Dlx2b Function in Zebrafish Tooth Development

In the Jackman lab we study the molecular signals responsible for the early stages of tooth development. This summer I studied the role of the transcription factor dlx2b on zebrafish tooth development. Zebrafish teeth are a particularly useful means for examining the effects of molecular signals on development. Teeth, unlike many other organs, develop relatively independently of surrounding tissue, thus experimental manipulations can be observed and quantified. Zebrafish are a useful model organism because it is a vertebrate with a relatively short generation time that produces large clutches of transparent embryos. These characteristics are beneficial because as a vertebrate, findings can be related and applied to other vertebrates, such as humans, and external, quickly developing embryos allow for frequent experimental manipulations, close observation during all aspects of development, and frequent acquisition of results. The role of dlx2b in tooth development has been elucidated through loss of function experiments in mice and zebrafish. Knockdown of Dlx1 and Dlx2 in mouse hinders molar development (Thomas, 1997) and similar hindrance of tooth development was found in zebrafish. Knockdown of dlx2b alone had little effect on teeth, but as progressively more dlx genes were eliminated, teeth became smaller and misshapen (Jackman, 2006). These results suggest that dlx genes are necessary for tooth development. Through my project I explored if dlx genes are sufficient for tooth development. The gene dlx2b is a member of the Dlx family, which is a group of transcription factors that are thought to function by controlling the expression of target genes. Dlx2 is known to be expressed in mouse dental epithelium and to play a role in tooth development. Particular interest in dlx2b stems from evidence showing that it was the first gene discovered to be involved in zebrafish tooth development, but be absent from the oral region, where, during evolution, zebrafish have lost their teeth. Its expression is very specific to teeth; it is not present in surrounding tissue. I began my summer using mRNA injections as my method to overexpress dlx2b to test sufficiency in inducing tooth development. These injections, into one-cell staged embryos, are beneficial because mRNA injection allows for widespread uptake of a gene product into dividing embryonic cells. By synthesizing capped mRNA that mimics eukaryotic mRNA that is synthesized in vivo, I hoped to successfully overexpress dlx2b and observe its effect on tooth development. Apart from one seemingly promising result, that appeared similar to other results seen in our lab during overexpression experiments, teeth consistently looked wild-type. I verified dlx2b overexpression through in situ hybridizations, which allows for visualization of specific DNA and RNA sequences. This process suggested that I was overexpressing dlx2b, I was just unable to see an effect on the teeth. I also tried overexpression through injection of inducible DNA constructs. DNA injections are another form of overexpression that is useful because DNA constructs allow for temporal and spatial determination of expression. The first of my two constructs is dlx2b DNA that is the equivalent of my dlx2b mRNA. The second construct is VP16, an activator of transcription, attached to a dlx homeodomain, which should activate all of the targets of dlx genes. Preliminary experiments have yielded wild-type teeth, so I would like to continue experiments to increase numbers of results. Throughout the summer, I have also researched downstream targets of dlx2b, which could potentially serve as probes for dlx2b function during in situ hybridization. Four potential targets have been found, of which I hope to synthesize probes to use during in situs. Lastly, I recently began synthesizing GFP mRNA to use as a control for mRNA injections. I intend to ligate the GFP into the T7TS vector, which is the vector in which my dlx2b mRNA is located. Depending on the expression of GFP after injection, I will have a better understanding if my negative results are a result of dlx2b itself or the translation vector in which it is located. If the negative results are real, it would suggest that dlx2b, although necessary for tooth development, is not sufficient to induce development. The downstream targets of dlx2b, however, may be directly involved in tooth development. As a result, future directions include examining these dlx2b targets more directly, such as by overexpression. This would suggest the role these target genes play in the signaling pathway and reveal whether or not they are sufficient for tooth development.