1 Research Institute of Molecular Pathology, Vienna Biocenter, Dr Bohr-Gasse 7,
1030 Vienna, Austria
2 McGill Cancer Centre, Biochemistry Department, McGill University, 3655
Promenade Sir-William-Osler, Montreal, Quebec, H3G 1Y6, Canada
3 Department of Molecular Biology, Genentech, 1 DNA Way, South San Francisco, CA
94080, USA
Author for correspondence (e-mail:
maxime.bouchard{at}mcgill.ca)
Accepted 22 March 2005
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SUMMARY |
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Key words: Mid-hindbrain development, Pax2-regulated genes, Sef, Tapp1, Ncrms, En2, Brn1, Fgf8 regulation, Mouse
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Introduction |
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The initiation of IsO development crucially depends on the transcription
factor Pax2 (Favor et al.,
1996; Brand et al.,
1996
), which shares similar DNA-binding and transactivation
functions with Pax5 and Pax8 of the same paired domain protein subfamily
(Kozmik et al., 1993
;
Dörfler and Busslinger,
1996
). Pax2 is the earliest known gene to be expressed
throughout the prospective mid-hindbrain region in late gastrula embryos
(Rowitch and McMahon, 1995
).
The initially broad expression pattern of Pax2 is progressively
refined to a narrow ring centered at the mid-hindbrain boundary by embryonic
day 9.5, while the related Pax5 and Pax8 genes are activated
in the same region at 3-4 and 6-7 somites, respectively
(Urbánek et al., 1994
;
Rowitch and McMahon, 1995
;
Pfeffer et al., 1998
).
Consistent with this sequential gene induction, mutation of the Pax2
gene leads to the loss of the midbrain and cerebellum in mouse and zebrafish
embryos (Favor et al., 1996
;
Brand et al., 1996
;
Bouchard et al., 2000
), whereas
the inactivation of Pax5 or Pax8 results in a mild
cerebellar midline defect or no brain phenotype at all
(Urbánek et al., 1994
;
Mansouri et al., 1998
). The
severe mid-hindbrain deletion is, however, only observed in
Pax2/ mouse embryos on the C3H/He genetic
background (Bouchard et al.,
2000
), where the compensating Pax5 and Pax8
genes fail to be activated at the mid-hindbrain boundary
(Pfeffer et al., 2000
;
Ye et al., 2001
) similar to
the Pax2.1 (noi) mutant embryos of the zebrafish
(Pfeffer et al., 1998
). In the
absence of Pax2, Otx2, Gbx2 and Wnt1 are normally
transcribed at early somite stages, while the expression of En1 is
reduced in the developing mid-hindbrain region
(Ye et al., 2001
).
Importantly, Fgf8 expression is never initiated at the mid-hindbrain
boundary of Pax2/ C3H/He embryos
(Ye et al., 2001
), resulting
in the complete absence of IsO activity and subsequent apoptotic loss of the
mid-hindbrain tissue starting at the 12-somite stage
(Pfeffer et al., 2000
;
Chi et al., 2003
).
To further investigate the role of Pax2 at the onset of mid-hindbrain development, we searched for novel Pax2-regulated genes by gene expression profiling of mid-hindbrain cells isolated by FACS sorting from wild-type and Pax2/ E8.5 embryos. This unbiased approach identified the En2, Brn1 (Pou3f3 Mouse Genome Informatics), Sef (Il17rd Mouse Genome Informatics), Tapp1 (Plekha1 Mouse Genome Informatics) and non-coding Ncrms genes as genetic Pax2 targets that are totally dependent on Pax2 function for their expression in the mid-hindbrain region. The transcription factors En2 and Brn1, as well as the signaling modifiers Sef and Tapp1, implicate Pax2 in the establishment of distinct transcriptional programs and the control of intracellular signaling during mid-hindbrain development. Biochemical and transgenic analyses demonstrated that Pax2 directly activates the mid-hindbrain-specific expression of Brn1 by interacting with two functional Pax2/5/8-binding sites in the promoter and an upstream regulatory element of the Brn1 gene. Moreover, ectopic expression of a dominant-negative Brn1 protein in chick embryos implicated Brn1 as a novel regulator of Fgf8 expression. The identification of new Pax2-regulated genes has thus provided important insight into the role of Pax2 in mid-hindbrain development.
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Materials and methods |
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FACS sorting and linear RNA amplification
The mid-hindbrain region of GFP+ E8.5 embryos from
Pax2+/ Pax2GFP intercrosses was
dissected with 26-gauge needles and dissociated into single cells at 37°C
for 15 minutes in 24-well plates containing 500 µl of 1% trypsin in PBS.
The reaction was stopped by transferring the single-cell suspension into 4 ml
of cold DMEM containing 10% fetal calf serum followed by centrifugation and
resuspension in phenol red-free DMEM containing 10% fetal calf serum and 1
µg/ml propidium iodide (PI). Live PI GFP+
cells of individual embryos were sorted with a FACSVantage TSO flow-cytometer
(Becton-Dickinson) directly into the Trizol Reagent (Gibco-BRL), vortexed for
1 minute and then stored in liquid nitrogen. This sorting protocol yielded
5000-10,000 GFP+ cells per embryo. Total RNA from selected samples
was submitted to linear amplification as described
(Hoffmann et al., 2003) with
some modifications. Briefly, the total RNA from a minimum of 5000 cells was
reverse-transcribed with an oligonucleotide consisting of d(T)15
linked to a T7 RNA polymerase recognition site. Following second-strand
synthesis, the samples were amplified by T7 polymerase-mediated in vitro
transcription. The resulting aRNA was reverse-transcribed using random nonamer
oligonucleotides [pd(N)9] and used for a second round of cDNA
synthesis and in vitro transcription. Two rounds of amplification from 5000
cells typically yielded 30 to 80 µg of aRNA.
cDNA microarray hybridization
The cDNA microarray screening was essentially performed as described
(Cheung et al., 1999). A
detailed description of the method used can be found as supplementary
information. Briefly, aRNA was reverse-transcribed into cDNA in the presence
of Cy3-dUTP or Cy5-dUTP using the Gibco-RT kit. The Cy3- and Cy5-labeled cDNA
probes were pooled and ethanol-precipitated together with poly-dA, tRNA and
mouse Cot.1 DNA. The precipitated cDNA probes were washed, prehybridized at
50°C for 1 hour in a solution containing 35% formamide, 4 xSSPE,
0.5% SDS, 5 x Denhardt's solution and 10 µg/ml denatured salmon sperm
DNA and then added to microarray slides for overnight hybridization at
50°C. Post-hybridization washes were carried out for 10 minutes in 0.2
xSSC, 0.1% SDS and 10 min in 0.2 xSSC. The slides were dried and
scanned using an Axon GenePix 4000 scanner. The hybridization results were
normalized using the marray-package of Bioconductor
(http://www.bioconductor.org)
and the `Print Tip Loess' algorithm (Yang
et al., 2002
). The cDNA microarrays contained 26,000 spotted EST
clones (11,000 BMAP clones from Research Genetics and 15,000 NIA clones from
the National Institute of Aging), which corresponded to 17,000 UniGene
clusters.
In situ hybridization
Embryos were dissected and processed for in situ hybridization with
digoxigenin-UTP-labeled RNA probes as described
(Henrique et al., 1995). The
En2 probe was previously described
(Davis et al., 1988
). The
Sef probe contained a 750 bp cDNA sequence extending from the PCR
oligonucleotide 5'-GGAGCCTGACTGGTTTGAGAA-3' to the NdeI
site. The Tapp1 and Ncrms probes were derived from the
identified ESTs (Tapp1, BC020017; Ncrms, BE655589). The
mouse Brn1 probe (643 bp) was cloned into the pGEM-Teasy plasmid
(Promega) following RT-PCR from E10.5 head cDNA using the primers
5'-GGGCAGAAGTCAAGGGAAGTG-3' and
5'-TGGCGTCGTCGGTGGAGAACA-3' and the chick Brn1 probe (429
bp) following RT-PCR amplification from chick embryo RNA with the primers
5'-ATGGT(G/C)CAGAG(C/T)GACTTCATGCAGGG-3' and
5'-GCT(C/T/G)AGCAT(G/A/T)CCGTT(C/T)AC(C/A)GTGAA-3'. The partial
chick Brn1 cDNA sequence was submitted to GenBank (Accession Number
DQ002393).
5'-RACE
The transcriptional start sites of the Brn1 gene were identified
by 5'-RACE, using the SMART RACE cDNA amplification kit (BD Bioscience)
according to the manufacturer's instructions. RNA isolated from the head of
E10.5 embryos was reverse-transcribed into cDNA with the
Brn1-specific primer 5'-GCTTCCACGGCAGCGGCGGCGGCAGCAG-3'
followed by PCR amplification with the oligonucleotides
5'-ACGGGAGACAACAAAGGACGAAGCGGTTCC-3' (outer) and
5'-GGAAGAAGAGTGCATTGGTGGAGGTGGAGA-3' (inner) in combination with
the primers provided with the RACE kit.
Electrophoretic mobility shift assay
The Pax2 protein was synthesized by coupled in vitro
transcription/translation and used for EMSA analysis with published
CD19 and Blnk oligonucleotide probes as described
(Kozmik et al., 1992;
Schebesta et al., 2002
). The
competitor fragments C, D and P were cloned by PCR with the following
primers:
The following double-stranded Brn1 oligonucleotides were used as competitor DNA:
Brn1 transgenes
lacZ transgenes were generated by insertion of a 3.2-kb
BglII-NotI fragment or a 6.2-kb AflII-NotI
fragment from the 5' flanking region of Brn1 into the
BglII-NotI sites of pTRAP-PL, which is a modified version of
pTRAP (Pfeffer et al., 2000)
lacking the minimal promoter. The mutant lacZ transgenes were
obtained by site-directed mutagenesis, using the QuikChange kit (Stratagene)
together with the following oligonucleotides:
Plasmid-free DNA of the transgene was injected into pronuclei followed by
the transfer of zygotes into pseudopregnant females. Transgenic embryos were
stained for ß-galactosidase activity as described
(Pfeffer et al., 2000).
In ovo electroporation
cDNAs for electroporation were cloned into the expression vector pCIE
containing a chick ß-actin promoter, a polylinker and an internal
ribosomal entry sequence (IRES) linked to a GFP gene. Full-length
mouse Otx2, chick Gbx2 and chick Pax2 constructs have been described
(Ye et al., 2001). VP16-Brn1
and EnR-Brn1 constructs were generated by fusing the POU homeodomain of rat
Brn1 (amino acids 301-497) in-frame C-terminal to the transcriptional
activator domain of VP16 or the repressor domain of Drosophila
Engrailed (amino acids 1-298), respectively. These constructs were
unilaterally electroporated into chick embryos at HH stage 8-10 as described
(Hynes et al., 2000
). Briefly,
DNA at 3-6 mg/ml was microinjected into the central canal of the neural tube,
and platinum electrodes flanking the neural tube delivered six square pulses
of 28 V with a duration of 40 mseconds and an interpulse interval of 45
mseconds. Two days later, chick embryos were analyzed by in situ
hybridization.
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Results |
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As the expression of mid-hindbrain-specific genes is upregulated during
somitogenesis (Wurst and Bally-Cuif,
2001), we determined the gene expression increase during
mid-hindbrain development and used this information, in addition to the
genotype comparison, as a second criterion for the identification of
Pax2-regulated genes. To this end, we prepared aRNA from
Pax2GFP-expressing mid-hindbrain cells of control
(wild-type or Pax2+/) embryos at the 0-2 somite and
8-9 somite stages. The different aRNAs were reverse-transcribed in the
presence of Cy3-dUTP or Cy5-dUTP, and the labeled cDNA probes were hybridized
to microarrays, which contain 26,000 cDNA clones corresponding to 17,000
UniGene clusters. Only genes with expression levels that were more than
fourfold above background were chosen for further analysis. Among the selected
13,200 ESTs, putative Pax2-regulated genes were identified (1) by an
expression ratio of more than 1.7 in at least one genotype comparison of
six-somite-stage embryos (103 ESTs) and (2) by an expression difference of
more than 2.0 between the 0-2 and 8-9 somite stages (168 ESTs). Selection
according to both criteria resulted in 12 candidate genes, including the known
Pax2 target gene Pax5 (Pfeffer et
al., 2000
). Six of these genes (shown in
Table 1) could subsequently be
validated as Pax2-activated genes by in situ hybridization analysis (see
below).
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|
The five genes En2, Brn1, Sef, Tapp1 and Ncrms are
expressed at E8.5 in a broad domain corresponding to the prospective midbrain
and anterior hindbrain (Fig.
2C,E,G,I,K), which reflects Pax2 expression at this
developmental stage (Fig. 2A).
At E9.5, the expression patterns of the putative Pax2-regulated genes have
started to diverge in the mid-hindbrain region, while novel expression domains
have emerged in other parts of the embryo. At this stage, only Ncrms
is expressed like Pax2 in a narrow stripe centered at the
mid-hindbrain boundary (Fig.
2B,L). En2 and Sef are broadly expressed in the
posterior midbrain and anterior hindbrain
(Fig. 2D,H), as previously
published (Davis et al., 1988;
Fürthauer et al., 2002
).
Tapp1 expression is observed in the anterior midbrain
(Fig. 2J), whereas
Brn1 is strongly expressed from the forebrain throughout the entire
midbrain to the hindbrain (Fig.
2F). In summary, these expression data suggest that Pax2 may
control the initiation, but not the maintenance of expression of the candidate
genes in the mid-hindbrain boundary region.
We directly tested this hypothesis by comparing the expression pattern of the five putative Pax2 target genes in wild-type and Pax2/ embryo at the five- to eight-somite stage (E8.5). All five genes failed to be expressed in the developing mid-hindbrain region of Pax2/ embryos (Fig. 3B,D,F,H,J), in contrast to stage-matched control embryos (Fig. 3A,C,E,G,I), while other expression domains remained unaffected by the Pax2 mutation. These results unequivocally demonstrate that Pax2 controls the mid-hindbrain-specific expression of En2, Brn1, Sef, Tapp1 and Ncrms during early somitogenesis.
|
Gene promoters are often located within CpG islands
(Antequera and Bird, 1999),
which are also present in the first 4 kb upstream of the Brn1 start
codon (Fig. 4A). The
PromoterInspector program (Scherf et al.,
2000
) identified elements D and P as potential promoter regions.
To test this possibility, we determined the start sites of Brn1
transcription by using primers located in the two putative 5'
untranslated regions for 5'-RACE analysis of E10.5 head RNA. PCR
fragments could readily be amplified with an element P primer
(Fig. 4B) in contrast to
element D sequences (data not shown). Cloning and sequence of 63 RACE products
demonstrated that the 5' ends of 41 clones clustered at five sites
spanning a 17 bp region within element P
(Fig. 4C). As expected for
heterogeneous transcription initiation, no TATA box could be found upstream of
these start sites, which are located between positions 1400 and
1383 relative to the initiation codon
(Fig. 6D). Taken together,
these results identified a single Brn1 promoter, giving rise to
heterogeneous transcription initiation in embryonic brain cells.
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|
Both Pax2-binding sites are essential for mid-hindbrain-specific expression of Brn1
We next investigated by transgenic analysis whether the Pax2-binding sites
Dd and Pc are important for Brn1 expression in the developing
mid-hindbrain region. For this, we generated the transgenes
3.2wt-lacZ and 6.2wt-lacZ by inserting 3.2 kb and 6.2 kb
5' flanking sequences of Brn1 (starting at position 59
relative to the ATG codon) upstream of a lacZ reporter gene.
Transgenic embryos were generated by pronuclear DNA injection and analyzed by
X-gal staining for lacZ expression at E9.5. The shorter
3.2wt-lacZ transgene was unable to drive reporter gene expression,
indicating that the promoter (P) and upstream element D with their
Pax2-binding sites are not sufficient for activating Brn1 expression
in the embryo (not shown). By contrast, the 6.2wt-lacZ transgene
containing the promoter (P) and all four conserved upstream elements (A-D)
gave rise to localized lacZ expression in the posterior forebrain
(diencephalon), mid-hindbrain boundary region and spinal cord
(Fig. 6A). This expression
pattern differs from that of the endogenous Brn1 gene at E9.5, as the
6.2wt-lacZ transgene failed to be expressed in the mesonephros and
throughout the entire forebrain-hindbrain region (compare
Fig. 2F with
Fig. 6A). Hence, the
6.2wt-lacZ transgene contains the control elements for initiating
Brn1 expression in the mid-hindbrain boundary region, while lacking
regulatory sequences for maintaining Brn1 expression throughout the
entire midbrain and hindbrain. To further study the role of the Pax2-binding
sites Dd and Pc in the initiation of mid-hindbrain-specific Brn1
expression, we mutated each Pax2 recognition sequence individually in the
6.2wt-lacZ transgene by introducing the two-nucleotide substitutions
that abrogate Pax2 binding (Fig.
5B-D). Both mutant transgenes, 6.2Ddm-lacZ and
6.2Pcm-lacZ, failed to be expressed above the detection limit in the
mid-hindbrain boundary region, although they gave rise to strong
ß-galactosidase staining in the diencephalon of the forebrain and along
the entire spinal cord. The two high-affinity Pax2-binding sites in the
promoter and upstream element D are therefore essential for initiating the
mid-hindbrain-specific expression of Brn1 during early somitogenesis.
These data unequivocally identify the Brn1 gene as a direct target of
Pax2 during mid-hindbrain development.
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Discussion |
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Control of intracellular signaling by Pax2
Fgf receptor stimulation by the IsO signal Fgf8 activates the
Ras/mitogen-activated protein kinase (MAPK) pathway
(Kouhara et al., 1997;
Corson et al., 2003
), which
controls cell proliferation and differentiation
(Marshall, 1995
). Fgf8
signaling also activates the expression of the transmembrane protein Sef
(similar expression to Fgf genes), which acts as a negative feedback regulator
to limit the duration of Ras-MAPK signaling by inhibiting the tyrosine
phosphorylation of Fgf receptors
(Fürthauer et al., 2002
;
Tsang et al., 2002
;
Kovalenko et al., 2003
). Here,
we have demonstrated that the expression of Sef fails to be induced
in the mid-hindbrain region of Pax2/ mouse
embryos, similar to Pax2.1 (noi) mutant embryos of the
zebrafish (Tsang et al.,
2002
). As Sef expression is also not activated at the
mid-hindbrain boundary of Fgf8 (ace) mutant embryos
(Fürthauer et al., 2002
),
it is possible that the absence of Sef transcripts in
Pax2/ embryos is an indirect consequence of
the failed induction of Fgf8 expression
(Ye et al., 2001
). However, it
is equally likely that Pax2 directly activates the Sef gene in
cooperation with Fgf signaling.
Pax2 also regulates the midbrain-specific expression of the tandem
PH-domain-containing protein 1 (Tapp1) gene. Tapp1 was identified as
an adaptor molecule that specifically binds to the lipid phosphatidylinositol
3,4-bisphosphate [PI(3,4)P2] via its C-terminal pleckstrin
homology (PH) domain (Dowler et al.,
2000). PI(3,4)P2 is generated by the inositol
5'-phosphatase SHIP from PI(3,4,5)P3, which in turn
is produced through phosphorylation of PI(4,5)P2 by the
phosphatidylinositol 3'-kinase (PI3K)
(Rohrschneider et al., 2000
).
Both PI3K and SHIP are activated by stimulatory and inhibitory tyrosine kinase
receptors, respectively, in agreement with the role of their second messengers
PI(3,4,5)P3 and PI(3,4)P2 in
recruiting different PH domain-containing effector proteins to the plasma
membrane (Rohrschneider et al.,
2000
). The PI(3,4,5)P3-dependent recruitment and
activation of the Akt/PKB, PDK1 and Btk kinases promotes cell survival and
proliferation (Rohrschneider et al.,
2000
). SHIP antagonizes these pathways by metabolizing the lipid
ligand PI(3,4,5)P3 of these kinases to
PI(3,4)P2, which functions as a membrane docking site for
adaptors such as Tapp1 (Dowler et al.,
2000
; Kimber et al.,
2002
; Marshall et al.,
2002
). Tapp1 is constitutively associated though its PDZ
domain-binding motif with the protein tyrosine phosphatase-like protein 1
(PTPL1/FAP1), which dephosphorylates receptors and adaptor proteins at the
plasma membrane, thus further inactivating PI3K signaling
(Kimber et al., 2003
). Hence,
the Pax2-dependent expression of Tapp1 may contribute to feedback inhibition
of PI3K signaling during midbrain development.
The non-coding RNA Ncrms
The Pax2-regulated gene Ncrms is transcribed into a non-coding RNA
that was initially identified because of its higher abundance in alveolar
rhabdomyosarcoma compared with the embryonic subtype of this pediatric muscle
tumor (Chan et al., 2002).
Recently, it has been shown that non-coding RNA genes are almost as prevalent
as protein-coding genes in the mammalian genome
(Cawley et al., 2004
). Among
these RNAs, the Ncrms transcript belongs with its size of 1.25 kb to
the family of long non-coding RNAs, which include the H19, Air and
Xist transcripts (Reik and
Walter, 2001
; Sleutels et al.,
2002
; Wutz et al.,
2002
). Analogous to the regulatory functions of these known
non-coding RNAs, it is conceivable that the Ncrms transcript is
involved in the control of mid-hindbrain-specific gene expression.
Pax2-dependent regulation of the En2 transcription factor gene
Pax2 also controls the expression of the transcriptional regulators En2 and
Brn1 in addition to the transcription factors Pax5 and Pax8 in the developing
mid-hindbrain region. These data indicate a key role for Pax2 in the
activation of distinct transcriptional programs at the onset of mid-hindbrain
development. The homeodomain protein En2 is required for normal development of
the cerebellum (Joyner et al.,
1991; Millen et al.,
1994
). Interestingly, a 1.0 kb enhancer of the En2 gene
contains two Pax2/5/8-binding sites that are essential for directing
lacZ transgene expression at the mid-hindbrain boundary
(Song et al., 1996
). However,
mutation of these two sites in the En2 locus only minimally affects
the initiation of endogenous En2 transcription
(Song and Joyner, 2000
). Our
observation, that the mid-hindbrain-specific expression of En2
completely depends on Pax2 function, points to the presence of yet
unidentified functional Pax2/5/8-binding sites that must lie outside of the
1.0 kb enhancer in the En2 locus. In contrast to En2, En1
expression is reduced but not absent at the mid-hindbrain boundary of
Pax2/ embryos
(Ye et al., 2001
). Hence, Pax2
acts upstream of En genes in the genetic cascade of mid-hindbrain development,
consistent with the fact that both En1 and En2 are not required for the
initiation, but for the maintenance, of mid-hindbrain-specific gene expression
(Liu and Joyner, 2001b
) in
marked contrast to Pax2 (Pfeffer et al.,
2000
; Ye et al.,
2001
) (this study). Moreover, the combined inactivation of
En1 and En2 results in a similar mid-hindbrain phenotype
(Liu and Joyner, 2001b
) as
mutation of Pax2 (Favor et al.,
1996
; Bouchard et al.,
2000
) in agreement with the regulation of both En genes by
Pax2.
|
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/11/2633/DC1
* These authors contributed equally to this work
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