1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto M5G 1X5,
Canada
2 Max-Planck Institute of Immunobiology, Department of Molecular Embryology,
Freiburg 79108, Germany
3 Kyoto University Graduate School of Medicine, Department of Pharmacology,
Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
4 University of Toronto, Department of Medical Genetics and Microbiology,
Toronto M5S 1A8, Canada
Author for correspondence (e-mail:
rossant{at}mshri.on.ca)
Accepted 30 March 2005
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SUMMARY |
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Key words: Wnt/ß-catenin signaling, Gastrulation, RNA interference (RNAi), Target genes, Expression profiling, Grsf1, Fragilis2, Functional genomics
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Introduction |
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Mutations in a number of Wnt genes and Wnt signaling components highlight
the crucial role of the Wnt/ß-catenin signaling pathway in the initiation
of primitive streak formation, as well as in the patterning and morphogenesis
of the gastrulation-stage embryo
(Beddington and Robertson,
1999; Lu et al.,
2001
). Mutant mouse embryos that lack functional Wnt3 or
ß-catenin fail to establish an anterior-posterior (A-P) axis and do not
form a primitive streak, thus they fail to generate endoderm and mesoderm,
resulting in an arrest of development before gastrulation
(Liu et al., 1999
;
Haegel et al., 1995
;
Huelsken et al., 2000
). Both
Wnt3a null mutants and Lef1;Tcf1 compound homozygous null
mutants fail to differentiate paraxial mesoderm, do not form somites caudal to
the forelimb buds and exhibit severe posterior truncations
(Takada et al., 1994
;
Galceran et al., 1999
). In
addition, Wnt3a controls directly the expression of Axin2
and Dll1 in the paraxial mesoderm, and thereby, links the Notch and
Wnt signaling pathways in the processes of somitogenesis
(Aulehla et al., 2003
;
Galceran et al., 2004
;
Hofmann et al., 2004
). By the
end of gastrulation and the beginning of neurulation, secreted Fgf8 and Wnt1
molecules from the isthmic organizer play an important role in patterning the
mid/hindbrain region along the A-P axis (reviewed by
Liu and Joyner, 2001
;
Wurst and Bally-Cuif,
2001
).
Recently, using the Cre/loxP system, we have conditionally
inactivated ß-catenin in the visceral endoderm (VE) and the anterior
primitive streak (APS) by using a Cytokeration 19 (K19)-driven Cre
(Lickert et al., 2002).
Similar to in Wnt3 and ß-catenin null mutants, A-P axis formation was
affected; however, the conditional ß-catenin mutants proceeded through
gastrulation. This revealed a crucial function for ß-catenin during later
developmental processes, such as posterior axis elongation and somite
formation, processes affected in other Wnt mutants. Additionally, the node, an
embryonic structure functionally equivalent to the Spemann/Mangold organizer
in frog, failed to form in these mutants. Taken together, these results are
consistent with the hypothesis that Wnt/ß-catenin signaling is important
for the induction of the mouse embryonic organizing centers, the formation of
somites, and the proper morphogenesis of the gastrulating embryo.
Here, we have used a functional genomic approach combining Affymetrix GeneChip analysis, whole-mount in situ screening and rapid functional assessment by RNAi in embryonic stem (ES) cell-derived embryos to dissect the Wnt/ß-catenin signaling pathway during gastrulation. Intriguingly, the knock-down phenotypes of two potential target genes, Grsf1 and Fragilis2 (Ifitm1 Mouse Genome Informatics), recapitulate specific but distinct aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. In summary, this approach represents a highly efficient and rapid methodology with which to unravel developmental pathways in the mouse.
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Materials and methods |
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Microarray experiments
Microarray experiments have been submitted to GEO in a MIAME compliant
format (Minimum Information About a Microarray Experiment)
(Brazma et al., 2001). The
accession number is GSE2519.
Sample preparation
Embryos from the above described intercrosses were dissected in PBS and
separated into embryonic and extraembryonic portions. The embryonic portions
were stored in PBS at 80°C until the PCR genotyping was carried out
using the extraembryonic portions. The pooled embryos were homogenized in 250
µl of TRIzol Reagent (Invitrogen), and total RNA was extracted according to
the manufacturer's protocol.
Probe preparation and GeneChip hybridization
For each sample, 5 µg total RNA was used for cDNA synthesis according to
the Expression Analysis Technical Manual (Affymetrix). The in vitro
transcription and labeling of cRNA was carried out using the BioArray
High-Yield Transcription Labeling Kit (Enzo). Then 25 µg of labeled cRNA
was used to hybridize all three GeneChips from the Affymetrix U74v2 according
to the standardized Affymetrix protocol.
Data analysis
MAS 5.0 software was used to generate the expression data set for each
GeneChip.dat file and scale normalized to a target value of 150. Comparisons
were made to calculate signal log ratios of expression between mut:wt and
mut:het using either wild type (wt) or heterozygotes (het) as a baseline,
respectively. The resulting table was exported to Microsoft Excel to filter
out probe sets with absent calls across all samples (P-detection
>0.04) and probe sets with no change in expression (0.003 >
P-signal log ratio <0.997) in both mut:het and mut:wt comparisons.
Illogical combinations of absent and present calls with a significant change
call were deleted, e.g. an absent call in baseline and present call in mutant
with a decrease change. Genes that were consistently up- and downregulated in
both comparisons (absolute signal log ratio of 0.5) were hierarchically
clustered using Cluster 3.0 software
[http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/software.htm,
based on the method of Eisen et al. (Eisen
et al., 1998
)].
Using the Affymetrix Gene Ontology (GO) analysis software (www.affymetrix.com) the numbers of probe sets on U74v2A GeneChip corresponding to the GO terms `Transcription' (GOID:6350), `Cell Communication' (GOID:7154), `Pattern Specification' (GOID:7389) and `Morphogenesis' (GOID:9653) were calculated as of the November 2003 annotation build. The total numbers of all other probe sets with a GO term were denoted as `Others'. Additionally, the numbers for the same GO terms were calculated for the 49 downregulated genes from the U74Av2 GeneChip. Percentiles for each GO term category were calculated by dividing the numbers in each category by the total number of probe sets with any GO term.
Whole-mount in situ hybridization, histology and alkaline phosphatase staining
For whole-mount in situ hybridization and histology, embryos were processed
as described previously (Lickert et al.,
2002). Sense and antisense in situ probes were in vitro
transcribed using ESTs available from ATCC (Manassas, VA, USA) with the
following I.M.A.G.E. Clone IDs: Fragilis2, 657273; Zic3,
5120056; Scap2, 3599914; Punc, 3514346; EST10,
3980327; EST16, 1328681; Sox2, 5707193. Additionally, ESTs
from the NIA 15K Mouse cDNA Clone Set with the following H3 clone IDs were
used for probe synthesis: Grsf1, H3046G05; EST6, H3074B02.
Additional probes for known genes were as follows: Axin2
(Aulehla et al., 2003
),
Wnt3a (Gavin et al.,
1990
), Wnt8 (Bouillet
et al., 1996
), Frzb1
(Hoang et al., 1998
),
Notch1 (Conlon et al.,
1995
), Dll1 (Hrabe de
Angelis et al., 1997
), Gbx2
(Wassarman et al., 1997
),
Hoxb1 (Marshall et al.,
1992
), T (Herrmann et
al., 1990
), Tbx6
(Chapman et al., 1996
),
PAPC (Rhee et al.,
2003
), and Krox20
(Swiatek and Gridley,
1993
).
To identify germ cells, embryos were fixed in 4% paraformaldehyde in PBS
for 30 minutes and stained for tissue non-specific alkaline phosphatase for 5
minutes [25 mM Tris-maleic acid (pH 9.0), 0.4 mg/ml -naphthyl phosphate
(Sigma), 1 mg/ml Fast Red TR salt (Sigma), 8 mM MgCl2, 0.01%
Na-desoxycholate, 0.02% NP-40].
shRNA targeting of ES cells and embryos and northern blot analysis
For construction of the Grsf1 and Fragilis2 shRNA
transgenes, we have used the pcDNA3.1 RasGAP shRNA plasmid described
recently (Kunath et al.,
2003). RasGAP shRNA was released from the plasmid by
Asp718, XbaI digestion, and annealed oligonucleotides
corresponding to the target sequence were introduced into the same sites using
the following sense- and antisense-strand oligonucleotides (target sequence in
bold):
Grsf1 shRNA forward, 5'-GT ACC AAA GCA CAG GGA AGA AAT TGG TA C AAG AGA TA CCA ATT TCT TCC CTG TGC TTT TTT TTGG AAA T-3' and Grsf1 shRNA reverse, 5'-CTA GAT TTC CAA AAA AAA GCA CAG GGA AGA AAT TGG TA T CTC TTG TA CCA ATT TCT TCC CTG TGC TTT G-3' (corresponding to bases 982-1004 of the murine Grsf1 gene, NCBI accession no.: NM_178700); and
Fragilis2 shRNA forward, 5'-GT ACC GAA CAT CAG CTC CCT GTT CTT CA C AAG AGA TG AAG AAC AGG GAG CTG ATG TTC TTT TTT TGG AAA T-3' and Fragilis2 shRNA reverse, 5'-CTA GAT TTC CAA AAA AA GAA CAT CAG CTC CCT GTT CTT CA T CTC TTG TG AAG AAC AGG GAG CTG ATG TTC G-3' (corresponding to bases 295-317 of the murine Fragilis2 gene, NCBI accession no.: BK001123).
The resulting shRNA targeting constructs were confirmed by DNA sequencing.
Transgenic ES-cell lines were established as described
(Kunath et al., 2003). The
mRNA expression level of the individual ES-cell lines for Grsf1 and
Fragilis2 was determined by northern blotting using the
NorthernMax-GlyTM Kit (Ambion), according to the manufacturer's protocol.
Pre-selected ES-cell lines were used to generate totally ES cell-derived
embryos using the tetraploid aggregation technique as described previously
(Nagy et al., 1993
;
Kunath et al., 2003
). Embryos
with any contribution of tetraploid EGFP-positive cells were excluded from the
analysis. Experimental animals were treated according to guidelines approved
by the Canadian Council for Animal Care.
Co-culture of ES cells with Wnt1-expressing fibroblasts
The co-cultivation of ES cells with NIH3T3 fibroblasts was carried out
essentially as described previously
(Lickert et al., 2000), with
the exception that the ES cells were seeded in transwell filters
(Transwell-COL, collagen-treated, 0.4 µm pore-size; Costar #3491).
After 18 hours of co-cultivation, total RNA was isolated from the ES cells
using the RNeasy Mini Kit (Qiagen). For each sample, 2 µg of RNA
was treated with DNaseI and then reverse transcribed using oligo (dT)-primers
and SuperSriptII reverse transcriptase (Invitrogen).
Quantitative PCR was performed using the LightCycler Fast Start DNA MasterPlus SYBR Green I Kit (Roche) according to the manufacturer's protocol. The following primers were used to amplify mRNAs for Gapdh, Fragilis2 and Grsf1: Gapdh-fwd, 5'-ACCACAGTCCATGCCATCACT-3'; Gapdh-rev, 5'-GTCCACCACCCTGTTGCTGTA-3'; Fragilis2-fwd, 5'-GGGCTCCTCGACCACACCTCTT-3'; Fragilis2-rev, 5'-CCCAGTCGTATCACCCACCATCT-3'; Grsf1-fwd, GATATTCGGCCTATGACGGCT-3'; Grsf1-rev, CAAAATCGACAGCCTCTGGAAG.
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Results |
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Expression screening identifies novel genes regulating embryonic development
To discover new components of Wnt/ß-catenin signaling in developing
embryos, we investigated further the expression of 16 downregulated genes
(EST1-16) for which there was little or no published evidence concerning their
developmental roles at the time the screen was conducted. Upon in situ
hybridization in wild-type embryos at E7.5 and E8.5, eight of the genes showed
expression patterns that strongly overlapped with known regions of high Wnt
reporter activity (Fig. 3)
(Mohamed et al., 2004). All
genes except EST6 showed expression in the PS region at gastrulation stages
(Fig. 3). In addition,
Grsf1, Punc and Zic3 showed expression in the mid/hindbrain
region at E8.5, while Scap2, Punc, Zic3, Fragilis2 and EST16 were
expressed in the paraxial mesoderm, somites or neurectoderm of the tailbud
region (Fig. 3). Interestingly,
EST6 showed strong expression in the extraembryonic ectoderm and in a row of
cells anterior to the node, known regions of organizing activity
(Fig. 3C). EST10 showed a
specific expression pattern in the definitive endoderm around gastrulation by
whole-mount in situ hybridization and histological sectioning of stained
embryos (Fig. 3F and data not
shown).
For further analysis, we used transgenic RNA interference (RNAi)
(Kunath et al., 2003) to
analyze the function of the newly identified potential Wnt/ß-catenin
target genes, Irx3, Scap2, Smarcd3, Fragilis2 and Grsf1
(Table 1). Because the results
for the Smarcd3 knock-down analysis was recently published
(Lickert et al., 2004
) and
because in a first attempt we were not able to knock down Scap2 and
Irx3, we focus here on the analysis of Grsf1 and
Fragilis2 (Fig.
3A,G).
Knock down of potential Wnt/ß-catenin targets, Grsf1 and Fragilis2
Grsf1 codes for the mouse ortholog of the human G-rich sequence
specific binding factor1 (GRSF1), which was previously shown to bind to a
specific consensus sequence in the 5'-UTR of influenza virus
nucleocapsid mRNA and thereby act positively on translation
(Park et al., 1999;
Kash et al., 2002
).
Fragilis2 belongs to a family of interferon-inducible genes with five
members (Fragilis1-5), clustered on 68 kb of mouse chromosome 7 and
associated with germ-cell differentiation
(Tanaka and Matsui, 2002
;
Lange et al., 2003
).
Both Grsf1 and Fragilis2 contain several putative
Wnt-responsive elements in inter- and intragenic regions (see Fig. S1 in
supplementary material). To test the hypothesis that Grsf1 and
Fragilis2 are components of the Wnt-signaling response, we first
monitored mRNA expression in ES cells that had been co-cultured in transwell
filters on top of Wnt1-expressing 3T3 fibroblasts
(Fig. 4A)
(Lickert et al., 2000).
Quantitative RT-PCR revealed that Wnt1 induced endogenous Grsf1 mRNA
expression in ES cells by twofold, but had no effect on Fragilis2
expression, which is already highly expressed in ES cells per se
(Fig. 4D). We then used in situ
hybridization analysis to test the mRNA expression level of both genes in
wild-type and CKO embryos at E7.5 (Fig.
4B,C). Consistent with the GeneChip experiments, the expression of
both genes is absent in the PS of CKO embryos, but weak Fragilis2
expression remained at the base of the allantois, where the primordial germ
cells (PGCs) are located. Because PGCs and ES cells share many molecular
markers and cellular properties, this might suggest that the regulation of
Fragilis2 expression in PGCs and ES cells is independent of
Wnt/ß-catenin, but is dependent on this signaling system in the paraxial
mesoderm emerging from the PS. This idea is further supported by transgenic
enhancer studies of a PGC-specific enhancer located in intron1 of
Fragilis2, which does not depend on two Wnt-responsive elements (data
not shown). Taken together with the co-expression of Grsf1 and
Fragilis2 in the regions of high Wnt activity, these results suggest
that both genes represent good candidate Wnt/ß-catenin target genes.
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Grsf1 and Fragilis2 knock downs recapitulate Wnt signaling phenotypes
Grsf1 shRNA-silenced ES cell-derived embryos did not show any
obvious phenotype at E7.5 (clone 5, n=7; clone 6, n=5). At
E9.5 (clone 5, n=10; clone 6, n=25), we consistently found
two phenotypes: (1) truncation of the posterior axis with formation of a large
allantois; and (2) abnormal mid/hindbrain development
(Fig. 5A). The phenotypes
observed were restricted to the expression domains of Grsf1 in the
posterior epiblast and in the mid/hindbrain region, suggesting that the
effects seen are specific for Grsf1 gene knock down
(Fig. 3A). Shortening of the
tailbud region was clearly seen at E8.5 (clone 5, n=6; clone 6,
n=8), when the mid/hindbrain region still looked morphologically
normal (Fig. 5B). The
abnormally large allantois is most likely a secondary effect due to the
truncation of the posterior axis and failure of chorio-allantoic fusion. A
more detailed histological analysis of the mutants at E9.5 revealed an
overgrowth of neurepithelium in the mid/hindbrain region of
Grsf1-silenced embryos (Fig.
5C). Loss of epithelial integrity was seen in the neural tube from
the level of the septum transversum in the midtrunk region of
Grsf1-silenced embryos and extending posteriorly
(Fig. 5D), which might be due
to secondary effects, because the neural tube rapidly degenerates following a
failure of allantoic placental development. To avoid studying a secondary
degeneration phenotype at this late stage, we analyzed the expression of
marker genes in knock-down embryos prior to the 13-somite stage. At the 7- to
8-somite stage, Grsf1-silenced embryos showed slightly reduced levels
of Fgf8 expression in the isthmus region, whereas expression in the
tailbud was strongly reduced (Fig.
5E) (Crossley and Martin,
1995). By the 10-somite stage, Grsf1-silenced embryos
showed barely detectable levels of Fgf8 in the midline of the
mid/hindbrain region and almost no expression in the lateral regions of the
neural tube (Fig. 5F). Expression of Gbx2 in rhombomeres (r) 1 and 2 of knock-down embryos
was strongly reduced at the 7- to 8-somite stage
(Fig. 5G), whereas the
hindbrain marker Krox20 appeared to be normally expressed in r3 and
r5 in these mutants (Fig. 5H).
Interestingly, Gbx2 expression in the tailbud appeared to be normal
(Fig. 5G), in contrast to the
reduced level of Fgf8 in this region
(Fig. 5E), which indicates that
the knock down of Grsf1 selectively alters the expression of marker
genes. Onset of expression of the potential ß-catenin target
Gbx2 (Fig. 2G) in the
epiblast seemed to be unaffected at head-fold stage
(Fig. 5I), indicating that
Grsf1 is important for maintaining Gbx2 expression at later
developmental stages in the mid/hindbrain region.
|
Fragilis2-silenced embryos also did not show any obvious phenotype
at E7.5 (clone 1, n=6; clone 2, n=4). Embryos analyzed
between E9.0 and E9.5 (clone 1, n=8; clone 2, n=13) revealed
problems in somite formation and a truncation of the posterior axis
(Fig. 6A). Similar to
Grsf1-silenced embryos, Fragilis2-silenced embryos also
developed a large allantois, presumably due to the posterior truncation. In
Fragilis2-silenced embryos at E8.5 (clone 1, n=12; clone 2,
n=8), the somites appeared hollow and were irregular in shape and
smaller in size (Fig. 6B, see
b',b''). Additionally, the neural tube appeared kinked, a phenotype
frequently seen in mutants affecting somite formation
(Conlon et al., 1995).
Histological analysis of the Fragilis2-silenced embryos at E8.5
revealed abnormalities in epithelialization and/or maintenance of epithelial
integrity of the somites (Fig.
6C). As the Fragilis gene family is implicated in PGC development
and Fragilis2 is expressed in this cell population, we stained
Fragilis2-silenced embryos for tissue non-specific alkaline
phosphatase (AP). No difference in the AP staining between wild-type and
knock-down embryos was observed, suggesting that the formation of PGCs was
normal at head-fold stage (Fig.
6D).
Wnt/ß-catenin signaling is implicated in both the formation of
paraxial mesoderm (Takada et al.,
1994; Galceran et al.,
1999
) and the subsequent segmentation of presomitic mesoderm into
somites (Auhlela et al., 2003; Galceran et
al., 2004
; Hofmann et al.,
2004
). To discriminate between possible defects in these two
processes in the Fragilis2 knock-down embryos, we analyzed T
and Tbx6, genes implicated in paraxial mesoderm formation, and
PAPC, a gene important for epithelialization of the somites
(Rhee et al., 2003
).
Expression of T and Tbx6 was unaffected in mutant embryos at
head-fold stage, but decreased in the tailbud region at E8.5
(Fig. 5E). Expression of PAPC
in wild-type embryos is restricted to two stripes at the anterior end of the
presomitic mesoderm corresponding to the next two presumptive somites
(Fig. 5E; somite 0 and
1). Analysis of Fragilis2 knock-down embryos revealed that
PAPC expression is decreased at head-fold stage and was barely detectable in
the two presomitic stripes at 10-somite stage
(Fig. 5E). Taken together,
these results demonstrate that knock down of Fragilis2 predominantly
affects the epithelialization of somites, and to a lesser extent the formation
of paraxial mesoderm, suggesting that Fragilis2 acts downstream of
Wnt/ß-catenin to regulate these processes.
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Discussion |
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|
The objective of target gene screens is not only to identify characterized
and functionally annotated genes, but also to add new players and their
respective function to the gene regulatory network. All the genes we tested,
whether well characterized or less well annotated, showed the expected
expression differences between wild type and ß-catenin mutants by in situ
hybridization. Thus, we expect that our dataset will provide a rich resource
for future data mining to characterize Wnt/ß-catenin pathways in
gastrulation. Ideally, the relative importance of target genes needs to be
tested by assessing their function during development. Using shRNA-mediated
gene silencing in ES cells and then in ES-derived embryos
(Kunath et al., 2003), we have
identified and characterized two novel putative Wnt/ß-catenin target
genes, Grsf1 and Fragilis2, whose expression is required for
normal development. Both genes, when knocked down, recapitulate specific but
distinct aspects of the conditional ß-catenin mutant phenotype,
implicating them as crucial downstream mediators of the Wnt/ß-catenin
signaling pathway.
The human ortholog GRSF1 is a sequence-specific RNA-binding protein, and
has been shown to act positively on translation in vitro and in a cell-culture
system (Park et al., 1999;
Kash et al., 2002
). This
raises the interesting possibility that Wnt induction of Grsf1 selectively
activates the translation of other mRNA transcripts in the primitive streak
and/or the mid/hindbrain region. Using a computational approach for predicting
possible target genes of Grsf1, we have screened the genes expressed at late
gastrulation stage according to our U74A Affymetrix wild-type data set. From
4694 annotated 5' UTRs in the ENSEMBL database, we found 386
non-redundant genes with at least one high-affinity Grsf1 consensus binding
site (5'-AGGGU-3'; see Table S3 in the supplementary material).
Interestingly, among these genes we found developmental regulatory factor
genes, such as T, Hoxb1, Hoxb8 and Frzb1, which are
co-expressed with Grsf1 at gastrulation stage. We also found genes
regulating cell proliferation, such as p53, cyclin B1, cyclin A2 and
Cdk2, and genes regulating apoptosis, Bcl2 and Bax,
as candidate Grsf1 target genes. In-depth analysis of these potential target
genes will be required to dissect the mechanisms by which Grsf1 regulates
mid/hindbrain development, posterior elongation and axial mesoderm
specification.
Importantly, the observed Grsf1 knock-down phenotypes remarkably
recapitulate distinct aspects of the CKO mutant phenotype and other Wnt
pathway mutants (Lickert et al.,
2002; McMahon and Bradley,
1990
; Thomas and Capecchi,
1990
; Brault et al.,
2001
), suggesting that Grsf1 is a crucial mediator of the
Wnt/ß-catenin signaling cascade. Interestingly, the lack of T
expression in the anterior primitive streak of Grsf1 knock-down
embryos is comparable to lack of T expression in Wnt3a mutants
(Yamaguchi et al., 1999
),
offering an explanation for the axis truncation in both mutants. The normal
expression of the Wnt/ß-catenin target genes, Cdx1 and
Grsf1, in Grsf1 knock-down embryos suggests that Grsf1 acts
downstream of the Wnt/ß-catenin signaling pathway selectively on target
mRNAs and is not involved in signal transduction, e.g. by stabilizing
components of the pathway. This might also be the case for mid/hindbrain
development, where Grsf1 is necessary for maintaining Fgf8
and Gbx2 expression, two factors important for the establishment of
the mid/hindbrain boundary. The comparison of putative mRNA targets of the
RNA-binding factor Grsf1 (see Table S3 in the supplementary material) with all
the deregulated genes from the ß-catenin target gene screen (Table S1 in
supplementary material) revealed several potentially coregulated transcripts
(see Table S4 supplementary material), which might explain similarities in the
Grsf1 and CKO mutant phenotypes.
Fragilis2 is expressed in the primitive streak, including the base
of the allantois, where the PGCs are localized at late gastrulation stage, and
in the paraxial and lateral mesoderm, as well as in the first forming somites
at E8.5. Studies in the immune system suggest a role for Fragilis2 (human
orthologs Leu13/9-27/IFITM1) as part of a transmembrane multiprotein signaling
complex implicated in inhibition of cell proliferation and homotypic cell
adhesion (Knight et al., 1985;
Deblandre et al., 1995
;
Sato et al., 1997
).
Histological analysis of Fragilis2-silenced embryos revealed a defect in
epithelialization of the somites, consistent with a function in homotypic cell
adhesion. Additionally, marker gene analysis revealed that Fragilis2
knock-down embryos show reduced expression of PAPC, a gene implicated in
somite epithelialization, and reduced expression of the paraxial mesoderm
markers T and Tbx6 at tailbud stage. These phenotypes are
very similar to the paraxial mesoderm and somite segmentation defects seen in
several different Wnt mutants (Lickert et
al., 2002
; Takada et al.,
1994
; Galceran et al.,
1999
; Aulehla et al.,
2003
; Galceran et al.,
2004
; Hofmann et al.,
2004
), thus it seems likely that Fragilis2 is a crucial
downstream mediator of the Wnt/ß-catenin signaling cascade in these
processes, mediating homotypic cell adhesion.
By using RNAi-mediated gene functional studies in ES cell-derived embryos, we have shown that it is possible to rapidly evaluate the relative importance of putative target genes of developmental pathways identified from expression profiling of mutant versus wild-type embryos. The potential for parallel functional analyses of several candidate genes in a relatively high throughput manner is an important component of genome-wide approaches to developmental genomics in the mouse.
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ACKNOWLEDGMENTS |
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Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/11/2599/DC1
* Present address: GSF National Research Center for Environment and
Health, Institute of Stem Cell Research, Neuherberg 85764, Germany
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