Pitx2 Overexpression: Effects on the Early Development of Zebrefish Teeth

Abstract

Objectives: To address the effects of pitx2 overexpression on the early development of teeth in zebrafish.

Methods:

The mRNA of pitx2 was synthesized in vitro via cloning the CDS of pitx2 by RT-PCR. The DNA fragment was subjected to adding in restriction enzyme sites to become a template. The zebrafish embryos were divided into four experimental groups, with each embryo accepting a microinjection of pitx2 mRNA to establish the overexpression model. Development of embryos within 72hr was observed under microscope. Semi-quantitative RT-PCR and grayscale value analysis were adapted to detect possible changes in the gene expression related to the tooth development.

Results: Morphological observation of 72-hour embryos of zebrafish revealed arrest developments, pericardium edema, notochord deformation, and eye malformation. Semi-quantitative RT-PCR and grayscale value analysis indicated that over-expression of pitx2 down-regulated bmp4, while up-regulated fgf8, dlx2b, and lef1. No significant effect was detected in the expression levels of runx2a.

Conclusions:

Over-expression of pitx2 could result in morphological changes in the embryos of zebrafish. Genes related to early development of teeth of zebrafish were up- or down-regulated, or not effected by pitx2 overexpression. The changes were similar as what we observed in mice.