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First published online 15 April 2009
doi: 10.1242/dev.032714

Development 136, 1665-1674 (2009)
Published by The Company of Biologists 2009
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A cluster of three long-range enhancers directs regional Shh expression in the epithelial linings

Tomoko Sagai1, Takanori Amano1, Masaru Tamura1, Yoichi Mizushina1, Kenta Sumiyama2 and Toshihiko Shiroishi1,*

1 Mammalian Genetics Laboratory, National Institute of Genetics, Yata-1111 Mishima Shizuoka-ken 411-8540, Japan.
2 Population Genetics Laboratory, National Institute of Genetics, Yata-1111 Mishima Shizuoka-ken 411-8540, Japan.


Figure 1
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  		Fig. 1. Location of CNCSs in the interval between the Shh-coding sequence and Lmbr1. (A) The genomes of five evolutionarily distant species: mouse (chr5), human (chr7), chicken (chr2), Xenopus (chr28) and medaka (chr20). The scale is in kb and is expanded to 2.5-fold in the chicken and Xenopus genomes, and 10-fold in the medaka genome compared with the mammalian genomes. To compare the relative positions of the CNCSs, MFCS1 was right-aligned at the same position for all species. The colored ovals indicate the locations of MRCS1 (blue), MFCS3 (orange), MFCS4 (red), MACS1 (green), MFCS2 (purple) and MFCS1 (yellow). Putative medaka MACS1 is depicted with a white oval rimmed with green. (B) VISTA plots of the six CNCSs. Mouse CNCSs are compared with the homologs of the human, cow, opossum, platypus, chicken, lizard, Xenopus, fugu and medaka CNCSs. Constraints used are a 100 bp window length and 70% conservation level.

 

Figure 2
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  		Fig. 2. lacZ reporter expression driven by MRCS1 and MFCS4 in transgenic mouse embryos. (A-G) Reporter signal in MRCS1 transgenic embryos. X-gal staining is detected in the dental placode of the maxilla (A) and mandible (B) at E12.5. Signal is also detected in the fungiform papillae (B), whisker buds, teeth, palatal rugae and fungiform papillae (C,D) at E13.5. At E15.5, expression is detected in the whisker, hair and nail buds (E); teeth, primary palate and palatal rugae (F); molar teeth and fungiform papillae (G). (H-M) Reporter signal in MFCS4 transgenic embryos. (H) At E11.5, transient signal is detected in the dental placode, and intense signal around the pituitary fossa, the tympanic tube and recess. Punctate signal is detected on the tongue surface (inset in H). (I) At E13.5, expression is detected in the soft palatal edge and tympanic tube in the skull base. (J) Strong signal is detected in the epiglottis and arytenoid swelling. (K,L) At E15.0, expression is observed in the midline of the soft palate (K), but the intensity of the signal is depressed in the epiglottis and arytenoid swelling (L). (M) A mid-sagittal section of an E13.5 embryo shows reporter signal in the epithelia of the tongue, epiglottis, arytenoid swelling and dorsal pharyngeal wall. a, arytenoid swelling; d, dental placode; e, epiglottis; it, incisor tooth; fp, fungiform papillae; mt, molar tooth; n, nail bud; pf, pituitary fossa; pp, primary palate; r, palatal rugae; s, soft palate; t, tongue; tr, trachea; tt, tympanic tube; w, whisker bud.

 

Figure 3
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  		Fig. 3. MACS1-driven lacZ reporter expression in transgenic embryos. (A) lacZ reporter expression is observed throughout the entire gut tube at E9.0. (B) The condensed signal is observed in the tracheal diverticulum at E9.5. (C) A mid-sagittal section at E9.5 shows that signal is confined to the epithelial lining of the endoderm. (D) At E10.5, strong caudal expression disappears and signal is detected in the gut endoderm and cloaca. (E) Clear signal is detected in the primary lung buds. (F) A mid-sagittal section of an E10.5 embryo shows reporter signal in the epithelia of the ventral pharyngeal wall, laryngotracheal tube and cloaca. (G,H) At E11.5, reporter signal is widely observed in the laryngotracheal tube, lung, gut (G) and genital tubercle (H). (I) A mid-sagittal section of an E12.5 embryo shows strong signal in the epithelia of the laryngotracheal tube and lung. c, cloaca; g, gut; gt, genital tubercle; l, lung; lb, lung bud; lt, larybgotracheal tube; s, stomach; td, tracheal diverticulum.

 

Figure 4
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  		Fig. 4. Shh is expressed in the epithelial linings along the anteroposterior axis. (A-H) Endogenous Shh and Ptch1 expression in sagittal (A,B,E,F) and cross (C,D,G,H) sections of wild-type embryos. At E13.5, Shh expression is widely detected in the epithelia of the teeth, palatal shelf, lingual papillae, epiglottis, arytenoid swelling, pharyngeal wall, lung, gut and urogenital tract (A,C). Ptch1 expression is mainly detected in the mesenchyme adjacent to the Shh-expressing epithelia (B,D). (E-L) Magnified images in the pharyngeal apparatus. Shh expression is detected in the epithelia of the epiglottis, arytenoid swelling and laryngotracheal tube (E,G). Ptch1 expression is mainly observed in the mesenchyme adjacent to the Shh-expressing epithelia (F,H). (I-L) Reporter expression in MFCS4 and MACS1 transgenic embryos. MFCS4 reporter expression (I,K) partially overlapped with MACS1-driven expression (J,L) in the epithelia of the arytenoid swelling. (M) Reporter expression in the transgenic mice with a tandem array of MFCS4 and MACS1. The linked sequence drives the signal in both expression domains by MFCS4 and MACS1. a, arytenoid swelling; b, bladder; e, epiglottis; it, incisor tooth; in, intestine; l, lung; la, laryngeal aditus; lp, lingual papillae; lt, laryngotracheal tube; mt, molar tooth; n, nasal septum; p, pharynx; ps, palatal shelf; t, tongue; tr, trachea; u, urethra; uc, umbilical cord.

 

Figure 5
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  		Fig. 5. Phenotypes of {Delta}MFCS4/{Delta}MFCS4 mice. (A) Many {Delta}MFCS4/{Delta}MFCS4 mice had a dilated belly (asterisk). (B) A {Delta}MFCS4/{Delta}MFCS4 neonate whose stomach is not filled with milk (asterisk) (B). (C-M) Phenotypes of embryos at E18.5. (C) Wild-type oro- and naso-pharyngeal junction, upper right. The epiglottis and arytenoid are clearly recognizable. (D,E) Lower aspect of the hard and soft palates, and the naso-pharyngeal opening. In wild-type mice, the hard palate (hp, double arrow) and soft palate (sp, double arrow) are well formed (D). In {Delta}MFCS4/{Delta}MFCS4 mice, the palates are fused at the midline. The hard palate is formed normally, but the soft palate terminates prematurely. As a consequence, the naso-pharyngeal opening in {Delta}MFCS4/{Delta}MFCS4 mice is misaligned to the anterior side (E). (F-I) Views of the dorsal aspect of the tongue and the oro-pharyngeal opening. (G,I) Magnified images of the oro-pharyngeal opening. In {Delta}MFCS4/{Delta}MFCS4 mice, the tongue is misshapen (H) compared with wild-type (F), the epiglottis and arytenoid are hypoplastic (I) compared with wild-type (G). (J,K) Ventral view of the skull. Mutant animals have minor morphological anomalies in the basisphenoid and basioccipital bones, and have a hole in the spheno-occipital synchondrosis just inferior to the pituitary (K). (L,M) Dorsal views of the laryngeal cartilages. In mutant animals, the hyoid bone is malformed and the thyroid cartilage appeared to be hypoplastic (M). (N,O) Mid-sagittal sections of the oral and pharyngeal structures of heterozygous and homozygous mutant animals at E18.5. In {Delta}MFCS4/+ mice, the posterior edge of the soft palate extends alongside the posterior pharyngeal wall and is situated close to the epiglottis. The epiglottis and arytenoid are well formed (N). By contrast, in {Delta}MFCS4/{Delta}MFCS4 mice, the soft palate terminates prematurely, and the posterior tongue, epiglottis and arytenoid are severely deformed. The cross-sectional diameter of the body of the hyoid bone is decreased (thinner), and the bone and cartilage beneath the pituitary gland show morphological abnormalities (O). Red outline indicates the affected area in the {Delta}MFCS4/{Delta}MFCS4 mutant. a, arytenoid; bo, basioccipital bone; bs, basisphenoid; cc, cricoid cartilage; e, epiglottis; es, esophagus; hb, hyoid bone; hp, hard palate; it, incisor tooth; m, mandible; p, palate; pg, pituitary gland; sp, soft palate; t, tongue; tc, thyroid cartilage; tr, tracheae.

 

Figure 6
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  		Fig. 6. MFCS4 is a cis-acting regulator of Shh. (A-D) Shh expression in wild-type and {Delta}MFCS4/{Delta}MFCS4 embryos. (A) In wild-type embryos, Shh expression is detected in the epiglottis and arytenoid swelling at E13.5. (B) In {Delta}MFCS4/{Delta}MFCS4 embryos, Shh expression is lost in both structures. (C,D) Section in situ hybridization for Shh in wild-type and {Delta}MFCS4/{Delta}MFCS4 embryos at E13.0. (C) In wild-type embryos, Shh signal is broadly detected in the pharyngeal epithelium. (D) In {Delta}MFCS4/{Delta}MFCS4 embryos, Shh expression is downregulated in the epithelium of the pharyngeal wall, which is the future oro-pharynx area, and expression almost completely disappears in the epithelium of the epiglottis, the thyroid duct and the arytenoid swelling. Shh signal is detected at wild-type levels in the incisor tooth primordium of {Delta}MFCS4/{Delta}MFCS4 embryos (D). Boxed areas indicate Shh expression in the pharyngeal region of the wild type and the {Delta}MFCS4/{Delta}MFCS4 mutant. (E-H) Genetic test for cis-activity of MFCS4. (E) Diagram shows how cis or trans regulation of Shh expression by MFCS4 would function in the pharyngeal epithelium. If MFCS4 were a trans-acting regulator of Shh expression, an intact MFCS4 sequence located in a cis position relative to the Shh KO allele should be able to activate transcription of the intact Shh gene on the trans allele. Therefore, the {Delta}MFCS4/{Delta}MFCS4 phenotype should be rescued in Shh KO: MFCS4 KO compound heterozygotes. Conversely, if MFCS4 were a cis-acting regulator, the intact MFCS4 sequence would not be able to activate transcription of the Shh gene on the trans allele, and the compound heterozygotes would have the same phenotype as individuals with the single MFCS4 mutation. (F-H) Phenotypes of E18.5 embryos from the cross mating of Shh+/– mice to {Delta}MFCS4/+ mice. The four expected genotypes of the progeny segregated in a Mendelian fashion. Among them, the wild-type and the Shh+/– or {Delta}MFCS4/+ mice had normal phenotypes with respect to the oro-pharyngeal openings (G). However, all compound heterozygotes had abnormal openings, as did single {Delta}MFCS4/{Delta}MFCS4 mutants (H). a, arytenoid swelling; e, epiglottis; it, incisor tooth; m, mandible; t, tongue.

 

Figure 7
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  		Fig. 7. Genomic map of Shh enhancers. (A) The 840 kb genome region upstream of the Shh transcriptional start site contains numerous regulatory elements. Previously described enhancers directing Shh expression in the CNS are also shown (Jeong et al., 2006Go). (B) Schematic diagram showing the expression domains of Shh in epithelial lining regulated by MRCS1 (blue), MFCS4 (red) and MACS1 (green) along the anteroposterior axis.

 

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© The Company of Biologists Ltd 2009