Max-Planck Institut für Entwicklungsbiologie, Abt. Evolutionsbiologie, Spemannstr. 37-39, D-72076 Tübingen, Germany
* Author for correspondence (e-mail: ralf.sommer{at}tuebingen.mpg.de)
Accepted 9 November 2002
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SUMMARY |
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Key words: Hox genes, Evolution, Ray formation, Caenorhabditis elegans, Pristionchus pacificus
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INTRODUCTION |
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The observation that functional changes of Hox proteins are only rarely
associated with the evolution of the protein sequences is surprising because
the sequence conservation of most transcription factors, in particular Hox
proteins, is restricted to the DNA binding region. Most other parts of the
proteins are highly divergent and often, only a few islands of conserved amino
acids exist. One obvious question therefore is how Hox proteins provide
functional specificity. So far, this question has mainly been addressed by
studies using insects and vertebrates
(Graba et al., 1997) and only
rarely in nematodes (Hunter and Kenyon,
1995
; Maloof and Kenyon,
1998
).
In the nematode Caenorhabditis elegans, Hox genes play an
important role during the formation of multiple developmental processes
(Kenyon et al., 1997).
However, nematode Hox clusters and Hox genes show several characteristics that
differ from their counterparts in most other animal phyla. First, the Hox
cluster of C. elegans contains only four core members: ceh-13,
lin-39, mab-5 and egl-5, the labial, Deformed,
Antennapedia and Abdominal-B orthologs, respectively
(Fig. 1A). Second, these genes
are scattered over an approximately 300 kb interval with many unrelated genes
being interspersed (The Caenorhabditis
elegans Sequencing Consortium, 1998
). Third, two additional
Abd-B-like Hox genes, php-3 and nob-1, are located
more than 1 Mb away on the same chromosome
(Fig. 1A) (Van Auken et al., 2000
).
Fourth, the two anterior genes ceh-13 and lin-39 deviate
from the colinearity rule in that lin-39 is more distal than the
labial-like gene ceh-13
(Fig. 1A). To our knowledge,
this represents the only proven case in the animal kingdom, in which the
colinearity rule is broken. Furthermore, some C. elegans Hox genes,
such as ceh-13, have taken over essential functions during
embryogenesis, whereas other core members provide positional information
during pattern formation, but are not essential for development. Finally,
nematode Hox proteins show only limited sequence conservation. In addition to
sequence divergence in the N-terminal and C-terminal regions, the N-terminal
arm and helices I and II of the homeodomain, also show sequence differences to
an extent that is not seen in most other animal phyla
(Fig. 1B,C). Thus, nematode Hox
genes show several special features unknown in Hox genes of other organisms.
These features are most probably secondary modifications of Hox gene
organization, function and sequence, many of which might be the result of the
adaptation to the small body size and the simpler bauplan. Given these
differences in nematode Hox genes, the question arises of how specificity is
provided to particular Hox functions. In insects, original studies had
suggested that most of the functional specificity resides within the
homeodomain (Furukubo-Tokunaga et al.,
1993
; Zeng et al.,
1993
). However, more recent studies have clearly indicated that
regions outside of the homeodomain are also of importance
(Chauvet et al., 2000
).
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One simple developmental process in nematodes, in which the evolutionary
diversification of Hox proteins can be studied in detail, is the formation of
the ray sensilla in males. Rays are sensory structures that are generated
specifically in the male by the lateral hypodermis. Generally, the lateral
hypodermis is generated by six bilateral pairs of V cells, called V1-V6, which
are located along the anteroposterior body axis
(Fig. 2) (Sulston and Horvitz, 1977).
The anterior cells V1-V4 undergo a stem cell division pattern and form seam
cells that produce epidermal ridges in the adult cuticle, called alae. V5 and
V6 have initially a division pattern similar to that of V1-V4. However,
instead of generating alae, V5 and V6, together with the post-anal blast cell
T, generate nine pairs of copulatory sensillae, rays R1-R9 in the male. V5
generates the anterior ray R1 and V6 generates the rays R2-R6, whereas the
posterior rays R7-R9 are formed from the T cell
(Fig. 2B). The homeotic gene
mab-5 provides positional information during ray formation. In the
absence of MAB-5 protein, V5 and V6 generate alae instead of rays and only the
posterior T rays R7-R9 are formed (Costa et
al., 1988
; Kenyon,
1986
). The mab-5 gene is switched on and off several
times in the V5 and V6 lineage, indicating that the gene is regulated in a
complex manner during ray specification
(Salser and Kenyon, 1996
).
Thus, MAB-5 not only has a global patterning role for V-rays, but also
specifies several V-sublineages giving rise to individual rays. One downstream
target of MAB-5 is the Abdominal-B ortholog egl-5, which is
required in certain sublineages of V6
(Chisholm, 1991
;
Ferreira et al., 1999
).
Together, the role of MAB-5 during C. elegans ray formation provides
an easy test system for the functional specificity of Hox proteins: MAB-5 is a
central regulatory control gene for V-rays. In addition, the ray system is
simple and easy to manipulate by transgenesis, thereby providing the basis for
a detailed study of the evolution of protein function.
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We address how orthologous, paralogous and chimeric nematode Hox proteins provide functional specificity by testing their rescuing activity during ray formation in Cel-mab-5 mutants. We show, that orthologous nematode MAB-5 proteins, but not the paralogous Hox proteins from C. elegans, can functionally replace Cel-MAB-5. Studies with chimeric and truncated Hox proteins suggest that the specificity is conferred by the homeodomain. In vitro mutagenesis experiments further indicate that the N-terminal arm and helix I of MAB-5 are sufficient to provide ray identity. Similar mutagenesis experiments with the neighboring Hox protein LIN-39 suggest that protein domains other than the N-terminal arm and helix I provide specificity of this Hox protein during vulva formation. Together, our data indicate that different domains of nematode Hox proteins provide functional specificity in individual developmental decisions.
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MATERIALS AND METHODS |
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Plasmid constructs
All DNA constructs were made using standard techniques
(Sambrook and Russell, 2001)
and verified by restriction mapping or sequencing. In addition, all exons
amplified by PCR were verified by sequencing. The pAG1 Cel-mab-5
construct was generated by PCR amplification from C. elegans genomic
DNA and cloning of the product into pBIIKS (Stratagene). This construct
contains 8.9 kb upstream of the ATG, the 7.6 kb gene and 1.5 kb downstream of
the stop codon. A 3.5 kb fragment, which contains the start codon, was
isolated from pAG1 by digestion with NcoI and SacI and was
modified to create a unique XhoI site in front of the start codon
using the Quikchange technique (Stratagene) according to the manufacturers
instructions. This fragment was reintroduced into the basic construct to
generate pAG2. A frame-shift mutation in exon 3 was generated by
NcoI-mediated digestion of pAG2, followed by fill-in and religation,
leading to the final pAG3 vector. The Cel-mab-5, Ppa-mab-5,
Cel-lin-39 and Cel-egl-5 cDNAs were amplified by PCR, are
supposed to represent full length cDNAs and have been verified by sequencing.
These cDNAs were amplified by using 5' primers introducing XhoI
sites in front of the start ATG and 3' primers with XhoI sites
downstream of the stop codons. These modified cDNAs were cloned into pAG3 to
generate pAG4, pAG5, pAG6 and pAG7, respectively. We have used a
Cel-mab-5 cDNA of 603 bp in size. This cDNA is shorter than
originally reported and was recently corrected in the database (Grandien and
Sommer). To create pAG9, the GFP-coding sequence was amplified using PCR from
the plasmid pPD95.70 (a gift from A. Fire) and cloned in-frame with the
hexapeptide of Cel-mab-5. The GFP-mediated expression of pAG9 is in
large parts similar to the previously reported Cel-mab-5 expression
pattern (Cowing and Kenyon,
1992
). pAG10 contains a truncated cDNA, which lacks the C-terminal
end of Cel-mab-5, the region 3' of the homeodomain. Throughout
the text we refer to the protein coding part that is located 3' of the
homeodomain as the `C-terminal end'. Plasmids pAG11 and pAG12 are chimeric
constructs carrying the N-terminal part of Cel-lin-39 fused with the
C-terminal part (hexapeptide, homeodomain and C-terminal end) of
Cel-mab-5 (pAG11) and vice versa (pAG12). Finally, the N-terminal arm
helix I and helix II of Cel-lin-39, were modified to the
corresponding sequences of Cel-mab-5 by PCR using the Quikchange
technique to generate pAG13 and pAG14, respectively.
In the case of Cel-lin-39, we used the construct pKG11
(Grandien and Sommer, 2001) to
insert Cel-mab-5 (pLK1) and the modified cDNAs (pLK2 and pLK3). pLK2
and pLK3 were generated in the similar way as pAG13 and pAG14 by modifying the
N-terminal arm and helix I (pLK2) and helix II (pLK3) of Cel-mab-5
into the corresponding sequences of Cel-lin-39 by in-vitro
mutagenesis.
Germline transformation
Germline transformation and generation of transgenic C. elegans
strains was performed as described (Mello
and Fire, 1995; Mello et al.,
1991
). Rescue constructs were injected into mab-5(e1239);
him-5 (e1490) with the co-injection marker pTG96
(Gu et al., 1998
), encoding
sur-5::GFP (a gift from M. Han). The co-injection marker was kept at
a concentration of 50 ng/µl, whereas rescuing constructs were injected 10
ng/µl. The total DNA concentration was kept constant by addition of pBIIKS.
It should be noted that transgenic nematode animals contain multiple copies of
the transgene.
Ray rescue assay
Transgenic adult males were picked and the tail region was analyzed using
Nomarski microscopy as described elsewhere
(Kenyon, 1986).
Egg laying rescue assay
The strain MT4498, with the genotype Cel-lin-39 (n1880), was used
for rescue experiments. Transgenic L2/L3 animals were picked singly to plates
at day 1 and incubated at 25°C. Eggs were counted daily until day 5, where
normally no or only a few eggs were still laid. Animals laying at least one
egg were scored as rescued (non-Egl). Eggs and newly hatched larvae were
counted and removed until the end of the experimental period or until the
mother died from internal hatching of progeny. Cel-lin-39(n1880)
mutant animals are completely egg-laying defective and no eggs are laid.
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RESULTS |
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We generated a basic construct pAG1 containing all exons and introns, as
well as the 3' UTR and 9 kb of upstream regulatory sequences of
Cel-mab-5. When this 19 kb construct was introduced as a transgene
into CB3531, rescue of the ray defect in males was seen in all transgenic
animals of five independent transgenic lines
(Fig. 3). On average, 3.6
V-rays were seen per side of the animal (in the following, the given ray
numbers are the observed V-rays, per side and transgenic animal)
(Fig. 3). Although most rays
had wild-type ray-like morphology, some had the morphology of fused rays as
often seen in various mutants. These malformations in the rescued ray pattern
are most probably the result of an incorrect temporal and/or spatial
expression of Cel-MAB-5 in the transgenic arrays. Similar results
have also been described in the functional analysis of Cel-mab-5
(Costa et al., 1988;
Hunter and Kenyon, 1995
;
Salser and Kenyon, 1996
;
Salser et al., 1993
).
The observed rescuing activity of pAG1 specifies the included regulatory elements as sufficient for driving Cel-mab-5 expression in the V lineage and certifies this construct as a backbone construct for further analysis. To generate a Cel-mab-5 expression vector, pAG1 was modified in several ways. We introduced a unique XhoI restriction site immediately upstream of the start ATG (Fig. 3). This construct, pAG2, retains a rescuing activity of 3.4 V-rays (Fig. 3). To diminish endogenous Cel-mab-5 gene activity of the pAG2 construct, a frameshift mutation was introduced in the hexapeptide (Fig. 3). The resulting construct, pAG3 did not show any rescuing activity of V-rays, i.e. not a single V-ray was seen (Fig. 3). Ray formation was restored by introducing a Cel-mab-5 cDNA into pAG3 (pAG4) (Fig. 4B). On average, transgenic animals carrying pAG4 formed 3.2 V-rays (Fig. 3). Thus, pAG3 provides an expression vector, in which open reading frames of genes of interest can be expressed in a way similar to Cel-mab-5. Such a construct can be used to test orthologous, paralogous or chimeric Hox proteins.
Ppa-mab-5 mutants have a ray phenotype similar to
Cel-mab-5
Previous studies on vulva formation in the nematode Pristionchus
pacificus indicated an important role for the Hox genes
Ppa-lin-39 and Ppa-mab-5
(Eizinger and Sommer, 1997;
Jungblut et al., 2001
;
Jungblut and Sommer, 1998
;
Jungblut and Sommer, 2000
;
Sommer et al., 1998
). However,
these studies did not investigate the role of Ppa-mab-5 during ray
formation. P. pacificus contains nine rays with a spatial pattern
that differs from the one seen in C. elegans
(Fig. 5A) (Sommer, 1996).
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To study the role of mab-5 during ray formation in P.
pacificus, we used the Ppa-mab-5(tu31) allele that represents a
strong reduction-of-function mutation
(Jungblut and Sommer, 1998).
R1-R6 were absent in Ppa-mab-5 mutant animals, whereas R7-R9 were
formed normally (Fig. 5B).
These results suggest that Ppa-MAB-5 provides positional information
for ray specification in a way similar to Cel-MAB-5.
Ppa-mab-5 expression restores ray formation in
Cel-mab-5 mutants
Next, we asked whether the Ppa-MAB-5 protein, when expressed under
the control of the Cel-mab-5 regulatory elements, could rescue the
Cel-mab-5 mutant phenotype. The similarity between the
Cel-mab-5 and Ppa-mab-5 mutant ray phenotypes would argue in
favor of rescue. However, a sequence comparison between both proteins
indicates substantial sequence differences inside and outside of the
homeodomain (Fig. 1D).
When a Ppa-mab-5 cDNA was driven by Cel-mab-5 regulatory elements, the resulting construct rescued the ray phenotype nearly as well as a Cel-mab-5 cDNA. On average, 2.8 V-rays were observed (Fig. 3, Fig. 4C). Specifically, all transgenic animals tested had more than two V-rays and no animals were observed without V-rays. These results indicate that the orthologous MAB-5 protein from P. pacificus can functionally replace Cel-MAB-5 when expressed appropriately.
From orthologs to paralogs: Cel-lin-39 and
Cel-egl-5 cannot restore ray formation in Cel-mab-5
mutants
Orthologous nematode Hox proteins when compared with the paralogous Hox
proteins within C. elegans, can often only be aligned properly by
comparing the homeodomain region (Fig.
1B,C). The non-DNA binding regions of Cel-mab-5, Ppa-mab-5,
Cel-lin-39 and Ppa-lin-39, respectively, show only limited
similarities. In particular, the degree of amino acid identity between
Cel-MAB-5 and Ppa-MAB-5 is only 19% and 29% in the
N-terminal and C-terminal region, respectively. At the same time,
Cel-MAB-5 and Cel-LIN-39 have in the corresponding region an
amino acid identity of 18% and 12%, respectively. In addition to the non-DNA
binding regions, the homeodomains of paralogous Hox proteins in C.
elegans also differ much more from one another than their counterparts in
insect Hox proteins. For example, the homeodomain sequences of Dfd,
Antp and Ubx in Drosophila differ at only five amino
acid positions (92% identity), nearly all of which are located in the
N-terminal arm and helix I (Fig.
1C). By contrast, the homeodomains of LIN-39, MAB-5 and EGL-5 in
C. elegans differ at several amino acids in the N-terminal arm and
helix I, but also in helix II (Fig.
1C). As a result, the sequence identity is only 65% between
Cel-MAB-5 and Cel-LIN-39, and 45% between Cel-MAB-5
and Cel-EGL-5 (Fig.
1B). The most important difference between the paralogous proteins
in Drosophila and C. elegans is that sequence differences in
Drosophila occur only in the N-terminal arm and helix I, whereas in
C. elegans also helix II differs substantially. As helix II has been
suggested to be crucial for protein-protein interactions
(Mann and Affolter, 1998;
Mann and Chan, 1996
), these
sequence differences might be important to provide functional specificity.
We sought to determine if the paralogous Hox proteins from C.
elegans could take over the function of Cel-mab-5 during ray
formation when expressed in a similar way to the Cel-MAB-5 protein.
First, we tested a construct containing the Cel-lin-39 cDNA (pAG6)
(Fig. 6). We have shown
previously that this Cel-lin-39 cDNA is sufficient to rescue a
Cel-lin-39 mutant (Grandien and
Sommer, 2001). When expressed under the regulatory elements from
the Cel-mab-5 gene, the Cel-lin-39 construct pAG6 showed
poor rescue of ray structures with an average of 0.4 V-rays
(Fig. 6). Specifically, 68% of
transgenic animals showed no rescue (0 V-rays), a result never seen in
transgenic animals of constructs containing orthologous MAB-5 proteins. Next,
we tested a construct containing a cDNA of Cel-egl-5 (pAG7). This
construct also showed poor rescue of ray structures. On average, 0.5 V-rays
were formed (Fig. 6). Together,
these results indicate that the paralogous Hox proteins Cel-LIN-39
and Cel-EGL-5 cannot substitute for Cel-MAB-5 and suggest
the existence of important functional differences between these paralogous Hox
proteins. Finally, we tested a construct containing a cDNA of the
Drosophila gene Ubx under the control of Cel-mab-5
regulatory elements (pAG8) (Fig.
6). The construct pAG8 also showed poor rescue of ray structures
with on average 0.8 V-rays (Fig.
6).
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The homeodomain of MAB-5 provides functional specificity
Given the different rescuing activities of orthologous and paralogous
nematode Hox proteins, we next asked which parts of the proteins are providing
ray specificity. Is the N-terminal part of the MAB-5 protein dispensable for
function? Can chimeric Hox proteins function properly during ray formation?
Are there several regions within the homeodomain that are of importance? To
address such questions, we generated two sets of constructs. In one construct,
we truncated the MAB-5 protein and fused the C-terminal part containing the
homeodomain to GFP (pAG9). pAG9 does not contain the N-terminal part of a Hox
protein and can therefore be used to determine whether or not this part of the
protein is dispensable for function. In a second set of constructs, we
generated chimeric proteins containing the N-terminal part of one Hox protein
and the C-terminal part including the homeodomain of another.
We found that the GFP-MAB-5 N construct pAG9 rescued the ray
phenotype of Cel-mab-5. On average, we observed 2.5 V-rays in
transgenic pAG9 animals (Fig.
6). This rescuing activity is very similar to that of the
Ppa-mab-5 construct pAG5, indicating that the C-terminal part of
MAB-5 is sufficient for ray function. However, this result does not rule out
the possibility that regions other than the homeodomain also contribute to
protein function. Recent studies in insect have indicated an important role
for a transcriptional repression domain located in the C-terminal end of the
Ubx protein involved in the diversification of thoracic and anterior
abdominal segments (Galant and Carroll,
2002
; Ronshaugen et al.,
2002
).
To study if the C-terminal end of MAB-5 has any role in ray formation, we generated a construct with a truncation of the C-terminal end (pAG10). However, pAG10 showed rescue of ray structures, with (on average) 2.9 V-rays (Fig. 6). This result is a further indication of the importance of the homeodomain and suggests that the very C-terminal end of the MAB-5 protein is dispensable for ray formation.
To study the importance of the N-terminal and the C-terminal half of MAB-5 in a different way, we generated two chimeric Hox proteins. The construct pAG11 contains the N-terminal domain of Cel-LIN-39 and the C-terminal region, including the homeodomain of Cel-MAB-5. We found that pAG11 has a rescuing activity very close to that of the pAG4 and pAG5 constructs. Specifically, 2.6 V-rays were generated and there were no animals without V-rays (such as in the Cel-lin-39 construct) (Fig. 6). This result confirms that the C-terminal part of Cel-MAB-5 is sufficient for ray formation.
The opposite construct pAG12 contains the N-terminal part of
Cel-MAB-5 and the C-terminal part of Cel-LIN-39. Testing the
construct pAG12, we observed an intermediate rescuing activity of 1.6 V-rays,
which was not seen in any other construct
(Fig. 6). Transgenic animals
differed strongly in their rescuing ability showing no rescue, i.e. no V-rays
to good rescue (three V-rays) (Fig.
6). Specifically, in 33% of transgenic animals no rescue was
observed. These results suggest that the N-terminal region of
Cel-MAB-5 can also contribute to ray specificity.
The N-terminal arm and helix I of the homeodomain provide most of the
specificity of Cel-MAB-5
The comparison of the rescuing activities of the orthologous, paralogous
and chimeric Hox proteins described above, indicates that the C-terminal part
of Cel-MAB-5 is sufficient for ray formation. In particular the
comparison between pAG6 and pAG7 with that of pAG9 and pAG10 suggests the
specificity to be conferred by the homeodomain. As indicated above, C.
elegans paralogous Hox proteins differ not only in the N-terminal arm and
the neighboring region of helix I as in Drosophila, but also in helix
II (Fig. 1). To determine if
both regions of the homeodomain provide specificity to MAB-5 function, we
tested both regions by in vitro mutagenesis. Specifically, we mutated the
N-terminal arm and helix I of Cel-LIN-39 to the sequence of
Cel-MAB-5. The resulting construct pAG13 contains this small sequence
motif characteristic for Cel-MAB-5 in an otherwise
Cel-LIN-39 protein (Fig.
6). The second construct was generated using the same strategy,
this time modifying helix II of Cel-LIN-39 towards Cel-MAB-5
(pAG14).
pAG13 and pAG14 differed strongly in their rescue activity. Whereas pAG13 with the N-terminal arm and helix I of Cel-MAB-5 showed a strong rescue with 2.6 V-rays, the rescue of pAG14 is poor (Fig. 6). Most pAG14 transgenic animals show no rescue, a result that is reminiscent of the Cel-lin-39 construct itself. By contrast, all transgenic animals of the pAG13 construct showed a good rescue, similar to the Cel-mab-5 constructs pAG1 and pAG4. Together, these results suggest that although the N-terminal arm and helices I and II show similar sequence differences between paralogous Hox proteins, the functional specificity for ray formation resides mostly within the N-terminal arm and helix I.
MAB-5 protein cannot substitute for LIN-39 during vulva
formation
The experiments described above clearly indicate that paralogs of C.
elegans mab-5 cannot restore ray formation in Cel-mab-5 mutants.
One obvious question therefore is, whether this result holds true for other
nematode Hox functions as well. To answer this question, we performed similar
experiments using vulva formation as a test system. The Hox gene
Cel-lin-39 plays a crucial role during vulva cell fate specification
and has been studied in detail in C. elegans and P.
pacificus (Eizinger and Sommer,
1997; Maloof and Kenyon,
1998
; Salser and Kenyon,
1996
; Sommer et al.,
1998
). As Cel-lin-39 and Ppa-lin-39 differ in
their functional specificity during vulva formation in both species, we have
previously used a similar assay to replace Cel-lin-39 in the vulva
and to identify those parts of the gene, regulatory versus protein-coding
regions, that provide species-specific functions
(Grandien and Sommer,
2001
).
To study if paralogous Hox genes of C. elegans can substitute for
Cel-lin-39 during vulva formation, we have used the assay system
previously established (Grandien and
Sommer, 2001). We generated a construct (pLK1) in which the
Cel-mab-5 cDNA is introduced into the Cel-lin-39 backbone
construct pKG11 (Grandien and Sommer,
2001
) that contains all regulatory elements of the
Cel-lin-39 gene (Fig.
7A,B). Consistent with our data on ray formation, pLK1 does not
rescue vulva formation. All transgenic animals tested are egg-laying defective
with no eggs being laid (Fig.
7B). By contrast, if Cel-lin-39 itself is expressed under
the control of Cel-lin-39 (pKG12) vulva formation and the egg-laying
defect is restored in 63% of transgenic animals
(Fig. 7B).
|
The second major conclusion of our work described above is that the functional specificity for ray formation resides mostly within the N-terminal arm and helix I of Cel-MAB-5. Is the specificity conferred by the N-terminal arm and helix I a general property of Hox proteins in C. elegans or do different domains of Hox proteins provide specificity in individual developmental decisions? To determine if the N-terminal arm/helix I and/or helix II of the homeodomain provide specificity to LIN-39 function during vulva development, we tested both regions by in vitro mutagenesis. In the constructs pLK2 and pLK3, we mutated the N-terminal arm/ helix I and helix II of Cel-MAB-5 to the sequence of Cel-LIN-39, respectively. pLK2 shows poor rescue of egg-laying, whereas pLK3 shows no rescue at all (Fig. 7B). Specifically, 6% of transgenic animals carrying the construct pLK2 were egg-laying positive with, on average, 23 eggs being laid. This is a poor rescue when compared with pKG12, in which 63% of transgenic animals are egg-laying positive with on average 84 eggs being laid (Fig. 7B). In addition, the rescuing activity of pLK2 in the vulva is severely lower than the rescuing activity of pAG13 in the rays (Fig. 6). Together, these data indicate that the functional specificity of the Hox proteins Cel-lin-39 and Cel-mab-5 during vulva and ray formation is provided by different domains of the Hox proteins.
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DISCUSSION |
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We have addressed the question of the functional specificity of nematode
Hox genes by investigating ray formation in males. To overcome the problems
often associated with overexpression studies of constitutive promoters, we
have used a strategy recently developed for the analysis of the function of
the neighboring Hox gene lin-39
(Grandien and Sommer, 2001).
cDNAs and chimeric Hox genes were expressed in a 19 kb vector backbone that
contains sufficient cis-regulatory elements for rescue of a Cel-mab-5
mutant by the Cel-mab-5 gene itself. Cel-mab-5 is
transcriptionally regulated in the V5/V6 lineage and is required multiple
times in various sublineages (Salser and
Kenyon, 1996
). As a result, none of the tested cDNAs, not even
Cel-mab-5 itself, was able to restore ray formation completely,
reflecting the complex requirements of Cel-mab-5. Although some of
the rays of transgenic animals were malformed, the average number of rays
generated as well as the range of ray numbers seen in transgenic animals
provided an easy measurement of the rescuing activity of individual
transgenes.
In a previous study, Hunter and Kenyon
(Hunter and Kenyon, 1995)
analyzed the ability of Drosophila Hox proteins to specify cell fates
in C. elegans by overexpression under a heat shock promoter. Although
this study mainly focused on neuronal cells, rays have also been investigated.
However, the limited control of gene expression from constitutive promoters
did not provide the sensitivity to distinguish between good and poor rescuing
activity in the comparison of mab-5, Antp and lin-39
(Hunter and Kenyon, 1995
). The
average rescue of V-rays was lower than in constructs using endogenous
regulatory elements as reported in this study, and no significant difference
was observed between orthologous and paralogous proteins. The lack of
sensitivity most probably results from the limited control of gene expression
in a spatiotemporal way.
Orthologous versus paralogous proteins: sequence and function evolve
differently
The comparison of orthologous and paralogous Hox proteins clearly shows
that only MAB-5 orthologs, but not MAB-5 paralogs can functionally substitute
for Cel-MAB-5 when expressed similar to the endogenous gene.
Considering the sequence similarity of orthologous and paralogous Hox proteins
in nematodes (Fig. 1), these
results suggests that it is not the overall sequence similarity that
determines functional specificity. Rather, discrete small domains of the
protein are of fundamental importance.
Our work strongly suggests that most of the functional specificity for ray formation is conferred by the N-terminal arm and helix I of the homeodomain. This result is surprising as the sequence comparison of the homeodomains of nematode Hox genes shows substantial sequence divergence not only in the N-terminal arm and helix I, but also in helix II. Thus, only a subset of those parts of the homeodomain that differ in sequence between nematode paralogous Hox proteins provide functional specificity, whereas other evolving regions are not required for ray specification at all.
Two different explanations might account for this observation. Helix II
might be of importance in the functional specificity of other developmental
structures. Indeed, nematode Hox genes are known to be required in multiple
developmental decisions. In the case of mab-5, previous studies
indicated a role in posterior Pn.p-cell specification in males, in P(7,8).p
specification in hermaphrodites, in Q cell migration, in the regulation of
cell fusion in the ventral epidermis and in the specification of various
neuronal cells (Costa et al.,
1988; Hunter and Kenyon,
1995
; Kenyon,
1986
; Kenyon et al.,
1997
; Salser et al.,
1993
). Furthermore, similar in vitro mutagenesis experiments of
Cel-lin-39 in the hermaphrodite vulva indicate that regions other
than the N-terminal arm and helices I and II are of importance for generating
vulva specificity (see below). In addition, recent studies in insects have
shown that the functional requirement of the very C-terminus of Ubx
is only required in a small subset of Ubx functions
(Grenier and Carroll,
2000
).
Another potential explanation for the absence of helix II function during ray formation could be that the observed sequence differences between nematode Hox proteins might be neutral. As long as amino acid substitutions do not interfere with any of the important functions of a protein, they might be tolerated. Therefore, mutations resulting in such amino acid substitutions might be fixed in natural populations.
Although the functional specificity of the chimeric MAB-5/LIN-39 proteins is largely determined by which homeodomain they express, chimeric proteins retain activities characteristic of their non-homeodomain parts. In particular, the chimeric protein containing the N-terminal part of MAB-5 and the homeodomain of LIN-39 had an intermediate rescuing activity of on average 1.6 V-rays, a value not seen with any other construct. This result suggests a role for the N-terminal part in ray specificity. However, the mechanism by which the N-terminal part of MAB-5 confers ray specification remains unknown. At the same time, our data also indicate that when the C-terminal part of MAB-5 is fused to GFP, ray rescue is observed in the absence of the N-terminal part of the protein. Thus, a completely artificial construct is able to rescue if the DNA-binding and protein-protein interaction domains are present.
The structural analysis of Hox proteins has provided valuable insight into
how these transcription factors function during development. The homeodomain
folds into three -helices, the third one of which binds to the major
groove of the DNA. It is this region of the homeodomain that is most highly
conserved with regard to sequence. Helices I and II, however, are not involved
in DNA binding and have been suggested to be required for protein-protein
interactions (Mann and Affolter,
1998
; Mann and Chan,
1996
). The N-terminal part of the homeodomain, the N-terminal arm
contacts the minor groove of the DNA and has also been indicated to form
protein-protein interactions. Thus, the region that confers specificity to
MAB-5 function during ray formation is involved in DNA binding and
protein-protein interactions. Therefore, one can speculate that the
association with other proteins strongly affects the ability of nematode Hox
proteins to act upon downstream target genes. Recently, the
extradenticle homologs ceh-20 and ceh-40, as well
as the Homothorax homolog unc-62 have been shown to interact
with the posterior-group Hox proteins NOB-1 and PHP-3
(Van Auken et al., 2002
). In
addition, an overlapping role of Cel-mab-5 and Cel-lin-39
with Cel-ceh-20 has been described for the diversification of the
postembryonic mesoderm (Liu and Fire,
2000
). Other recent studies in insects, however, indicate that
some Hox functions act independently of extradenticle
(Galant et al., 2002
).
Finally, it should be noted that, although the Ppa-mab-5 cDNA can rescue the formation of rays, this cDNA is not sufficient to produce the P. pacificus pattern of rays in a C. elegans background. Thus, although MAB-5 is instructive for ray formation, the responding pattern depends on the genetic architecture of the receiving species, in this case C. elegans.
Cel-MAB-5 evolution is not representative for other C.
elegans Hox genes
We have addressed the question of whether the specificity conferred by the
N-terminal arm and helix I is a general property of Hox proteins in C.
elegans. After studying Cel-LIN-39 during vulva formation by in
vitro mutagenesis, there is no indication that the specificity of this Hox
gene is conferred in the same way as in Cel-MAB-5. The critical
construct pLK2, in which the N-terminal arm and helix I of Cel-MAB-5
have been mutated into the corresponding region of Cel-LIN-39, shows
poor rescue when compared with Cel-lin-39 (pKG12) itself and
Cel-mab-5 (pAG13) during ray formation. Thus, our data support the
hypothesis that Hox specificity in different developmental decisions is
provided by different parts of the proteins. This observation also holds true
for insects, in which different parts of the Hox proteins have been shown to
be required for individual functions
(Chauvet et al., 2000). In the
case of Cel-LIN-39, additional studies will be required to localize
those regions of the protein responsible for providing vulval specificity.
Although our previous work showed that the C-terminal end of the protein is
dispensable for vulva formation (Grandien
and Sommer, 2001
), studies as detailed as those described here for
Cel-mab-5 will be necessary to determine the regions that provide
specificity to Cel-lin-39.
Differential evolution of Ppa-mab-5 functions
P. pacificus is a second nematode besides C. elegans, in
which large-scale mutagenesis screens have been carried out to identify genes
involved in pattern formation and cell fate specification
(Eizinger et al., 1999).
Ppa-mab-5 was one of the first genes to be studied in detail in
P. pacificus (Jungblut et al.,
2001
; Jungblut and Sommer,
1998
; Jungblut and Sommer,
2000
). In contrast to C. elegans, Ppa-mab-5 mutants have
a strong vulva phenotype, resulting in the ectopic differentiation of P8.p, a
vulval precursor cell that remains epidermal in wild-type animals. We have
recently shown that the ectopic differentiation is not dependent on the
inductive signal from the somatic gonad but rather relies on signaling from
the misspecified mesoblast M (Jungblut et
al., 2001
). The blastomere M gives rise to all mesodermal
structures formed during postembryogenesis, including body wall muscles, sex
muscles and coelomocytes. Although Cel-mab-5 mutants also have M cell
lineage defects, the cell lineage alterations in Ppa-mab-5 are much
stronger and affect nearly the complete M lineage. Thus, Cel-mab-5
and Ppa-mab-5 mutants differ strongly in both, their vulval and
muscle phenotypes.
The ray phenotype of Ppa-mab-5 as shown in this study, represents the first mab-5 function that is conserved between Cel-mab-5 and Ppa-mab-5. R1-R6 are missing in mab-5 mutants in both species and the severity of the phenotype is also similar. Together, the comparison of the mab-5 phenotypes in vulva, muscle and ray specification indicates that genes can evolve with regard to certain functions, while other functions are retained. Most likely, such patterns of evolutionary diversification of gene function are achieved by changing gene regulation. It remains unknown, however, if some of the novel functions of Ppa-mab-5 also require a different function of the Ppa-MAB-5 protein. A direct analysis of these functions awaits the establishment of transgenic technology in P. pacificus.
In summary, our work provides the first detailed analysis of the functional specificity of nematode Hox genes by studying orthologous, paralogous and chimeric proteins for their role in ray formation. We have shown that besides the limited sequence conservation of nematode Hox proteins, the N-terminal arm and helix I of MAB-5 are sufficient to induce ray formation when provided in an otherwise LIN-39 protein. Thus, although the homeodomain is the most highly conserved part of nematode Hox proteins, it is this part of the protein that confers most of the functional specificity. At the same time, similar studies with LIN-39 during vulva formation suggest the importance of regions other than the homeodomain in providing functional specificity. Although still in their infancy, these studies support the view that there is no common mechanism in providing specificity to nematode Hox proteins.
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ACKNOWLEDGMENTS |
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