In many organisms, the initial stages of organ formation involve, first, the formation of a bud-like structure from epithelial placode, or a local thickening of the endoderm . This placode will eventually develop into organs such as mammary glands, kidneys, hair, and teeth. On this page, we will be looking specifically at the development of teeth in mammals. The following video provides a brief overview of tooth development in mammals:
For tooth development in mice specifically, as shown in Figure 1C , the odontogenic ectoderm, or tooth forming tissue, thickens to form the dental region. After the dental region is formed, a signal cascade causes an invagination of the dental region to form the “bud” stage . Further along in the development, this bud structure will flatten out and the cells at its center will form a primary enamel knot (EK) . The EK functions as the center where tooth patterning and structure is initiated.Eventually, the bud structure will grow outward and encapsulate the dental mesenchyme, resulting in the cap stage . The bud-to-cap stage is vital in the development of teeth with proper morphogenesis and patterning and can be regulated by several transcription factors, one of which is Msx1 . This transcription factor will stop the bud-to-cap stage development, resulting in bud stage arrest .
From previous research on Msx1 mutant mice, it has been determined that Bmp4, a signaling protein, is necessary for successful bud-to-cap stage transition . Recent research has also shown that the introduction of Bmp4 to Msx1 knockout mice can rescue the bud stage arrest. Further studies completed by Saadi et al.  have shown that Tbx2, a T-box protein, is also expressed in the dental mesenchyme at the bud stage and may be involved with Bmp4 expression in dental development.
In this paper, Saadi et al. sought to determine the relationship between Msx1 and Tbx2, and their roles in regulation of Bmp4 expression during the bud-to-cap stage transition in tooth development in mice.
To obtain the necessary data, the following experimental procedures were implemented:
1. Embryo production, in which mice were intercrossed to produce compound mutants with genotype Msx1-/-;Tbx2+/-. The resulting compound mutant will allow observations of whether there is a difference between wild-type, singular mutants, and compound mutants during dental development.
2. Immunohistochemistry, in which sample tissues from dental mesenchyme were used to analyze the distribution and localization of specifically expressed protein. In this case, Tbx2 expression was located within the tissue sample.
3. Bead implantation assay, in which agarose beads were incubated with Bmp4 and placed on top of the dental mesenchyme sample tissue of both wild-type and Msx1 mutant mice. This assay allows for observation of Tbx2 expression in the mesenchyme due to Bmp4 presence.
4. GST pull-down assay, in which a pull-down assay was conducted with a GST labeled Msx1 protein and a 35S labeled Tbx2 protein. This assay allows for observation of whether a complex can be formed, which would indicate any physical interactions.
5. Co-immunoprecipitation assay, in which mammalian dental mesenchyme cells were incubated with anti-Msx1 antibody and analyzed through Western Blot. This assay allows for the observation of any protein-protein interactions between Msx1 and Tbx2 to further confirm any physical interactions between the two.
6. Lentiviral knockdown and qPCR analysis, in which lentiviral particles (a virus-based method of gene delivery) that expressed genes against Tbx2 were used in order to observe whether Tbx2 and Msx1 expression is altered in the presence of the other. The qPCR analysis was used to further asses whether there was a significant change in protein expression.
For more details on the methods of the experiment, click on the links provided above or check out the links provided at the bottom of this page.
Results & Discussion
Tbx2 is expressed in dental mesenchyme and can be induced by Bmp4
Through immunohistochemistry assays, it was determined that Tbx2 is expressed in the dental mesenchyme in both upper and lower molars, and that the localization of expression overlaps with the expression of Msx1 (Figure 2B). In the samples where Msx1 is not expressed, Tbx2 expression is maintained, indicating that Msx1 is not necessarily required for Tbx2 expression in the bud stage (Figure 2F) .
Bead implantation experiments using Bmp4 soaked beads resulted in the activation of Tbx2 expression (Figure 2G). Activation was also present in the Msx1 knockout mice (Figure 2H), indicating that Msx1 is not required to mediate the Bmp4 effect on Tbx2 expression .
Tbx2 physically interacts with Msx1
The GST pull-down assay was conducted to determine whether Msx1 and Tbx2 have physical interaction. When in the presence of GST tagged Msx1, there was retention of Tbx2 (Figure 3A), however, when in the presence of GST alone, there was no retention of Tbx2. This indicates that Msx1 and Tbx2 forms a complex and confirms that there is a physical interaction between the two .
A co-immunoprecipitation assay was also conducted within a C3H10T1/2 mammalian mesenchymal cell line, using anti-Msx1 antibody against endogenous Msx1 (Figure 3C). The resulting Western blot shows that Tbx2 is present when the anti-Msx1 antibody is present, indicating that there is a physical interaction between Msx1 and Tbx2 .
These experiments can confirm a physical interaction because Msx1 and Tbx2 showed co-expression in similar areas in the bud. This is consistent with a hypothesis that Tbx2 and Msx1 are co-regulatory during the bud-to-cap stage transition period .
Tbx2 gene dosage reduction partially rescues Msx1-/- bud stage arrest
To determine whether there is a genetic interaction between Tbx2 and Msx1, the Tbx2 and Msx1 mouse mutants were crossed, resulting in a compound mutant of genotype Msx1-/-;Tbx2+/-. There were no significant deviations from expected Mendelian ratios, and the compound mutant mice did not show any obvious signs of differences in molar or incisor development (Figures 4A, B, C, D). The resulting embryos from this cross were expected to show a similar bud stage arrest phenotype to the wild type .
To determine whether gene dosage reduction has a significant change in phenotype, the compound mice were analyzed. The compound mice showed partial rescue of bud stage arrest. The upper molars showed development beyond bud stage (Figure 4E compared to 4G), but the lower molars showed no significant development (Figure 4I compared to Figures 4K, L).
These experiments confirm that there is a genetic interaction between Msx1 and Tbx2, and that the upper molars show more sensitivity to Tbx2 gene dosage reduction . This difference between upper and lower molars can be attributed to their distinct signaling pathways .
Mesenchymal Bmp4 expression is restored in Msx1-/-;Tbx2+/- rescued molars
Figure 5A, shows that Bmp4 expression is restored in the compound mutant mice and that it shows a similar expression pattern to that of wild type mice.
Since there was also a restoration of the EK within the compound mutant mice, there is the possibility that Tbx2 negatively regulates Msx1 activity and Bmp4 expression in upper molar development . In the Tbx2 knockdown experiments in the C3H10T1/2 mesenchymal cells, there is a significant upregulation of Bmp4 expression compared to the wild type, although Msx1 expression remains unchanged (Figure 5B). These results are consistent with the assertion that Tbx2 is repressive for Bmp4 regulation .
From these results, Saadi et al. were able to create a basic model in order to explain the relationship between Msx1, Tbx2, and Bmp4 (Figure 5C).
From these experiments, Saadi et al. were able to conceive a model at which they believe Msx1, Tbx2, and Bmp4 interact during dental development in mice. This model (Figure 5C) involves an initial Bmp signaling, which activates factors within the mesenchyme. These factors can either go on to activate or maintain Bmp4 signals or repress them. This indicates a more complex equilibrium system in order to regulate the proper morphogenesis and patterning during dental development.
Further experiments must be conducted in order to illustrate a more detailed picture of the relationship between Msx1 and Tbx factors in other tissue and whether they play a consistent role in the development of other tissues, such as cardiac or craniofacial.
Overall, this journal article was well written. The article was clear and detailed in explaining how the results lead to the conclusions made by the research team. Saadi et al. used a variety of different experimental methods and analyses in order to obtain very strong evidence for their conclusions. Saadi et al. also mentioned that their results were consistent with the results obtained by other researchers in previously published literature. This a good way to ensure that previous researchers were credited, but this also lends credence to the validity of their own study. Although the authors were thorough in explaining the results and discussion, the methods could have been more elaborate in explaining the necessity of each assay to the experiment as a whole.
Helpful Links/Resources (Methods)
1. Fay, D. (2006, June 14). Genetic mapping and manipulation: Chapter 7-Making compound mutants*. Genetic mapping and manipulation: Chapter 7-Making compound mutants. Retrieved April 3, 2014, from http://www.wormbook.org/chapters/www_makingcompdmutants.2/makingcompdmutants.html
2. Ramos-Vara, J. A., & Miller, M. A. (2014). When Tissue Antigens and Antibodies Get Along: Revisiting the Technical Aspects of Immunohistochemistry-The Red, Brown, and Blue Technique.Veterinary Pathology, 51(1), 42-87. doi:10.1177/0300985813505879
3. Sneider, J. (n.d.). Overview of Immunohistochemistry. Overview of Immunohistochemistry. Retrieved April 3, 2014, from http://www.piercenet.com/method/overview-immunohistochemistry
4. Gurdon, J. B., Ryan, K. K., Stennard, F. F., McDowell, N. N., Crease, D. D., Dyson, S. S., & … Carnac, G. G. (1996). Long range signalling process in embryonic development. The International Journal Of Developmental Biology, Suppl 157S-58S.
5. Sneider, J. (n.d.). Pull-Down Assays. Pull-Down Assays. Retrieved April 3, 2014, from http://www.piercenet.com/method/pull-down-assays
6. Sneider, J. (n.d.). Co-immunoprecipitation (Co-IP). Co-Immunoprecipitation (Co-IP). Retrieved April 3, 2014, from http://www.piercenet.com/method/co-immunoprecipitation-co-ip
7. Lentiviral and adenoviral delivery of RNAi vectors. (n.d.). Lentiviral shRNA and miRNA analysis. Retrieved April 3, 2014, from http://www.lifetechnologies.com/us/en/home/life-science/rnai/virus-based-rnai-analysis/lentiviral-shrna-and-mirna-analysis.html
1. Jussila, M. M., & Thesleff, I. I. (2012). Signaling networks regulating tooth organogenesis and regeneration, and the specification of dental mesenchymal and epithelial cell lineages. Cold Spring Harbor Perspectives In Biology, 4(4), doi:10.1101/cshperspect.a008425
2. Saadi, I., Das, P., Zhao, M., Raj, L., Ruspita, I., Xia, Y., & … Bei, M. (n.d). Msx1 and Tbx2 antagonistically regulate Bmp4 expression during the bud-to-cap stage transition in tooth development.Development, 140(13), 2697-2702.