does not divide in P. pacificus and
is not competent to adopt a vulva fate, whereas it is a VPC in C.
elegans. However, P8.p is involved in cell fate specification of the VPCs
by inhibiting P(5,7).p from taking a 1° fate, an interaction that has been
designated as lateral inhibition (Jungblut
and Sommer, 2000).
Genetic and molecular studies revealed that the VEG in P.
pacificus is also specified by the Hox gene lin-39
(Eizinger and Sommer, 1997). In
P. pacificus lin-39 (Ppa-lin-39) mutants, P(5-8).p die from
PCD like their anterior and posterior lineage counterparts, indicating that
the ground state of ventral epidermal cells in P. pacificus is PCD
(Fig. 1D,
Table 1B). Thus,
Ppa-LIN-39 prevents PCD, whereas Cel-LIN-39 prevents cell
fusion, indicating that Ppa-LIN-39 must be part of a different
regulatory network than Cel-LIN-39 during VEG formation.
|
). Pax proteins contain up to three highly conserved
protein domains: an N-terminal paired domain (PD) and a C-terminal homeodomain
(HD), both of which are involved in DNA-binding, as well as an octapeptide.
Pax proteins were shown to function as both transcriptional activators and
repressors. For example, in mammals, Pax5 (also known as BSAP in humans) is
converted from a transcriptional activator to a repressor via interactions
with the co-repressors of the Groucho family in an octapeptide-dependent
manner (Eberhard et al., 2000).
At the sequence level, the presence or absence of the HD and/or the
octapeptide, and the sequence similarities within the PD, allow the
subdivision of Pax proteins into specific subfamilies
(Chi and Epstein, 2002).
Although there is a general conservation of Pax genes throughout the animal
kingdom, many differences can be found in individual taxa. For example, the
genome of the nematode C. elegans contains five bona-fide Pax genes
(Hobert and Ruvkun, 1999).
vab-3 belongs to the Pax-6 subfamily and is involved in head
and tail development (Chisholm and Horvitz,
1995; Zhang and Emmons,
1995). egl-38 is a member of the Pax-2/5
subfamily and regulates uterine and tail development
(Chamberlin et al., 1997). The
highly related gene K06B9.5 results from a recent gene duplication.
Interestingly, the gene F27E5.2 is the only gene present in the
C. elegans genome that contains all three protein domains, the PD, HD
and octapeptide. Here, we describe, by mutant analysis, the first specific postembryonic developmental function of a nematode Pax-3-type gene. We show that Ppa-pax-3 is involved in the formation of the VEG and that Ppa-pax-3 has distinct functions in cell fate specification of epidermal cells. Whereas Ppa-pax-3 regulates cell survival of the central Pn.p cells, it regulates cell death of posterior epidermal cells. Additional data indicate that Ppa-pax-3 is a direct target of Ppa-LIN-39. Together, these data show a function for a nematode Pax-3-type gene during vulva development and indicate a different regulatory network for the formation of the VEG.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
The following strains were used in this study: P. pacificus PS312
(the wild-type strain) is a derivative of an isolate from Pasadena, California
and represents the wild-type strain used for genetic analysis; P.
pacificus PS1843, isolated from Port Angeles, Washington represents the
polymorphic reference strain for mapping
(Srinivasan et al., 2002).
Other mutant strains used in this study were Ppa-lin-39(tu29)
(Eizinger and Sommer, 1997) and
Ppa-mab-5(tu357) (Jungblut and
Sommer, 1998).
Mutagenesis
TMP/UV mutagenesis was carried out as described elsewhere
(Jungblut and Sommer, 2001).
In short, mixed-stage animals were washed off the plates in M9 buffer and were
incubated for 20 minutes with 33 µg/ml TMP and then UV irradiated for 50
seconds with an intensity of 500 µW/cm2. In the F2 generation,
egg-laying defective mutants were isolated and their progeny were reanalyzed
for vulva defects using Nomarski microscopy. Mutant hermaphrodites were
backcrossed multiple times using wild-type males. Complementation tests were
carried out using dpy-marked Gev mutants
(Kenning et al., 2004).
Mapping and SSCP detection
For mapping, mutant hermaphrodites in the California background were
crossed with males of the Washington strain. To extract genomic DNA, F2 mutant
animals were picked to single tubes containing 2.5 µl of lysis buffer (50
mM KCl; 10 mM Tris-HCl pH 8.3; 2.5 mM MgCl2; 0.45% NP-40; 0.45%
Tween; 0.01% gelatin; 5 µg/ml Proteinase K) and incubated for 1 hour at
65°C, followed by inactivation of the Proteinase K at 95°C for 10
minutes. To assign linkage of a mutation to a certain chromosome, two
representative single-strand conformational polymorphism (SSCP) markers per
chromosome were tested against 42 Washington-backcrossed mutant animals. For
SSCP detection, PCR samples were diluted 1:1 in denaturing solution (95%
formamide, 0.1% xylene cyanol, 0.1% bromophenol blue), denatured at 95°C
for 5 minutes and loaded onto a GeneGel Excel prepoured 6% acrylamide gel
(PharmaciaBiotech, Piscataway, NJ). Gels were fixed and silver stained to
detect the DNA.
Morpholino experiments
Oligonucleotides (Gene Tools) were dissolved in water and subsequently
diluted to a concentration of 100 µM. Primer sequences are available on
request.
Quantitative PCR experiments
A total of 120 J1 animals were picked into 15 µl of 1:10 diluted single
worm lysis buffer. RNA was extracted with 100 µl of TRIZOL using a repeated
freeze-thaw protocol in liquid nitrogen. RNA was reverse transcribed in 20
µl total volume reaction (Invitrogen) with negative controls without
reverse transcriptase included for each sample. Quantitative PCR was performed
on a Roche LC480 LightCycler using the manufacturer's SYBR green PCR mix.
Primer concentrations were 0.5 mM.
|
32P]-ATP using T4 polynucleotide kinase (NEB, Beverly, MA). Labeled
oligonucleotides (1.5 pmol) were used in bandshift experiments.
Electrophoresis was carried out at 4°C on a 6% non-denaturing
polyacrylamide gel.
Bioinformatic analysis of Hox-binding sites
We used the SITEBLAST software (Michael
et al., 2005) and consensus sequences from the JASPAR database to
identify conserved putative Hox target enhancers in the P. pacificus
genome.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gev-2 mutant animals were strongly egg-laying defective.
More-detailed cell lineage analysis in the ventral epidermis of gev-2
mutants revealed two independent defects in Pn.p cell fate specification
(Table 1). First, the central
cells, P(5-8).p, died from PCD in many gev-2 mutant animals,
resulting in the absence of a vulva (Fig.
3). Although both alleles had a similar PCD phenotype, they showed
a slight posterior bias in that P8.p survived with a higher frequency than
P5.p (Table 1C,D). Second,
mutations in gev-2 resulted in a distinct phenotype in the posterior
body region. In wild-type and Ppa-lin-39 mutant hermaphrodites
P(9-11).p die from PCD (Table
1A,B) (Sommer and Sternberg,
1996). However, P(9-11).p survive in gev-2 mutant animals
with an frequency of 10-20% (Table
1C,D). Thus, gev-2 has different functions in Pn.p cell
fate specification in the central and the posterior body region: it positively
regulates cell survival of P(5-8).p and induces the PCD of P(9-11).p
(Fig. 1E).
gev-2 is required for vulva induction
Mutations in lin-39 result in a Gev phenotype in both P.
pacificus and C. elegans
(Clark et al., 1993;
Wang et al., 1993;
Eizinger and Sommer, 1997).
However, Ppa-lin-39 and Cel-lin-39 differ with regard to
their involvement in vulva induction. Whereas Cel-lin-39 is crucial
for vulva induction and was shown to represent a downstream target of EGF/RAS
and WNT signaling (Maloof and Kenyon,
1998; Wagmaister et al.,
2006), Ppa-lin-39 is dispensable for vulva induction: in
Ppa-lin-39; Ppa-ced-3 double mutants, the PCD of Pn.p cells is
rescued and the surviving VPCs undergo normal vulva differentiation
(Table 1E)
(Sommer et al., 1998).
To determine whether gev-2 has a role in P. pacificus
vulva induction, we generated similar double mutants with a
cell-death-defective mutant to overcome the PCD of P(5-8).p. Because genetic
mapping of gev-2 revealed that it is located on chromosome II in
close proximity to the Ppa-ced-3 gene (see below for details), we
generated double mutants containing gev-2 with a second
cell-death-defective mutant previously described as ipa-2
(Sommer et al., 1998).
ipa-2 shows complete cell survival of all Pn.p cells, similar to
Ppa-ced-3. ipa-2 maps to chromosome III and has been shown to be the
Ppa-ced-4 gene, which encodes an ortholog of the cell-death adaptor
CED-4/APAF-1 (Dinkelacker, Lee and Sommer, unpublished information).
Interestingly, the phenotype of the Ppa-gev-2; Ppa-ced-4 double
mutant was allele-specific: whereas Ppa-gev-2(tu358);
Ppa-ced-4(tu324) mutant animals mostly formed a normal vulva
(Table 1F),
Ppa-gev-2(tu214); Ppa-ced-4(tu324) double mutants were slightly
induction vulvaless. Specifically, 20-30% of the VPCs remained un-induced in
Ppa-gev-2(tu214); Ppa-ced-4(tu324) double-mutant animals
(Table 1G). This is consistent
with findings in the gev-2(tu214) single mutant, in which the
majority of the surviving VPCs remained epidermal. Only 12% of P5.p cells, 31%
of P6.p cells and 17% of P7.p cells, respectively, formed vulva tissue in
tu214 mutant animals, although a higher percentage of cells did
survive (Table 1C). By
contrast, nearly all of the surviving VPCs underwent vulva differentiation in
the tu358 allele (Table
1D). When we scored vulva induction in transheterozygous
tu214/tu358 animals, we found vulva differentiation in most of the
surviving VPCs (Table 1H).
Together, these results suggest that gev-2(tu214) has an
allele-specific effect indicating a function of gev-2 during vulva
induction.
|
|
|
). To study whether Ppa-mab-5 is involved in
P(9-11).p determination in males, we investigated Ppa-mab-5(tu357)
mutant males. Indeed, P(9-11).p underwent PCD in Ppa-mab-5(tu357)
males, indicating that the role of MAB-5 in P(9-11).p male-specification is
conserved between P. pacificus and C. elegans
(Fig. 1G,I;
Table 2C). Similarly, P(9-11).p
also underwent PCD in gev-2(tu214); Ppa-mab-5(tu357) double mutants
(Table 2D). Taken together, the
analysis of P(9-11).p determination in P. pacificus males suggests
strong similarities to C. elegans, but does not provide any evidence
for a role of gev-2. Thus, gev-2 represents a
hermaphrodite-specific regulator of Pn.p cell fate specification with distinct
functions in the central and the posterior body region.
gev-2 is the Ppa-pax-3 gene
To identify the molecular nature of gev-2, we mapped the locus
using the polymorphic reference strain P. pacificus var. Washington
(Srinivasan et al., 2002).
gev-2 maps to chromosome II between the molecular markers S264 and
S152, and is most closely linked to the marker S98, which is associated with
the BAC clone BACPP17D-2 (Fig.
4A). Fine mapping determined recombination breakpoints to the left
and right of S98, and established an interval of three BAC clones. Light
shotgun sequencing of all three BAC clones and BLAST searches of these
sequences with the draft of the P. pacificus genome sequence
identified the pax-3 gene as a potential candidate gene for
gev-2 (Fig. 4B). When
we sequenced the pax-3 gene as a candidate for gev-2, we
found that it is mutated in both alleles of gev-2
(Fig. 4C). We identified a
deletion of 830 bp from intron 7 to exon 9 in tu214. Sequence
analysis of mutant cDNA clones revealed the use of a novel splice acceptor
site, which caused skipping of the complete exon 9, resulting in a deletion of
the HD and the 3' UTR (Fig.
4C, Fig. 5A).
tu358 results in an amino acid replacement of His to Arg, which is
one of the highly conserved residues in the octapeptide
(Fig. 4C,D). We conclude that
gev-2 is Ppa-pax-3.
Although Ppa-pax-3(tu214) represents a strong
reduction-of-function allele, the available alleles do not necessarily
indicate the null phenotype of Ppa-pax-3. To identify a potential
null allele of Ppa-pax-3, we used reverse genetic deletion library
screens, but were unable to identify a third allele after screening more than
4 million gametes (data not shown). To study the function of
Ppa-pax-3 further, we used morpholino (MO) knockdown experiments,
which were previously shown to function in sensitized genetic backgrounds
(Zheng et al., 2005).
Consistent with these previous findings, we saw no effect of a
Ppa-pax-3 MO when applied in wild-type animals
(Table 1I). However, a
Ppa-pax-3 MO in a Ppa-pax-3(tu358) mutant background showed
a strong enhancement of the cell-death and the vulva-differentiation phenotype
(Table 1J), further indicating
that the observed effects are due to a reduction of Ppa-pax-3
function.
gev-2 is expressed in the embryo and in the early larval stages
Ppa-PAX-3 has bona-fide PD and HD domains and an octapeptide, as
known from other PAX-3-type proteins (Fig.
4B) (Chi and Epstein,
2002). The overall amino acid sequence identity between
Ppa-PAX-3 and Cel-PAX-3 is 49%, and is highest in the three
conserved domains (Fig. 4D).
Interestingly, Ppa-pax-3 and Cel-pax-3 differ in gene
structure and isoform formation. Whereas Cel-pax-3 has six exons and
forms a single isoform, Ppa-pax-3 contains nine exons and two
isoforms were isolated in reverse transcriptase (RT)-PCR experiments. Using
the spliced leader SL1 as the forward primer in RT-PCR experiments, two
alternatively spliced forms were obtained
(Fig. 5A). The second
Ppa-pax-3 isoform misses the N-terminal part of the PD, but contains
the octapeptide and the HD (Fig.
5A).
|
Ppa-lin-39 acts upstream of Ppa-pax-3, and Ppa-LIN-39 binds to the Ppa-pax-3 promoter in vitro
Given that mutations in Ppa-pax-3 and Ppa-lin-39 have a
similar PCD phenotype in P(5-8).p, we wanted to know whether these two loci
interact with one another. First, we generated a Ppa-pax-3(tu214);
Ppa-lin-39(tu29) double mutant and analyzed the cell survival frequencies
of P(5-8).p. Ppa-pax-3(tu214); Ppa-lin-39(tu29) double mutants had a
cell survival index of 0.9 cells per animal, which is similar to the survival
index of the Ppa-lin-39(tu29) single mutant
(Table 1B,K). This result
indicates that the frequency of cell survival in Ppa-pax-3;
Ppa-lin-39 double mutants is not additive, providing a first indication
that these genes might act in a linear pathway.
To obtain further evidence, we analyzed the expression of Ppa-pax-3 in Ppa-lin-39 mutant and wild-type animals, and the expression of Ppa-lin-39 in Ppa-pax-3 mutant and wild-type animals, by quantitative RT-PCR. Whereas the expression of Ppa-lin-39 was unchanged between wild-type and Ppa-pax-3(tu214) mutant animals, Ppa-pax-3 expression was drastically reduced in Ppa-lin-39(tu29) mutant animals when compared with wild-type (Fig. 5C,D). Specifically, the relative pax-3 transcript level was reduced eightfold in Ppa-lin-39 mutants. These results suggest that Ppa-lin-39 might act upstream of Ppa-pax-3.
To test whether Ppa-pax-3 is directly regulated by
Ppa-LIN-39, we searched for Hox-binding sites in the
Ppa-pax-3 promoter. Hox-binding sites have been studied intensively
by in vivo and in vitro studies, and Hox proteins are known to interact with
cofactors, such as Extradenticle-like proteins of the PBC class in
Drosophila (Pearson et al.,
2005). A common property of Hox target enhancers is the
requirement for multiple Hox-monomer-binding sites, most of which possess an
ATTA (or TAAT) core sequence (Pearson et
al., 2005). In addition, many Hox-response elements require
Hox-PBC heterodimer binding sites. We searched for potential binding sites in
the Ppa-pax-3 promoter using SITEBLAST software
(Michael et al., 2005) and
found one putative Hox-PBC-binding site, which conformed to the sequence
TGATNNAT (Fig. 6A).
Next, we tested whether Ppa-LIN-39 could bind this putative binding site by using electrophoretic mobility shift assays (EMSA) (Fig. 6B). Ppa-LIN-39 caused a shift in mobility of an oligonucleotide containing a high-affinity binding site for Drosophila Antennapedia, but not for an oligonucleotide containing the binding site of the Ppa-pax-3 promoter (Fig. 6B). However, when we added the PBC-type cofactor Ppa-CEH-20 to Ppa-LIN-39, the oligonucleotide with the binding site of the Ppa-pax-3 promoter was shifted in a similar way as the Drosophila Antennapedia control oligonucleotide (Fig. 6B). At the same time, Ppa-CEH-20 alone did not cause a shift of the oligonucleotide (Fig. 6B). When the binding site of the Ppa-pax-3 promoter had been mutated, the shift of the oligonucleotide by Ppa-CEH-20 and Ppa-LIN-39 was abolished. We conclude that a heterodimer of Ppa-LIN-39 and Ppa-CEH-20 can bind to the Ppa-pax-3 promoter in vitro.
HOX-PBC-binding sites in the Ppa-pax-3 promoter are phylogenetically conserved
Finally, we wanted to know whether the HOX-PBC-binding site in the
Ppa-pax-3 promoter is evolutionarily conserved. P. pacificus
and C. elegans are members of different nematode families, and
promoter elements cannot be compared over such evolutionary distances
(Grandien and Sommer, 2001).
Therefore, we cloned the pax-3 gene, including the 5'
regulatory regions, from Pristionchus sp. 11 and Pristionchus
maupasi, two additional members of the genus Pristionchus
(Herrmann et al., 2006;
Herrmann et al., 2007). In
these species, cell fate specification of the VPCs is identical to P.
pacificus (data not shown). Although the pax-3 coding regions
were found to be highly conserved between all three Pristionchus
species, the 5' regulatory regions were much less conserved. We found
that the HOX-PBC-binding site that caused the shift in the EMSA assay is
nearly completely conserved between all three Pristionchus species,
further supporting the significance of the Ppa-pax-3 promoter element
for regulation by Ppa-LIN-39 (Fig.
6C).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This study provides the first detailed analysis of the function of a
Pax-3-type gene in nematode postembryogenesis and indicates a role for
Ppa-pax-3 in a Hox gene-regulated developmental process, namely the
establishment of the VEG. Ppa-pax-3 mutants are Gev and represent the
second complementation group besides Ppa-lin-39 with such a
phenotype. Also, in C. elegans, two complementation groups with a Gev
phenotype have been identified - Cel-lin-39
(Wang et al., 1993;
Clark et al., 1993) and
Cel-ceh-20, the latter of which encodes a PBC-like gene
(Liu and Fire, 2000;
Takacs-Vellai et al., 2007).
Although Ppa-ceh-20 was originally a candidate for the genetic locus
described in this study, further mapping analysis indicated that
Ppa-ceh-20 and gev-2 are located on different
chromosomes.
pax-3 is unique among the nematode Pax genes with regard to the
presence of all three functional domains: the PD, the HD and the octapeptide.
Our mutant analysis revealed that Ppa-pax-3(tu358) represents a point
mutation in the octapeptide, resulting in an amino acid substitution of the
first amino acid from histidine to arginine. This histidine is highly
conserved in PAX-3-type proteins throughout the animal kingdom. The fact that
the cell-survival phenotype of tu358 is nearly identical to the one
of the deletion mutant tu214, which eliminates part of the HD,
supports the idea that the octapeptide is of functional importance for the
repression of PCD. Interestingly, studies of the mammalian Pax5 protein BSAP
indicate that the octapeptide is only required for the repressive function of
BSAP, whereas it is dispensable for the activator function
(Eberhard et al., 2000). Given
that surviving VPCs have no vulva phenotype in the tu358 allele,
whereas mutant animals of the deletion allele tu214 result in vulva
differentiation defects, we speculate that the octapeptide is dispensable for
the role of Ppa-pax-3 in vulva induction, which is most probably
involved in transcriptional activation.
Our analysis of Ppa-pax-3 indicates significant differences in the
regulation of the VEG between P. pacificus and C. elegans.
Whereas studies in C. elegans suggest an indirect role of LIN-39 in
the regulation of the cell fusion effector eff-1 via the GATA
transcription factors egl-18 and elt-6
(Shemer and Podbilewicz, 2002;
Koh et al., 2002;
Cassata et al., 2005), our
studies show that Ppa-LIN-39 is part of a different regulatory
network. Our genetic and molecular results suggest that Ppa-LIN-39
acts upstream of the Ppa-pax-3 gene
(Fig. 2B). We speculate that
Ppa-PAX-3 is directly involved in the suppression of PCD of P(5-8).p.
However, the exact molecular mechanism of this suppression remains to be
identified and several distinct mechanisms might be at work. One potential
target for Ppa-PAX-3 might be the ortholog of egl-1, a key
regulator of PCD in C. elegans (for a review, see
Yuan, 2006). So far, we have
been unable to detect an ortholog of egl-1, which encodes a small
peptide, in the P. pacificus genome assembly. However, alternative
mechanisms could also be involved in the regulation of PCD. Park et al. have
recently shown that the Pax2/5/8 member egl-38 and pax-2
promote cell survival in C. elegans
(Park et al., 2006). These
studies suggest a role for egl-38 and pax-2 in the
regulation of ced-9 (Park et al.,
2006). Future work in P. pacificus will indicate whether,
and how, Ppa-pax-3 is coupled to the PCD machinery.
Besides its role in vulva formation, Ppa-PAX-3 plays a role during
the regulation of the PCD of P(9-11).p. This finding indicates for the first
time that P(9-11).p are regulated in a different way than the ventral
epidermal cells in the anterior body region. Interestingly, the function of
Ppa-pax-3 in P(9-11).p specification is independent of
Ppa-LIN-39, indicating that Ppa-pax-3 is regulated
differently in these cells. Indications about the mechanism of this regulation
might come from the analysis of a second gene, ped-9, which, when
mutated, causes the survival of P(9-11).p, and which is currently being
analyzed further (R. Molnar and R.J.S., unpublished observation). With regard
to P(1-4).p, recent studies have shown that P(3,4).p are regulated differently
from P(1,2).p; mutations in Ppa-hairy, a gene that has no counterpart
in the C. elegans genome, or in Ppa-groucho, the ortholog of
C. elegans unc-37, result in the survival of P(3,4).p, but not of
P(1,2).p (Schlager et al.,
2006). Genetic and biochemical studies revealed that
Ppa-HAIRY and Ppa- GROUCHO downregulate the activity of
Ppa-lin-39 and thereby restrict the size of the VEG to P(5-8).p. The
finding that Ppa-pax-3 can rescue P(9-11).p, but not P(1,2).p, from
PCD indicates that at least five different types of positional information
subdivide the 12 ventral epidermal cells of P. pacificus
(Fig. 2C): in the central body
region, P(5-8).p survive based on positional information provided by
Ppa-LIN-39 and Ppa-PAX-3
(Eizinger and Sommer, 1997)
(this study). In the posterior body region, P(9-11).p die from PCD and
Ppa-PAX-3 has a role distinct from the one described for P(5-8).p.
P12.pa is a special cell that might be regulated by the Hox gene
Ppa-egl-5, as occurs in C. elegans. However, a
Ppa-egl-5 mutant has not been identified yet. In the anterior region,
P(3,4).p die because of the downregulation of Ppa-lin-39 by
Ppa-HAIRY and Ppa-GROUCHO
(Schlager et al., 2006). The
regulation of PCD in P(1,2).p is distinct from the PCD of P(3,4).p and
P(9-11).p, but no specific mutants have been isolated that rescue P(1,2).p in
a specific manner. Because all Pn.p cells survive in Ppa-ced-3 and
Ppa-ced-4 mutants, the PCD of P(1,2).p is dependent on the normal PCD
machinery (Sommer et al.,
1998). One might speculate that the fate of P(1,2).p represents
the ground state of Pn.p cells and that no specific positional information is
required for the determination of this fate. Taken together, the seven
cell-death events in the ventral epidermis of the P. pacificus
hermaphrodite are highly regulated and require three distinct genetic
mechanisms in the different regions of the animal
(Fig. 2C). In C.
elegans, the distinct cell fates of Pn.p cells in the hermaphrodite are
also regulated in a region-specific manner. In particular, cell fusion of
P(1,2).p and P(9-11).p are regulated by distinct genetic programs
(Alper and Kenyon, 2001).
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alper, S. and Kenyon, C. (2001). REF-1, a protein with two bHLH domains, alters the pattern of cell fusion in C. elegans by regulating Hox protein activity. Development 128,1793 -1804.[Abstract]
Cassata, G., Shemer, G., Morandi, P., Donhauser, R.,
Podbilewicz, B. and Baumeister, R. (2005).
ceh-16/engrailed patterns the embryonic epidermis of
Caenorhabditis elegans. Development
132,739
-749.
Chamberlin, H. M., Palmer, R. E., Newman, A. P., Sternberg, P. W., Baillie, D. L. and Thomas, J. H. (1997). The PAX gene egl-38 mediates developmental patterning in Caenorhabditis elegans. Development 124,3919 -3928.[Abstract]
Chi, N. and Epstein, J. A. (2002). Getting your Pax straight: Pax proteins in development and disease. Trends Genet. 18,41 -47.[CrossRef][Medline]
Chisholm, A. D. and Horvitz, H. R. (1995). Patterning of the Caenorhabditis elegans head region by the Pax-6 family member vab-3. Nature 377, 52-55.[CrossRef][Medline]
Clark, S. G., Chisholm, A. D. and Horvitz, H. R. (1993). Control of cell fates in the central body region of C. elegans by the homeobox gene lin-39. Cell 74,43 -55.[CrossRef][Medline]
Dieterich, C., Roeseler, W., Sobetzko, P. and Sommer, R. J. (2007). Pristionchus.org: a genome-centric database of the nematode satellite species Pristionchus pacificus. Nucleic Acids Res. 35,D498 -D502.[CrossRef]
Eberhard, D., Jimenez, G., Heavey, B. and Busslinger, M. (2000). Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family. EMBO J. 19,2292 -2303.[CrossRef][Medline]
Eizinger, A. and Sommer, R. J. (1997). The
homeotic gene lin-39 and the evolution of nematode epidermal cell
fates. Science 278,452
-455.
Grandien, K. and Sommer, R. J. (2001).
Functional comparison of the nematode Hox gene lin-39 in C.
elegans and P. pacificus reveals evolutionary conservation of
protein function despite divergence of primary sequences. Genes
Dev. 15,2161
-2172.
Herrmann, M., Mayer, W. E. and Sommer, R. J. (2006). Nematodes of the genus Pristionchus are closely associated with scarab beetles and the Colorado potato beetle in Western Europe. Zoology Jena 109,96 -108.[Medline]
Herrmann, M., Mayer, W., Hong, R., Kienle, S., Minasaki, R. and Sommer, R. J. (2007). The nematode Pristionchus pacificus (Nematoda: Diplogastridae) is associated with the Oriental beetle Exomala orientalis (Coleoptera: Scarabaeidae) in Japan. Zool. Sci. In press.
Hobert, O. and Ruvkun, G. (1999). Pax genes in Caenorhabditis elegans: a new twist. Trends Genet. 15,214 -216.[CrossRef][Medline]
Hong, R. L. and Sommer, R. J. (2006). Pristionchus pacificus: a well-rounded nematode. BioEssays 28,651 -659.[CrossRef][Medline]
Jungblut, B. and Sommer, R. J. (1998). The Pristionchus pacificus mab-5 gene is involved in the regulation of ventral epidermal cell fates. Curr. Biol. 8, 775-778.[CrossRef][Medline]
Jungblut, B. and Sommer, R. J. (2000). Novel cell-cell interactions during vulva development in Pristionchus pacificus. Development 127,3295 -3303.[Abstract]
Jungblut, B. and Sommer, R. J. (2001). The nematode even-skipped homolog vab-7 regulates gonad and vulva position in Pristionchus pacificus. Development 128,253 -261.[Abstract]
Kenning, C., Kipping, I. and Sommer, R. J. (2004). Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus. Genesis 40,176 -183.[CrossRef][Medline]
Kenyon, C. (1986). A gene involved in the development of the posterior body region of C. elegans. Cell 46,477 -487.[CrossRef][Medline]
Koh, K., Peyrot, S. M., Wood, C. G., Wagmaister, J. A., Maduro, M. F., Eisenmann, D. M. and Rothman, J. H. (2002). Cell fates and fusion in the C. elegans vulval primordium are regulated by the EGL-18 and ELT-6 GATA factors - apparent direct targets of the LIN-39 Hox protein. Development 129,5171 -5180.[Medline]
Liu, J. and Fire, A. (2000). Overlapping roles of two Hox genes and the exd ortholog ceh-20 in diversification of the C. elegans postembryonic mesoderm. Development 127,5179 -5190.[Abstract]
Maloof, J. N. and Kenyon, C. (1998). The Hox gene lin-39 is required during C. elegans vulval induction to select the outcome of Ras signaling. Development 125,181 -190.[Abstract]
Michael, M., Dieterich, C. and Vingron, M.
(2005). SITEBLAST-rapid and sensitive local alignment of genomic
sequences employing motif anchors. Bioinformatics
21,2093
-2094.
Park, D., Jia, H., Rajakumar, V. and Chamberlin, H. M.
(2006). Pax2/5/8 proteins promote cell survival in C.
elegans. Development
133,4193
-4202.
Pearson, J. C., Lemons, D. and McGinnis, W. (2005). Modulating Hox gene functions during animal body patterning. Nat. Rev. Genet. 6, 893-904.[CrossRef][Medline]
Schlager, B., Roseler, W., Zheng, M., Gutierrez, A. and Sommer, R. J. (2006). HAIRY-like transcription factors and the evolution of the nematode vulva equivalence group. Curr. Biol. 16,1386 -1394.[CrossRef][Medline]
Shemer, G. and Podbilewicz, B. (2002).
LIN-39/Hox triggers cell division and represses EFF-1/fusogen-dependent vulval
cell fusion. Genes Dev.
16,3136
-3141.
Sigrist, C. B. and Sommer, R. J. (1999). Vulva formation in Pristionchus pacificus relies on continuous gonadal induction. Dev. Genes Evol. 209,451 -459.
Sommer, R. J. (2005). Evolution of development in nematodes related to C. elegans. In WormBook (ed. The C. elegans Research Community), WormBook, doi/10.1895/wormbook.1.46.1, http://www.wormbook.org.
Sommer, R. J. and Sternberg, P. W. (1996). Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis. Curr. Biol. 6,52 -59.[CrossRef][Medline]
Sommer, R. J., Eizinger, A., Lee, K. Z., Jungblut, B., Bubeck, A. and Schlak, I. (1998). The Pristionchus HOX gene Ppa-lin-39 inhibits programmed cell death to specify the vulva equivalence group and is not required during vulval induction. Development 125,3865 -3873.[Abstract]
Srinivasan, J., Sinz, W., Lanz, C., Brand, A., Nandakumar, R.,
Raddatz, G., Witte, H., Keller, H., Kipping, I., Pires-daSilva, A. et al.
(2002). A bacterial artificial chromosome-based genetic linkage
map of the nematode Pristionchus pacificus.
Genetics 162,129
-134.
Srinivasan, J., Sinz, W., Jesse, T., Wiggers-Perebolte, L., Jansen, K., Buntjer, J., van der Meulen, M. and Sommer, R. J. (2003). An integrated physical and genetic map of the nematode Pristionchus pacificus. Mol. Genet. Genomics 269,715 -722.[CrossRef][Medline]
Sternberg, P. W. (2005). Vulval development. In WormBook (ed. The C. elegans Research Community), WormBook, doi/10.1895/wormbook.1.6.1, http://www.wormbook.org.
Takacs-Vellai, K., Vellai, T., Chen, E. B., Zhang, Y., Guerry, F., Stern, M. J. and Muller, F. (2007). Transcriptional control of Notch signaling by a HOX and a PBX/EXD protein during vulval development in C. elegans. Dev. Biol. 302,661 -669.[CrossRef][Medline]
Wagmaister, J. A., Gleason, J. E. and Eisenmann, D. M. (2006). Transcriptional upregulation of the C. elegans Hox gene lin-39 during vulval cell fate specification. Mech. Dev. 123,135 -150.[CrossRef][Medline]
Wang, B. B., Muller-Immergluck, M. M., Austin, J., Robinson, N. T., Chisholm, A. and Kenyon, C. (1993). A homeotic gene cluster patterns the anteroposterior body axis of C. elegans. Cell 74,29 -42.[CrossRef][Medline]
Yuan, J. (2006). Divergence from a dedicated cellular suicide mechanism: exploring the evolution of cell death. Mol. Cell 23,1 -12.[CrossRef][Medline]
Zhang, Y. and Emmons, S. W. (1995). Specification of sense-organ identity by a Caenorhabditis elegans Pax-6 homologue. Nature 377, 55-59.[CrossRef][Medline]
Zheng, M., Messerschmidt, D., Jungblut, B. and Sommer, R. J. (2005). Conservation and diversification of Wnt signaling function during the evolution of nematode vulva development. Nat. Genet. 37,300 -304.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  R. Rae, B. Schlager, and R. J. Sommer Pristionchus pacificus: A Genetic Model System for the Study of Evolutionary Developmental Biology and the Evolution of Complex Life-History Traits CSH Protocols, October 1, 2008; 2008(11): pdb.emo102 - pdb.emo102. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
