Figure 1.
Enamelin expression depicted by β-galactosidase whole-mount staining of Enam knockout lacZ knockin mice.
(A) Embryonic day 13.5, (B) newborn, and (C) PN7 mice were processed with and without the removal of intestines. Separately, internal organs from PN7 Enam−/− mice were processed for cryosectioning and β-gal staining; no positive staining was observed in (D) lung, (E) liver, (F) kidney, (G) stomach, (H) small and (I) large intestines, or (J) cartilage from long bones.
Figure 2.
Beta-galactosidase staining of maxillary molars from PN5 wild type, Enam+/− and Enam−/− mice.
(A, unstained; D, H&E counter stained) No positive staining is consistently observed in wild type samples. (B, E) Positive staining is observed in the Enam+/− molars localized to the well-polarized ameloblast layer outlining the developing enamel space and showed no detectable differences from wild type molars in terms of ameloblast organization, cell height and thickness of the enamel layer. (C, F) In the Enam−/− molars, abnormal accumulations of enamel matrix and changes in ameloblast morphology and alignment were evident on PN5. (K) Ameloblasts lost polarity and were unable to maintain an even enamel space soon after enamel formation would have normally begun (arrow). (L–N) Irregular aggregation of ameloblasts and extracellular matrix material is apparent.
Figure 3.
Beta-galactosidase staining of maxillary molars from PN7 collected from wild type, Enam+/−, and Enam−/− mice.
(A–C, H&E counter stained) No positive staining is consistently observed in wild type samples. (D–F, unstained) There are no detectable differences between wild type and Enam+/− molars in terms of ameloblast organization, size of the ameloblasts and thickness of the enamel layer. (G–I) In Enam−/− molars, bulges of enamel matrix along the cuspal slopes are associated with flattening ameloblasts. Non-polarized, lacZ positive cells are seen instead of ameloblasts and extending into the stratum intermedium area, sometimes incorporated in the abnormal aggregation of matrix and cellular components near the cusp tips in the null mouse samples.
Figure 4.
Beta-galactosidase staining of maxillary molars from PN14 collected from wild type, Enam+/−, and Enam−/− mice.
(A–C) No positive staining is observed in wild type samples. (D–F) There are no appreciable abnormalities in Enam+/− molars. In Enam−/− molars, abnormal aggregations are present along the cuspal slopes (arrow). (G–I) The enamel organ is highly disorganized compared to expected appearance for normal maturation stage and the dentin is covered by an abnormally thin disorganized matrix.
Figure 5.
Immunohistochemical staining of PN7 and PN14 molars using amelogenin rm179 polyclonal and enamelin mENAM223–236 antipeptide antibodies.
(A–V) Weak amelogenin positive staining can be detected in PN7 and PN14 molars of wild type (A–B and M–N, arrowhead), Enam+/− (E–F and Q–R, arrowhead), and Enam−/− (I–J and U–V, arrowhead) mice. (C–D versus G–H) A similar distribution pattern but with slightly different intensities of enamelin staining is apparent comparing wild type to Enam+/− samples. The enamelin positive staining is evenly distributed across the entire thickness of enamel layer in wild type and Enam+/− samples. (K–L) However, no positive staining can be detected in Enam−/− molars. (I, K, U, W) Abnormal accumulations of organic matrix are evident on the mesial and distal cusp slopes in Enam−/− samples. (M–V) The trend continued into PN14, with amelogenin signals becoming more intense in all three genotypes. (O–P) Enamelin expression is evident in the wild type, trace amounts of expression in the Enam+/− molars (S–T) but complete absence in Enam−/− molars (W–X).
Figure 6.
SEM of enamel layer, enamel rods, DEJ, and dentin from 7-week-old mandibular incisors of wild type (A–D), Enam+/− (E–H) and Enam−/− (I–L).
Although no major differences in enamel and dentin seem to exist in the light microscope, under the higher resolution of SEM, the mineral crystals in the enamel rods of Enam+/− samples are more distinct and they fracture differently from the wild type crystals (C, G). Amorphous enamel surface (I), unevenly thin enamel layer absent of decussating rods (J, K) but seemingly normal dentin mineralization (L) are observed consistently in Enam−/− incisor samples. Amorphous mineral deposition and occasionally plate-like minerals are seen in the pseudo enamel layer of Enam−/− mouse samples. Blue line indicates enamel thickness.
Figure 7.
TEM crystal morphology and expression of amelogenin in Enam+/− and Enam−/− mice.
(A–D) Longitudinal and cross sections of forming enamel crystals in Enam+/− molars where typical enamel crystals (A–B) and less well-defined, amorphous crystals (C–D) can be detected in different areas of the developing enamel. (E, F) Low magnification of ameloblasts and the dystrophic enamel from PN7 Enam−/− molar showing a scalloped zone of irregular, mineralized tissue at the dentinoenamel junction (DEJ) where layered mineral of different textures is apparent. (G–H) Immunogold labeling for amelogenin appeared irregularly associated with mineralized masses/layers and between disorganized ameloblasts at occasional ectopic calcification sites (G), or as small and large pools of amelogenin between ameloblasts (H). Ectopic, mineralizing cellular debris (arrowheads in G), is also consistently observed. Enlarged rough endoplasmic reticulum is readily observed in the ameloblasts (arrowheads in H). Bars = 100 nm in A–B, 50 nm in C–D, 10 µm in E, and 1 µm in F–H.
Figure 8.
Enamelin transgene expression as determined by ELISA.
Graph A presents the enamelin expression as detected by mENAM223–236 anti-peptide antibodies in wild type, Enam+/−, Enam−/−, Enam+/−,tg, Enam−/−,tg mouse molars from 5 different transgenic lines. Lines 7 and 11 are low expressers, lines 2 and 12 are medium expressers, and line 3 is high expresser. Graph B demonstrates GAPDH expression levels of test samples, which were assessed to be comparable among all test samples.
Figure 9.
Comparison of incisor enamel in wild type mice with incisor enamel from littermates of a cross between Enam+/−,tg and Enam−/− mice.
(A) The Enam+/− mice without the Enam transgene have chalky enamel that readily chips from dentin, although it has slightly decreased thickness and seemingly normal rod organization except for the outer enamel layer. The Enam−/− mouse has a mineral crust covering dentin that is very thin, and shows none of the characteristic of enamel. (B, BEI and fractured surface SEM of line 12). The Enam+/−,tg from line12(M) had normal looking enamel that was similar to the wild type in thickness, prism pattern, and resistance to wear but in line 12(M) Enam−/−,tg incisors and molars, the structural defect of enamel was not recovered based on SEM observation. (B, lines 3 and 7) The fractured surface SEMs of lines 3(H) and 7(L) in the lower half of panel B show the thickness of incisor enamel fractured at the alveolar crest. No rescue of enamel defect can be observed in line 3(H) Enam−/−,tg but partial recovery of thickness and structure can be seen in line 7(L) Enam−/−,tg. Blue line depicts thickness of the presumed enamel layer.
Figure 10.
Survey of enamel formation in presecretory, secretory and early maturation stages on maxillary and mandibular incisors from 7-week-old mice.
(A, B) Wild type incisors showed normal matrix deposition and enamel development. Matrix protein stained blue in the enamel space during secretory stage was absorbed in the maturation stage allowing crystal maturation to take place (arrowheads). (C) Maxillary incisors in Enam+/− mice appear similar to wild type. (D) However, the mandibular incisors in these mice consistently demonstrated disturbed ameloblasts resulting in cyst formation within the enamel organ beginning in late secretory stage (arrow). (E) In Enam−/− maxillary incisors, there was no apparent enamel matrix deposition; (F) in the mandibular incisors, soon after the onset of the secretory stage, ameloblasts showed pooling of secreted material at their apices and gradual loss of polarity and organization within the enamel organ (arrowheads). (G, H) In line 2(M), Enam−/−,tg incisors, ameloblasts were polarized and deposited matrix during early secretory stage. Soon after that ameloblast layer became detached and cystic formation was evident on both maxillary and mandibular incisors (arrows). These 0.5 µm thick toluidine blue stained sections of EDTA-decalcified incisors were obtained from mice perfused with 2.5% GA and embedded in Epon. Bar = 500 µm for all panels.
Figure 11.
Von Kossa staining of PN4 first molars from wild type, Enam−/−, Enam+/−,tg, and Enam−/−,tg.
(A) Positive von Kossa stain was present in bone, dentin and enamel of wild type molars. (B) Although dentin was positively stained, there is no enamel layer evident in Enam−/− molars. Molars of Enam+/−,tg mice from lines 2(M) (C) and 3(H) (E) both demonstrated positively stained enamel and dentin layers. Molars of Enam−/−,tg mice from line 2(M) (D) showed positively stained enamel in all cusps but mice from line 3(H) (F) showed positively stained enamel only on major cusps. A positively stained enamel layer is evident on the major cusp slope of molars from wild type (G) and Enam−/−,tg mice in line 3(H) (H) but not in Enam−/− mice (I). En: enamel, De: dentin. Bar = 100 µm.