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doi: 10.1242/10.1242/dev.00337

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Targeted deletion of a branchial arch-specific enhancer reveals a role of dHAND in craniofacial development

Hiromi Yanagisawa1, David E. Clouthier2, James A. Richardson1,3, Jeroen Charité1,* and Eric N. Olson1,{dagger}

1 Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, TX 75390-9148, USA
3 Department of Pathology, University of Texas Southwestern Medical Center at Dallas, TX 75390-9148, USA
2 Department of Molecular, Cellular and Craniofacial Biology, Birth Defects Center, School of Dentistry, University of Louisville, Louisville, KY 40292, USA
* Present address: Department of Cell Biology and Genetics, Erasmus Medical Center Rotterdam, Dr Molewaterplein 50, 3015GE Rotterdam, The Netherlands



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  		Fig. 1. Generation of BAenh–/– mice. (A) Targeting strategy. A 754 bp XhoI-BamHI fragment containing a 208 bp branchial arch-specific enhancer was replaced with a neor cassette flanked by loxP sites (triangles), introducing an XbaI site at the 5' end of the cassette. The +neoBAenh mutant allele contains a neor cassette. Cre-mediated recombination of this allele generated the {Delta}neoBAenh mutant allele. tk, thymidine kinase; S, SacI; E, EcoRI; Xh, XhoI; B, BamHI; N, NotI; Nd, NdeI; P, PstI; Xb, XbaI. (B) Southern blot analysis of tail DNA digested with SacI and hybridized with a 5' probe (upper panel), or digested with NdeI and XbaI and hybridized with a 3' probe (lower panel). The genotype is listed on the top of each lane. (C) PCR genotype of tail DNA. Each panel shows a PCR reaction amplifying a 500 bp neomycin gene (neo), a 300 bp fragment containing the branchial arch enhancer of dHAND (BAen), a 300 bp Cre-recombinase fragment (cre) and a 302 bp control sequence (cont).

 


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  		Fig. 2. Gross abnormalities of BAenh–/– mice. (A) Gross appearance of wild-type and BAenh–/– mutant mice at P1. The BAenh–/– mouse has a hypoplastic jaw (arrow) and an empty stomach. The wild-type littermate shows milk in the stomach (double arrows). (B) View of the palate of wild-type and BAenh–/– mutant mice at P1. The cleft palate in the mutant is indicated by arrows.

 


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  		Fig. 3. Histology of wild-type (A,C,E) and BAenh–/– mutant (B,D,F) mice at P1. (A,B) The secondary palate formed by fusion of bilateral palatine bones (pa) in the wild-type mouse (A) is absent in the BAenh–/– mutant mouse (arrows in B). Muscle fibers of the tongue (tg) are irregular and sparse in the mutant mouse. (C,D) Wild-type and homozygous mutant mice both show symphysis of Meckel's cartilage (asterisk) and lower incisors (i). (E,F) The junction between the malleus (m) and Meckel's cartilage (mc) is observed in wild-type and mutant mice. oc, otic capsule. Bars indicate 300 µm.

 


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  		Fig. 4. Craniofacial analysis of wild-type and BAenh–/– mice at P1. (A,C,E,G,I,K,M,O) wild-type; (B,D,F,H,J,L,N,P) BAenh–/– mutant mice. (A,B) Lateral view, (C-F) ventral view, (G-P) isolated bones and cartilages. (A-D) The mandible (ma) is hypoplastic and deformed, and Meckel's cartilage (arrows in C and D) is truncated in the mutant mouse (B,D). (E,F) Tympanic rings (ty) are thickened and deformed in the mutant mouse (F, compare arrows in E and F). Fusion of the bilateral palatine processes observed in the wild type mouse (E, dashed line) is absent in the mutant mouse (F, dashed lines). Asterisk in F indicates the presphenoid bone. (G,H) In the wild-type mouse, the secondary palate is formed by the fusion of bilateral palatine processes (arrows). In the BAenh–/– mouse, the palatine processes are not formed so that the presphenoid (ps) becomes visible. (I,J) Palatine bones viewed end on. Compare black arrows in I and J, which indicate fused palatine processes and absence of palatine processes, respectively. An asterisk indicates the presphenoid bone. (K,L) Ventral views of the mandibles. Note that the mandible in the BAenh–/– mouse is shorter and deformed. The angle between the left and right mandible is wider than that in the wild-type mouse. (M,N) Lateral views of the mandibles. Note that the articular process (ap) in the BAenh–/– mouse is severely hypoplastic and an ectopic process (black arrow) is formed. (O,P) Lateral views of middle ear cartilages and tympanic ring. In the mutant mouse, Meckel's cartilage (mc) is truncated, and the tympanic ring (ty) is shortened and thickened. Black and red arrows indicate the gonial bone and manubrium of the malleus, respectively. Note that the gonial bone is hypoplastic and the projection of the manubrium is abnormal in the BAenh–/– mutant. bs, basisphenoid; crp, coronoid process; h, hyoid; I, incus; lo, lamina obturans; m, malleus; mc, Meckel's cartilage; oc, otic capsule; pa, palatine; pt, pterygoid; tc, thyroid cartilage.

 


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  		Fig. 5. Cartilage preparations of wild-type and BAenh–/– mutant embryos at E14.5. (A,C) Wild-type; (B,D) BAenh–/– mutant. In the mutant embryo, Meckel's cartilage is truncated (compare black arrows in A and B). Close examination shows that the distal primordium of Meckel's cartilage is formed in the mutant embryo (arrow in D). h, hyoid; mc, Meckel's cartilage.

 


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  		Fig. 6. In situ hybridization analysis of dHAND, eHAND and Dlx6 transcripts in wild-type (A,D,F,H), +neoBAenh–/– (B,E,G,I) and {Delta}neoBAenh–/– (C) embryos at E10.5. dHAND (A,B,C,D,E), eHAND (F,G) and Dlx6 (H,I) transcripts were detected by in situ hybridization to transverse (A-C,F-H) or sagittal (D,E) sections. Note that ventrolateral dHAND expression in the first branchial arch of mutant embryos was absent (arrows in B,C,E), whereas heart expression was not affected (h in E). Branchial arches 1 and 2 are indicated.

 


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  		Fig. 7. Whole-mount in situ hybridization analysis of wild-type and the BAenh–/– embryos at E10.5. Left panel, lateral views; right panels, frontal views. Transcripts for dHAND (A), eHAND (B), Msx1 (C), Msx2 (D), Gsc (E), Dlx5 (F) and Dlx6 (G) were detected by whole-mount in situ hybridization to wild-type and BAenh–/– mutant embryos, as indicated. flb, forelimb bud; np, nasal process. Branchial arches 1 and 2 are indicated.

 


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  		Fig. 8. A model for the regulation of dHAND expression during craniofacial development. The expression domains of dHAND, eHAND and Dlx6 in the mandibular branchial arch of an E10.5 embryo are shown on the right side. Subdomains of dHAND expression are shown on the left side. ET-1 is secreted from the surface epithelium of the branchial arch (shown in brown) and acts on a 208 bp dHAND enhancer to induce a ventrolateral domain of dHAND (shown in red). The ventral domain of dHAND (shown purple) is regulated by unknown branchial arch enchancer(s) via a transcription factor (shown as a question mark) in response to ET-1. The most ventral tip of dHAND expression is regulated in an ET-1-independent manner (shown in dark blue).

 

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© The Company of Biologists Ltd 2003