SHH signaling directed by two oral epithelium-specific enhancers controls tooth and oral development

Abstract

Interaction between the epithelium and mesenchyme coordinates patterning and differentiation of oral cavity structures including teeth, palatal rugae and tongue papillae. SHH is one of the key signaling molecules for this interaction. Epithelial expression of Shh in the tooth buds and tongue papillae is regulated by at least two enhancers, MRCS1 and MFCS4. However, it is unclear how the two enhancers cooperate to regulate Shh. Here, we found that simultaneous deletion of MRCS1 and MFCS4 results in the formation of a supernumerary tooth in front of the first molar. Since deletion of either single enhancer barely affects tooth development, MRCS1 and MFCS4 evidently act in a redundant fashion. Binding motifs for WNT signaling mediators are shared by MRCS1 and MFCS4, and play a central role in regulating Shh expression, indicating that the two redundant enhancers additively exert their Shh regulation by responding to WNT signal input.

Figure 1

Molar pattern in the MRCS1 KO homozygote and the compound heterozygote of MRCS1 and Shh KO alleles. Occlusal views of maxilla in the wild type mouse (a), the MRCS1 KO homozygote (b, ΔMRCS1/ΔMRCS1) and the compound heterozygote (c, ΔMRCS1/ShhKO) at one month old. Molar pattern in the mandible of the wild type (d,g,j), the MRCS1 KO homozygote (e,h,k) and the compound heterozygote (f,i,l). Three-dimensional reconstructions of X-ray micro CT images of molars are shown in g–l. Arrowheads mark supernumerary teeth. The alleles of the compound heterozygote of the ΔMRCS1 and the Shh coding sequence KO alleles are schematically illustrated in Supplementary Fig. 1d. M1, first molar; M2, second molar; M3, third molar.

Figure 2

Formation of the supernumerary tooth in the double KO homozygote of MRCS1 and MFCS4. Transverse sectional micro-CT images of the maxilla (a,b) and mandible (c–h) in the wild type (WT) (a,c,e,f) and the DKO mouse (b,d,g,h). Maxillary tooth in the WT (a) and the DKO mouse (b). Mandibular tooth in the WT (c,e,f) and the DKO mouse (d,g,h). Magnified pictures of the insets of c (e,f) and of d (g,h). Yellow arrows indicate supernumerary teeth. The double KO homozygote of MRCS1 and MFCS4 is schematically illustrated in Supplementary Fig. 1e. Expression of Shh mRNA in the dental placode of the WT at E12.5 (i) and high magnification of the dental placode (j, an open box in i). Nuclei of the dental placode were stained with DAPI. The depth half and the superficial half are shown in red and yellow, respectively (k). Signals for Shh nascent RNA in the dental placode of the WT and DKO (l,n), and cells were stained with DAPI (m,o). The %positive cells transcribing Shh in the depth and the surface of the dental placode (p). The black and open bars depict the wild type and the DKO, respectively. Error bars represent the standard deviations obtained from three independent samples. Two-tailed Student’s t-test was used to test significance of differences in number of signal-positive cells (**P < 0.001).

Figure 3

Motifs shared between MRCS1 and MFCS4. (a) Diagram of the upstream region of the mouse Shh locus. The two epithelial enhancers MFCS4 (blue) and MRCS1 (green) are located at 620–720 kb upstream of the Shh TSS. Comparison of mouse MRCS1 (b) and MFCS4 (c) in vertebrate taxa by Vista plots. A shaded region represents the core unit of the enhancer, which contains three shared motifs. The core sequences are shown at the bottom of (b). (d) Alignments of the three motifs in the MFCS4 and MRCS1 sequences, which were obtained from seven vertebrate species.

Figure 4

LacZ expression patterns in transgenic mice with MRCS1 deletion constructs. Diagram of deletion constructs of MRCS1 (a). Dotted lines show the region deleted in each construct. Arrowheads indicate the three motifs shown in Fig. 3. Numbers of LacZ-positive embryos among transgene-positive embryos in each organ are shown in the right column. LacZ expression patterns in the maxilla (b,d) and mandible (c,e) with deletion construct 1 (del1; b,c) and deletion construct 2 (del2; d,e). LacZ expression patterns in transgenic mice with deletions of motif1 (Δmo1: f,g; n = 6), motif2 (Δmo2: h,I; n = 8) and motif3 (Δmo3: j,k; n = 7) and a silent mutation of motif1 (mut mo1: l,m; n = 10). (n) EMSA with sequences at motif1 of MRCS1 and MFCS4 (MR-wt and M4-wt), and sequences with a silent mutation of motif1 (MR-mut and M4-mut, respectively). An arrowhead indicates a specific band shift. An open arrowhead indicates free probes. LNE is an abbreviation of nuclear extracts derived from Lef1-overexpressing cells. Cold oligos as a competitor, co. (o) Relative luciferase activity of the core MRCS1 (MR-wt), the core MRCS1 with a silent mutation of motif1 (MR-mut), the core MFCS4 (M4-wt) and the core MFCS4 with a silent mutation of motif1 (M4-mut) in HEK293T cells. The luciferase reporter constructs containing the core enhancer sequences were cotransfected with plasmid vectors expressing Gfp and Lef1. The reference value for cotransfection with Gfp-expressing and pGL4 empty plasmids was set as 1. Error bars represent the standard deviations obtained from three independent experiments. An asterisk shows a significant difference, as evaluated by Student’s t test (o, p < 0.05).

Figure 5

Functional similarity between mouse MRCS1 and Xenopus MFCS4. LacZ expression in the teeth buds of the maxilla (a–c) and papillae of the tongue (d–i). Expression directed by the whole fragments of mouse MRCS1 (a,d), mouse MFCS4 (b,e), Xenopus MFCS4 (c,f) or three tandem copies of the core sequence of mouse MRCS1 (g), mouse MFCS4 (h) or Xenopus MFCS4 (i). (j) Schematic diagram of Shh regulation by MRCS1 (MR) and MFCS4 (M4). MRCS1 primarily regulates Shh in the oral epithelium of mouse. MFCS4 alone regulates Shh in Xenopus.