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First published online August 12, 2008
doi: 10.1242/10.1242/dev.019778

Development 135, 2905-2916 (2008)
Published by The Company of Biologists 2008
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Dlx genes pattern mammalian jaw primordium by regulating both lower jaw-specific and upper jaw-specific genetic programs

Juhee Jeong1,*, Xue Li2, Robert J. McEvilly3, Michael G. Rosenfeld3, Thomas Lufkin4 and John L. R. Rubenstein1,*

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.


Figure 1
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  		Fig. 1. Branchial arch expression patterns of the genes downregulated in Dlx5/6-/-. Lateral views (A-L) or frontal views (M-X) of wild-type and Dlx5/6-/- E10.5 mouse embryos processed by whole-mount in situ hybridization. Arrows and arrowheads indicate changes in gene expression in mdBA1 and BA2, respectively.

 

Figure 2
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  		Fig. 2. Branchial arch expression patterns of the genes upregulated in Dlx5/6-/-. (A-W) Lateral views of wild-type, Dlx5/6-/- and Dlx1/2-/- E10.5 mouse embryos processed by whole-mount in situ hybridization. Arrows and solid arrowheads indicate upregulation of expression in Dlx5/6-/- mdBA1 and distal BA2, respectively; open arrowheads indicate downregulation of expression in Dlx1/2-/- mxBA1.

 

Figure 3
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  		Fig. 3. Identification of a Dlx-regulated enhancer upstream of Gbx2. (A) Evolutionary conservation of the genomic sequence upstream of Gbx2 analyzed using 30-way multiz alignment (Blanchette et al., 2004Go). Image generated using University of California Santa Cruz Genome Browser. Blue, degree of conservation among mammals; black vertical bars, conservation in each species as indicated; red bar, the Gbx2 enhancer used for the reporter assay in C; black box, coding region of an exon; white box, untranslated region. (B) ClustalW alignment (Larkin et al., 2007Go) of the 0.6 kb region of the Gbx2 enhancer conserved down to chicken. *, conserved nucleotides; putative Dlx-binding sites are highlighted in orange. (C) Results of the luciferase reporter activation assay. pGL, minimal promoter-reporter plasmid without an enhancer; Gbx2en, pGL plasmid with mouse Gbx2 enhancer; - Dlx5, co-transfected with empty expression vector; + Dlx5, co-transfected with Dlx5 expression vector.

 

Figure 4
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  		Fig. 4. Expression of Pou3f3 RNA during jaw development and craniofacial skeletal defects in Pou3f3-/- mutants. (A-E) In situ hybridization for Pou3f3 on the coronal sections of E10.5 (A,B) and E13.5 (C-E) wild-type heads. B and D are high-magnification views of the boxed areas in A and C, respectively. C and E are from the same head, but C is rostral to E. Arrow in C, mdBA1 expression of Pou3f3. (F-M) Head skeleton of E18.5 wild-type (F,H,J,L) and Pou3f3-/- (G,I,K,M) mice stained with Alcian Blue (cartilage) and Alizarin Red (bone). H and I are enlargements of the boxed areas in F and G, respectively. Asterisks in I indicate the absence of jugal and squamosal bone in the mutant. (J,K) Otic capsule and middle ear ossicles. Open arrowheads, incus phenotype of the mutant; solid arrowheads, abnormal attachment of stapes and styloid process in the mutant. L and M are the same pictures as H and I, but with individual skeletal elements highlighted by color: yellow, zygomatic process of maxilla; green, jugal; dark green, zygomatic process of squamosal; orange, squamosal; gray, lamina obturans. DE, dentary; FN, frontal bone; IN, incus; JG, jugal; LM, lower molar; LO, lamina obturans; MA, maleus; PA, parietal bone; PS, palatal shelf; rt, retrotympanic process of squamosal bone; SP, styloid process; SQ, squamosal bone; ST, stapes; TO, tongue; UM, upper molar; zpm, zygomatic process of maxilla; zps, zygomatic process of squamosal bone. Scale bars: 0.1 mm in A; 0.5 mm in C,E; 1 mm in F,J.

 

Figure 5
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  		Fig. 5. Comparison of Dlx5-/- and Dlx6-/- head skeletal phenotypes and branchial arch gene expression changes. (A) Structure of the Dlx6-lacZ (Dlx6-) allele. Black boxes, exon coding region; white boxes, untranslated region; red boxes, homeodomain; blue bars, PCR primers for genotyping. (B-J) Head skeleton and skeletal elements of E18.5-P0 mice stained with Alcian Blue (cartilage) and Alizarin Red (bone). (B-D) Lateral views of the whole head. (E-G) Dentaries. (H-J) Otic capsules and associated skeletal elements. Arrows and arrowheads indicate the skeletal abnormalities of the mutants; see text for details. (K-V) Lateral views of E10.5 mouse embryos processed by whole-mount in situ hybridization. Arrows and arrowheads, downregulation (K-P) or upregulation (Q-V) of expression in the mutant mdBA1 and BA2, respectively. agp, angular process; cdp, condylar process; crp, coronoid process; GN, gonial; IP, interparietal; MC, Meckel's cartilage; NA, nasal bone; OC, otic capsule; OP, os paradoxicum; TY, ectotympanic; for remainder, see legend to Fig. 4. Scale bar: 1 mm.

 

Figure 6
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  		Fig. 6. Dlx1-/-;6-/- and Dlx2-/-;6-/- head skeleton phenotypes. Head skeleton and skeletal elements of E18.5-P0 mice stained with Alcian Blue (cartilage) and Alizarin Red (bone). (A-F) Lateral views of the whole head. (G-L) Dentaries. (M-R) Skull base views; the right half is a mirror image of the left half with individual skeletal components highlighted by color. (S-X) Oblique lateral views of the head after removing dentary. (Y-d) Middle ear ossicles, ectotympanic and gonial. Note that os paradoxicum (OP) of Dlx1-/-;6-/- and Dlx2-/-;6-/- has been integrated into the skull base (see Fig. S6E,F in the supplementary material), and thus excluded from c and d. avp, alveolar process; BS, basisphenoid; E, eye; PT, pterygoid; for remainder, see legends to Figs 4 and 5. *, duplicates found in the mutants. Scale bars: 1 mm.

 

Figure 7
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  		Fig. 7. Gene expression phenotypes in Dlx1-/-;6-/-, Dlx2-/-;6-/- and Dlx2-/-;5-/- mdBA1. Lateral views (A-L) and frontal views (M-P) of E10.5 mouse embryos processed by whole-mount in situ hybridization. Note that the samples in M-P are each one half of a hemisected face, but are digitally modified into a full face to help visualization. Arrows, expression changes in mdBA1.

 

Figure 8
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  		Fig. 8. A model for the mechanism of jaw patterning by Dlx genes. In BA1 mesenchyme, Dlx5/6 are only expressed in mdBA1, whereas Dlx1/2 are expressed in both mdBA1 and mxBA1. Dlx5/6 induce and/or maintain expression of Group A genes (Dlx3, Dlx4, Hand1, Hand2, Gbx2, Gsc, Alx3, Alx4, Bmper, Cited1, Zac1, Unc5c, Hgf, Rgs5, A/S Dlx1 and Evf1/2) in mdBA1, while repressing Group B (Pou3f3, 2610016I09Rik and 2900092D14Rik) and Group C (Foxl2, E330015D05Rik, Cyp26a1 and Irx5) genes so that their expression is mostly confined to mxBA1. Dlx1/2 induce and/or maintain Group B genes in mxBA1. By contrast, in mdBA1, Dlx1/2 induce and/or maintain Group A genes and repress Group B genes. Presumably, Group A genes promote lower jaw development, whereas Group B and Group C genes promote upper jaw development.

 

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© The Company of Biologists Ltd 2008