1 ABI/Molecular Neurogenetics, LMU Munich, 80336 Munich, Germany
2 IFOM (Firc Institute of Molecular Oncology Foundation), 20139 Milan,
Italy
3 Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa,
Israel
4 BioIII/Bioinformatics and Molecular Genetics, University of Freiburg,
Schaenzlestrasse 1, D-79104 Freiburg, Germany
* Author for correspondence (e-mail: baumeister{at}celegans.de)
Accepted 6 December 2004
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SUMMARY |
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Key words: C. elegans, engrailed, Patterning
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Introduction |
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One striking fact is the similarity between the involvement of
engrailed in the specification of the posterior compartment in the
wing imaginal disc of Drosophila and in the specification of the
ventral ectoderm in the developing limb in mouse. It has been shown that
during embryonic development the anterior ectodermal ridge (AER) of the
developing limb remarkably resembles the anterior/posterior (A/P) compartment
boundary in the fly. Both the A/P boundary and the AER express
decapentaplegic (dpp)/BMP2 homologous proteins of the
TGF-ß family, which are repressed by en/EN1 in adjacent cells.
Additionally, in both cases the expression of dpp/BMP2 is induced by
hedgehog (hh)/Shh from neighboring cells. These neighboring
cells in Drosophila are the posterior cells, where hh
depends on engrailed. In the mouse limb, Shh is expressed posteriorly
adjacent to, and maintained by, the AER, depending indirectly on EN-1 function
(reviewed by Hidalgo, 1998).
In addition to the regulation of organizers, engrailed is required to
preserve compartment boundaries in Drosophila. When the selector gene
engrailed is removed, in vivo, from a posterior clone of cells in the
wing, those cells gain anterior affinity. They sort out from posterior cells
and, if in contact with anterior cells, sort into and mix with them
(Lawrence and Struhl, 1982
;
Morata and Kerridge, 1982
).
This segregation mechanism might be controlled in part by regulation of cell
adhesion molecules (Dahmann and Basler,
1999
). It has been suggested that the ancestral function of
engrailed may be neuronal targeting, because it regulates the
connectivity through the transcriptional regulation of cell adhesion molecules
in the central nervous system in arthropods and vertebrates
(Gibert, 2002
;
Vincent, 1998
). Moreover,
engrailed has been proposed to play a general role in segmentation of
protostomes (Prud'homme et al.,
2003
).
In Caenorhabditis elegans, the functions of engrailed
have not yet been described. Instead, the function of GATA and other
homeobox-containing transcription factors have been studied in the patterning
of the epidermis in roundworms. Briefly, the GATA factor ELT-1 specifies
general epidermal identity (Page et al.,
1997). The epidermis is subsequently patterned in three
morphologically distinguishable major areas during embryogenesis: (1) dorsal
cells that fuse to form the syncytia hyp6 and hyp7 during embryonic elongation
(Podbilewicz and White, 1994
),
(see Movie 1 in the supplementary material); (2) two single left and right
rows of lateral seam cells; and (3) the ventral P cells whose descendants
either fuse postembryonically to hyp6 and hyp7 or develop vulval structures
and the ventral nerve cord (Podbilewicz
and White, 1994
; Sulston et
al., 1983
). LIN-39/HoxD4/Dfd and CEH-20/Exd play a crucial role in
repressing the cell fusion of some posterior descendants of the P cells
(Clark et al., 1993
;
Maloof and Kenyon, 1998
;
Shemer and Podbilewicz, 2002
;
Wang et al., 1993
). In
addition, the operon encoding the two GATA factors ELT-5(=EGL-18) and ELT-6 is
important for differentiation/fusion-repression in the lateral seam cells
(Koh and Rothman, 2001
) and
for cell fusion-repression in the vulval precursor cells (VPCs), where it is
controlled by LIN-39/HoxD4/Dfd (Koh et
al., 2002
). A general effector for cell fusion in the epidermis of
the worm is the transmembrane protein EFF-1
(Mohler et al., 2002
).
Moreover, EFF-1 is both necessary and sufficient for epithelial and
myoepithelial cell fusion in C. elegans
(Shemer et al., 2004
). It has
also been shown that LIN-39 represses the expression of eff-1 in the
VPCs (Mohler et al., 2002
;
Shemer and Podbilewicz,
2002
).
In this study we show how ceh-16/engrailed controls the differentiation of the seam cells, thereby patterning the embryonic epidermis of C. elegans. ceh-16/engrailed represses the fusion of the seam cells with the neighboring epidermal cells by repressing the expression of the fusion effector eff-1. ceh-16/engrailed also triggers the expression of elt-5 and other seam cell markers and is indispensable for alae formation (a hallmark of seam cell differentiation). We also show that in the ceh-16/engrailed mutant the seam cells lose their lateral position and migrate either dorsally or ventrally, intermingling with these cells. Therefore, seam cells in the wild-type context seem to act by preventing cell migration and maintaining embryonic compartment.
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Materials and methods |
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Isolation of ceh-16 mutants
ceh-16(lg16)III, ceh-16(lg17)III were obtained by
screening ethylmethanesulfonate (EMS) mutagenized worm libraries via PCR
according to Anderson (Anderson,
1995); breakpoints of the deletions were sequenced twice
independently using standard procedures. The deletion in ceh-16(lg16)
spans 2218 bp and encompasses 560 bp of the promoter region, the
transcriptional start and the first four of the five exons. Exon 5 encodes the
C-terminal 51 amino acids. By molecular means this allele is predicted to be a
null allele. In ceh-16(lg17), the deletion spans 867 bp encompassing
471 bp of the promoter region, the transcriptional start and most of the
second exon including the start methionine and the first 56 amino acids. Since
the transcription start in ceh-16(lg17) is severely compromised and
the phenotype is identical in both ceh-16(lg16)
(Table 1) and
ceh-16(lg17), both alleles probably constitute a null allele.
Breakpoint for ceh-16(lg16): GATCGAAAAAGTAGTG/CAGTTGTTTTGGCATGA.
Breakpoint for ceh-16(lg17): TAATTCCCATGTTATATT/GCACAAGATATTCCGATC.
The sequences of the primers used for screening are available upon request.
Both mutants were out-crossed ten times prior to analysis.
|
Transgenic strains
Transgenic strains were obtained using standard procedures
(Mello et al., 1991), adapted
as in Cassata et al. (Cassata et al.,
2000
). ceh-16::gfp translational fusions were injected at
a concentration of 30 µg/µl along with 50 µg/µl pRF4
(rol-6 dm) plasmid. Roller lines were crossed into heterozygous
ceh-16(lg16) and tested for their rescue ability by selecting
homozygous transgenics (genotypization by PCR). Thereafter, the rescued strain
was crossed into jcIs1 for microscopical analyses. Transgenic Ex
[wrt-5::gfp] and Ex [wrt-2::gfp] strains were a generous
gift from T. Burglin (Aspock et al.,
1999
). The wrt-5::gfp and wrt-2::gfp
extrachromosomal arrays were integrated as follows: 50 transgenic L4 were
irradiated with UV using a Stratalinker (Model 1800) from Stratagene at 30,000
µJ/cm2. After irradiation the animals were singled. After
starvation the plates were chunked to let the worms crawl out of the agar; 250
were singled and analyzed for their ability to produce 100% transgenics in the
offspring. One integrated line of each transgenic was isolated in this way.
Both were out-crossed twice. Transgenic lines of nhr-73::gfp and
nhr-74::gfp were obtained as described in Miyabayashi et al.
(Miyabayashi et al.,
1999
).
Lethality tests
Candidate young adult heterozygous ceh-16(lg16) or
ceh-16(lg17) P0 animals were singled and grown on agar plates for 4-5
hours (this allowed each worm to lay 15-20 eggs). Thereafter they were removed
and the genotype was determined via PCR. The sum of all the eggs (of the
positive plates) was counted. The next day, the number of dead eggs was
determined. A similar procedure was adopted with
[ceh-16(lg16)/+]X[ceh-16(lg17)/+] crosses: males derived from a
ceh-16(lg17) cross with N2 males were crossed with
ceh-16(lg16) hermaphrodites. These hermaphrodites were used as P0 for
the lethality test. The presence of both alleles in the offspring was tested
via PCR. Dead eggs were counted as above.
RNAi experiments
The full-length ceh-16 cDNA was cloned into pBluescript II
SK-using HindIII and BamHI sites. In vitro transcribed ssRNA
from linearized vectors was produced using commercially available T3 and T7
RNA polymerase systems (Promega). Annealed dsRNA was injected into young adult
hermaphrodites at a concentration of 1 µg/µl. The offspring was analyzed
(by microscopy or lethality test as described above). elt-5(RNAi)
experiments were performed similarly using the F55A8.1 RNAi clone from the MRC
C. elegans RNAi bank.
Heatshock experiments
The PCR amplified full-length ceh-16 cDNA was inserted in frame
into the KpnI site of the heatshock promoter (hsp16-2)
contained in construct pPD49.78 (Stringham
et al., 1992) and sequenced.
To perform heatshock experiments, two independent lines of transgenic worms
were constructed by injecting 5 µg/µl of heatshock promoter (with or
without ceh-16 for control purposes) along with pMH86
(dpy-20+) (Han and Sternberg,
1991) into a dpy-20(e2017)IV; wIs66
[elt-5::gfp] strain. Plates containing transgenic animals were
heatshocked three times for 1 hour at 30°C (recover period between
heatshocks: 1 hour at 15°C). Transgenic offspring L1 was analyzed the next
day for ectopic expression of ELT-5::GFP.
Antibody stainings and microscopy
Antibody staining was performed according to Waddle et al.
(Waddle et al., 1994). Light
microscopy was performed using a Zeiss Axioplan2 imaging microscope, Zeiss
Axiocam HRc camera and Axiovision software. Confocal microscopy was performed
as in Shemer and Podbilewicz (Shemer and
Podbilewicz, 2002
), and confocal time-lapse movies were recorded
taking Z series projections each 5-10 minutes for 1.5 hours at 20°C
(Rabin and Podbilewicz,
2000
).
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Results |
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To further investigate the epidermal expression, an in situ staining with a
monoclonal antibody (4D9) that recognizes engrailed proteins in many
species was performed (Patel et al.,
1989). Localized immunoreactivity in the seam cells was detected
during the same stages of embryonic development
(Fig. 1E,F). This additionally
confirms the expression of ceh-16 in the nuclei of these cells.
In summary, ceh-16 encodes an engrailed homolog in C. elegans. ceh-16 is expressed in the AB lineage during early embryogenesis, in the lateral seam cells and neurons during morphogenesis, and in two motoneurons during postembryonic stages.
ceh-16/engrailed is required in the seam cells during embryonic development
To study the functions of ceh-16 in vivo, two deletion alleles
[ceh-16(lg16) and ceh-16(lg17)] were isolated from an EMS
deletion library. As both mutations delete the transcriptional start and a
large part of the coding region, they are predicted to be null alleles
(Fig. 1A; see Materials and
methods for details). Both mutant alleles are recessive embryonic lethal, do
not complement each other and show a very similar phenotype to worms subjected
to ceh-16 RNA interference (RNAi)
(Table 1; and see Movie 3 in
the supplementary material). As the penetrance of the phenotype of both
deletion alleles is identical, we used ceh-16(lg16) and
ceh-16(RNAi) for further experiments. The full-length translational
gfp construct used for expression studies
(Fig. 1A) was sufficient to
fully rescue the mutant phenotype (see below), whereas a shorter translational
gfp construct (Fig.
1A) that contained only the transcriptional start and the first
exon did not (data not shown), confirming the specificity of the
phenotype.
Microscopic analysis of the ceh-16() embryos revealed that
the epidermal cells were disorganized, causing severe morphological defects
and lack of elongation (Fig.
2C,D). To examine what led to this terminal phenotype the rescuing
transgene was used as a marker for ceh-16(+) cells in a
ceh-16 mutant background. As, in transgenic C. elegans,
extrachromosomal arrays are frequently lost, embryos that expressed the array
in a subset of the seam cells were analyzed (mosaic analysis). We found that,
in mosaic animals, seam cells lacking ceh-16 in their nuclei showed a
dorsal and/or ventral displacement with no obvious directional preference. In
addition to the positioning defects, the ceh-16() cells fused
to the dorsal or ventral epidermis (Fig.
2G,H; arrows). In some mosaic animals (8/63), mutant seam cells
projected ventrally in such a way that may have destabilized ventral closure
(George et al., 1998),
implying that embryonic lethality may be a result of leakage of internal
cells. In Fig. 2I,J we show an
example of such an embryo with free undetermined cells near the ventral
closure that may have leaked out of the embryo at the ventral side. In all the
mosaic animals, the seam cells expressing CEH-16::GFP [ceh-16(+)]
were rescued and those lacking CEH-16::GFP [ceh-16()] showed
the phenotype described above (displacement and/or ectopic fusion;
Fig. 2E,F). Partial rescued
animals manage to hatch showing an attenuated phenotype
(Fig. 2K,L). As AB precursors
that give rise to ventral and dorsal epidermis express ceh-16 at
earlier stages, we cannot rule out expression of ceh-16 below
detection level in dorsal and ventral epidermis, but we did not detect any
defects in the hypodermis of these areas (e.g. the vulvae were perfectly
formed in mosaic semi-rescued animals; not shown). We conclude from this
analysis that ceh-16 is required for embryonic seam cell
development.
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ceh-16/engrailed is required to maintain correct seam cell positioning during embryogenesis
We had observed that ceh-16() seam cells migrate
(Fig. 2). We decided to analyze
this phenotype in more detail. Of the mosaic embryos (n=20), 70%
displayed abnormal cell positioning phenotype (bean to comma stage), with no
directional preference (see Fig.
2E,F; arrows). Do ceh-16() cells in mosaic animals
leave their position (migration), or is the loss of collinear arrangement due
to earlier events, as ceh-16 is expressed earlier
(Fig. 1C,E)?
To answer this question, ceh-16 mosaic embryos in an eff-1() background were analyzed. In these animals ectopic fusions were not present (Fig. 3B; Table 2). As in a ceh-16();eff-1(+) background, ceh-16();eff-1() cells were not in their normal position. Of 40 embryos tested from bean to 1.5-fold stage of elongation, 33 embryos showed a seam cell-defective phenotype (83%). Out of 62 aberrant seam cells, 48 (77%) had either a minor or strong projection intercalating with the ventral P cells. The minor projection often preceded a more pronounced migration of the entire cell, visible when the embryo was re-analyzed at a later stage (1.8-fold). The remaining 23% of the ceh-16() seam cells displayed migration toward the dorsal side (when animals were analyzed at a later stage). Unlike in eff-1() and wild-type animals, in eff-1();ceh-16() double mutants the shape of the seam cells was not wild type and the cell belt margins were no longer straight (see Movie 2 in the supplementary material), but were dented or intercalated (Fig. 6; see Movie 3 in the supplementary material). Moreover, migration can be observed live in a time-lapse experiment, where a ceh-16() seam cell, marked by the ectopic expression of eff-1p::gfp, migrated dorsally (see Movie 3 in the supplementary material: in the depicted experiment, not all the seam cells expressed GFP, probably due to mosaic expression of the extrachromosomal array). We conclude from these experiments that ceh-16 is required for the maintenance of correct boundaries between the lateral rows of seam cells and the ventral and dorsal row of epidermal cells during embryonic development.
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Discussion |
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ceh-16/engrailed the only ortholog of the engrailed genes in the nematode C. elegans
CEH-16 shares the archetypal structure of engrailed class proteins
from arthropods, annelids, chordates and vertebrates (see Fig. 1 in the
supplementary material). Besides the homeodomain referred to as Engrailed
Homology domain 4 (EH4), CEH-16 possesses at least EH1, EH2 and EH3 [of the
five known EH domains (Gibert,
2002)]. EH1 is constituted by the engrailed repressor
domain, which binds to Groucho in Drosophila. Groucho can act as a
co-repressor of transcription (Jimenez et
al., 1997
), is conserved in C. elegans and binds to the
EH1 domain, which is also present in unc-4
(Winnier et al., 1999
).
Interestingly, we have shown that ceh-16/engrailed represses the
transcription of eff-1, but we do not know whether this repression is
direct or indirect, nor if it is dependent on Groucho-like genes. EH2 and EH3
are involved in the binding of Hox homeodomain co-factors such as Pbx/Exd in
Drosophila. These bind again to other homeodomain co-factors of the
HtH/Prep/Meis gene family; these interactions are important for nuclear import
(Abu-Shaar et al., 1999
;
Berthelsen et al., 1999
;
Kurant et al., 1998
;
Rieckhof et al., 1997
). Exd,
HtH and Engrailed can form a functional triple repressor complex in
Drosophila (Kobayashi et al.,
2003
). In C. elegans, like ceh-16/engrailed, the
orthologs of Exd (=ceh-20/40) and Hth (=unc-62) are also
involved in embryonic epidermal development
(Van Auken et al., 2002
). As
the EH2/3 domains in CEH-16 are present, CEH-20/40 and UNC-62 might also be
co-factors of CEH-16/Engrailed in C. elegans.
Cell fusion and differentiation two separable functions controlled by ceh-16/engrailed
Cell fusion has been shown to control cell fates. When cell fusion is
blocked during postembryonic development in eff-1 mutants, unfused
VPCs can also respond to neighboring signals and adopt vulval fates (reviewed
by Shemer and Podbilewicz, 2003). The result is an ectopic and non-functional
vulva (Mohler et al.,
2002).
We have shown that the regulation of epidermal cell fusion is also crucial during embryonic development. Seam cells act as a non-fusing `inter-zone' between the dorsal and the ventral areas of the embryo. Although fusion repression prevents the seam cells from becoming a part of the dorsal syncytium, we have shown by bypassing ectopic fusions in an eff-1 mutant background that ceh-16/engrailed is necessary for the differentiation of the seam cells, also in a fusion negative genetic background. In the seam cells that lack ceh-16/engrailed, eff-1 is de-repressed. The de-repression of eff-1 occurs simultaneously with expression of eff-1 in the other epidermal cells (e.g. in the forming hyp7 syncytium). ceh-16/engrailed therefore installs an additional program in a subset of cells otherwise committed to behaving like the surrounding epidermal cells. In summary, ceh-16/engrailed, during embryonic development, primes a transcriptional cascade necessary for seam cell differentiation. To allow this separate seam cell differentiation, and to maintain the lateral epidermal cell fate, ceh-16/engrailed also represses the fusion of the seam cells.
ceh-16/engrailed commits the lateral epidermis to seam cell fate
in part by regulating the expression of elt-5 (=egl-18). As
mutations of elt-5, like ceh-16, have also been shown to
prevent cell fusions and, to a minor extent, inappropriate cell migration of
the seam cells (Koh and Rothman,
2001), we suggest a regulatory cascade in which ceh-16
controls elt-5, which may repress eff-1 expression and
participates in anti-migratory mechanisms
(Fig. 7C,D). Although we are
able to ectopically express elt-5 by misexpression of ceh-16
we do not know if elt-5 is a direct target of ceh-16.
Moreover, supporting an indirect regulation, we found no putative
ceh-16/engrailed binding site in the elt-5 locus by
screening in silico, using the reported Drosophila engrailed
binding sequence (Solano et al.,
2003
). Are all the functions of ceh-16/engrailed mediated
by elt-5? Although the phenotype of elt-5 larvae is very
similar to the one seen in ceh-16 mutants, we think that this is not
the case. Koh et al. (Koh et al.,
2002
) showed that elt-5 controls many markers of the seam
cells. But nhr-73 and nhr-74 are not regulated by
elt-5, so the authors speculated that there must be an additional
factor X, which might act in parallel to elt-5. We have shown that
ceh-16 regulates nhr-73/74 and elt-5. So
ceh-16 may be the factor X, which is placed upstream of
elt-5 (Fig. 7C).
Interestingly, in the ventral region of the epidermis, cell fusions are
controlled by the expression of another homeobox repressor,
lin-39/HOXD4/Dfd and ceh-20/Exd
(Shemer and Podbilewicz,
2002
), which also act through elt-5
(Koh et al., 2002
). In this
region, elt-5, controlled by lin-39, is essential for vulva
formation. Therefore, it seems that a concerted spatial-temporal
(lateral-ventral) expression of different homeodomain proteins controls
differentiation of respective epidermal structures by recruiting in part the
same factors (such as elt-5 and eff-1;
Fig. 7D). The occurrence of
elt-5 in lateral and ventral domains of the epidermal cells might be
required for the regulation in both areas of eff-1
(Fig. 7D).
|
ceh-16/engrailed blocks ectopic cell migrations
The mosaic analyses and the time-lapse recordings show that the seam cells
form a straight cell line that acts as a migration barrier
(Fig. 7A,B). Differential cell
adhesion properties may account for cells segregating from this line, as in
mosaic animals ceh-16() cells invade the neighboring tissues.
At this stage we cannot say whether this phenomenon is cell-autonomous or not.
Since ceh-16/engrailed is expressed earlier in precursors of ventral
and dorsal epidermis, it may be necessary for correct migration events there
as well but not detectable by our experimental means. That
ceh-16/engrailed may elicit such a phenotype might be due to the
de-regulation of homophilic cell-surface molecules regulating adhesion and/or
cell motility. Analogously, in Drosophila such mechanisms have been
hypothesized to be involved in the formation/maintenance of compartment
boundaries, where the additional paracrine function of hh is required
for the boundary to be held in place
(Dahmann and Basler, 1999).
Studies based on rRNA have demonstrated that nematodes had previously been
misplaced, and their true position is in a sister group of the arthropods
(Adoutte et al., 2000
;
Aguinaldo et al., 1997
).
Therefore their evolutionary relationship to arthropods is closer than
expected. The question arises whether engrailed in nematodes is
controlling cell migration mechanisms by regulating the same molecules as in
arthropods (Drosophila). To answer this question the identification
in both species of these cell-adhesion molecules (controlled by
engrailed) is required.
In conclusion, this work shows how engrailed patterns the embryonic epidermis of C. elegans. For this purpose, ceh-16/engrailed acts as a differentiation factor, as a cell migration inhibitor, and we describe for the first time how an engrailed-like gene controls animal developmental processes also by the regulation of cell fusion.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/4/739/DC1
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