National Research Center (NCCR) `Frontiers in Genetics', Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland
* Author for correspondence (e-mail: denis.duboule{at}zoo.unige.ch)
Accepted 29 April 2005
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SUMMARY |
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Key words: Hox genes, Development, Penis, Clitoris, Sgk, Epha3, Mouse
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Introduction |
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Over the past 15 years, gene expression analyses and functional approaches
have largely substantiated the surprising similarity between the distal part
of developing limbs (the digits) and the genitalia. Indeed, the majority of
genes known to be of interest for developmental growth and patterning in
presumptive digits are also transcribed in the emerging genital bud. The
initial outgrowth of the limb bud is dependent on the production of retinoic
acid by the enzyme ALDH1A2 (RALDH2)
(Niederreither et al., 2002),
whose expression also suggests a role in the formation of the genital bud.
Fibroblast growth factor (FGF) signaling is required for both AER and DUE
function (Haraguchi et al.,
2000
; Sun et al.,
2002
). In the limb, expression of the sonic hedgehog gene
(Shh) from a posterior patch of mesodermal cells called the zone of
polarizing activity (ZPA) is required for both anteroposterior and
proximodistal patterning (Riddle et al.,
1993
). In developing genitals, Shh expression in the
urethral epithelium, rather than the mesoderm, is required for outgrowth of
the bud (Haraguchi et al.,
2001
; Perriton et al.,
2002
). The significance of these homologous expression patterns is
confirmed by gene inactivation experiments, which often cause phenotypic
effects in both structures (e.g. Dolle et
al., 1993
).
The patterning of the autopod domain of the limb, which corresponds to the
hands and feet, is of particular interest in this context. For example, the
expression patterns of the genes coding for the transcription factors of the
HOX family are comparable in both the genitalia and in distal limbs
(Dolle et al., 1991).
Specifically, the Hoxa and Hoxd genes related to the Drosophila Abd-B
gene, located at the 5' extremity of their respective clusters, are
necessary for patterning both the digits and genitalia. The most 5'
genes from each complex, Hoxa13 and Hoxd13, are the ones
that are most strongly expressed at the distal ends of limbs and in the
genital bud. The combined inactivation of Hoxa13 and Hoxd13
in the same animal leads to the agenesis of both external genitalia and distal
limbs (Fromental-Ramain et al.,
1996
; Kondo et al.,
1997
). Analyses of compound Hoxa13/Hoxd13
mutants revealed that effects of these null mutations are dose-dependent in
genitals, in the digestive and urogenital tracts
(Warot et al., 1997
), and in
limbs (Zakany et al., 1997
).
Likewise, a variety of human congenital syndromes involving mutations in
either HOXD13 or HOXA13 are characterized by morphological
defects in both digits and genitalia
(Goodman, 2002
).
These observations raise the question of how the same set of control genes,
active in similar developmental processes, ultimately generates
morphologically and functionally different structures. One possibility is that
Hox transcription factors regulate the expression of different target genes
depending on the ontogenetic context. Interestingly, there is little
information about the identity of HOX-dependent target genes during limb
patterning, and their relationships with either the FGF, WNT, SHH, BMP or
retinoid signaling pathways are unclear. Recent studies have begun to address
these questions. In one approach, candidate genes were chosen because of their
known role in the patterning pathways outlined above. In this way Knosp et al.
(Knosp et al., 2004)
identified Bmp2 and Bmp7 as being downstream of
Hoxa13 in limb development, and a related study showed that
Bmp7 and Fgf8 expression is dependent on Hoxa13 in
genitals as well (Morgan et al.,
2003
).
A more comprehensive method is to use microarray technology because in this
case no prior knowledge of candidate genes is required. One approach is to
express Hox genes in cell lines where they are normally not expressed, and
then compare expression profiles with and without Hox expression. In this way,
Itga8 was established as a target of Hoxa11 regulation in
the developing kidney (Valerius et al.,
2002). A perhaps more physiologically relevant method is to
compare expression in tissues from wild-type and Hox-mutant mice. With such a
technique, Hedlund et al. (Hedlund et al.,
2004
) identified candidate target genes by comparing embryonic
spinal cord tissue from wild-type and Hoxd10-null mice. In a related
approach, Pruett et al. (Pruett et al.,
2004
) used transgenic mice overexpressing the Hoxc13 gene
to identify keratin genes of the Krtap16 family as likely direct
targets of HOXC13. The use of such approaches is nevertheless complicated by
the severity of some Hox mutations and the functional redundancy of these
genes. If the mutation is severe enough to radically transform tissues,
expression profiling would undoubtedly identify many candidate genes as,
effectively, different structures would be being compared. However, the
relevance of these candidates would be unclear, because in this case the genes
immediately downstream would be difficult to differentiate from the many genes
far downstream that are necessary to form the structure. Therefore, we chose
not to use a mutant with a drastic phenotype such as the
Hoxa13/Hoxd13 double mutant described above. Using this or
comparable strains would preclude a comparative approach as external genitalia
and distal limbs do not develop in these mutants.
Instead, we analyzed a mutant with an intermediate phenotype in which the
genitalia and distal limbs were partially, but not completely, lost. We used a
mouse strain deleted for the entire Hoxd cluster
(HoxDDel1-13). When Hoxd genes are deleted, the digits and
external genitalia are reduced in size, but the basic pattern of both
structures, as well as the relative amount of cell types, apparently remains
intact (Zakany and Duboule,
1996; Zakany et al.,
1997
). Because of the common expression patterns of Hoxd genes in
genitals and digits, and the related phenotypic effects of their mutations, we
hypothesized that genes downstream of Hoxd could be similarly regulated in
both structures. Therefore, we simultaneously identified candidate genes in
distal forelimbs and genitals by analyzing global gene expression in both
tissues, in both the presence and absence of Hoxd genes. The advantage of this
comparative strategy is that in addition to identifying specific candidate
genes, we can also begin to characterize the general nature of gene expression
programs downstream of the Hoxd genes in two distinct structures, and
therefore address the effects on gene expression of an evolutionary-conserved
Hox expression pattern.
Candidate genes were identified by microarray analysis and subsequently validated by complementary approaches: real-time RT-PCR and in situ hybridization. Only a minority of candidate genes identified in either tissue appeared to be Hoxd dependent in just one of the two structures. This suggests that expression of the same Hox genes in two different developmental contexts modulates at least a subset of overlapping downstream genes. Furthermore many of the identified genes were not previously known to have any role in limb or genital development, and most are not part of any of the established limb or genital patterning pathways.
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Materials and methods |
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The RNA samples used for microarray analysis (herein referred to as primary samples) were extracted from pools of limb or genital tissue from five to 10 individual embryos of the same genotype, three replicate pools for each tissue and genotype. RNA was isolated using the RNeasy micro- or mini-kit (QIAGEN), following homogenization and disruption with a POLYTRON device (Kinematica) using the QIAGEN RLT solution. The yield of total RNA was 5-7 µg per pool of genital tissue and 20-45 µg per pool of limb tissue. The quality of all RNA pools was confirmed by analysis on a 2100 Bioanalyzer (Agilent). Secondary samples for quantitative real-time RT-PCR analysis were collected in triplicate from E11.5-E14.5 embryos and extracted as above, except that in most cases a sample from only one individual embryo was used per replicate.
Microarray analysis
Double-stranded cDNA was synthesized from 2-5 µg total RNA from each
pool, according to the GeneChip Expression Analysis Technical Manual
(Affymetrix), using the SuperScript Choice system (Invitrogen). cDNA was used
to synthesize biotin-labeled cRNA using the BioArray HighYield RNA Transcript
Labeling Kit (Enzo). After purification using a QIAGEN RNeasy column, 25 µg
of cRNA was fragmented. Each fragmented cRNA (15 µg) was then hybridized to
an Affymetrix U74Av2 GeneChip microarray. Hybridization, washing and scanning
were performed according to the Affymetrix manual.
Data from the scanned chips were analyzed using Affymetrix MAS 5.0 software
(Hubbell et al., 2002;
Liu et al., 2002
). The
significance of differential expression was determined by the number of
pairwise comparisons (out of nine total) having a significant change in the
same direction. The cutoff for significance was a P-value of 0.0025
by Wilcoxon's Signed Rank test for each pairwise comparison. The fold-change
values reported are the means±s.d. of the nine comparisons. The Hoxd
genes were not included in the reported results, but detection of their
differential expression served as controls for both the tissue samples and
detection by the microarrays. Hoxd genes had the greatest fold changes
measured, as much as 190-fold greater signal strength for Hoxd13 when
comparing wild-type and HoxDDel1-13 forelimb samples.
Candidate genes were prioritized into categories by the number of significant
change comparisons, and were categorized secondarily by the fold change. Gene
lists were further filtered by eliminating those candidates that were called
absent in both genotypes by the MAS 5.0 analysis. In some cases, probe sets
were eliminated from the analysis when extensive BLAST searching indicated
that they were not specific for an individual gene. The raw data were also
analyzed with other publicly available software, including dChip
(Li and Hung Wong, 2001
) and
GCRMA (Wu et al., 2003). The results from these complementary analyses largely
confirmed the lists of candidate genes obtained by MAS 5.0. According to MIAME
guidelines (Brazma et al.,
2003
), the complete microarray dataset was deposited in the public
data repository of the European Bioinformatics Institute (ArrayExpress) with
accession number E-MEXP-257.
Quantitative real-time RT-PCR
Single-stranded cDNA templates for real-time RT-PCR analysis were
synthesized from the same RNA pools used for the microarray analysis, and from
independently derived and extracted secondary samples as described above.
Sixty-five to 100 bp amplicons and Taqman probes were designed using Primer
Express 2.0 software (Applied Biosystems). Primer pairs were tested and
efficiencies were measured using standard curves from serial dilutions of
cDNA. Primer pairs having greater than 88% efficiency were judged to be
acceptable for subsequent measurements. Results and amplification efficiencies
were comparable using either Taqman or Sybr Green. Specificity of Sybr Green
reactions was determined by examination of product melting curves as described
(Ririe et al., 1997). cDNA was
PCR amplified in a 7900HT SDS System (Applied Biosystems) and raw
threshold-cycle (Ct) values were obtained from SDS 2.0 software (Applied
Biosystems). Relative quantities (RQ) were calculated with the formula
RQ=E-Ct using efficiencies (E) calculated for each run with the
DART-PCR algorithm, as described (Peirson
et al., 2003
). A mean quantity was calculated from triplicate PCR
reactions for each sample, and this quantity was normalized to two (for
primary samples) or three (for secondary samples) similarly measured
quantities of normalization genes as described
(Vandesompele et al., 2002
).
Normalized quantities were averaged for three replicates for each data point
and represented as the mean±s.d. The highest normalized relative
quantity was arbitrarily designated as a value of 1.0. Fold changes were
calculated from the quotient of means of these normalized quantities and
reported as ±s.d. The statistical significance of fold-changes was
determined by a paired Student's t-test. Taqman PCR was used to
quantify the expression of Hoxa11, Papss2, Aldh1a2, Stra6, Msx2, Prrx1,
Gfra2 and Epha3. Sybr Green PCR was used to quantify the
expression of Sgk, Gdf10, Odz4, Nr2f1, Pcdha, Foxp1, Shox2 and
Lisch7. Control genes for normalization, Rps9, Tbp and
Tubb4, were amplified either by Sybr Green or Taqman PCR, as
appropriate for each run. Primers and probe sequences can be retrieved at:
http://www.unige.ch/sciences/biologie/biani/duboule/index_st.htm.
In situ hybridization
All cDNA fragments used as whole-mount situ hybridization (WISH) probes
were prepared by RT-PCR using E12.5 genital or limb bud RNA, with the
exception of the Hoxa11 probe (gift of Catherine Fromental-Ramain).
Four hundred to 700 bp amplicons were designed with MacVector software
(Accelrys) using publicly available GenBank cDNA sequences. Specificity of the
primers and chosen cDNA fragment was confirmed by BLAST analysis. Specific
primer sequences are listed on the website indicated above. The various cDNAs
were cloned into the pGEM-T vector (Promega) and the correct identities of the
cloned fragments were confirmed by restriction enzyme analysis. DIG-labeled
antisense probes were prepared by in vitro transcription with SP6 or T7
polymerase (Promega). WISH using embryos fixed in 4% paraformaldehyde was
performed according to standard procedures. Because sex-specific differences
in external genitalia only begin to appear at E16.5
(Suzuki et al., 2002), the sex
of embryos was not routinely determined. Nevertheless, in some cases the
gender of embryos was determined by PCR as described
(Lavrovsky et al., 1998
) to
confirm that the expression patterns were not sex-dependent. All images
represent one representative staining of at least two replicates for each
condition.
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Results |
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Absence of shared candidate genes in E12.5 distal limbs and genitals by microarray analysis
We chose to use samples from E12.5 embryos as a starting point for our
analysis, as it is the first day in which the genital bud exists as a
structure that can be reliably dissected, and because Hoxd gene expression is
reaching high levels in both structures at this time
(Fig. 1A). Also E12.5 embryos,
unlike younger specimens, have presumptive autopods clearly distinguishable
from more proximal domains.
Triplicate wild-type and HoxDDel1-13 homozygote pools of E12.5 RNA were analyzed using Affymetrix microarrays that contain probe sets representing approximately 12,000 cDNAs. The resulting data sets allowed a total of nine comparisons between pools to be made for each type of tissue (Fig. 1B). Using the criteria described in Table 1, the best candidates were chosen by the number of comparisons in which differential expression was statistically significant, and by the magnitude of the fold change. Surprisingly, of the strongest candidates, in which at least seven out of nine comparisons showed significant differential expression, only four genes showed a greater than twofold change in expression (Table 1; first row). When the criteria were relaxed to include genes with a fold-change cutoff of 1.4-fold, only 13 more genes were identified with seven significant comparisons (Table 1; second row). A third, even less stringent category, yielded 28 more candidates in which the threshold for fold-change remained at 1.4-fold, but the number of significant differential comparisons was reduced to 6. Further relaxation of the criteria (Table 1; fourth row) expanded the list to hundreds of genes with less than six statistically significant comparisons. Strikingly, none of the candidate genes identified in the two tissues were found to be differentially expressed in both distal forelimbs and genitals.
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Because the same secondary RNA samples were to be used to characterize all
candidate genes, we first assayed for any systematic differences amongst
samples. Accordingly, we chose two genes, Prrx1 and Msx2,
which showed no significant difference in expression by microarray analysis.
Furthermore, these particular genes served as robust controls for either
random or systematic effects, as both are expressed in the same mesenchymal
tissues as the Hoxd genes and function in limb patterning
(Leussink et al., 1995;
Satokata et al., 2000
). The
resulting real-time RT-PCR expression profiles for these control genes
indicated no differential expression in either forelimb or genital buds (see
Fig. S1 in the supplementary material), supporting the use of these samples in
identifying truly differentially expressed genes.
We confirmed differential expression at E12.5 for 14 out of the 16 genes in the secondary samples, giving a final list of 14 validated candidate genes (Tables 2 and 3). Therefore, despite the relatively small fold changes detected, multiple rounds of verification of these genes rigorously confirmed their differential expression. Furthermore, among the 11 candidates identified in distal limbs, seven also had highly significant (P<0.01) differential expression in genital buds on at least one day of development (Table 3; last column). With the exception of Gdf10, the most significant differential expression in genitals was found in embryos older than 12.5 days (the differential expression of Gdf10 in E12.5 genitals was apparently not detected by the microarrays because of its weak signal). Similarly, of the three candidates initially identified in genitals, two also had highly significant differential expression in limbs (Table 2; last column) at stages other than E12.5. Ultimately, nine out of the 14 candidate genes were found to be differentially expressed in both presumptive digits and genitals.
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Odz4 and Epha3 are downregulated by Hoxd gene products
The expression patterns of all of the candidate genes in
Table 3 were determined
(Fig. 5, see also Figs S2-S4 in
the supplementary material) for the developmental stages in which they showed
significant differences by real-time RT-PCR. Odz4 and Epha3
RNA levels were increased in both HoxDDel1-13 limbs and
genitals (Fig. 5A,B),
suggesting that they could be targets for Hoxd repression (as are
Shox2 and Pcdha, see Fig. S4C,D). The increase in
Odz4 and Epha3 expression in mutant forelimbs was clearly
detected by WISH. At E12.5, the domain of Odz4 expression increased
both in its distal extent and intensity, and this increase was still visible
in E13.5 and E14.5 embryos (not shown). Similarly, the domain of
Epha3 expression extended slightly more distally, and was reinforced
towards the posterior and proximal parts of mutant digits at E12.5
(Fig. 5B). An increase of
Odz4 and Epha3 in genitals was difficult to visualize by
WISH, but was highly significant (P<0.01) by real-time RT-PCR in
both cases (Fig. 5A,B).
Candidates target genes for Hoxd activation in distal forelimb
In contrast to Odz4 and Epha3, Gdf10 is a candidate for
Hoxd upregulation (as are the six other genes initially identified in limbs
listed in Table 3). Because
Gdf10 is expressed broadly in both forelimbs and genital buds from
both genotypes, its loss of expression in the HoxDDel1-13
mice was difficult to document by WISH
(Fig. 5C), although the
difference was highly significant by real-time RT-PCR in both tissues. For
other genes, such as Papss2 (see Fig. S2A in the supplementary
material) and Aldh1a2 (see Fig. S3A in the supplementary material),
the in situ staining confirmed the differential expression as quantified by
microarray and real-time PCR, but their expression patterns were so dynamic
that an interpretation of their role downstream of Hoxd was difficult. The
patterns of the remaining six genes confirmed to varying degrees the
differential expression (see Figs S3, S4 in the supplementary material).
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The gain of expression of posterior Hoxd genes in HoxDDel1-10 mutant mice was also used to monitor expression of the Sgk gene, a candidate for transcriptional upregulation by Hoxd gene products. Unlike Epha3, Sgk expression in wild-type developing digits perfectly overlaps with that of Hoxd13 (Fig. 6; compare A with C). In particular, Sgk, like Hoxd13, was not detected in the most anterior aspect of the developing distal limb (Fig. 6). In mutant HoxDDel1-10 limbs, however, Sgk expression was detected concomitantly with the gain of expression of Hoxd13 in this anterior domain (Fig. 6A). This confirms the results obtained with both microarray and RT-PCR analyses, which placed Sgk downstream of HOXD function.
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Discussion |
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An intermediate situation is observed in both limbs and genitalia, where mostly two clusters (Hoxa and Hoxd) are involved in patterning. Consequently, the level of functional redundancy observed in these structures is probably not as important as that in the developing trunk, and removing the functions of two orthologous genes leads to drastic alterations. Therefore, the search for target genes should, in principle, be easier in developing limbs and genitalia than in the developing spinal cord or sclerotomes. This is, however, not necessarily the case, as removing Hox gene functions progressively deletes the corresponding limb and genital structures, rather than inducing distinct morphological alterations.
We tried to overcome this problem by using an intermediate genetic condition in which the limbs and genitalia, while being affected in their overall size and patterning, still display fairly good morphologies due to the function of the remaining Hoxa genes. Because limbs and genitals have comparable early developmental phases, we wondered whether their different morphological fates could be accounted for by distinct transcriptional outputs following the activation of the same Hox genes, i.e. whether the same HOX proteins would trigger the activation of different genetic programs.
Distal limbs and genitals share common genes downstream of Hoxd function
We identified and validated 14 genes as potential HOXD targets. Six of
these (Hoxa11, Sgk, Gfra2, Epha3, Odz4 and Gdf10) are
especially strong candidates. Interestingly, with the exception of
Hoxa11, these genes are similarly regulated in limbs and genitals,
which further indicates that these two structures display closely related
developmental strategies. Initially, these genes were not found to be
regulated simultaneously in both developing buds, until the analyses were
extended to various time-points. This revealed that the same variations in
gene regulation were observed in both structures, but usually slightly later
in genitalia than in limb buds (Gfra2 is an exception to this trend,
see below). This observation nicely fits the developmental delay that exists
between these buds, as the genital eminence emerges with a one to two day
delay with respect to forelimb budding.
Strikingly, except for Hoxa11, the function of the five other potential target genes has not yet been fully explored in limb or genital development. In fact, none of the 14 candidate genes are members of the classical FGF, BMP, WNT or SHH signaling pathways. Only the retinoid pathway (Aldh1a2, Stra6) is represented among the candidates. This observation supports the conclusion that, at the developmental stages we analyzed, the Hoxd genes act downstream or independently of most of the previously described limb and genital patterning pathways.
Regulatory crosstalk between Hox clusters
Post et al. (Post et al., 1999) previously reported that the expression
domain of Hoxa11 extends more distally into the autopod domain when
Hoxa13 is mutated. We have found a similar upregulation of
Hoxa11 in genitals devoid of Hoxd gene function. Therefore
Hoxa11 is a common downstream target of Hox proteins. In our study,
Hoxa11 did not increase significantly in forelimbs, probably because
of the presence of the Hoxa13 product. As Hoxa13 is also
expressed in developing genitals, it is unclear why we observe a clear
increase in Hoxa11 transcripts in the absence of Hoxd genes.
Nonetheless, the combined results from our study and the findings of Post et
al. (Post et al., 1999) identify Hoxa11 as similarly regulated by Hox
proteins in limbs and genitals. In both cases, one may wonder whether such an
upregulation of Hoxa11 could both weaken the phenotype and,
accordingly, reduce the changes seen in the expression of candidate target
genes through functional compensation.
Despite this clear effect on Hoxa11 regulation and a few other
reported cases of auto-regulation (Popperl
et al., 1995; Popperl and
Featherstone, 1992
), cross-regulatory and autoregulatory
interactions amongst Hox genes and their products does not appear to be the
rule, particularly for those posterior Hoxd genes involved in limb and
genitalia development. This is demonstrated well by the inability of Hoxd
transgenes to be faithfully expressed in distal limbs and genitalia whenever
integrated outside the Hoxd cluster, even in the presence of the full Hox gene
complement (see van der Hoeven et al.,
1996
).
Sgk in limb and genital development
The Sgk gene is certainly amongst the more unlikely candidates
identified in this work. No developmental role for this kinase has been
reported, even though the Sgk orthologous gene in C. elegans
has been identified as a critical determinant of life span and stress response
(Hertweck et al., 2004). A
previous study reported tissue-specific expression of Sgk during
development, but an analysis of limbs beyond E10.5 was not described, thus
overlooking the expression phase starting at E11.5
(Lee et al., 2001
). The mouse
genome encodes two other Sgk isoforms (Sgk2 and
Sgk3), which is likely to account for the virtual absence of a
phenotype in mice null for the gene (Wulff
et al., 2002
).
Among protein kinases, the SGK family is most closely related to the AKT
protein kinases. A developmental role for AKT kinases was not fully evident
until null mutations for Akt1 and Akt2 were combined
(Peng et al., 2003). Likewise,
the developmental role of the Sgk genes will probably require
detailed combined analysis of all three forms. Because both AKT and SGK
kinases act downstream of PDK1 (3-phosphoinositide-dependent protein kinase
1), SGK could participate in the recently reported involvement of AKT in the
regulation of apoptosis in the limb
(Kawakami et al., 2003
).
Biochemical studies have shown that SGK has an anti-apoptotic function
(reviewed by Lang and Cohen,
2001
), at least partially through its phosphorylation and
inactivation of the pro-apoptotic transcription factor FKHRL1
(Brunet et al., 2001
;
Mikosz et al., 2001
).
Intriguingly, the domain of Sgk expression we report, especially at
E13.5 (Fig. 3), is adjacent to
the domain of interdigital cell death occurring more distally
(Chen and Zhao, 1998
).
Likewise, a domain of apoptosis has been reported in the distal genital bud
(Haraguchi et al., 2001
;
Suzuki et al., 2003
),
immediately adjacent to the domain of Sgk expression. SGK could be
involved in regulating the domains of apoptosis or proliferation in both
structures and its misregulation could contribute to the smaller size of
appendages seen in the HoxDDel1-13 mutant.
Gfra2 and the innervation of distal structures
Gfra2 codes for a receptor for the neutrophic factor neurturin,
which signals through the RET receptor tyrosine kinase
(Buj-Bello et al., 1997). A
null mutation in this gene caused defective parasympathetic innervation of the
gut and penis (Laurikainen et al.,
2000
; Rossi et al.,
1999
) but, as for Sgk, the existence of closely related
family members makes full assessment of the developmental role difficult. We
report here that the Gfra2 gene is highly expressed from the onset of
genital budding, an expression that is markedly diminished in the absence of
Hoxd genes. Although the expression pattern correlates with the role in the
innervation of the genitalia, the breadth of Gfra2 expression
throughout the genital bud suggests an additional developmental role as well.
The expression outside of purely neuronal tissue has been previously noted,
but as yet no function has been assigned
(Klein et al., 1997
).
The Gfra2 expression pattern we report in limbs is significantly
more restricted than that in genitals, but is equally Hoxd dependent. In
contrast to most of the other candidates, differential expression of
Gfra2 appears later in limbs than in genitals. The only function for
Gfra2 that has been reported in limbs is a postnatal requirement for
the innervation of sweat glands on the ventral surface of the paws
(Hiltunen and Airaksinen,
2004). Although this function is likely to be required too late in
development to be assigned to the expression we see at E13.5-14.5, it suggests
a possible role in the innervation of this domain of the limb.
Eph genes as Hox targets?
Ephrins and their receptors have a well-established role in neuronal
pathfinding, cell migration and cell adhesion (reviewed by
Poliakov et al., 2004). Their
role in limb development has only recently begun to be explored, but the
redundancy and overlapping expression domains of the ephrin and Eph genes
(eight and 13 family members, respectively) makes this task particularly
challenging. Compagni et al. showed that females heterozygous for a null
mutation of ephrin B1 have digit duplications and bifurcations
(Compagni et al., 2003
).
Stadler et al. initially established a link between Hox gene expression and
ephrin signaling by showing that Epha7 expression is markedly lower
in Hoxa13-null forelimbs (Stadler
et al., 2001
). Another study showed that overexpression of ephrin
A2, one of the ligands of EPHA3, caused digit bifurcations and fusions in
chick limbs (Wada et al.,
2003
).
In addition, previous studies have reported that ephrin receptor (Eph)
genes are the direct targets of Hox proteins. HOXA9 has been shown to directly
bind to and regulate the expression of the Ephb4 gene in cultured
endothelial cells (Bruhl et al.,
2004). Similarly, when co-expressed in vitro along with PBX1,
HOXA1 and HOXB1 bind to and activate transcription from an enhancer sequence
that is known to direct rhombomere-specific expression of Epha2
(Chen and Ruley, 1998
). Taken
together these studies clearly establish a link between Hox genes and ephrin
signaling.
Our data show that Hoxd and Hoxa genes act in combination to downregulate
Epha3 expression in developing digits. However, the biological
relevance of this repression is unclear in part because Epha3-null
mice have no abnormal phenotype (Vaidya et
al., 2003). Our microarray data indicate that at least 12 of the
ephrin and Eph genes are expressed in developing limbs and genitals.
Therefore, defining a precise role for Eph and ephrin genes in limb
development will require comprehensive studies of all of these genes.
Regardless of its function, the Epha3 gene can serve as a model for
Hox regulation, as demonstrated by its complementary expression response in
three kinds of Hox mutant stocks.
A horizontal regulatory strategy
Like the Hox genes themselves, all of the best candidate genes identified
have multiple paralogs in the mouse genome, suggesting that functional
redundancy may prevent the rapid elucidation of the biological role of the
downstream genes. This complex situation is not unexpected considering the
global function of Hox genes during vertebrate development, which is to
modulate the fate of a given morphological module, rather than triggering the
activation of novel genetic pathways. For example, the difference between a
cervical and a lumbar vertebra is most likely to be due to subtle modulations
of the same genetic determinants, rather than to the function of distinct
pathways. In the case of both the limbs and genitalia, Hox genes participate
in both the elaboration and the specification of the structures, and therefore
affect the entire process rather than some specific parts of it.
In this view, the difficulty to assign clear target genes to the Hox proteins, at least during trunk, limb and genital development, is not surprising, but may be a precise indication of how the system works. This `horizontal' regulatory strategy, whereby subtle variations in the amounts of related proteins impact upon the balance between a large set of products is in marked contrast with the situation found in arthropods, where Hox genes seem to be part of more `vertical' regulatory processes. In the former case, various thresholds of target production, or combinations thereof, may induce a structure to produce a given morphology rather than another, related one. In the latter case, the presence or absence of a Hox product may trigger a chain of events leading to the choice of a given genetic pathway.
The vertebrate case will be difficult to solve with our current analytical
tools, given the difficulty of identifying global correlations out of multiple
parameters, rather than punctual downstream effectors. The analysis of HOX
protein-binding sites may help to some extent, but in vitro studies have shown
that Hox proteins by themselves bind DNA rather nonspecifically, using
core-binding sequences of only four nucleotides
(Phelan and Featherstone,
1997). The binding specificity apparently comes from Hox proteins
forming complexes with other proteins, including those of the Pbx and Meis
gene families, each with their own binding specificities. Indeed recent work
in Drosophila has shown that a complex of at least five protein
subunits represses the well-characterized Hox target gene Distalless
(Gebelein et al., 2004
). While
some of the binding sites for higher order complexes on mammalian DNA have
been determined (reviewed by Mann and
Affolter, 1998
), it is as yet difficult to reconcile these in
vitro studies with the in vivo evidence. In particular, we do not know which
complexes function in limbs and genitals, especially since MEIS1, which has
been reported to be the strongest in vitro binding partner for
AbdB-related HOX proteins (Shen
et al., 1997
), is not expressed in distal limbs
(Mercader et al., 1999
).
Therefore, although some of the genes identified here could be direct Hox
targets, demonstrating this conclusively will require future studies of the
Hox transcription complexes in limbs and genitals. When appropriate antibodies
become available, chromatin-immunoprecipitation experiments will be invaluable
for conclusively demonstrating Hoxd interactions with specific regulatory
regions. Our study provides a list of genes that can serve as substrates for
these future studies.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/13/3055/DC1
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ACKNOWLEDGMENTS |
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