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First published online August 12, 2008
doi: 10.1242/10.1242/dev.019778
1 Department of Psychiatry, Nina Ireland Laboratory of Developmental
Neurobiology, University of California San Francisco, 1550 4th street, San
Francisco, CA 94158, USA.
2 Department of Surgery/Urology and Department of Pathology, Children's Hospital
of Boston, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115,
USA.
3 Howard Hughes Medical Institute, Department of Medicine, University of
California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA
92093, USA.
4 Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672,
Singapore.
* Authors for correspondence (emails: john.rubenstein{at}ucsf.edu; juhee.jeong{at}ucsf.edu)
Accepted 26 June 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: Dlx, Gbx2, Pou3f3, Craniofacial, Branchial arch, Jaw, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Couly et al., 1993;
Osumi-Yamashita et al., 1994;
Köntges and Lumsden,
1996; Chai et al.,
2000). Subsequently, the FNP becomes the mid- and upper face,
while the first branchial arch (BA1) develops into most of the jaw, the
lateral skull, palate and the middle ear
(Köntges and Lumsden,
1996). BA1 is further divided into maxillary arch (mxBA1,
prospective upper jaw) on the proximal half, and mandibular arch (mdBA1,
prospective lower jaw) on the distal half (see Fig. S1A,B in the supplementary
material). The second branchial arch (BA2) mainly contributes to the ear and
neck skeleton.
How CNCCs recognize their positional information and develop accordingly is
beginning to be understood (reviewed by
Depew et al., 2002a;
Santagati and Rijli, 2003;
Chai and Maxon, 2006).
Interactions of CNCCs with the neighboring tissues result in the expression of
a diverse set of transcription factors in CNCCs, and the specific combination
of transcription factors provides a positional identity to the cells.
The vertebrate Dlx genes are homologs of Drosophila Distal-less;
they encode homeodomain transcription factors
(Panganiban and Rubenstein,
2002). Mice have six Dlx genes, which are organized as three
linked pairs in the genome (Dlx1/2, Dlx3/4 and Dlx5/6)
(Porteus et al., 1991;
Price et al., 1991;
Robinson and Mahon, 1994;
Simeone et al., 1994;
McGuinness et al., 1996;
Nakamura et al., 1996;
Liu et al., 1997).
During craniofacial development, mouse Dlx genes are regionally expressed
within BAs as well as in olfactory and otic placodes (see Fig. S1A,C in the
supplementary material) (Dolle et al.,
1992; Bulfone et al.,
1993; Robinson and Mahon,
1994; Simeone et al.,
1994; Qiu et al.,
1997; Depew et al.,
2002b). In the ectomesenchyme of BA1 in mid-gestation stage
embryos, Dlx1/2 are expressed in both mxBA1 and mdBA1, whereas
Dlx5/6 are expressed in mdBA1 only. Dlx3/4 expression is
further restricted to a narrow domain within mdBA1. The same proximodistal
arrangement is also found in BA2. Since Dlx3/4 expression is
dependent on Dlx5/6 (Depew et
al., 2002b) (this study), essentially two different combinations
of Dlx partition much of BA1: Dlx1/2 for mxBA1 and
Dlx1/2+5/6 for mdBA1. The functional importance of this `Dlx code' in
BA patterning has been investigated using mouse loss-of-function mutants.
Owing to the tight linkage in the genome, the double mutations of
Dlx1/2 and Dlx5/6 pairs were achieved by deleting both genes
in one allele (Qiu et al.,
1997; Merlo et al.,
2002; Depew et al.,
2002b). Inactivation of Dlx1 and/or Dlx2
(Dlx1-/-, Dlx2-/- and
Dlx1/2-/-) caused abnormalities in upper jaw skeleton with
little effect on the lower jaw (Qiu et
al., 1995; Qiu et al.,
1997; Depew et al.,
2005). By contrast, Dlx5-/- exhibited defects
in lower jaw development (Depew et al.,
1999). Most strikingly, the simultaneous inactivation of
Dlx5 and Dlx6 (Dlx5/6-/-) resulted in
homeotic transformation of the lower jaw into upper jaw
(Beverdam et al., 2002;
Depew et al., 2002b).
Therefore, the differential expression of Dlx genes along the proximodistal
axis is important for the regional specification of BA1; Dlx1/2 are
necessary for the proper development of mxBA1, whereas Dlx5/6 confer
mdBA1 identity.
Our current work addresses three important issues on how the Dlx genes regulate BA patterning. First, the mechanism through which Dlx5/6 specify lower jaw fate needs to be understood. To this end, we performed a genome-wide transcriptional profiling and obtained a comprehensive list of genes with altered expression in Dlx5/6-/- mdBA1. Second, we provide the first evidence of the upper jaw-specific genetic program and show that it is partly regulated by Dlx genes. Prior to this study, the abundance of mdBA1-specific markers but the lack of any known mxBA1-specific markers has been compatible with the idea that the upper jaw is the default state upon which the lower jaw fate is imposed. Finally, we investigated the functional relationship of different Dlx genes expressed in BA1 by comparing the morphological and molecular phenotypes of various combinations of Dlx single and double mutants. We found that Dlx1, 2, 5 and 6 act both partially redundantly and antagonistically, depending on the context, to achieve differential expression of their downstream genes in mxBA1 and mdBA1.
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Transcriptional profiling using DNA microarrays
Dlx5/6-/- and Dlx5/6+/+ littermates
were collected from Dlx5/6+/- intercrosses at E10.5. The
mdBA1s were dissected, flash-frozen and stored in liquid nitrogen until the
day of RNA extraction. The tissue was homogenized in Trizol reagent
(Invitrogen) using Pellet Pestle (Kontes). RNA was extracted using chloroform
and then concentrated by isopropanol precipitation. After a rinse with 80%
ethanol, the RNA pellet was dissolved in water and purified using the RNeasy
Mini Kit (Qiagen). From 13 Dlx5/6-/- and 13
Dlx5/6+/+ E10.5 embryos, we recovered 14.7 µg and 13
µg of mdBA1 total RNA, respectively.
All subsequent steps of the microarray experiment were performed by the Translational Genomics Research Institute (TGen, Phoenix, AZ), through the NIH Neuroscience Microarray Consortium. The RNA sample from each genotype was hybridized onto GeneChip Mouse Genome 430 2.0 arrays (Affymetrix) in triplicate. Data acquisition and analysis employed GeneChip Operating Software (GCOS, Affymetrix) version 1.2.
In situ hybridization and skeletal preparation
Whole-mount and section in situ hybridizations were performed using
digoxigenin-labeled RNA probes as described
(Jeong et al., 2004;
Jeong and McMahon, 2005),
except that 20 µm sections were used for the section in situ hybridization.
The control and mutant embryos were stage-matched using a combination of
several morphological criteria, including the size and shape of the limb buds,
morphogenesis of the eye, and somite numbers. Skeletons of E18.5 or P0 animals
were stained with Alcian Blue and Alizarin Red as described
(Jeong et al., 2004). For the
in situ hybridization and skeletal preparations shown in Figs
1,
2,
3 and
4, embryos of +/+ and
+/- genotypes were used indiscriminately and are referred to as `wild
type'. For Figs 5 and
6, `wild type' refers to +/+
for all the genes in question, or Dlx1+/-.
DNA templates for in situ hybridization probes were obtained by PCR from a wild-type mouse E10.5 BA1 cDNA library or from adult tail genomic DNA, purchased from companies, or kindly provided by other investigators. Further information on the probes is available upon request.
Luciferase reporter activation assay
The 1 kb putative Gbx2 enhancer (see
Fig. 3A) was amplified by PCR
from mouse tail genomic DNA using primers
5'-ACACCTCGAGAGAGGATGACAGCGAGCTTCG-3' and
5'-GTGTAAGCTTGAGCAAACATTCCAGTTTTAATGC-3', and cloned into
XhoI-HindIII sites of pGL4.23 (Promega). pGL4.23 contains a
minimal promoter and the firefly luciferase coding sequence. pCAGGS-Dlx5
(Stuhmer et al., 2002) was
used to express Dlx5 protein. pGL4.73 (Promega), a plasmid that constitutively
expresses Renilla luciferase, was used as a control for variations in
transfection efficiency. 3T3 cells were transfected with FuGene 6 (Roche), and
40 hours later the cells were lysed and analyzed for luciferase activity using
the Dual Luciferase Reporter Assay System (Promega). The experiments were
performed in triplicate and the results combined for statistical analysis.
Generation of Dlx6 mutant allele
To generate the Dlx6-lacZ (Dlx6-) allele, a 4.3 kb
SpeI-SnaBI genomic fragment spanning Dlx6 exon 3
(which encodes the homeodomain) was subcloned into the
SpeI-SmaI sites of pBS KS+. Site-directed mutagenesis was
performed to engineer a unique NruI site immediately following the
219th amino acid from the N-terminus of the Dlx6 protein (F of the sequence
VKIWFQNKRS). Flanking genomic sequences were added between the unique 5'
XhoI site (5.8 kb 5' homology arm) and the 3'
NotI site (3.5 kb 3' homology arm), and the reporter cassette
IRES-lacZ-PGKneo (Robledo et al.,
2002) was cloned into the unique NruI site to generate
the final targeting construct. The Dlx6-lacZ allele therefore
interrupts the Dlx6 protein immediately following the amino acid F as
described above. ES cell culture, screening, chimera generation and testing
were as previously described (Robledo et
al., 2002).
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;
Acampora et al., 1999) (see
Fig. S1 in the supplementary material), and the wild-type and mutant BA1 still
appear grossly comparable in size and morphology at E10.5 (see also Fig. S2 in
the supplementary material). The Dlx5/6-/- mutant
exhibited downregulation of 39 genes (see Table S1 in the supplementary
material) and upregulation of 24 genes (see Table S2 in the supplementary
material) with greater than a 2-fold change. Our results included most, but
not all, of the genes that were previously shown to be dysregulated in the
Dlx5/6-/- mutant
(Depew et al., 1999;
Depew et al., 2002b). Thus,
although it is a robust procedure, technical limitations exist (see Tables S1
and S2 in the supplementary material). The complete set of raw data from our transcriptional profiling experiment is available at NIH Neuroscience Microarray Consortium data repository (http://arrayconsortium.tgen.org) under accession ruben-affy-mouse-187820, and at GEO (accession number GSE 4774).
Genes encoding transcription factors, non-coding RNAs and signaling molecules exhibit decreased expression in Dlx5/6-/- mdBA1
The Dlx3, Hand2 and Alx4 transcription factors were previously identified
as being downstream of Dlx5/6
(Depew et al., 2002b). Our
screen found that their close relatives, Dlx4, Hand1 and Alx3, are also
downregulated in Dlx5/6-/- mdBA1 and BA2
(Fig. 1A,B,M-P). In the otic
vesicle, Dlx5/6 function is required for the expression of Gbx2, a
homeodomain transcription factor (Robledo
and Lufkin, 2006). We confirmed the same regulatory relationship
in mdBA1 (Fig. 1C,D). The
transcription factors Cited1 (Msg1)
(Shioda et al., 1996) and Zac1
(Lot1, Plagl1) (Abdollahi et al.,
1997; Spengler et al.,
1997) are expressed at high levels in the medial mdBA1 and BA2;
their expression was downregulated in Dlx5/6-/-
(Fig. 1Q-T).
|
). In BA1 and BA2, the expression of A/S Dlx1 was
restricted to the distal region (Fig.
1E), and was completely abolished in Dlx5/6-/-
(Fig. 1F). Evf1/2, the
two splicing variants of a ncRNA gene (Dlx6os1 - Mouse Genome
Informatics), map adjacent to Dlx5/6; their brain expression depends
on Dlx1/2 function (see Fig. S3B in the supplementary material)
(Faedo et al., 2004;
Kohtz and Fishell, 2004).
Evf1/2 expression was greatly reduced in both brain and BAs of
Dlx5/6-/- mutants (Fig.
1G,H). It should be noted that the promoter and the first exon of
Evf2 fall within the deletion in the Dlx5/6 null allele
(Robledo et al., 2002), and
thus the decreased Evf1/2 expression in Dlx5/6-/-
could simply be due to the loss of the Evf2 promoter. However, we
found reduced expression of Evf1/2 also in
Dlx5-/- and Dlx6-/- mutants (see
below), in which both Evf1 and Evf2 sequences are intact.
This result, and the fact that Dlx1/2 activity is necessary for the
expression of Evf1/2 in the brain, argue that Dlx5/6
regulate Evf1/2 transcription, directly or indirectly.
A secreted signaling molecule, hepatocyte growth factor (Hgf, also known as
scatter factor, SF) (Stoker et al.,
1987; Birchmeier and Gherardi,
1998), is expressed in BA1 and BA2; this expression was dependent
on Dlx5/6 function (Fig.
1I,J). Unc5c encodes one of the receptors for the axon
guidance molecule netrin 1 (Ackerman et
al., 1997). Unc5c was expressed in the medial domain of
mdBA1 and BA2, and was severely downregulated in Dlx5/6-/-
(Fig. 1U,V). BMP-binding
endothelial regulator (Bmper, also known as crossveinless-2, Cv2) is a
secreted molecule that binds to and enhances the signaling of bone
morphogenetic proteins (BMPs) (Coffinier
et al., 2002; Moser et al.,
2003; Coles et al.,
2004; Ikeya et al.,
2006). Bmper expression in distal BA1 and BA2 was
dependent on Dlx5/6 (Fig.
1K,L). The regulator of G-protein signaling 5 (Rgs5) gene
is expressed only at the rostromedial tip of mdBA1; this expression was lost
in Dlx5/6-/- (Fig.
1W,X). Rgs proteins are GTPase-activating proteins that attenuate
G-protein-coupled receptor signaling (Chen
et al., 1997; Xie and Palmer,
2007).
Since Dlx3/4 expression is lost in Dlx5/6-/-
mdBA1 (Depew et al., 2002b)
(Fig. 1A,B), it is possible
that at least some of the gene expression changes in
Dlx5/6-/- mutants are due to the loss of Dlx3/4
activity.
Pou3f3, Foxl2 and uncharacterized transcripts linked to them, are strongly repressed by Dlx5/6 in mdBA1
Pou3f3 (Brn1), a POU-domain transcription factor
(Hara et al., 1992), was
highly expressed in the entire mxBA1 and maxillary-mandibular junction, but
was absent from most of mdBA1 (Fig.
2A). It was also confined to the proximal region in BA2. In
Dlx5/6-/-, Pou3f3 expression expanded into mdBA1
and distal BA2 with the same intensity as in its normal expression domains
(Fig. 2B).
We identified two Riken cDNA clones (26100017I09Rik, 2900092D14Rik), both suspected to be non-coding, that are closely linked to and have very similar expression patterns as Pou3f3, in both wild-type and Dlx5/6-/- embryos (Fig. 2D,E,G,H; see Fig. S3C in the supplementary material). Because the proximal BA expression of Pou3f3, 2610017I09Rik and 2900092D14Rik overlaps with that of Dlx1/2 (see Fig. S1C in the supplementary material), we tested whether the expression of these genes is dependent on the activity of Dlx1/2 in the mxBA1. We found downregulation of all three genes in BA1 and BA2 of Dlx1/2-/-, demonstrating that Dlx1/2 are necessary for their normal expression (Fig. 2C,F,I).
Foxl2 encodes a winged helix/forkhead transcription factor
(Crisponi et al., 2001). It
was expressed strongly in the dorsal mxBA1 just below the eye, in addition to
a small domain at the maxillary-mandibular junction
(Fig. 2J). In
Dlx5/6-/-, the mxBA1 pattern of Foxl2 expression
was duplicated in mdBA1 (Fig.
2K). Riken cDNA E330015D05Rik is a poorly characterized
gene closely linked to Foxl2 (see Fig. S3D in the supplementary
material). Its expression pattern, both in wild type and
Dlx5/6-/-, was identical to that of Foxl2
(Fig. 2M,N).
Cyp26a1, a retinoic acid-metabolizing enzyme cytochrome P450
(White et al., 1996;
Fujii et al., 1997;
Ray et al., 1997), is normally
expressed along the border of BA1 and BA2 with higher expression in mxBA1 than
in mdBA1 (Fig. 2P). In BA2,
Cyp26a1 expression was also strong proximally and weak distally.
Removal of Dlx5/6 activity lead to upregulation of Cyp26a1
in distal BA1 and BA2 to the same intensity as in the proximal domains
(Fig. 2Q). Irx5, which
encodes an Iroquios-related homeodomain transcription factor
(Bosse et al., 2000;
Cohen et al., 2000), was
expressed strongly in the dorsal mxBA1 and weakly in the ventral mdBA1
(Fig. 2S). The latter
expression was moderately increased in Dlx5/6-/-
(Fig. 2T). Unlike Pou3f3,
2610017I09Rik and 2900092D14Rik, the mxBA1 expression of
Foxl2, E330015D05Rik, Cyp26a1 and Irx5 was not dependent on
Dlx1/2 (Fig.
2L,O,R,U).
The genes described above in this section are normally expressed in mxBA1
and upregulated in the Dlx5/6-/- mdBA1, making the mutant
mdBA1 molecularly similar to mxBA1. By contrast, transmembrane protein 30b
(Tmem30b), a homolog of yeast endosomal protein Cdc50
(Katoh and Katoh, 2004), was
barely detectable in either mxBA1 or mdBA1 in wild-type embryos
(Fig. 2V), but was strongly
upregulated in Dlx5/6-/- mdBA1, making the mutant mdBA1
molecularly different from mxBA1 (Fig.
2W).
|
) showed that Dlx6 binds to, and thus most likely directly
regulates, a Hand2 BA enhancer. Sumiyama and Ruddle
(Sumiyama and Ruddle, 2003)
showed that the BA enhancer for Dlx3/4 contains a consensus
Dlx-binding motif, and thus it is highly likely that Dlx5/6 directly regulate
Dlx3/4 expression in the BA. Thus, the fact that Hand2, Dlx3
and Dlx4 were identified by our microarray analysis supports the
validity of our approach, and suggests that additional genes listed in Table
S1 (see Table S1 in the supplementary material) are directly regulated by Dlx5
and Dlx6 proteins.
To identify direct targets of Dlx5/6, we performed in silico analyses of
the genomic sequences surrounding Dlx5/6-downstream genes.
Previously, a systematic in vitro binding assay determined that
(A/C/G)TAATT(G/A)(C/G) is a consensus binding motif for Dlx proteins
(Feledy et al., 1999). In
addition, various researchers have analyzed cis-regulatory elements from
several Dlx target genes, discovering 13 sequences to which Dlx proteins bind
directly, including some that do not conform to (A/C/G)TAATT(G/A)(C/G) (see
Table S3 in the supplementary material). Using rVISTA
(Loots and Ovcharenko, 2004),
we searched genomic sequences flanking several of the genes listed in Table S1
(see Table S1 in the supplementary material) for all the known Dlx-binding
motifs that are conserved between mouse and human. This analysis identified
0.6 kb of a highly conserved region (conserved down to chick) with a
cluster of three putative Dlx-binding sites located 10 kb upstream of
Gbx2 (Fig. 3A,B). We
tested a 1 kb fragment containing this region (red bar in
Fig. 3A, including the adjacent
0.25 kb sequence conserved down to opossum) for transcriptional enhancer
activity in 3T3 cells. Co-transfection with a Dlx5 expression vector resulted
in a >70-fold activation (Fig.
3C), demonstrating that the fragment contains a transcriptional
enhancer regulated by Dlx proteins.
Pou3f3 is required for the formation of the zygomatic arch and the maxillary component of the jaw joint
To our knowledge, Pou3f3, Foxl2, 26100017I09Rik, 2900092D14Rik and
E330015D05 are the first examples of genes that are expressed
specifically in the maxillary domain of BA1 at any stage of mouse development.
Therefore, they provide evidence of an upper jaw-specific genetic program (see
Discussion).
Among the five genes, Pou3f3 has the strongest and broadest expression in mxBA1 (Fig. 2), and thus we performed further analysis on its expression and function during upper jaw development (Fig. 4). Section in situ hybridization at E10.5 revealed that Pou3f3 is expressed only in the mesenchyme (Fig. 4A,B). At E12.5 (data not shown) and E13.5 (Fig. 4C-E), Pou3f3 expression was found in both upper and lower jaw; however, its expression in the condensed dental mesenchyme was restricted to upper molars (Fig. 4D). In addition, Pou3f3 was expressed in the caudal, but not rostral, palatal shelves (PS) (Fig. 4C,E).
Pou3f3-/- (McEvilly
et al., 2002) mutant skull revealed the essential role of this
gene in the development of a part of the upper jaw; mxBA1-derived squamosal
bone (SQ) normally articulates with mdBA1-derived dentary to make the
functioning jaw joint in mammals. In Pou3f3-/-, the
squamosal bone is largely missing (except the retrotympanic process, rt), and
thus the jaw joint does not exist (Fig.
4F-I,L,M). Jugal bone (JG), which forms the zygomatic arch on the
lateral skull together with the maxilla and squamosal bone, was also lost in
the mutant (Fig. 4H,I,L,M). In
the mutant middle ear, the malleus (mdBA1-derived) appeared normal, whereas
the incus (mxBA1-derived) had a slightly elongated short crus
(Fig. 4J,K). In addition, the
BA2-derived stapes was fused to the styloid process
(Fig. 4J,K). Other bones in the
skull, and the teeth and palate, appeared unaffected in the
Pou3f3-/- mutant (data not shown).
|
;
Acampora et al., 1999;
Beverdam et al., 2002;
Depew et al., 2002b). Based on
these results alone, it was not clear whether Dlx5 and Dlx6
are functionally redundant, or whether Dlx6 has a more prominent
role. To address this, we generated an allele that inactivated Dlx6
alone, by inserting an IRES-lacZ-neomycin resistance cassette within the
Dlx6 homeobox coding sequence
(Fig. 5A). This insertion
prevents translation of one-third of the homeodomain and the entire C-terminal
domain, including the nuclear localization signal [amino acids 220-228
(Cokol et al., 2000)]; thus,
this allele is likely to be null.
Dlx6-/- mice were born alive but died within a day with
aerophagia, as reported for Dlx2-/-, Dlx1/2-/-
and Dlx5-/- (Qiu et
al., 1995; Qiu et al.,
1997; Acampora et al.,
1999; Depew et al.,
1999). The head skeleton of Dlx6-/- neonates
had several abnormalities that are also found in Dlx5-/-
animals (Acampora et al., 1999;
Depew et al., 1999). The
mutant had a slightly reduced mandible
(Fig. 5B,D), in which the
dentary lacked the coronoid process and had a hypoplastic condylar process
(Fig. 5E-G, arrows and
arrowheads). The ectotympanic of the mutant ear was shortened
(Fig. 5H-J, arrows), and the
gonial bone was attached to an ectopic piece of bone [named os paradoxicum
(Depew et al., 1999)]
extending toward the ala temporalis on the skull base
(Figs 5H-J; see S4A-C,
arrowheads, in the supplementary material). The skeletal elements mentioned
thus far are thought to be derivatives of BA1. Therefore, allowing for some
individual variations in morphological details, Dlx6-/-
mice have BA1-associated defects that are very similar to those of
Dlx5-/-, but are much less severe than the homeotic
transformation observed in Dlx5/6-/-. This result
establishes that Dlx5 and Dlx6 are in large part
functionally redundant in lower jaw development.
In addition to the BAs, Dlx5/6 were expressed in otic and
olfactory placodes (see Fig. S1A,C in the supplementary material) and, as a
result, Dlx5-/- animals have dorsally deficient otic
capsule (Fig. 5H,I, open
arrowheads) and hypoplastic nasal cartilage (see Fig. S4D,E, arrowheads, in
the supplementary material) (Acampora et
al., 1999; Depew et al.,
1999). However, both structures appeared normal in
Dlx6-/- (Fig.
5J; see Fig. S4F in the supplementary material), which suggests
that Dlx6 is less important than Dlx5 in otic and olfactory
placode development.
Next, we examined the expression of the molecular markers that are affected in Dlx5/6-/- mdBA1 (Figs 1 and 2) in Dlx5-/- and Dlx6-/- single mutants. Most of the genes showed no, or moderate, changes in either mutant, indicating that they are directly or indirectly regulated by both Dlx5 and Dlx6 to similar degrees (see Fig. S5A-d in the supplementary material). Surprisingly, however, several genes were differentially changed in Dlx5-/- and Dlx6-/-; Gbx2, Bmper, A/S Dlx1, Dlx4 and Evf1/2 were all severely downregulated in Dlx5/6-/- BAs (Fig. 1), but Gbx2, Dlx4 and Evf1/2 were more downregulated in Dlx5-/- than in Dlx6-/-, whereas Bmper and A/S Dlx1 were more affected in Dlx6-/- than in Dlx5-/- (Fig. 5K-P; see Fig. S4G-I and Fig. S5e-j in the supplementary material). Similarly, for the genes upregulated in Dlx5/6-/-, Pou3f3, Foxl2, 2610017I09Rik and E330015D05Rik showed greater changes in Dlx5-/- than in Dlx6-/-, whereas Tmem30b was upregulated only in Dlx6-/- (Fig. 5Q-V; see Fig. S4J-L and Fig. S5k-p in the supplementary material). These results suggest that there are some differences between the transcriptional activities of Dlx5 and Dlx6, even though inactivation of each gene results in similar morphological consequences.
|
; Qiu et al.,
1997; Depew et al.,
2005), Dlx1/2 can contribute to mdBA1
development, as the lower jaw phenotypes of Dlx5-/- are
greatly exacerbated in Dlx1-/-;5-/- and
Dlx2-/-;5-/- mutants
(Depew et al., 2005). We tested whether Dlx6 is also at least in part functionally redundant with Dlx1/2 by generating Dlx1-/-;6-/- and Dlx2-/-;6-/- double mutants. The compound mutants exhibited far greater craniofacial defects than those found in any of the single mutants. The dentaries of Dlx1-/-;6-/- and Dlx2-/-;6-/- were shortened, fragmented, and bifurcated to become bones resembling the maxilla, jugal and pterygoid of the upper jaw (Fig. 6A-L; see S6A-F in the supplementary material). The ventrolateral side of the skull of Dlx1-/-;6-/- and Dlx2-/-;6-/- had what appears to be a duplicate lamina obturans juxtaposed to the endogenous one (Fig. 6S-X; LO* in Fig. 6W,X). In the ear, the ectotympanic and middle ear ossicles were progressively reduced or lost in Dlx1-/-;6-/- and Dlx2-/-;6-/- (Fig. 6Y-d). The basihyoid and lesser horn of the hyoid were also progressively more affected in Dlx1-/-;6-/- and Dlx2-/-;6-/- (see Fig. S6G-L in the supplementary material).
The phenotypes of Dlx1-/-;6-/- and
Dlx2-/-;6-/- are reminiscent of those reported
for Dlx1-/-;5-/- and
Dlx2-/-;5-/-
(Depew et al., 2005). In all
four cases, the mdBA1-derivatives are reduced in size and/or transformed into
structures resembling upper jaw elements, although the extent of
transformation is less than in Dlx5/6-/-.
Dlx1/2 function partially redundantly with Dlx5/6 in regulating mdBA1 gene expression
To elucidate the mechanisms underlying the morphological changes in
Dlx1-/-;6-/-,
Dlx2-/-;6-/- and
Dlx2-/-;5-/-, we examined the gene expression
patterns in their mdBA1.
The expression of Gbx2 and Bmper in mdBA1 was minimally
or moderately downregulated in Dlx5-/- and
Dlx6-/- (Fig.
5). However, their expression is severely reduced or completely
abolished in Dlx1-/-;6-/-,
Dlx2-/-;6-/- and
Dlx2-/-;5-/-
(Fig. 7A-H). Pou3f3
was repressed by Dlx5/6 in mdBA1, and was moderately upregulated in
Dlx5-/- and Dlx6-/-
(Fig. 5). Further removing
Dlx1 or Dlx2 activity expanded and intensified
Pou3f3 overexpression in mdBA1 (compare
Fig. 7J,K with
Fig. 5S, and
Fig. 7L with
Fig. 5R). Therefore, even
though Dlx1/2 activity is required for the normal expression of
Pou3f3 in mxBA1 (Fig.
2A,C), Dlx1/2 apparently share the repressive effects of
Dlx5/6 on Pou3f3 in mdBA1. Hand2 was severely
downregulated in Dlx5/6-/-, but unaffected in
Dlx5-/- and Dlx6-/-
(Depew et al., 2002b) (see
Fig. S5A-D in the supplementary material). Hand2 expression appeared
normal in Dlx1-/-;6-/-, but was gradually
reduced and became restricted to caudomedial mdBA1 in
Dlx2-/-;6-/- and
Dlx2-/-;5-/-
(Fig. 7M-P).
The expression changes of Gbx2, Bmper and Pou3f3 in Dlx1-/-;6-/-, Dlx2-/-;6-/- and Dlx2-/-;5-/- are either similar or identical to the changes in Dlx5/6-/- (Figs 1 and 2), in line with the overall similar morphological defects of these four mutants. By contrast, Hand2 expression was more severely reduced in Dlx5/6-/- than in any of Dlx1-/-;6-/-, Dlx2-/-;6-/- or Dlx2-/-;5-/- (see Fig. S5D in the supplementary material). This could explain why the morphological transformations of lower jaw into upper jaw in the latter three mutants are incomplete compared with Dlx5/6-/-.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Therefore, it is possible that Evf2 similarly controls the activities of Dlx
proteins in mdBA1 and thus the downregulation of Evf1/2 expression in
Dlx5/6-/- (Fig.
1) contributes to the gene expression changes observed in
Dlx5/6-/- mutants. To date, none of the Dlx5/6-downstream genes has been shown to recapitulate the phenotypes of Dlx5/6-/- when mutated in mice. Given that Dlx5/6 directly or indirectly regulate the expression of dozens of genes, it is likely that Dlx5/6 achieve their function through the combined efforts of many genes.
Among the Dlx5/6-downstream genes listed in Tables S1 and S2 (see
Tables S1 and S2 in the supplementary material), BA enhancers have been
characterized for only three (Hand2 and Dlx3/4), all of
which showed some evidence of direct regulation by Dlx proteins
(Charite et al., 2001;
Sumiyama and Ruddle, 2003).
Our identification of a Dlx5-regulated enhancer near Gbx2 suggests
that Gbx2 might also be a direct target of Dlx5/6. Since
Gbx2 is downstream of Dlx5/6 in BA1 and the otic vesicle,
this enhancer is likely to function in one or both of these tissues.
|
We found that the normal level of Pou3f3 expression in mxBA1 requires Dlx1/2 activity, whereas Foxl2 expression does not (Fig. 2). Therefore, the upper jaw-specific program in mxBA1 has both Dlx-dependent and Dlx-independent components. In addition, the mxBA1-specific genes that we identified show altered expression in Dlx5/6-/- mdBA1. It is possible that there are genes, the expression of which is restricted to mxBA1 but which is not upregulated in Dlx5/6-/- mdBA1; our screen was not designed to identify these.
Functional comparison of different Dlx genes in jaw patterning
Morphological analysis of the Dlx5 and Dlx6 single
mutants indicates that they have very similar roles in mdBA1 development,
despite the intriguing differences in their transcriptional activities
(Fig 5; see Fig. S4 in the
supplementary material). In addition, the analysis of
Dlx1-/-;5-/-,
Dlx2-/-;5-/-
(Depew et al., 2005),
Dlx1-/-;6-/- and
Dlx2-/-;6-/- (Figs
6 and
7) establishes that the
activity of Dlx5/6 to specify lower jaw fate is shared by
Dlx1/2. However, Dlx1/2 are clearly less important
than Dlx5/6 in this process because lower jaw development is
essentially normal in Dlx1/2-/- mutants
(Qiu et al., 1997;
Depew et al., 2005). Also,
among Dlx1 and Dlx2, Dlx2 appears to have a greater
influence on lower jaw development because the defects seen in
Dlx2-/-;5-/- and
Dlx2-/-;6-/- are more severe than those in
Dlx1-/-;5-/- and
Dlx1-/-;6-/-, respectively
(Depew et al., 2005) (this
study). These differences could be due to the molecular properties of each Dlx
protein (as determined by amino acid sequence), but they could also be owing
to differences in expression level, pattern or timing.
Another important conclusion from our analysis of
Dlx1-/-;6-/- and
Dlx2-/-;6-/- mutants concerns the functional
relevance of the previous classification of Dlx genes based on their sequence
homology (Stock et al., 1996)
into type A (Dlx2, 3, 5) and type B (Dlx1, 4, 6). If two Dlx
genes of the same type were functionally closer than those of different types,
then removing one gene of each type would result in milder phenotypes than
removing two genes of the same type, owing to compensation. Whereas
Dlx1-/-;5-/- has milder phenotypes than
Dlx2-/-;5-/-
(Depew et al., 2005),
Dlx2-/-;6-/- has more severe phenotypes than
Dlx1-/-;6-/- (Figs
6 and
7), contrary to the prediction.
Therefore, it appears that the greater sequence divergence between Dlx genes
of different types does not result in more-dissimilar functions.
|
;
Qiu et al., 1997;
Depew et al., 2002b) is
compatible with two different hypotheses on the nature of the codes: the
qualitative hypothesis invokes the unique activities of Dlx1/2 versus Dlx5/6
proteins in specifying each domain of BA1, whereas the quantitative hypothesis
proposes that the higher level of total Dlx protein in mdBA1 differentiates it
from mxBA1. Although we do not have positive proof for the quantitative
theory, a growing body of data has accumulated that cannot be explained by the
qualitative theory, at least on its own. First of all, a recent study
(Depew et al., 2005) and the
present work (Fig. 6) showed
that both Dlx1/2 and Dlx5/6 can regulate lower jaw
development, and that Dlx1/2 and Dlx5/6 co-regulate several
genes in mdBA1, directly or indirectly
(Fig. 7). More importantly,
Dlx1/2 perform opposing roles in mxBA1 versus in mdBA1: in mxBA1,
Dlx1/2 positively regulate Pou3f3 and other mxBA1-specific
genes to guide upper jaw development (Fig.
2); whereas in mdBA1, Dlx1/2 act partially redundantly
with Dlx5/6 to repress Pou3f3 (and mxBA1 fate), and
upregulate mdBA1-specific genes to promote lower jaw fate
(Fig. 7). These results suggest
that the patterning activity of a particular Dlx protein is context-dependent.
For example, the activities of Dlx1/2 proteins might be modulated by some
factors that are unevenly distributed between mxBA1 and mdBA1. Alternatively,
Pou3f3 might be induced by the moderate level of Dlx proteins found
in mxBA1, but repressed by the high level of Dlx proteins in mdBA1.
|
;
Ruest et al., 2004;
Miller et al., 2007;
Verzi et al., 2007), Dlx genes
are expressed such that Dlx5/6 are restricted to mdBA1, while
Dlx1/2 are in both mdBA1 and mxBA1. Dlx5/6 induce and/or
maintain expression of the genes that promote the development of the lower jaw
(Group A; see Fig. 8 legend).
At the same time, Dlx5/6 repress other sets of genes (Group B and
Group C) so that their expression is mostly confined to mxBA1. Here, Group B
and Group C genes promote upper jaw development. Dlx1/2 participate
in BA1 patterning by inducing and/or maintaining Group B genes in mxBA1. By
contrast, in mdBA1, Dlx1/2 positively regulate Group A genes and
repress Group B genes, directly or indirectly, to specify lower jaw fate.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2905/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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