William Go1,2 and Vladimir Korzh1,3
1- Institute of Molecular and Cell Biology, A-STAR, Singapore
2- Department of Biochemistry, National University of Singapore
3- Department of Biological Sciences, National University of Singapore
Published in Bone 54 (2013) pp. 48-57
The zebrafish transgenic lines provide a possibility to observe development of tissues and organs in real time. Using the reporter line for the zebrafish plasma membrane Ca2+ATPase (SqET4), we detected its expression in the epithelium of pharyngeal teeth and analyzed its role in their calcification and that of cranial bones. atp2b1a's expression in the pharyngeal epithelium is faithfully recapitulated in the SqET4 transgenics by GFP expression. We showed by morpholino knockdown of Atp2b1a translations as well as chemical inhibition of Atp2b1a pump activity using carboxyeosin, that its activity is required to facilitate calcification of the developing pharyngeal teeth by the dental epithelium. Atp2b1a could be required during calcification of endochondral bones, where it acts at two levels: 1) by exporting Ca2+ from ameloblasts, it provides raw material for calcifying the pharyngeal teeth by adjacent odontoblasts; and 2) by regulating terminal differentiation of pharyngeal epithelial cells, including ameloblasts required for tissue hyper-mineralisation. atp2b1a's expression in the pharyngeal epithelium is regulated by the homeodomain transcription factor dlx2b.
Atp2b1a is required for calcification of early-forming bones. (A & B), bone calcification pattern (3-7 dpf) in control and atp2b1a knock-down morphant larvae as detected by Alcian Blue (AB) and Alizarin Red (AR) dual staining at the level of fifth ceratobranchial bone (Cb5). In controls, bone calcification starts in the pharyngeal teeth 4V1 and followed by Cb5 and later pharyngeal teeth 3/5V1. In morphants only a low level of calcification was detected with signal found at the tip of 4V1 and not in Cb5 and 3/5V1 (N = 10). (C, D) Shows the progression of calcification in developing operculum (op) using differential-live staining larvae by Alizarin red (AR) and Calcein (green) in controls and morphants. Calcification of morphant operculum was reduced at ventral-posterior (vp) edges. (E, F) show distribution of osteoblasts at operculum of Tg(osx-mCherry) larvae. In control larvae, osteoblasts were closely associated with the growing joint socket (js) and vp edges and outline the expanding edges of jp and jv apices (E: red arrows) unlike that in morphant, where osteoblasts were found only at the js apex (F). E & F are pseudo-colored confocal images at maximal intensity projections of 70 µm z-stack.
Abbreviations: Cb5: 5th ceratobranchial bone; 4V1: 1st-forming pharyngeal teeth; 3/5V1: later-forming pharyngeal teeth; e: eye; ie: inner ear; cl: cleithrum; op: operculum; js: joint socket; v: ventral apex; p: posterior apex
For more information on Vladimir KORZH's laboratory, please click here.