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. 2017 Apr;96(4):421-429.
doi: 10.1177/0022034516683674. Epub 2017 Jan 12.

Loss of Function of Evc2 in Dental Mesenchyme Leads to Hypomorphic Enamel

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Free PMC article

Loss of Function of Evc2 in Dental Mesenchyme Leads to Hypomorphic Enamel

H Zhang et al. J Dent Res. .
Free PMC article

Abstract

Ellis-van Creveld (EvC) syndrome is an autosomal-recessive skeletal dysplasia, characterized by short stature and postaxial polydactyly. A series of dental abnormalities, including hypomorphic enamel formation, has been reported in patients with EvC. Despite previous studies that attempted to uncover the mechanism leading to abnormal tooth development, little is known regarding how hypomorphic enamel is formed in patients with EvC. In the current study, using Evc2/ Limbin mutant mice we recently generated, we analyzed enamel formation in the mouse incisor. Consistent with symptoms in human patients, we observed that Evc2 mutant mice had smaller incisors with enamel hypoplasia. Histologic observations coupled with ameloblast marker analyses suggested that Evc2 mutant preameloblasts were capable of differentiating to secretory ameloblasts; this process, however, was apparently delayed, due to delayed odontoblast differentiation, mediated by a limited number of dental mesenchymal stem cells in Evc2 mutant mice. This concept was further supported by the observation that dental mesenchymal-specific deletion of Evc2 phenocopied the tooth abnormalities in Evc2 mutants. Overall, our findings suggest that mutations in Evc2 affect dental mesenchymal stem cell homeostasis, which further leads to hypomorphic enamel formation.

Keywords: Hedgehog signaling; Limbin; ameloblast; cilium; odontoblast; stem cells.

Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

Figure 1.
Figure 1.
Evc2 mutant mice showed hypomorphic enamel formation. (A) Mutant alleles of Evc2/Limbin used in this study. Ex12-Stop (a global knockout allele), a stop codon, along with an IRES-LacZ cassette has been inserted into exon 12 to mimic a nonsense mutation identified in human patients. Floxed (a conditional allele) and 2 loxP sites have been introduced to flank exon 13 and exon 14. A Cre-mediated recombination results in a frame shift downstream of exon 12 of Evc2. dE, a Cre recombined allele, produces a similarly truncated EVC2 protein with the one from Ex12-Stop allele, if any. (B) LacZ staining of Evc2 ex12/+ incisor indicates that Evc2 is expressed in dental epithelium, mesenchyme, and surrounding bone tissue. (C) Lateral X-ray radiogram of 2-mo-old Evc2 mutant mice shows hypomorphic maxilla and mandible incisor formations (top). Frontal view and side view of the same mice demonstrate possible hypomorphic enamel in Evc2 mutant mice (bottom). Yellow dashed lines indicate maxilla incisors and blue dashed lines indicate mandible incisors. (D) Lateral X-ray radiogram of 2-mo-old dissected maxilla and mandible shows hypomorphic incisor and molar formation in Evc2 mutant. Yellow dashed lines indicate maxilla incisors and blue dashed lines indicate mandible incisors.
Figure 2.
Figure 2.
Evc2 mutant incisors showed delayed ameloblast differentiation and hypomorphic enamel formation. (A) Sagittal sections of embryonic day 18.5 (E18.5) embryonic mandible incisors. Regions in boxes in both controls and mutants were enlarged and shown on the right. Arrows indicate the ameloblasts with polarized nuclei and * indicates the odontoblasts with polarized nuclei. (B) Sagittal sections of postnatal day 2 (P2) mandible incisors. Regions in boxes in both control and mutant were enlarged and shown on the right. Arrowheads indicate ameloblasts with polarized nuclei in both controls and Evc2 mutants and * indicates the odontoblasts with polarized nuclei. (C) Sagittal sections of postnatal day 1 molar. Regions in boxes in both control and mutant were enlarged and shown. (D) Immunohistochemistry of amelogenin in mandible incisors at P2 indicates a delayed ameloblast differentiation. Posterior regions of incisors of both genotypes were enlarged and shown. Red dashed lines indicate the epithelial cells without amelogenin immunosignals. The immediate next section was processed for hematoxylin and eosin staining. (E) Sagittal sections of postnatal day 8 (P8) mandible incisors. Regions in boxes in both control and mutant were enlarged and shown on the right. Arrows indicate enamel formation in both control and Evc2 mutants.
Figure 3.
Figure 3.
Evc2 mutant incisors showed delayed odontoblast differentiation. (A) Mandible incisors from postnatal day 2 (P2) were sectioned and processed for alkaline phosphatase (ALP) staining. Black dashed lines indicate a boundary between dental epithelium and mesenchyme. Yellow dashed lines indicate ALP-stained cells in the odontoblast layer. (B, C) Mandible incisors from P2 were sectioned and processed for immunohistochemistry for Osterix (OSX). White dashed lines separate dental epithelium and mesenchyme. Red dashed lines indicate OSX-expressing dental mesenchymal cells. Yellow dashed lines indicate OSX-stained cells in the odontoblast layer. The number of OSX-expressing dental mesenchymal cells was quantified and is shown in B (n = 4, P < 0.01).
Figure 4.
Figure 4.
Evc2 mutant incisors showed decreased dental epithelial and mesenchymal stem cells. (A) A Gli1-LacZ reporter was introduced to label dental epithelial and mesenchymal stem cells in the incisor. E18.5 mandible incisors of indicated genotypes were processed for LacZ staining. (B) Quantification of Gli1-LacZ–positive cells in A. n = 3, **P< 0.01. (C) Immunohistochemistry of Ki-67 was applied to mark the transient amplifying (TA) cells in controls and Evc2 mutants. Red arrowheads in the left panels indicate the 2 ends of the zone of Ki-67–positive cells in the odontoblast layers. White dashed lines in the right panels indicate a boundary between dental epithelium and mesenchyme. Red dashed lines indicate areas of Ki-67–positive cells in preodontoblasts (areas between 2 red arrowheads shown in the left panels). Yellow dashed lines indicate Ki-67–positive cells in preameloblasts. (D) Quantification of Ki-67–positive cells in B. n = 4, *P < 0.05; #not significant. (E) Immunohistochemistry of phospho-histone3 (P-H3) depicts proliferating cells in control and Evc2 mutant incisors at postnatal day 2 (P2). White dashed line indicates a boundary between dental epithelium and dental mesenchyme. (F) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining indicates apoptotic cells in control and Evc2 mutant incisor at P2. (G) Quantification of P-H3–positive cells in C. n = 4, #not significant.
Figure 5.
Figure 5.
Mesenchyme-specific deletion of Evc2 phenocopied hypomorphic tooth formation found in Evc2 global knockout incisors. (A) Frontal view of maxilla and mandible incisor of Evc2 conditional knockout mice at postnatal day 28 (P28). (B) X-ray radiogram of Evc2 conditional mutants showed hypomorphic tooth formation. Mandibles of Evc2 conditional mutants and littermate controls at P28 were dissected and sagittal X-ray radiography was performed. Yellow dashed lines indicate incisors. (C) Micro–computed tomography (CT) of Evc2 conditional mutants and littermate controls at P28 indicates hypomorphic enamel in molars. (D) Mandible incisors of mesenchyme-specific Evc2 mutants and littermate controls were sagittally sectioned and processed for hematoxylin and eosin staining. Regions in boxes in both control and mutant were enlarged and shown on the right. (E) Quantitative reverse transcribed polymerase chain reaction from incisor epithelium and mesenchyme indicated that Wnt1-Cre–mediated deletion of Evc2 is specific for dental mesenchyme. EPI and MES stand for epithelial and mesenchymal tissues, respectively. n = 3, **P < 0.001. (F) A model to explain how mesenchymal function of Evc2 secondarily affects enamel formation. EP-TA, epithelial transient amplifying cells; ESC, dental epithelial stem cells; MC-TA, mesenchymal transient amplifying cells; MSC, dental mesenchymal stem cells. See text for details.

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