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. Author manuscript; available in PMC: 2010 Mar 2.
Published in final edited form as: Genesis. 2003 Dec;37(4):188–194. doi: 10.1002/gene.10247

Dlx5/6-Enhancer Directed Expression of Cre Recombinase in the Pharyngeal Arches and Brain

Louis-Bruno Ruest 1, Robert E Hammer 2, Masashi Yanagisawa 3, David E Clouthier 1,*
PMCID: PMC2830754  NIHMSID: NIHMS164293  PMID: 14666512

Summary

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in β-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.

Keywords: Distal-less, homeobox gene, Cre recombinase, neural crest, craniofacial development, cell fate analysis, transgenic mouse

Signaling cascades initiated by cell–cell or tissue–tissue interactions during development are often conserved between various tissue types. This is especially true for craniofacial, limb, and brain development (Bulfone et al., 1993; Depew et al., 1999). Dlx5 and Dlx6, two of the six mammalian orthologs of the Drosophila Distalless (Dll) gene, are intimately involved in development of all three tissues (for reviews, see Merlo et al., 2000; Panganiban and Rubenstein, 2002; Zerucha and Ekker, 2000). These genes are found in a tail–tail tandem in mammals (Simeone et al., 1994) and are expressed in a variety of tissues during embryogenesis, including the developing pharyngeal arches, brain and apical ectodermal ridge (AER) of the limbs. (Acampora et al., 1999; Depew et al., 1999; Qiu et al., 1997), Dlx5−/− mice die shortly after birth, with defects in the facial skeleton and middle ear ossicles (Acampora et al., 1999; Depew et al., 1999). More extensive developmental defects are observed following targeted inactivation of both Dlx5 and Dlx6, including severe facial and limb defects similar to human Split Hand/Foot Malformation Type 1 (Beverdam et al., 2002; Depew et al., 2002; Robledo et al., 2002). As Dlx5/6−/− embryos have an apparent homeotic transformation of the lower jaw into a maxilla-like structure, Dlx5 and Dlx6 likely play a crucial role in conferring mandibular identity to cells in the mandibular pharyngeal arch (Beverdam et al., 2002; Depew et al., 2002; Robledo et al., 2002)

The signaling cascades that include Dlx5 and Dlx6 in facial, limb, and brain development appear quite complex. For example, expression of Dlx5 and Dlx6 in the pharyngeal arches is at least partially induced through endothelin-A (ETA) receptor signaling (Charité et al., 2001; Yanagisawa et al., 2003), with both required for expression of the gene encoding the bHLH transcription factor dHAND (Charité et al., 2001; Depew et al., 2002). In the limb bud, BMP4 is crucial for initiating Dlx5 transcription, as blocking BMP4 signaling disrupts Dlx5 expression (Merlo et al., 2002). Further, Dlx1, Dlx2, and Dlx5 can induce expression of a Dlx5/6-lacZ construct in isolated slices from an embryonic mouse forebrain (Stuhmer et al., 2002a).

To further characterize the potential roles of Dlx5 and Dlx6 in embryogenesis, we have undertaken a fate lineage analysis of Dlx5 and Dlx6 daughter cells. We created transgenic mice in which an enhancer from the mouse Dlx5/6 intergenic region, termed mI56i, was used to direct Cre recombinase expression to Dlx5/6 expression domains (Fig. 1A). Two highly conserved enhancers, mI56i (~440 bp) and mI56ii (~310bp), lie in the intergenic region between Dlx5 and Dlx6 (Ghanem et al., 2003). The mI56i enhancer is closest to Dlx6, ~1.5 kb from the 3′ end of exon 3 and is identical to the human ortholog (hI56i), excluding a 3 bp insertion in the human gene (Zerucha et al., 2000; Ghanem et al., 2003). Further, this enhancer is 81–99% identical in pairwise comparisons between mouse, human, zebrafish, Sphoeroides nephelus, and Takifuga rubripes (Ghanem et al., 2003). When used in transgenic constructs, the mI56i enhancer directs transgene expression to the pharyngeal arches and brain in mice in a Dlx5/6-specific pattern (Zerucha et al., 2000; Ghanem et al., 2003). In contrast, the mI56ii enhancer directs variable expression to the brain and very little expression to the arches (Ghanem et al., 2003). By crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain, in which Cre expression permanently activates lacZ expression, we have been able to follow the fate of the Dlx5/6 cell lineage by staining for β-galactosidase (β-gal) activity.

FIG. 1.

FIG. 1

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Early Dlx5/6-Cre expression in Dlx5/6-Cre;R26R mouse embryos. A: The Dlx5/6-Cre transgene consisted of a minimal β-globin promoter fused to a Cre cDNA containing a nuclear localization signal. The mouse id6/id5 enhancer 1 (mI56i) fragment provided tissue-specific expression. B–M: Lateral views of E9.5 (B,E,H,K), E10.5 (C,F,I,L), and E11.5 (D,G,J,M) Dlx5/6-Cre; R26R embryos stained in whole mount for β-galactosidase (β-gal) activity (B–D) or processed for whole mount in situ hybridization using cRNA probes against Cre (E–G), Dlx5 (H–J), or Dlx6 (K–M). B–D: Between E9.5 and E11.5, β-gal-labeled cells (blue) are observed in the mandibular (1) and second (2) pharyngeal arches (red arrows), with staining area and intensity increasing with age. Labeled cells are also observed in the diencephalon (di) telencephalon (te), and olfactory placode (op). Insets in C and D illustrate staining in the apical ectodermal ridge (aer). E–G: At E9.5, Cre expression is observed in a similar pattern to β-gal activity. However, at E10.5, most expression is absent, although small areas near the first pharyngeal cleft and caudal second arch still show Cre expression (arrows in F). Labeled cells are also present in the diencephalon and telencephalon. By E11.5, Cre expression is absent in the arches. H–J: Dlx5 expression mirrors that of β-gal staining between E9.5 and E11.5. Staining at E10.5 is strongest near the first arch cleft and caudal arch 2 (arrows in I), correlating to areas in which Cre expression is still observed. Note expression in the otic placode (otp), a site in which the mI56 enhancer does not direct transgene expression. K–M: Dlx6 expression appears similar to that of β-gal and Dlx5 expression between E9.5 and E11.5.

Microinjection of the transgenic construct in one-cell eggs resulted in the generation of 14 founder transgenic mice. Four male founders with transgene copy number between 10–20 copies per cell were test-bred with R26R female mice, with embryos collected at E (embryonic day) 10.5 and stained for β-gal activity. While similar staining patterns were obtained for all four lines, embryos from the 6-1 and 6-2 lines showed the strongest staining in the pharyngeal arches, brain, and limbs and were thus used in our subsequent analysis. In general, expression of the Dlx5/6-Cre transgene in the 6-1 line was stronger than in the 6-2 line in all areas other than the brain.

We first examined the extent of Cre expression in Dlx5/6-Cre;R26R embryos through analysis of β-gal staining. To better determine whether the observed staining was due to active expression of the mI56i enhancer, we also examined Cre expression through in situ hybridization analysis. We also compared β-gal staining with Dlx5 and Dlx6 expression to understand the extent to which the mI56i enhancer recapitulated endogenous Dlx5/6 expression. β-Gal staining was first obvious at E9.5 within the first and second pharyngeal arches (Fig. 1B), a pattern mirrored by the expression patterns of Cre (Fig. 1E), Dlx5 (Fig. 1H), and Dlx6 (Fig. 1K). These cells are presumably neural crest-derived, although expression in resident non-neural crest mesenchyme cannot be excluded. While DLX5 expression has been found along the border of the chick neural plate prior to crest cell migration (McLarren et al., 2003), we did not detect β-gal staining or Cre expression within the hindbrain or in streams extending from the hindbrain towards the pharyngeal arches. This supports previous studies in which neither Dlx5 nor Dlx6 expression has been observed in premigratory neural crest cells (Acampora et al., 1999; Depew et al., 1999; Robledo et al., 2002; Zerucha et al., 2000).

By E10.5, β-gal staining within arches 1 and 2 was more intense than observed at E9.5, with staining also observed in the diencephalon, telencephalon, olfactory placode, and apical ectodermal ridge (Fig. 1C). In contrast, Cre expression was found only near the first pharyngeal cleft and in the caudal portion of arch 2 (Fig. 1F), two places of strong Dlx5 expression (arrows in Fig. 1I). Cre expression was detected in other Dlx5 (Fig. 1I) and Dlx6 (Fig. 1L) expression domains, including the diencephalon, telencephalon, and AER. By E11.5, β-gal staining in Dlx5/6-Cre;R26R embryos (Fig. 1D) resembled the expression patterns of Dlx5 (Fig. 1J) and Dlx6 (Fig. 1M) in the pharyngeal arches, where Cre expression was not observed (Fig. 1G). The Dlx5/6 enhancer did not direct Cre expression in the Dlx5/6 expression domain within the otic placode, an aspect previously noted for this enhancer (Zerucha et al., 2000). While our findings suggest that most staining within the pharyngeal arches and arch-derived structures of Dlx5/6-Cre;R26R embryos after E9.5 represents a true fate of Dlx5/6 daughter cells, previous reports have shown that the mI56i enhancer directs lacZ expression in the mandibular arch of transgenic embryos at E10.5, as judged by β-gal staining (Zerucha et al., 2000; Ghanem et al., 2003). However, without actual in situ hybridization analysis of lacZ expression, it is not possible to determine whether the β-gal staining in these other mI56i transgenic embryos was due to active expression of the lacZ transgene or remaining β-gal enzyme, the latter having a half-life of at least 20–30 h (Bachmair et al., 1986).

While the staining patterns in Dlx5/6-Cre;R26R embryos closely matched the endogenous expression patterns of Dlx5 and Dlx 6 (Acampora et al., 1999; Depew et al., 1999; Robledo et al., 2002; Zerucha et al., 2000) and the β-gal staining in Dlx5/6-lacZ transgenic mouse embryos staining (Zerucha et al., 2000; Ghanem et al., 2003), the overall intensity of staining in most structures excluding the mandible appears to be less. This suggests that the enhancer unit required for Dlx5/6 expression in other sites may only be partially encompassed by the mI56i enhancer, resulting in only marginal expression in these sites. It is also likely that this enhancer requires other noncontiguous sequences to drive endogenous expression in specific tissues. Ghanem et al. (2003) recently illustrated that the mI56i enhancer drove more robust expression of a GFP construct in the forebrain of transgenic zebrafish when attached to the dlx6 promoter and a portion of the 5′ UTR rather than to the β-globin promoter.

By E13.5, labeled cells in the lower jaw were observed in the primary condensation of the mandibular bone and in the dental mesenchyme surrounding the tooth buds of the lower molars (Fig. 2A) and incisors (data not shown). Labeled cells were also present in the chondrocytes and surrounding perichondrium of Meckel’s cartilage (Fig. 2A) the mesenchyme surrounding the future glandular epithelium of the submandibular gland and tongue (data not shown). Labeled cells were not observed in the maxillary bone, although a few β-gal-positive cells were observed in the olfactory epithelium and in oral cavity epithelium overlying the maxillary incisors (data not shown).

FIG. 2.

FIG. 2

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Craniofacial contribution of Dlx5/6 lineage cells in Dlx5/6-Cre;R26R embryos. A–F: Transverse (A) and frontal (B–F) sections of E13.5 (A) and E18.5 (B–F) embryos through the mandible and nasal cavity of Dlx5/6-Cre;R26R embryos. After β-gal labeling, sections were counterstained with nuclear fast red. A: At E13.5, β-gal-labeled cells are observed in the mandibular bone (md) and dental mesenchyme (arrows) surrounding the mandibular molars (*), but not the maxillary molars (**). Faint staining is also observed in Meckel’s cartilage (mc) and surrounding perichondrium. B–E: By E18.5, labeled cells are observed in the mandibular bone and are mixed with unlabeled cells in the dental papilla (dp) of the mandibular molars (B,C) and mandibular (D) and maxillary (E) incisors (i). The oral epithelium (oep) remains unlabeled (B–D) except for a few labeled cells underlying the upper incisors (E). F: In the nasal cavity, labeled cells are observed in the columnar epithelial cells, although contribution is patchy. ns, nasal septum.

As observed at E10.5 and E11.5, Cre expression was absent in these structures (data not shown), even though Dlx5 is expressed within these areas and structures (our unpubl. data; Acampora et al., 1999; Robledo et al., 2002). That Dlx5/6 daughter cells are observed in the developing jaw in a Dlx5/6-specific manner even though the mI56i enhancer is downregulated by E10.5 likely indicates that the mI56i enhancer is mediating Dlx5/6 expression during early patterning of mandibular crest cells (Depew et al., 2002). Other enhancers would then be required for later events involving Dlx molecules (Acampora et al., 1999; Depew et al., 1999; Ferrari and Kosher, 2002). These findings also illustrate that comparison of β-gal staining in Cre transgene:R26R crosses with endogenous gene expression patterns may not be the most accurate method to determine whether β-gal staining is due to fated or active transgene expression.

Although it is thought that tissues and structures within the lower jaw of Dlx5/6-Cre;R26R embryos containing labeled cells are all neural crest derivatives, we cannot exclude the possibility that some stained cells are non-neural crest-derived. This is especially true in the mesenchyme surrounding the incisors and molars, as this tissue is composed of both neural crest and non-neural crest-derived cells (Chai et al., 2000). However, our overall staining pattern correlates well with that of Chai et al., suggesting that, within the mandible, stained cells represent postmigratory neural crest cells.

At both E16.5 and E18.5, labeled cells were most abundant in the ossified mandible, where they were observed along the entire proximal-distal axis (Figs. 2B–D, 3A,B,D–F). In whole mount-stained E16.5 embryos, labeled cells were also observed along the midline of the snout just distal to the maxillary incisors (arrows, Fig. 3B). When these embryos were cleared to better visualize staining, labeled cells were observed in the mandibular incisors and entire mandible, including the articular, coronoid, and condylar processes, as well as in the ear pinna, olfactory epithelium, maxillary incisors, and oral epithelium distal to the incisors (Fig. 3D–F). Sections through the lower jaw illustrated the large contribution of stained cells to the mandible, while labeled cells in the mandibular incisor and molar dental papilla were extensively mixed with unlabeled cells (Fig. 2B–D). Labeled cells were also observed in the maxillary incisor dental papilla (Fig. 2E); maxillary molars did not contain labeled cells (Fig. 2C). More scattered staining was observed in the lamina propria of the tongue and Meckel’s cartilage (Fig. 2B,D) and the columnar (Fig. 2F) and stratified (Fig. 2E) epithelium of the nasal cavity. Within the middle ear and throat, neural crest-derived structures, including the malleus, incus, stapes, hyoid, and thyroid cartilage, all contained labeled cells, although their contribution to these structures was limited (data not shown).

FIG. 3.

FIG. 3

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Contribution of Dlx5/6 daughter cells to mandibular and maxillary structures. β-gal staining in E16.5 Dlx5/6-Cre;R26R embryos. Lateral (A,C–F) and ventral (B) views of an E16.5 Dlx5/6-Cre; R26R embryo stained in whole mount and either photographed (A–C) or cleared in benzyl benzoate:benzyl alcohol before photographing. A,B: Strong staining is observed throughout the mandible (md), ear pinna (ep), nasal pit (black arrow in B), and medial maxilla epithelium (yellow arrows in B; see also inset). C: An enlarged view of the forelimb (lb) shows β-gal cells along the tips of the digits (arrows). D–F: Embryo in A cleared, illustrating a contribution of labeled cells throughout the mandible, including the articular (ar), condylar (cp), and coronoid (crp) processes (F) and mandibular incisors (i* in E). Staining in the upper jaw is observed in the olfactory epithelium, maxillary incisors (i**) and oral epithelium just distal to the maxillary incisors (arrow in E).

Labeled cells were also found in areas of the developing brain. In E13.5 Dlx5/6-Cre;R26R embryos, labeled cells were restricted in the diencephalon in areas that will eventually form the thalamus and hypothalamus and in the ganglionic eminences and future caudate nucleus of the telencephalon (Fig. 4A). This pattern of staining reflected the endogenous expression patterns of Dlx5 and Dlx6 (Acampora et al., 1999; Eisenstat et al., 1999; Liu et al., 1997; Simeone et al., 1994; Stuhmer et al., 2002a; Zerucha et al., 2000). In the brain of E18.5 Dlx5/6-Cre;R26R embryos, labeled cells were observed in telencephalon-derived structures, including the caudate nucleus (cn), basal layer of the cortex (bc), and medial nucleus of the amygdala (am) (Fig. 4B–D). In the thalamus, labeled cells were restricted to specific areas that included the bed nucleus (bn), ventral nucleus (vn), and posterior nucleus (pn) (Fig. 4C,D). In the hypothalamus, labeled cells were observed in the lateral (data not shown) and medial preoptic (po) nuclei and premamillary nucleus (pmn) (Fig. 4C,D). Scattered labeled cells were also observed in the olfactory bulb and cerebral cortex (data not shown). These areas represent areas in the mouse brain in which Dlx5 and Dlx6 are expressed (Long et al., 2003) and where zfdlx4/6-LacZ (Stuhmer et al., 2002b) and Dlx5/6-Cre (Stenman et al., 2003) transgenes have been previously reported to be expressed. This active expression prevents any conclusions from being drawn about the specific fate of Dlx5/6 daughter cells within the CNS. However, our results do illustrate that Dlx5/6-Cre expression may be useful in inactivating conditionally targeted alleles within the CNS.

FIG. 4.

FIG. 4

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Analysis of β-gal staining in the brain of Dlx5/6-Cre;R26R embryos. Transverse (A), sagittal (B) and frontal (C,D) sections of E13.5 (A) and E18.5 (B–D) Dlx5/6-Cre;R26R embryos. A,B: β-gal-labeled cells are observed in parts of the diencephalon (di) and ganglionic eminences of the telencephalon (te) at E13.5 (A) and in the thalamus (th), hypothalamus (hy) and caudate nucleus (cn) at E18.5 (B). C,D: In frontal views showing the right half of brain sections from an E18.5 embryo, labeled cells are observed in areas derived from the telencephalon, including the caudate nucleus (cn), globus pallidus (gp), and basal cortex (bc) (C). In areas derived from the diencephalon, β-gal-labeled cells are observed in the bed nucleus (bn) and preoptic nuclei (po), and are scattered in the middle forebrain bundle (mfb). Labeled cells are observed in the medial nucleus of the amygdala (am) and in parts derived form the diencephalon, including the reticularis nucleus (rn), ventral nucleus (vn) and posterior nucleus (pn) of the thalamus, and zona incerta (zi) and premamillary nucleus (pmn) of the hypothalamus (D). oc, otic capsule; sl, lateral nucleus of the septum; tm, medial nucleus of the thalamus.

Labeled cells were also observed along the AER and its remnants of the limbs at both E13.5 (data not shown) and E16.5 (Fig. 3C), with β-gal staining in the latter composing the tips of the limb digits (data not shown). Other sites of β-gal labeling at E18.5 included scattered cells in the neural layer of the eye, kidney, and intestine (data not shown). β-Gal staining was not observed in the membranous bone of the ribs, limbs, and digits, all of which are affected in Dlx5/Dlx6−/− embryos. This further illustrates that other enhancers in the Dlx5/6 locus work in combination with the enhancer used in this study to direct proper temporospatial expression of Dlx5 and Dlx6. Intermittent staining was observed along the smooth muscle of the outflow tract of the heart (data not shown). This was considered to be due to ectopic transgene expression, as the occurrence, extent, and intensity of β-gal staining differed between E18.5 embryos from the 6-1 line and was not observed in the 6-2 line. Further, when the lacZ gene is knocked into the Dlx5/6 locus, β-gal staining is not observed in the outflow tract (Robledo et al., 2002).

In conclusion, we have shown, using a Dlx5/6-Cre transgene, that Dlx5/6 daughter cells are prominently observed throughout the bone and teeth of the lower jaw and in the maxillary incisors of near-term embryos. Fewer numbers of labeled cells are also observed within specific regions of the developing brain and in the AER of the developing limbs. These sites correspond to areas in which Dlx5 and Dlx6 are known or hypothesized to have functions during embryonic development. While a previous study used a Dlx5/6-Cre transgene to examine Dlx5/6 daughter cells within the developing brain (Stenman et al., 2003), this is the first report using this enhancer to fate-map a subset of presumably cephalic neural crest cells during craniofacial development. These Dlx5/6-Cre transgenic mice should be useful both in better understanding the roles of Dlx5 and Dlx6 during development of the mandible, dentition, and the brain and as a valuable tool for creating tissue-specific gene deletions in mice containing conditionally targeted alleles. This is especially true in the mandible, as our mI56i enhancer appears to be downregulated in the mandibular arch by E10.5, allowing a tight temporal inactivation of genes within that tissue.

MATERIALS AND METHODS

Generation of the Dlx5/6-Cre Transgenic Construct

The plasmid pCre contains a β-globin minimal promoter (Yee and Rigby, 1993) driving the expression of a Cre cDNA containing a nuclear localization signal. To construct the Dlx5/6-Cre transgene, a mouse genomic XhoI fragment containing the mouse id6/id5 enhancer 1 (mI56i; Zerucha et al., 2000) was subcloned into the XhoI site of pCre, 3′ of the SV40 poly A cassette (both the enhancer and pCre were kind gifts from M. Ekker). This 440 bp enhancer is one of two highly conserved enhancers in the intergenic region between Dlx5 and Dlx6 and lies closer to Dlx6 than the second enhancer, mI56ii (Zerucha et al., 2000; Ghanem et al., 2003). The resulting plasmid, pDlx5/6-Cre, was then restricted with KpnI and SacI. This digest liberated the complete 1.8 kb Dlx5/6-Cre transgene, which was separated on a 1% agarose gel, purified by perchlorate elution, and quantified.

Production of Transgenic Mice

To construct Dlx5/6-Cre transgenic mice, the purified Dlx5/6-Cre construct was injected at 3 ng/μl into C57BL6/J × SJL F2 fertilized one-cell eggs and two-cell embryos were transferred to pseudopregnant females as previously described (Clouthier et al., 1997). Fourteen founder transgenic mice were identified by dot blot hybridization, with transgene copy number ranging from 2–40 copies per cell. Four of these founders were bred to R26R mice (strain 129/Sv-Gtrosa26tm1Sor, The Jackson Laboratory, Bar Harbor, ME) (Soriano, 1999). Embryos were collected at E10.5 and stained for β-gal activity to evaluate Cre expression. Two lines (6-1 and 6-2) that produced compound hemizygous pups with β-gal activity in a Dlx5/6-specific pattern were used for this study.

Genotyping of Dlx5/6-Cre Transgenic Mice and Embryos

PCR genotyping was performed using genomic DNA isolated from tail biopsy or embryonic yolk sac. Detection of the Dlx5/6-Cre transgene was determined using the primers 5′-AGGGCAGAGCCATCTATTGC and 5′-GCATAACCAGTGAAACAGCATTGCTG. The presence of the R26R allele was determined using primers 5′-GCGAAGAGTTTGTCCTCAACC-3′ (R1295) and 5′-AAAGTCGCTCTGAGTTGTTAT-3′ (R26RF) as previously described (Soriano, 1999). Bands were visualized on 1% agarose gels.

β-Gal Staining and Histological Analysis

β-Gal staining was performed as previously described (Ruest et al., 2003). In short, E9.5–11.5 embryos and E16.5 embryos were fixed in 4% paraformaldehyde, rinsed in lacZ buffer, and stained overnight at room temperature. Embryos were analyzed and photographed on an Olympus SZX12 stereomicroscope fitted with a DP11 digital camera. After analysis and photography, some E16.5 embryos were dehydrated in methanol before clearing in 2:1 benzyl benzoate: benzyl alcohol to allow better visualization of staining patterns. For staining at E13.5 and E18.5, embryos were embedded in OCT (TissueTek) and sectioned at 14 μ. Sections were fixed in 0.5% glutaraldehyde in PBS, rinsed, and stained at 37°C as described above. Slides were counterstained either with eosin or nuclear fast red, coverslipped with DPX mounting media (BDH), and analyzed with a Nikon E600 microscope and attached SPOT RT digital camera.

Acknowledgments

Contract grant sponsor: NIDCR/NIH, (Career Development Award to D.E.C.), Contract grant sponsor: National Institutes of Health, Contract grant number: DE14181, Contract grant sponsor: Kentucky Excellence in Education Research Trust Fund (to D.E.C.).

The authors thank Bo Mason, Jennifer Beavin, Tinisha Taylor, Tim Morgan, and Shelly Dixon for technical assistance, Marc Ekker for the Dlx5/6 enhancer element and insightful comments, and Hiromi Yanagisawa and Mengsheng Qiu for critical reading and discussion of the manuscript. M.Y. is an investigator of the Howard Hughes Medical Institute.

LITERATURE CITED

  1. Acampora D, Merlo GR, Paleari L, Zerega B, Postiglione MP, Mantero S, Bober E, Barbieri O, Simeone A, Levi G. Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5. Development. 1999;126:3811–3821. doi: 10.1242/dev.126.17.3795. [DOI] [PubMed] [Google Scholar]
  2. Bachmair A, Finley D, Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue. Science. 1986;234:179–186. doi: 10.1126/science.3018930. [DOI] [PubMed] [Google Scholar]
  3. Beverdam A, Merlo GR, Paleari L, Mantero S, Genova F, Barbieri O, Janvier P, Levi G. Jaw transformation with gain or symmetry after Dlx5/Dlx 6 inactivation: mirror of the past. genesis. 2002;34:221–227. doi: 10.1002/gene.10156. [DOI] [PubMed] [Google Scholar]
  4. Bulfone A, Kim HJ, Puelles L, Porteus MH, Grippo JF, Rubenstein JLR. The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain face and limbs in midgestation mouse embryos. Mech Dev. 1993;40:129–140. doi: 10.1016/0925-4773(93)90071-5. [DOI] [PubMed] [Google Scholar]
  5. Chai Y, Jiang X, Ito Y, Bringas P, Han J, Rositch DH, Soriano P, McMahon AP, Sucov HM. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000;127:1671–1679. doi: 10.1242/dev.127.8.1671. [DOI] [PubMed] [Google Scholar]
  6. Charité J, McFadden DG, Merlo GR, Levi G, Clouthier DE, Yanagisawa M, Richardson JA, Olson EN. Role of Dlx6 in regulation of an endothelin-1-dependent, dHAND branchial arch enhancer. Genes Dev. 2001;15:3039–3049. doi: 10.1101/gad.931701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clouthier DE, Comerford SA, Hammer RE. Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-β1 transgenic mice. J Clin Invest. 1997;100:2697–2713. doi: 10.1172/JCI119815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Depew MJ, Liu JK, Long JE, Presley R, Meneses JJ, Pedersen RA, Rubenstein JL. Dlx5 regulates regional development of the branchial arches and sensory capsules. Development. 1999;126:3831–3846. doi: 10.1242/dev.126.17.3831. [DOI] [PubMed] [Google Scholar]
  9. Depew MJ, Lufkin T, Rubenstein JL. Specification of jaw subdivisions by Dlx genes. Science. 2002;298:381–385. doi: 10.1126/science.1075703. [DOI] [PubMed] [Google Scholar]
  10. Eisenstat DD, Liu JK, Mione M, Zhong W, Yu G, Anderson SA, Ghattas I, Puelles L, Rubenstein JL. DLX-1, DLX-2 and DLX-5 expression define distinct stages of basal forebrain differentiation. J Comp Neurol. 1999;414:217–237. doi: 10.1002/(sici)1096-9861(19991115)414:2<217::aid-cne6>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]
  11. Ferrari D, Kosher RA. Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification. Dev Biol. 2002;252:257–270. doi: 10.1006/dbio.2002.0862. [DOI] [PubMed] [Google Scholar]
  12. Ghanem N, Jarinova O, Amores A, Long Q, Hatch G, Park BK, Rubenstein JLR, Ekker M. Regulatory roles of conserved intergenic domains in vertebrate Dlx bigene clusters. Genome Res. 2003;13:533–543. doi: 10.1101/gr.716103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Liu JK, Ghattas I, Liu S, Chen S, Rubenstein JL. Dlx genes encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev Dyn. 1997;210:498–512. doi: 10.1002/(SICI)1097-0177(199712)210:4<498::AID-AJA12>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  14. Long JE, Garel S, Depew MJ, Tobet S, Rubenstein JLR. DLX5 regulates development of peripheral and central components of the olfactory system. J Neurosci. 2003;23:568–578. doi: 10.1523/JNEUROSCI.23-02-00568.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McLarren KW, Litsiou A, Streit A. DLX5 positions the neural crest and preplacode region at the border of the neural plate. Dev Biol. 2003;259:34–47. doi: 10.1016/s0012-1606(03)00177-5. [DOI] [PubMed] [Google Scholar]
  16. Merlo GR, Zerega B, Paleari L, Trombino S, Mantero S, Levi G. Multiple functions of Dlx genes [Review] Int J Dev Biol. 2000;44:619–626. [PubMed] [Google Scholar]
  17. Panganiban G, Rubenstein JL. Developmental functions of the Distal-less/Dlx homeobox genes [Review] Development. 2002;129:4371–4386. doi: 10.1242/dev.129.19.4371. [DOI] [PubMed] [Google Scholar]
  18. Qiu M, Bulfone A, Ghattas I, Meneses JJ, Christensen L, Sharpe PT, Presley R, Pedersen RA, Rubenstein JLR. Role of the Dlx homeobox genes in proximodistal patterning of the branchial arches: mutations of Dlx-1, Dlx-2, and Dlx-1 and -2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches. Dev Biol. 1997;185:165–184. doi: 10.1006/dbio.1997.8556. [DOI] [PubMed] [Google Scholar]
  19. Robledo RF, Rajan L, Li X, Lufkin T. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev. 2002;16:1089–1101. doi: 10.1101/gad.988402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ruest L-B, Dager M, Yanagisawa H, Charité J, Hammer RE, Olson EN, Yanagisawa M, Clouthier DE. dHAND-Cre transgenic mice reveal specific potential functions of dHAND during craniofacial development. Dev Biol. 2003;275:263–277. doi: 10.1016/s0012-1606(03)00068-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Simeone A, Acampora D, Pannese M, Desposito M, Stornaiuolo A, Gulisano M, Mallamaci A, Kastury K, Druck T, Huebner K. Cloning and characterization of two members of the vertebrate Dlx gene family. Proc Natl Acad Sci USA. 1994;91:2250–2254. doi: 10.1073/pnas.91.6.2250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Gen. 1999;21:70–71. doi: 10.1038/5007. [DOI] [PubMed] [Google Scholar]
  23. Stenman J, Toresson H, Campbell K. Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis. J Neurosci. 2003;23:167–174. doi: 10.1523/JNEUROSCI.23-01-00167.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stuhmer T, Anderson SA, Ekker M, Rubenstein JLR. Ectopic expression of the Dlx genes induces glutamic acid decarboxylase and Dlx expression. Development. 2002a;129:245–252. doi: 10.1242/dev.129.1.245. [DOI] [PubMed] [Google Scholar]
  25. Stuhmer T, Puelles L, Ekker M, Rubenstein JLR. Expression from a Dlx gene enhancer marks adult mouse cortical GABAergic neurons. Cereb Cortex. 2002b;12:75–85. doi: 10.1093/cercor/12.1.75. [DOI] [PubMed] [Google Scholar]
  26. Yanagisawa H, Clouthier DE, Richardson JA, Charité J, Olson EN. Targeted deletion of a branchial arch-specific enhancer reveals a role of dHAND in craniofacial development. Development. 2003;130:1069–1078. doi: 10.1242/dev.00337. [DOI] [PubMed] [Google Scholar]
  27. Yee S-P, Rigby PWJ. The regulation of myogenin gene expression during the embryonic development of the mouse. Genes Dev. 1993;7:1277–1289. doi: 10.1101/gad.7.7a.1277. [DOI] [PubMed] [Google Scholar]
  28. Zerucha T, Ekker M. Distal-less-related homeobox genes of vertebrates: evolution, function, and regulation. Biochem Cell Biol. 2000;78:593. [PubMed] [Google Scholar]
  29. Zerucha T, Stuhmer T, Hatch G, Park BK, Long Q, Yu G, Gambarotta A, Schultz JR, Rubenstein JLR, Ekker M. A highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between Dlx genes in the embryonic forebrain. J Neurosci. 2000;20:709–721. doi: 10.1523/JNEUROSCI.20-02-00709.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]

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