RESEARCH ARTICLE
65
Development 138, 65-74 (2011) doi:10.1242/dev.058727
© 2011. Published by The Company of Biologists Ltd
Cdx1 refines positional identity of the vertebrate hindbrain
by directly repressing Mafb expression
Kendra Sturgeon1,2, Tomomi Kaneko1,*, Melissa Biemann1, Andree Gauthier1,
Kallayanee Chawengsaksophak1,† and Sabine P. Cordes1,2,‡
SUMMARY
An interplay of transcription factors interprets signalling pathways to define anteroposterior positions along the vertebrate axis.
In the hindbrain, these transcription factors prompt the position-appropriate appearance of seven to eight segmental structures,
known as rhombomeres (r1-r8). The evolutionarily conserved Cdx caudal-type homeodomain transcription factors help specify the
vertebrate trunk and tail but have not been shown to directly regulate hindbrain patterning genes. Mafb (Kreisler, Krml1,
valentino), a basic domain leucine zipper transcription factor, is required for development of r5 and r6 and is the first gene to
show restricted expression within these two segments. The homeodomain protein vHnf1 (Hnf1b) directly activates Mafb
expression. vHnf1 and Mafb share an anterior expression limit at the r4/r5 boundary but vHnf1 expression extends beyond the
posterior limit of Mafb and, therefore, cannot establish the posterior Mafb expression boundary. Upon identifying regulatory
sequences responsible for posterior Mafb repression, we have used in situ hybridization, immunofluorescence and chromatin
immunoprecipitation (ChIP) analyses to determine that Cdx1 directly inhibits early Mafb expression in the neural tube posterior
of the r6/r7 boundary, which is the anteriormost boundary of Cdx1 expression in the hindbrain. Cdx1 dependent repression of
Mafb is transient. After the 10-somite stage, another mechanism acts to restrict Mafb expression in its normal r5 and r6 domain,
even in the absence of Cdx1. Our findings identify Mafb as one of the earliest direct targets of Cdx1 and show that Cdx1 plays a
direct role in early hindbrain patterning. Thus, just as Cdx2 and Cdx4 govern the trunk-to-tail transition, Cdx1 may regulate the
hindbrain-to-spinal cord transition.
INTRODUCTION
Early anteroposterior (AP) patterning of vertebrates occurs through
the interplay of transcription factors that interpret fibroblast growth
factor (Fgfs), cysteine-rich secreted Wnt glycoprotein and retinoic
acid (RA) signalling pathways. These transcription factors prompt
the position-appropriate appearance of segmental and, thereby,
segmentally derived structures: the rhombomeres in the hindbrain,
which give rise to specific cranial motor nerves, and the somites,
from which vertebra in the trunk and tail are derived. Hindbrain
segmentation has been a particularly useful tool for understanding
early AP neural patterning. The developing hindbrain is segmented
early in embryogenesis along its AP axis into seven to eight
lineage-restricted compartments, the rhombomeres (Lumsden,
2004; Lumsden and Krumlauf, 1996; Schneider-Maunoury et al.,
1998; Tumpel et al., 2009). The first seven segments (r1-r7) form
from the midbrain/hindbrain boundary to the first somite pair, and
pseudo-rhombomere eight (r8) forms next to somites two to five
(Lumsden, 2004; Schneider-Maunoury et al., 1998). Many gene
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue,
Toronto, ON M5G 1X5, Canada. 2Department of Molecular Genetics, University of
Toronto, 1 King’s Crescent, Toronto, ON M5S 1A8, Canada.
*Present address: Department of Legal Medicine, School of Medicine, Tokyo
Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
†
Present address: Division of Genetics and Developmental Biology, Institute for
Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia
‡
Author for correspondence ([email protected])
Accepted 25 October 2010
expression patterns, including those of Hox genes, the most studied
segmentation genes, respect rhombomere boundaries and are
limited to specific subsets of rhombomeres (Lumsden, 2004;
Lumsden and Krumlauf, 1996; Schneider-Maunoury et al., 1998;
Tumpel et al., 2009). Their physical compartmentalization and
defined rhombomere-specific gene expression patterns facilitate
identification of regulatory regions and, thereby, molecules
required for establishment, maintenance and refinement of the
positional identity of individual rhombomeres. This provides an
exceptional model for the study of vertebrate AP patterning.
RA, Fgfs and Wnts have all been implicated in AP patterning of
the brain (Diez del Corral and Storey, 2004; Nordstrom et al., 2006).
Many studies have identified RA, a product of vitamin A (retinol)
metabolism, as the dominant posteriorizing factor in hindbrain
development (reviewed in Glover et al., 2006; McCaffery et al.,
2003). It is thought that the RA gradient is set up by a posterior
source and an anterior sink. The source of RA is provided by
retinaldehyde dehydrogenase 2 (Raldh2), which synthesizes RA in
the presomitic posterior mesoderm that flanks the future r7, r8 and
spinal cord. The sink is provided by RA-catabolizing enzymes,
cytochrome P450s (Cyp26s), expressed in the floor plate region of
the forebrain and midbrain (Dupe and Lumsden, 2001;
Niederreither et al., 2000; Wendling et al., 2001). RA can act
directly to induce transcription of some segmentation genes via RA
receptors (RARs) and the retinoid X receptors (RXRs), which are
ligand-dependent transcription factors that bind as homo- or
heterodimers to specific sequences, known as RA response elements
(RAREs). RAREs have been found to govern the hindbrain-specific
expression of some Hox genes, such as Hoxa1, Hoxb1, Hoxb4 and
DEVELOPMENT
KEY WORDS: Caudal related proteins (Cdx), Mafb (Kreisler, valentino), Axial patterning, Developmental gene regulation, Hindbrain
patterning, Rhombomere
RESEARCH ARTICLE
Hoxd4 (Frasch et al., 1995; Gould et al., 1998; Langston and Gudas,
1992; Marshall et al., 1994; Moroni et al., 1993; Nolte et al., 2003;
Popperl and Featherstone, 1993; Studer et al., 1998; Whiting et al.,
1991). These, along with many other studies performed in the chick,
quail, mouse, fish and frog have identified RA as a posteriorizing
factor in vertebrate hindbrain patterning.
In response to the RA gradient, initial segmentation genes are
expressed in subsets of prospective rhombomeres and induce a
molecular cascade that promotes further refinement of regionspecific expression patterns and segment identity (Lumsden and
Krumlauf, 1996; Schneider-Maunoury et al., 1998). One of the
earliest genes to be expressed segmentally in the posterior hindbrain
is Mafb [previously known as kreisler, Krml1, valentino (Val)], a
basic leucine zipper transcription factor (Cordes and Barsh, 1994).
Mafb is expressed in the future rhombomere five and six (r5-r6)
domain and is required for formation of these rhombomeres (Cordes
and Barsh, 1994; Frohman et al., 1993). The original X-ray-induced
Mafb mouse mutation, kreisler (kr), is a near-perfect chromosomal
inversion that abolishes rhombomere-specific Mafb expression
(Cordes and Barsh, 1994). In kr/kr mutants and in mouse embryos
homozygous for the severely hypomorphic or null allele krenu, the
r5-r6 domain does not develop properly, Hox genes expressed in the
hindbrain are misregulated, and morphological segmentation in the
posterior hindbrain (r4-r7) is lost (Cordes and Barsh, 1994; Frohman
et al., 1993; McKay et al., 1994; Sadl et al., 2003). Mafb has been
shown to directly activate rhombomere-specific expression of
Hoxa3 and Hoxb3 (Manzanares et al., 1999; Manzanares et al.,
1997; Yau et al., 2002), but whether it directly regulates other Hox
genes is unknown.
Expression of Mafb is regulated positively and negatively by RA
(Dupe and Lumsden, 2001; Gavalas and Krumlauf, 2000; GrapinBotton et al., 1998; Hernandez et al., 2004). Mafb activation in the
hindbrain depends on RA. For example, application of an RAR
pan-agonist at the 0-somite stage, eliminates Mafb expression in
mouse and chick embryos. In mouse embryos that are Raldh2–/–
or RAR–/–;RAR–/–, Mafb expression is lost completely
(Niederreither et al., 2000; Wendling et al., 2001). RA levels also
control the position of the posterior boundary of Mafb expression.
So, reducing RA signalling in the posterior hindbrain and neural
tube allows Mafb expression to expand further posteriorly beyond
the r6/r7 boundary. For instance, in Raldh2 mutant zebrafish, RA
signalling is decreased (Grandel et al., 2002), and embryos show
posterior expansion of Mafb (val) (Grandel et al., 2002; Maves and
Kimmel, 2005). These results suggest that Mafb induction by RA
is concentration dependent: higher RA levels in the spinal cord and
r7-r8 suppress it, whereas lower ones activate it in r5-r6.
RA-dependent activation of Mafb depends, at least in part, on
the homeodomain protein, variant hepatic nuclear factor 1
(vHnf1/HNF1) (Aragon et al., 2005; Hernandez et al., 2004; Kim
et al., 2005; Lecaudey et al., 2004; Maves and Kimmel, 2005;
Wiellette and Sive, 2003). At the 0-somite stage, Hnf1b expression,
which is induced by RA, extends from the future r4/r5 boundary
posteriorly into the posterior hindbrain and spinal cord of mouse,
chick and zebrafish embryos (Aragon et al., 2005; Kim et al., 2005;
Lecaudey et al., 2004; Maves and Kimmel, 2005). In the mouse,
identification of the r5-r6 specific S5 enhancer from Mafb
illustrated that vHNF1 is essential, but not sufficient, for driving
r5-r6 expression in 0- to 10-somite-stage embryos. In vHNF1–/–
zebrafish, Mafb(val) is not expressed (Sun and Hopkins, 2001).
Treatment of zebrafish with an RAR panagonist leads to loss of
vHnfl and Mafb(val) expression, but Mafb(val) expression can be
restored through the addition of exogenous Hnf1b (Hernandez et
Development 138 (1)
al., 2004). Thus, vHNF1 directly regulates RA-dependent, r5-r6specific Mafb activation. However, although the anterior expression
limits of Hnf1b and Mafb coincide at the r4/r5 boundary, Hnf1b
expression extends more posteriorly in the neural tube than Mafb
and, therefore, does not establish the posterior boundary of Mafb
expression (Kim et al., 2005). The initial posterior boundary of
Mafb expression could be established by two simple mechanisms:
(1) An unknown activator, expressed in r5 and r6, but not
posteriorly of the r6/r7 boundary, collaborates with vHNF1 to
activate Mafb in r5-r6; or (2) a repressor with an anterior
expression limit at the r6/r7 boundary inhibits Mafb expression in
the posterior neural tube (Kim et al., 2005). Because the earliest
genes expressed in the hindbrain have well-defined anterior
expression boundaries and show diffuse expression posteriorly
(Cordes, 2001; Deschamps et al., 1999; Hunt and Krumlauf, 1992)
and Mafb is the earliest gene confined to r5-r6, we hypothesized
that a repressive mechanism might be likely. Given the absence of
RARE elements in the S5 enhancer, such a repressive mechanism
might depend directly on an RA-inducible transcription factor(s).
The RA-responsive caudal-type homeodomain transcription
factors, which have conserved roles in posterior axial patterning of
animals, are candidate Mafb repressors (Young and Deschamps,
2009). In the early Drosophila embryo, Caudal protein is present
in a posterior to anterior gradient and initiates expression of
posterior gap and pair rule genes that establish embryonic
segmentation and induce Hox gene expression (Hader et al., 1998;
La Rosee et al., 1997; Ochoa-Espinosa et al., 2005; Rivera-Pomar
et al., 1995). In the mouse, there are three Cdx genes, Cdx1, Cdx2
and Cdx4, that are expressed in a stepwise manner along the AP
axis of the embryo, with Cdx1 being the most anterior, followed by
Cdx2, then by Cdx4 (Beck et al., 1995; Gamer and Wright, 1993;
Meyer and Gruss, 1993). Although their anterior limits are
staggered, they are all expressed into the posterior of the embryo,
essentially creating a posterior-to-anterior gradient of Cdx protein
(Gaunt et al., 2003; Gaunt et al., 2005). This nested pattern of
expression is highly conserved across vertebrates (Charite et al.,
1998; Deschamps et al., 1999; Gaunt et al., 2005; Houle et al.,
2000; Lohnes, 2003; Savory et al., 2009a). Cdx genes are regulated
by signalling molecules involved in AP patterning, including RA,
Fgfs and Wnts (Gaunt et al., 2005; Keenan et al., 2006; Lohnes,
2003; Pilon et al., 2006). In the mouse, RA directly regulates Cdx1
through an upstream RARE (Houle et al., 2000). Once Cdx1
expression is initiated, Wnt3a, the expression of which overlaps
with that of Cdx1, acts to maintain Cdx1 expression through two
response elements for the lymphoid enhancer-binding factor
(LEF)/T-cell factor (TCF), the nuclear interpreters of canonical
Wnt signaling (Ikeya and Takada, 2001; Prinos et al., 2001). Cdx1
binds as a heterodimer with LEF1 at the LREs to maintain its own
expression (Beland et al., 2004). Loss of the RARE and the LREs
strongly reduces Cdx1 expression (Pilon et al., 2007), indicating
that RA and Wnt3a signalling act synergistically to regulate Cdx1
expression (Beland et al., 2004; Lohnes, 2003; Pilon et al., 2007;
Prinos et al., 2001). Experiments in Xenopus and chick indicate that
Cdx1 is also induced by Fgfs (Bel-Vialar et al., 2002; Keenan et al.,
2006). Thus, Cdx1 responds to and integrates these signalling
pathways during AP patterning (Young and Deschamps, 2009).
Cdx proteins specify the vertebrate trunk and tail by activating
posteriorly expressed Hox genes. For example, loss of Cdx genes
causes a posterior shift in Hox gene expression and anterior
transformations of vertebra in Cdx1–/– and Cdx1–/–; Cdx2+/– mouse
embryos (Allan et al., 2001; Subramanian et al., 1995; van den
Akker et al., 2002). The homeotic transformations seen in Cdx1–/–
DEVELOPMENT
66
Cdx1 directly regulates hindbrain patterning
MATERIALS AND METHODS
Mice
Transgenic mice were generated by pronuclear injection into fertilized
oocytes from superovulated mice from the FVB/NJ strain. The 240 bp
containing the predicted Cdx1 sites (S5-LacZ) was removed from the S5
regulatory element by digestion with XbaI and partial digestion with BglII
and, after filling in, was cloned into the NotI site of the hsp68LacZ
expression construct. The S5Hox mutation was introduced using
Quickchange Site directed mutagenesis (Stratagene, La Jolla, CA, USA)
using the oligonucleotides: 5⬘-GTATCCCCCAATCATAAAGTTATCAGGCCTAGGACTTCATAACTGTAATTTTGC-3⬘ and 5⬘-GCAAAATTACAGTTATGAAGTCCTAGGCCTGATAACTTTATGATTGGGGGATAC-3⬘ to generate the S5Hoxmut-LacZ reporter. For microinjection, SalI
digestion followed by DNA electrophoresis separated inserts from vector
sequences, and inserts were purified using GeneClean beads (Bio101,
Vista, CA). LacZ expression was analyzed in embryos from two
67
independent transgenic lines. Embryos were stained for LacZ as previously
described (Whiting et al., 1991). Transgenic lines were maintained by
crossing with CD1 mice.
Cdx1–/– mice, first described by Subramanian et al. (Subramanian et al.,
1995), were a generous gift from D. Lohnes (University of Ottawa) and
P. Gruss, and were maintained by intercrossing. Cdx2–/– embryos were
generated by tetraploid aggregation of Cdx2–/– embryonic stem cells
(Chawengsaksophak et al., 2004; Nagy et al., 2010) and harvested at E8.0E8.5. All animal husbandry and embryo harvesting were performed
according to Canadian Council on Animal Care guidelines and approved
by the Samuel Lunenfeld Research Institute animal care committee.
Immunofluorescence and RNA in situ hybridization analyses
RNA in situ hybridization and LacZ staining were performed as previously
described (Kim et al., 2005). Images were captured with QImaging
MicroPublisher 3.3 RTV.
Immunofluorescence was performed on 10 mM frozen sections prepared
as previously described (Sadl et al., 2003). Imaging was performed on
Nikon Eclipse 80i fluorescence microscope with Nikon DS-Ri1 Camera.
Electrophoretic mobility shift assays
Mouse Cdx1 was in vitro transcribed and translated from the
Cdx1/pRcCMV plasmid, provided by J. Rossant, using the TNT
reticulolysate system (Promega). DNA-binding reactions using in vitro
transcribed and translated Cdx1 protein and radiolabelled Cdx1-S5A,
Cdx1-S5B or Cdx1-S5C double-stranded oligonucleotides were performed
and resolved by gel electrophoresis as described by Kim et al. (Kim et al.,
2005). The forward strand oligonucleotide sequences used in these
experiments were: Cdx1-S5A: 5⬘-CCAATCATAAAGTTATTTTTATTGC-3⬘, Cdx1-S5A-Mut1: 5⬘-CCAATCATGCCGTTATTTTTATTGC-3⬘,
Cdx1-S5A-Mut2: 5⬘-CCAATCATAAAGTTATTGCGATTGC-3⬘, Cdx1S5A-Mut1+2: 5⬘-CCAATCATGCCGTTATTGCGATTGC-3⬘, Cdx1-S5B:
5⬘-GCTACTTCATAACTGTAATTTTGC-3⬘, Cdx1-S5B-Mut1: 5⬘-GCTACTTCAGCCCTGTAATTTTGC-3⬘, Cdx1-S5B-Mut2: 5⬘-GCTACTTCATAACTGTGCCTTTGC-3⬘, Cdx1-S5B-Mut1+2: 5⬘-GCTACTTCAGCCCTGTGCCTTTGC-3⬘, Cdx1-S5C: 5⬘-TTGCTTTTGTTATAAACCATAAGGT-3⬘, Cdx1-S5C-Mut1: 5⬘-TTGCTTTTGTTGCGGCCATAAGGT-3⬘,
Cdx1-S5C-Mut2: 5⬘-TTGCTTTTGTTATAAACCAGCCGGT-3⬘, Cdx1S5C-Mut1+2: 5⬘-TTGCTTTTGTTGCGGCCAGCCGGT-3⬘, Cdx-Sif1: 5⬘TGAAGCAATAAACTTTATGGCCGGA-3⬘, Cdx-MutSif1: 5⬘-TGAAGCAATGCCCTTGCGGGCCGGA-3⬘.
ChIP on embryos
ChIP was performed on sets of ~150 embryos at the 0- to 10-somite stage
that had been fixed in 4% paraformaldehyde and stored in 100% methanol
according to a protocol provided by D. Lohnes and previously described
by Pilon et al. (Pilon et al., 2006). To verify IP of Cdx1 protein, western
blots were performed on a tenth of each IP using standard protocols.
Remaining samples were eluted from agarose beads; formaldehyde crosslinks were reversed. Samples were purified using Qiagen QIAquick PCR
Purification Kit. Real-time PCR was performed on the Applied Biosystems
7900HT Real time PCR system. Samples were prepared using SYBR
Green PCR Master Mix (Applied Biosystems, Foster City, CA) with a total
volume of 20 l and were run for a total of 40 cycles with an annealing
temperature of 60°C. Each experiment was performed a minimum of twice
from the embryo stage and PCRs were performed in triplicate. ChIP
assays were analyzed by a two-way ANOVA using GraphPad Prism 4.0
(GraphPad Software, La Jolla, CA). ChIP values with IgG and with Cdx1
antibody were entered as percent of input for two separate IP reactions. The
ANOVA test compared each group with every other group followed by a
Bonferroni post-hoc test. Primers used were: Cdx1-autoF 5⬘-GAGGAGTCGGAACCCCAAGCTG-3⬘, Cdx1-autoR 5⬘-GCGGCTTTGCATTTCAAAGCGG-3⬘, S5–2kb F 5⬘-CCACCGAAGGTCAGTCTCCAAA-3⬘,
S5–2kbR 5⬘-AGAGGTCTGTCCTGTCCTTCAG-3⬘, S5F 5⬘-CTCCTTTCTCTTCCTGTTTCTCTG-3⬘, S5R 5⬘-CACTACCTCTGCTGTAGAGTGCAT-3⬘,
S5+2kbF
5⬘-AGCCTCATTGATATGGGCTGGA-3⬘,
S5+2kbR 5⬘-ATCTCAGGGCAGGGTCATCACA-3⬘, krprF 5⬘-ATCGCTAAAAGCGAGGCTCAG-3⬘, krprR 5⬘-CTAGAGCCAAAGAGTCGGAGAG-3⬘.
DEVELOPMENT
Cdx2+/– mice are more severe than either of the single mutants,
indicating an overlapping role in vertebral patterning (van den
Akker et al., 2002). Conversely, ectopic or overexpression of Cdxs
expands trunk regions at the expense of the posterior hindbrain in
Xenopus, zebrafish and mice (Epstein et al., 1997; Gaunt et al.,
2008; Isaacs et al., 1998; Shimizu et al., 2006; Skromne et al., 2007;
Young et al., 2009). For example, ectopic expression of Cdx1a or
Cdx4 in the zebrafish eliminates expression of posterior hindbrain
specific markers, including Mafb (val) and the Mafb target Hoxb3
(Shimizu et al., 2006; Skromne et al., 2007). These and other
observations suggest that Cdx proteins might activate trunk-specific
Hox genes and may also repress Mafb in the hindbrain. Of course,
such repression of hindbrain character could be a secondary
consequence of misexpressing trunk-specific Hox genes.
Cdx proteins might transduce AP positional information to Hox
and other segmentation genes in several ways. First, in the mouse,
Cdx proteins have been shown to directly regulate some trunkspecific Hox genes, including Hoxa5 (Tabaries et al., 2005), Hoxa7
(Gaunt, 2001), Hoxb7 (Charite et al., 1998; Subramanian et al.,
1995), Hoxc8 (Shashikant et al., 1995) and Hoxc9 (Papenbrock et
al., 1998). Alternatively, Cdxs might regulate another layer of
transcription factor(s), such as vertebrate equivalents of the ‘Gap’
genes in the fly, which then govern Hox gene expression directly
(Rivera-Pomar and Jackle, 1996). Finally, Cdxs can affect Hox
gene expression indirectly by regulating RA and Wnt3a signalling.
For example, in Cdx2–/–; Cdx4–/+ mouse embryos, the RAdegrading enzyme Cyp26a1 is downregulated, RA localization is
shifted posteriorly and Wnt3a expression is also decreased (Young
et al., 2009). So, Cdx proteins might affect Mafb expression by
altering RA signalling.
Based on the role of Cdx1 in segmental skeletal development,
its expression at the appropriate region of the hindbrain, its
proposed role as a transducer of RA signalling, and given that Mafb
expression is highly sensitive to positive and negative regulation
via RA, we hypothesized that Cdx1 might repress Mafb, thereby
establishing the posterior boundary of Mafb expression in the
hindbrain. Here we show that Cdx1 is present at the right time and
place to act as a posterior repressor of Mafb; that Cdx1-binding
sites are present in a regulatory element required to repress reporter
gene expression in the posterior hindbrain and spinal cord. Mafb
expression is shifted posteriorly in Cdx1–/– embryos. Chromatin
immunoprecipitation (ChIP) experiments demonstrate that in
embryos at embryonic day 8.0-8.5 (E8.0-E8.5) Cdx1 binds within
the Cdx1-binding site containing S5 enhancer. Together, these data
reveal that Cdx1 acts as an early posterior repressor of Mafb and,
thus, plays a direct role in refining posterior hindbrain identity.
RESEARCH ARTICLE
RESEARCH ARTICLE
RESULTS
A Cdx-binding-site-containing region restricts
posterior S5-LacZ reporter expression
To address the molecular mechanisms that act to exclude Mafb
expression from the neural tube posterior of the r6/r7 boundary in
the hindbrain, we analyzed the early-acting, rhombomere-specific
S5 enhancer from the Mafb gene, which directs expression of a
LacZ reporter in r5-r6 in 0- to 10-somite-stage embryos (Fig. 1A,B)
(Kim et al., 2005). Upon dissection of this regulatory region, we
found that removing a 240 bp sequence from S5 (S5-LacZ)
resulted in expansion of LacZ expression past the r6/r7 boundary
into the posterior neural tube in 0- to 12-somite-stage embryos
from two out of two transgenic lines (Fig. 1C,D). These
observations suggest that binding site(s) for a repressor may reside
within this region.
Analysis of the transcription-factor-binding sites within the
deleted segment identified multiple candidate Cdx-binding sites
and one candidate Hox/Pbx-complex-binding site, referred to as
S5Hox (Fig. 1F). Hox proteins achieve high-affinity DNA binding
by forming heterodimers with the Pbx family of transcription
factors (Chan et al., 1997; Monica et al., 1991). Such Hox/Pbx
complexes have been shown to directly govern some rhombomerespecific genes (Gould et al., 1997; Popperl et al., 1995; Saleh et al.,
2000). Hoxb4 and Hoxd4 genes show expression up to the r6/r7
boundary in E8.5 embryos (Brend et al., 2003; Chan et al., 1997;
Folberg et al., 1999; Gould et al., 1998; Morrison et al., 1997;
Zhang et al., 2000; Zhang et al., 1997), when the posterior
boundary of Mafb expression is first established, and thus, might
be candidate Mafb repressors. To test possible involvement of the
S5Hox site in Mafb repression, we introduced mutations, which
had been shown to eliminate Hox/Pbx binding in other studies, to
generate the S5Hox-mut reporter (Di Rocco et al., 1997; Gould et
al., 1997; Kutejova et al., 2005; Serpente et al., 2005). In mouse
embryos, from three out of three lines carrying the S5Hox-mut
transgene, LacZ expression was still restricted to r5 and r6 (Fig.
1E). A few speckles of LacZ expression were observed posterior of
the r6/r7 boundary, but an equivalent amount of staining was also
seen in the S5-LacZ reporter (Fig. 1B) and in embryos carrying an
enhancerless hsp86-LacZ reporter (data not shown). Thus, Hox/Pbx
complexes do not play a significant role in directly establishing the
posterior boundary of Mafb expression via the S5Hox site. Of
course, these findings do not exclude the possibility that Hox
proteins could influence Mafb repression by unconventional sites.
Because the 240 bp deleted region contains multiple consensus
Cdx-binding sites (Margalit et al., 1993) (Fig. 1F,G) and such
arrays of Cdx-binding sites have been shown to regulate Hox gene
expression within the trunk (Tabaries et al., 2005; Gaunt, 2001;
Charite et al., 1998; Subramanian et al., 1995; Shashikant et al.,
1995; Papenbrock et al., 1998), we tested whether any of these
could bind Cdx proteins in vitro in electrophoretic mobility shift
assays (EMSAs). In vitro Cdx proteins are thought to preferentially
bind as homo- or heterodimers to two closely juxtaposed Cdx sites.
Hence we refer to pairs of juxtaposed Cdx sites as homodimer
sites. Out of four predicted Cdx homodimer binding sites, three
homodimer sites, which we named Cdx1-S5A, Cdx1-S5B and
Cdx1-S5C, bound in vitro transcribed-translated Cdx1 protein and
did so specifically. Cdx1 binding could be competed away by
addition of a double-stranded oligonucleotide containing an
established Cdx1-binding site from the Sif1 gene (Suh et al., 1994)
or the Cdx1-S5A, Cdx1-S5B or Cdx1-S5C sites themselves (Fig.
2A-C: lanes 3-6). Mutating a single Cdx site within each
homodimer binding site did not abrogate its ability to compete for
Development 138 (1)
Fig. 1. Loss of a sequence containing predicted Cdx sites
abolishes the posterior r6/r7 boundary of S5 enhancerdependent LacZ expression. (A)Mafb expression is detected in r5-r6
of 0- to 12-somite-stage embryos by whole-mount RNA in situ
hybridization. (B)The 2 kb S5 enhancer from the Mafb gene drives LacZ
expression in r5-r6. (C,D)Removal of a 240 bp sequence from the S5
enhancer (Cdx-S5-LacZ) causes LacZ expression to expand throughout
the posterior neural tube in transgenic embryos from two out of two
transgenic founder lines. (C)High magnification view of a Cdx-S5-LacZ
embryo; (D) full embryo view. (E)Embryos transgenic for the
S5Hoxmut-LacZ reporter show LacZ expression in r5-r6. (F)The 240 bp
sequence deleted from the S5 enhancer in Cdx-S5-LacZ mice contains
three predicted Cdx homodimer-binding sites, indicated in bold red
type and labelled Cdx1-S5A, Cdx1-S5B and Cdx1-S5C. A yellow box
denotes the predicted Hox/Pbx consensus site, and the point mutations
introduced to eliminate possible Hox/Pbx binding and generate the
S5Hox-mut transgenic lines is shown. Consensus Cdx half-binding sites
are indicated in bold type underneath the three CDX-S5 homodimer
sites. (G)The Cdx consensus site, as determined by Margalit et al.
(Margalit et al., 1993) is shown.
Cdx1 binding (Fig. 2A-C: lanes 7-10). However, simultaneously
mutating both single binding sites in each of the Cdx1-S5A, Cdx1S5B or Cdx1-S5C oligonucleotides eliminated the ability of these
oligonucleotides to effectively compete for Cdx1 binding (Fig. 2AC: lanes 11, 12). Competition with oligonucleotides containing a
mutated SIF1 site, that had previously been shown to no longer
bind Cdxs, had no effect on Cdx1 binding to any of the S5-Cdx
sites (data not shown). These three Cdx homodimer sites were also
bound specifically by Cdx2 and Cdx4 in EMSA assays and testing
overlapping fragments spanning the 240 bp region demonstrated
that Cdx binding localized only to the 70 bp containing these three
Cdx homodimer sites (data not shown). Thus, Cdx proteins bind
specifically to all three S5-Cdx homodimer sites in vitro and these
sites may be involved in restricting Mafb expression.
The anterior boundary of Cdx1 and posterior
boundary of Mafb expression coincide
In order for Cdx1 to establish the posterior boundary of Mafb
expression Cdx1 protein must be present in the hindbrain at the
r6/r7 boundary and more posteriorly early in embryogenesis.
Previous reports determined that Cdx1 is present in the posterior
hindbrain from the 1- to 10-somite stages (E8.0-E8.75) (Houle et
al., 2000; Meyer and Gruss, 1993), a period that coincides with the
presence of Hnf1b expression and Mafb induction (Kim et al.,
DEVELOPMENT
68
Cdx1 directly regulates hindbrain patterning
RESEARCH ARTICLE
69
Fig. 2. Cdx1 protein binds to three predicted Cdx homodimer-binding sites from the 240 bp deleted S5 sequence in vitro. (A-C)EMSA
using in vitro transcribed-translated Cdx1 protein detect binding to oligonucleotides containing each of three predicted Cdx1 homodimer-binding
sites: (A) Cdx1-S5A, (B) Cdx1-S5B and (C) Cdx1-S5C. Adding 10- or 100-fold molar excess of the SIF1 oligonucleotide containing the known Cdx1binding sites from SIF1 gene (lanes 3,4) abolished binding, as did competition with 10- or 100-fold excess of Cdx-S5A (A, lanes 5,6), Cdx1-S5B
(B, lanes 5,6) and Cdx1-S5C (C, lanes, 5,6). Oligonucleotides containing mutations within in only one half binding site still effectively competed for
binding (lanes 7-10). However, oligonucleotides, in which both Cdx1 half binding sites were mutated, could no longer compete for Cdx1 binding
(lanes 11,12). (D)Sequences of the predicted normal and mutated Cdx1-S5A, Cdx1–S5B and Cdx1-S5C binding sites are shown.
Posterior expansion of Mafb expression in Cdx1–/–
embryos
If the posterior extension of LacZ staining seen in the S5-LacZ
reporter were due to the loss of Cdx1-binding sites then, in the
absence of Cdx1 protein, posterior expansion of Mafb expression
should be observed. To test this hypothesis, we performed in situ
hybridization analyses on Cdx1–/– embryos at the 0- to 22-somite
stages (E7.5-E9.5). In Cdx1–/– embryos, expansion of Mafb
expression into the posterior neural tube was observed (Fig. 4).
However, this expansion was seen only in 0- to 8-somite stage
(E8.0-E8.5) embryos (Fig. 4A-D), after which, by the 10-somite
stage, ectopic posterior Mafb expression disappeared and the
normal r5-r6 expression pattern was regained (Fig. 4E,F). This
expansion was most pronounced at four somites (E8.0), and
thereafter expression began to regress towards the anterior.
Expansion of Mafb protein expression was also seen in Cdx1–/–
embryos at the four-somite stage (E8.0) (Fig. 4G,H). We also
examined whether lowering overall Cdx dosage in the posterior
neural tube might affect Mafb expression by examining Cdx2–/–
embryos. Cdx2 is not expressed in the hindbrain, but is expressed
early in a domain that extends from the tailbud to the anterior
spinal cord. Cdx2–/– embryos die at preimplantation because of
placental defects, which can be rescued in embryos generated by
tetraploid aggregation. When we examined Mafb expression in
Cdx2–/– embryos generated by tetraploid aggregation, Mafb was
expressed essentially normally in r5 and r6 in 0- to 18-somite stage
embryos (see Fig. S1 in the supplementary material; data not
shown). So, Cdx2 on its own is not required for repressing Mafb in
the posterior neural tube. Thus, Cdx1 directly or indirectly
establishes the initial posterior boundary of Mafb expression and a
Cdx1-independent mechanism acts to maintain r5-r6-specific Mafb
expression in r5-r6 after the 10-somite stage.
Cdx1 binds at the S5 enhancer in vivo
To test whether Cdx1 might bind within the S5 regulatory element
in embryos, we performed ChIP analyses with an anti-Cdx1
antibody on 0- to 12-somite-stage embryos (E8.0-E8.5). The
embryo ChIP samples were analyzed with quantitative PCR
(qPCR) using primers within and flanking the S5 Cdx-binding sites
as well as primers at the cdx1 auto-regulatory region and the Mafb
promoter as positive and negative controls, respectively. Cdx1
DEVELOPMENT
2005). Expression is maintained throughout the posterior neural
tube from the appearance of the first somite to the 35-somite stage
(E8.0-E10.5), but the anterior boundary of expression regresses
into the spinal cord during development (Meyer and Gruss, 1993).
Cdx2 and Cdx4 expression does not extend into the hindbrain
(Beck et al., 1995; Gamer and Wright, 1993). To localize the Cdx1
expression domain relative to the Mafb-expressing r5-r6 region, we
performed immunofluorescence experiments with Cdx1- and
Mafb-specific antibodies on serial sections of embryos at the 0- to
12-somite stages (E8.0-E8.5). In these experiments we found that
the anterior boundary of Cdx1 expression coincided with the
posterior boundary of Mafb expression at 0- to 8-somite stages
(E8.0-E8.5) (Fig. 3A-D). At the 10-somite stage (E8.75), there was
a gap between the posterior boundary of Mafb and the anterior
boundary of Cdx1 expression (data not shown). By the 12-somite
stage, Cdx1 expression had receded from the hindbrain and anterior
spinal cord altogether (Fig. 3E,F). These findings are in line with
other analyses of Cdx1 expression (Meyer and Gruss, 1993; Gaunt
et al., 2003; Gaunt et al., 2005; Houle et al., 2000; Lohnes, 2003;
Béland et al., 2004; Pilon et al., 2007). Moreover, analyses of E9.5
embryos doubly transgenic for the Z/AP reporter, in which LacZ
expression can be permanently activated by Cre recombinasemediated excision, and a Cre recombinase under the direction of
the Cdx1 regulatory region that appears to recapitulate early
endogenous regulation, but, of course, may not be entirely
complete, further supports that Cdx1 is unlikely to be expressed
anteriorly of the r6/r7 boundary (Hierholzer and Kemler, 2009).
The presence of Cdx1 at the r6/r7 boundary at early somite stages
supports its possible role as a repressor of Mafb in the posterior
hindbrain.
RESEARCH ARTICLE
Fig. 3. Cdx1 is present at the posterior limits of Mafb expression.
Immunofluorescence on sequential sections of embryos using Mafb(A,C,E) and Cdx1- (B,D,F) specific antibodies. r5-r6 localization of Mafb
protein is seen at the 4- (A), 8- (C) and 12-somite stages (E). Cdx1
protein is localized at the caudal limit of Mafb at the 4- (B) and 8somite stages (D). By the 12-somite stage, no Cdx1 staining is seen in
the hindbrain (F).
bound at its auto-regulatory region as expected (P<0.001), but not
at the Mafb promoter, or 2 kb upstream or downstream of the S5
Cdx1-binding sites (Fig. 5). Cdx1 binding was detected within the
S5 enhancer in embryos confirming an in vivo interaction
(P<0.001) (Fig. 5). This is the first evidence for in vivo binding of
a transcription factor to the endogenous S5 rhombomere-specific
Mafb enhancer and is consistent with a direct role of Cdx1 in
regulating early Mafb expression. All of these data point towards a
direct, but transient, role for Cdx1 in repressing Mafb expression
in the posterior hindbrain and spinal cord and, thereby, preventing
these from assuming more anterior r5-r6 like identity.
DISCUSSION
Here we have uncovered a role for Cdx1 in the direct repression of
the r5-r6 specific hindbrain segmentation gene Mafb. Ectopic or
overexpression of Cdx genes in Xenopus, mouse and zebrafish
eliminates expression of posterior hindbrain patterning genes,
including Mafb in some cases (Epstein et al., 1997; Gaunt et al.,
2008; Isaacs et al., 1998; Shimizu et al., 2006; Skromne et al.,
2007; Young et al., 2009). Conversely, reducing Cdx gene dosage
leads to expansion of the posterior hindbrain and Mafb expression
into the posterior neural tube. However, these effects on Mafb
expression could have been mediated indirectly by Cdx target
genes or by effects of Cdx dosage on overall Wnt and RA
signalling. Notably, transplantation experiments in zebrafish
Development 138 (1)
Fig. 4. Mafb expression in Cdx1+/+ and Cdx1–/– embryos. Wholemount RNA in situ hybridization detects (A,C,E) Mafb expression in
rhombomeres 5 and 6 in +/+ embryos. At the 4- (A,B) and 8- (C,D)
somite stage Mafb expression extends beyond r6 into the posterior
neural tubes of Cdx1–/– embryos (B,D). Posterior limits of expression are
indicated by arrows. Dashed arrows mark the r8/spinal cord boundary.
At the 10-somite stage (E,F), Mafb expression in Cdx1–/– embryos (F) is
restricted to rhombomeres 5 and 6 and is comparable to normal
expression (E). (G,H)Immunofluorescence with a Mafb-specific antibody
in normal four-somite stage embryos (G) detects Mafb protein in r5-r6.
Lines demark the boundaries of these rhombomeres. In Cdx1–/–
embryos (H), cells positive for Mafb appear beyond the posterior
boundary of r6.
showed that simultaneous reduction of both Cdx1a and Cdx4 leads
to cell-autonomous Mafb derepression in the posterior neural tube
and, thus, suggested that either Cdxs or Cdx targets might normally
repress Mafb in the posterior neural tube (Skromne et al., 2007).
All past observations are consistent with our discovery of direct
Mafb repression by Cdx1. Thus, Mafb is the earliest AP patterning
gene known to be directly regulated by Cdx1 – aside from Cdx1
autoregulation. Cdx1 and Cdx2 are known to interpret the RA
signal to Hox genes expressed along the spinal cord (Allan et al.,
2001; Gaunt et al., 2008; Tabaries et al., 2005; van den Akker et
al., 2002). Similarly, Cdx1 appears to be responsible, at least in
part, for the early RA-dependent repression of Mafb expression
posterior of the r6/r7 boundary.
A redundancy in the actions of Cdx1 and Cdx2 could explain the
differences in expansion observed for S5-LacZ in wild-type
embryos when compared with endogenous Mafb expression in
Cdx1–/– embryos. Although there is obvious posterior expansion of
Mafb expression in Cdx1–/– embryos, it is not as dramatic as that
observed for the S5-LacZ reporter in which all Cdx sites were
deleted. A simple explanation is that, when all Cdx binding is
eliminated, the LacZ reporter is free to be expressed throughout the
posterior neural tube. By contrast, when only binding of Cdx1 is
eliminated, Mafb expansion is limited to the posterior neural tube
and anterior spinal cord, where Cdx1 is the only Cdx gene
expressed. In the posterior neural tube, levels of Cdx2 and Cdx4
DEVELOPMENT
70
Fig. 5. Cdx1 binds at the S5 enhancer in vivo. (A)Representative
qPCR results for ChIP experiments performed with an anti-Cdx1
antibody on 0- to 12-somite-stage embryos detect Cdx1 binding at the
Cdx1 autoregulatory region (Cdx1 auto) of the Cdx1 gene, as a positive
control, and at the Cdx1-site-containing region of the S5 enhancer
(*P<0.001). No Cdx1 binding can be detected in regions 2 kb 5⬘ (S52kb) or 2 kb 3⬘ (S5+2kb) of the Cdx1 sites within S5 or at the Mafb
promoter (Mafb prom). qPCR products are expressed as percent of
input (sonicated embryo DNA before IP) and compared with
background levels (+IgG) using a two-way Anova and post-hoc t-test.
Error bars indicate the standard error of the mean (s.e.m.). (B)A
schematic diagram shows the relative locations of the primer pairs used
in ChIP-qPCR analyses.
suffice to suppress endogenous Mafb in Cdx1–/– animals. Although
Cdx1, Cdx2 and Cdx4 differ in their expression patterns and some
structural details, at least in the case of Cdx1 and Cdx2 they have
been shown to act redundantly in vertebral patterning (Savory et
al., 2009b; van den Akker et al., 2002). In transgenic mice, where
Cdx1 was replaced by Cdx2 at the endogenous locus, Cdx2 could
effectively replace Cdx1 function (Savory et al., 2009b). Aside
RESEARCH ARTICLE
71
from redundancy among Cdx genes, the number of Cdx sites
within the S5 enhancer are likely to make it exquisitely responsive
to even low levels of Cdx. Thus, Cdx1, Cdx2 and Cdx4 may all
collaborate to repress Mafb in the posterior neural tube, and the
three Cdx homodimer sites present in S5 may require only low
levels of Cdx; for example in the hindbrain only Cdx1 is required
to mediate repression. Although our findings are all in line with a
pivotal repressive role for Cdx1 in the posterior hindbrain and
possibly Cdx1, Cdx2 and Cdx4 in the spinal cord, we cannot
exclude the possibility that other sites within the 240 bp region
and/or additional regulators other than Cdxs may contribute to the
posterior repression of Mafb. Nonetheless, our data do ascertain
direct involvement of Cdx1 in establishing the early r6/r7 boundary
of Mafb expression.
Although Mafb is now the earliest known axial Cdx target, it is
not the only segmentation gene repressed by Cdxs. Previously,
Cdxs have been implicated in defining the posterior boundary of
Hoxa5 expression in the spinal cord (Tabaries et al., 2005). Hoxa5
expression extends from the caudal end of the posterior
myelencephalon (the r8/spinal cord boundary) in the neural tube to
prevertebra 3 in the preaxial skeleton. In the mouse, two Cdxbinding sites in a Hoxa5 enhancer, essential for mesodermal
expression in cervical and upper thoracic regions of E12.5
embryos, were required for establishing the posterior boundary of
LacZ reporter gene expression in transgenic mice. In contrast to our
findings, in vivo data did not corroborate a possible repressive role
for Cdxs in defining the Hoxa5 expression domain, as endogenous
Hoxa5 expression was not affected in Cdx1–/– or Cdx2–/– embryos.
Because Cdx1, Cdx2 and Cdx4 are co-expressed posterior of the
normal Hoxa5 expression domain, redundancy among Cdxs may
have obscured any effects of reducing Cdx dosage in these
experiments. In the case of Mafb, the in vivo binding of Cdx1
detected by ChIP analyses and the posterior expansion of Mafb
expression in Cdx1–/– embryos strongly support Cdx1 involvement
in direct Mafb repression.
Cdxs may play a more global role in establishing posterior
expression boundaries of segmentation genes. Previously, HoxB
genes were placed into two groups based on their reciprocal
sensitivity to RA or Fgf during early axial patterning (Bel-Vialar et
Fig. 6. Two-step regulation of rhombomere-specific
Mafb expression. (A)During the 0- to 8-somite stages, an
RA gradient establishes vHNF1 expression (green) up to the
r4/r5 boundary and Cdx1 (orange) expression to the r6/r7
boundary. Mafb is expressed in r5-r6 and Hoxb4 in r7-r8 in
Cdx1+/+ embryos. In Cdx1–/– embryos, Mafb expression
extends from the r4/r5 boundary posteriorly into the future
spinal cord and Hoxb4 expression is shifted posteriorly.
(B)During the ~10- to 20-somite stages, Hnf1b and Cdx1 are
absent and a mechanism that depends on Mafb
autoregulation and probably an unidentified Fgf-responsive
activator maintains Mafb expression in r5-r6 in both Cdx1+/+
and Cdx1–/– embryos. Hoxb4 expression, which is
autoregulated, but not known to depend on Fgf signalling
at these stages, remains shifted posteriorly in Cdx1–/–
embryos (van den Acker et al., 2002). (C)Schematic outline
of action of Mafb regulators on the S5 enhancer and lateracting, unidentified rhombomere-specific regulatory
elements.
DEVELOPMENT
Cdx1 directly regulates hindbrain patterning
RESEARCH ARTICLE
al., 2002). Exogenous Fgf expands the expression domains of the
Cdx genes and 5⬘ members from the HoxB complex (Hoxb6Hoxb9), which are normally trunk specific, anteriorly in the neural
tube up to the level of the otic vesicle. Conversely, the more
anteriorly expressed 3⬘ Hoxb genes (Hoxb1 and Hoxb3-Hoxb5) are
sensitive to RA, but not Fgf, treatment at these stages. These
analyses focused on RA and Fgf sensitivity of Hox gene activation
and anterior expression boundary establishment. Extrapolation
from our findings suggests that Fgfs and RA via Cdxs may also
selectively repress specific Hox genes and establish posterior
expression boundaries. Given the observations of direct Cdxdependent repression of Hoxa5 and Mafb, perhaps the repressive
effects of Cdx proteins may be specific to ‘anterior’ segmentation
genes. Such repressive influences could either be masked or
enhanced by subsequent regulatory mechanisms, e.g. inter-Hox
gene crossregulation or autoregulatory loops. Thus, as with Mafb
and Hoxa5, such mechanisms might only become apparent in
enhancer dissection experiments that reveal transient regulatory
events and developmental stages.
Our findings indicate that Cdx1-dependent repression is only
temporary, suggesting that a later-acting mechanism stabilizes
Mafb expression in r5 and r6 after the 10-somite stage. Initial
rhombomere-specific Mafb expression occurs through the S5
enhancer, which is active from the 0- to 10-somite stages. The
activities of Cdx1 and Hnf1b in Mafb regulation are limited to the
early induction period, because these molecules regress towards the
posterior during embryonic development and are absent from the
hindbrain by the 10-somite stage (Fig. 6) (Houle et al., 2000; Kim
et al., 2005; Meyer and Gruss, 1993), long before Mafb expression
is extinguished (Cordes and Barsh, 1994; Frohman et al., 1993;
Theil et al., 2002). Regulatory elements governing Mafb expression
between the 10- and 20-somite stages have not yet been described.
However, post-initiation Mafb(val) expression depends on
functional Mafb(val) protein in both mice and zebrafish, indicative
of an autoregulatory loop (Hernandez et al., 2004; Huang et al.,
2000; Moens et al., 1998; Sadl et al., 2003). For some Hox genes,
such as Hoxb4 (Gould et al., 1998), an RA-responsive early
enhancer establishes rhombomere-specific expression and,
subsequently, an autoregulatory enhancer helps maintain
expression. Consistent with this model, in Cdx1–/– mice, Hoxb4
expression is shifted posteriorly throughout the 0- to 20-somite
stages (van den Acker et al., 2002) (Fig. 6). However, the Mafb
autoregulatory loop is unique in that it appears to depend not only
on Mafb but also on the presence of a regionally restrictive signal,
possibly an Fgf (Marin and Charnay, 2000; Maves et al., 2002;
Walshe et al., 2002; Wiellette and Sive, 2003). This regionally
restricted signal might affect either Mafb itself by perhaps a posttranslational modification, such as phosphorylation, or may act via
inducing another transcriptional activator. In any case, it is
probably the regionally dependent signal that promotes the postinitiation retraction of Mafb expression into its normal r5-r6
domain in Cdx1–/– mice.
The molecular mechanism by which Cdx1 acts to repress Mafb
in the same regions in which it activates trunk-specific Hox genes,
is unknown. It seems probable that Cdx actions depend on the
sequence-specific context of the Cdx-binding sites within the S5
enhancer. In addition to its roles in embryonic patterning, Cdx2
plays a role in tumorigenicity in a subset of human colorectal
cancer cell lines (Chawengsaksophak et al., 1997). In an
established model for human colon cancer, the human colonic
adenocancinoma cell line (LOVO), Cdx2 directly represses the
insulin-like growth-factor-binding protein 3 gene (Chun et al.,
Development 138 (1)
2007). Here the existence of unknown repressive interactor(s) has
been invoked as a possible explanation and provides a plausible
mechanism for Cdx1-dependent repression of Mafb as well. Core
promoter composition might further influence the choice between
Cdx-dependent activation and repression. In flies, Caudal
preferentially activates promoters containing a downstream core
promoter element (DPE) (Juven-Gershon et al., 2008). The
promoters of Drosophila Hox genes are TATA-less, except for
those from the abdominal-B (Abd-B) and Ultrabithorax (Ubx)
genes, and can be activated by Caudal in preference to TATAcontaining promoters. Whereas generally the focus in
developmental gene expression has been on enhancers, there has
been precedence, in studies on mouse Hoxb2 and Hoxb4
regulation, for promoter selectivity among Hox gene enhancers
(Gould et al., 1997). The functional composition of vertebrate Hox
and Mafb gene promoters have not been examined. Although Cdxs
have not been shown to block the use of specific promoters, it is,
theoretically, conceivable that core promoter composition in
combination with Cdx interactors acting via specific enhancers
might influence the choice between Cdx-responsive activation or
repression.
In conclusion, we have shown for the first time that Cdx1
promotes not just trunk specification, but plays a role in defining
the r7-r8 domain of the hindbrain by directly repressing Mafb
expression. Thus, the role of Cdx1 within the hindbrain is an
active but transient one. Our observations suggest that other
transient effects of Cdxs may have been missed. Do such transient
perturbations subtly alter neuronal fate assignments or
distribution? Many important neuronal subtypes and neural
circuits are established during hindbrain patterning. Thus, it is not
difficult to imagine that subtle gene expression perturbations –
even when apparently corrected later on – may have consequences
later in life. Thus, further analyses of Mafb and hindbrain
regulation will continue to offer scientific and possibly clinically
relevant insights.
Acknowledgements
We thank D. Lohnes for providing the anti-Cdx1 antibody and Cdx1–/– mice,
originally generated by Subramanian et al. (Subramanian et al., 1995). We are
grateful to M. Gerstenstein, S. Tondat and the Samuel Lunenfeld transgenic
core facility for generating transgenic mice and embryos using tetraploid
aggregation. This research was funded by CIHR grants MOP 14312 and MOP
97966 to S.P.C.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058727/-/DC1
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