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Journal of Dental Research
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XBP1 May Determine the Size of the Ameloblast Endoplasmic Reticulum

M. Tsuchiya1,2, C.E. Tye1, R. Sharma1, C.E. Smith3 and J.D. Bartlett1,*

1 Department of Cytokine Biology, The Forsyth Institute, and Department of Developmental Biology, Harvard School of Dental Medicine, 140 The Fenway, Boston, MA 02115, USA;
2 Division of Aging and Geriatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai, 980-8575, Japan; and
3 Laboratory for the Study of Calcified Tissues and Biomaterials, Faculté de médecine dentaire, Université de Montréal, Montreal, QC, Canada H3C 3J7


Figure 1
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  		Figure 1. Percent volume per ameloblast of the ER, mitochondria, and Golgi at 6 defined developmental stages. Selected values presented in the TableGo were graphed so that the reader may better observe the magnitude of the differences among the various stages. Each percent volume represents data generated from approximately 4 ameloblasts per incisor from 2 incisors, performed on 10 different animals per stage. Error bars represent the standard error of the mean. Abbreviations: Sec, secretory stage; Mat, maturation stage; Regress, regression stage of enamel development.

 

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  		Figure 2. Identification of active IRE1 in ameloblast cells of the enamel organ as determined by immunohistochemical methods. (A) Staining for the active phosphorylated form of IRE1 in an adult mouse mandibular incisor [20X magnification]. (B) Enlargement of the indicated secretory-stage ameloblasts from panel A [40X magnification]. (C) Enlargement of the indicated maturation-stage ameloblasts from panel A [40X magnification]. (D) Negative control section treated with the secondary, but not primary, antisera [20X magnification]. Note that staining for active IRE1 was reduced in the maturation stage as compared with the secretory stage of enamel development. Scale bar in panel A represents 100 µm for panels A and D. Scale bar in panel C represents 50 µm for panels B and C. am, ameloblasts.

 

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  		Figure 3. Quantification of total Xbp1 and spliced Xbp1(S) expression in mouse enamel organ during the secretory and maturation stages of enamel development. (A) Top: the 26-bp region spliced out of the Xbp1 RNA. This results in a reading frame shift that encodes the active form of XBP1(S). Also shown is the PstI site present within the spliced region. Bottom: RT-PCR of the Xbp1 mRNA. Primers for PCR spanned the splice region so that, after digestion with PstI, the spliced Xbp1(S) and unspliced Xbp1(U) were identified. Note that more Xbp1(S) was present in the secretory stage compared with the maturation stage of enamel development. (B) Top: Total Xbp1 and Xbp1(S) expression as assessed by qPCR. Primers for qPCR were designed to amplify total Xbp1 or were designed such that elimination of the splice region was necessary for amplification of only Xbp1(S). The results of the secretory stage (first molar enamel organ from 4-day-old mice) are expressed relative to the results of the maturation-stage (first molar enamel organ from 11-day-old mice) expression levels. Note that the secretory stage expressed approximately two-fold more total Xbp1 than the maturation stage. However, this difference increased to approximately five-fold for Xbp1(S). The kidney served as an epithelium-derived negative control. Bottom: Enamelin served as a positive control, because its expression drops dramatically during the maturation stage, and RAMP-4 also served as a positive control, because its expression is induced by Xbp1(S). All qPCR results were as a function of the stably expressed internal reference control gene EF1{alpha}1 (Kubota et al., 2005) and were calculated as previously described (Pfaffl, 2001; Kubota et al., 2005). Six samples were assessed for each developmental stage, and the results were repeated three times. Error bars represent the standard error of the mean. Abbreviations: Sec, secretory stage; Mat, maturation stage.

 

Journal of Dental Research, Vol. 87, No. 11, 1058-1062 (2008)
DOI: 10.1177/154405910808701115
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