DEV019778 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF

• Supplemental Table S3 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Facial primordia of E10.5 mouse embryos and normal expression patterns of Dlx genes therein. (A) Schematic of E10.5 mouse embryo head indicating the names of various parts. BA2, the second branchial arch; FNP, frontonasal prominence; mdBA1, mandibular component of the first branchial arch; mxBA1, maxillary component of the first branchial arch; OLP, olfactory placode; OTP, otic placode. (B) Schematic showing the definition of the proximo (P)-distal (D) axis in BA1 and BA2. A and B are modified from Depew et al. (Depew et al., 1999) with permission. (C) E10.5 wild-type embryos processed by whole-mount in situ hybridization for the Dlx genes indicated.

• Supplemental Figure S2 -

  Fig. S2. Expression of Dlx5 and Dlx6 in BA1 of E8.5-9.0 wild-type embryos. Wild-type embryos processed by whole-mount in situ hybridization. The developmental stages of the embryos are indicated by somite numbers. (A,F) Dorsal views. Expression of Dlx5, but not Dlx6, is detected in hindbrain neural crest at the 7- to 8-somite stage. (B-E,G-J) Lateral views. In the first branchial arch, expression of both Dlx5 and Dlx6 begins around the 13- to 14-somite stage, with Dlx5 showing much stronger hybridization signal than Dlx6. The expression levels of both genes increase gradually over the next ∼10 hours. Arrows, BA1; arrowhead in A, hindbrain neural crest; open arrowheads, anterior neural ridge. Scale bar: 0.2 mm.

• Supplemental Figure S3 -

  Fig. S3. Genomic structures of the putative non-coding RNA (ncRNA) genes that are downstream of Dlx5/6. Genomic loci around Dlx1 (A), Dlx6 (B), Pou3f3 (C) and Foxl2 (D) indicating putative ncRNA genes. Arrows, direction of transcription; boxes, exons; orange bars, in situ hybridization probes for A/S Dlx1, Evf1/2, 2610017I09Rik, 2900092D14Rik and E330015D05Rik used in this study. Antisense Dlx1 (A/S Dlx1) transcripts contain short open reading frames (ORFs, 126-249 bp) and have no homology to any protein-coding genes, and so they are suspected to be ncRNAs. One of them (NR_002854) has been described previously in the brain (McGuiness et al., 1996; Liu et al., 1997). 26100017I09Rik is transcribed from the opposite strand of DNA from Pou3f3 and has at least six exons that undergo extensive alternative splicing (C). 2900092D14Rik, immediately downstream of Pou3f3, is a single exon with a 1.1 kb transcript (2C). Since both Riken genes have short ORFs (<250 bp) and the putative products have no homology to any known protein, their transcripts are likely to be ncRNAs. E330015D05Rik, transcribed in the opposite direction from Foxl2 (D), is an unclassifiable gene with an ORF for a hypothetical protein of 82 amino acids.

• Supplemental Figure S4 -

  Fig. S4. Comparison of Dlx5^−/− and Dlx6^−/− head skeletal phenotypes and branchial arch gene expression changes (additional data). (A-F) Head skeleton of E18.5-P0 animals stained with Alcian Blue (cartilage) and Alizarin Red (bone). (A-C) Skull base views after removing dentary. The proximal part of the mutant Meckel's cartilage is kinked, unlike in the wild type, which is smooth and straight (arrows). Arrowheads indicate the os paradoxicum of the mutants. (D-F) Basal views of the nasal cartilage and associated bones. Arrows and arrowheads indicate the skeletal abnormalities of the mutants; see text for details. (G-L) Lateral views of E10.5 embryos processed by whole-mount in situ hybridization. Arrows and arrowheads, downregulation (G-I) or upregulation (J-L) of expression in the mutant mdBA1 and BA2, respectively. AT, ala temporalis; BO, basioccipital; BS, basisphenoid; NC, nasal capsule; NS, nasal septum; PL, palatine; VM, vomer; for remainder, see legends to Figs 4 and 5. Scale bar: 1 mm.

• Supplemental Figure S5 -

  Fig. S5. Expression analysis of the genes regulated by Dlx5/6 in Dlx5^−/− and Dlx6^−/− single mutants. Frontal views (A-X) and lateral views (Y-p) of E10.5 embryos processed by whole-mount in situ hybridization.

• Supplemental Figure S6 -

  Fig. S6. Dlx1^−/−;Dlx6^−/− and Dlx2^−/−;Dlx6^−/− head skeleton phenotypes (additional data). Head skeleton and skeletal elements of E18.5-P0 animals stained with Alcian Blue (cartilage) and Alizarin Red (bone). (A-F) Skull base views after removing dentary; the right half is the mirror image of the left half with colors to highlight individual skeletal components. The maxilla of upper jaw normally extends one process laterally to connect to jugal, which in turn articulates with squamosal, and the second process medially that articulates with the palatine-pterygoid-basisphenoid series on the skull base. The dentaries of Dlx1^−/−;Dlx6^−/− and Dlx2^−/−;Dlx6^−/− appear similar to the maxilla and other upper jaw elements in their shape and articulations; for the mutant dentaries, one branch articulates with squamosal as with the wild-type dentary, but the other process grows medially to articulate with basisphenoid (see Fig. 6M-R, colored). In this view, the small fragments of Dlx1^−/−;Dlx6^−/− and Dlx2^−/−;Dlx6^−/− dentaries can be considered duplicate jugals (JG*, see Fig. 6Q,R, lavender) and pterygoids (PT*, see Fig. 6Q,R, lime green). (G-L) Laryngeal cartilage. Basihyoid is elongated in Dlx1^−/−;Dlx6^−/−, and is split into two normal-length pieces in Dlx2^−/−;Dlx6^−/− (arrows in G,L). The lesser horn of hyoid in Dlx1^−/−;Dlx6^−/− changes its angle toward the greater horn of hyoid, and in Dlx2^−/−;Dlx6^−/−, the two are fused to make an apparent duplicate set of greater horns. BA2, branchial arch 2; BH, basihyoid; CC, cricoid cartilage; GH, greater horn of hyoid; LH, lesser horn of hyoid; mdBA1, mandibular branchial arch; MX, maxilla; OLP, olfactory placode/pit; OTP, otic placode/pit; TC, thyroid cartilage; for remainder, see legends to Figs 4-6 and S5. Scale bar: 1 mm.