Introduction
Tooth development involves a complex process whereby teeth are formed from embryonic cells, and then eventually mature after erupting as evident in the mouth (Eckhardt, Jágr, Pataridis, & Mikšík, 2014). The development usually originates from mesoderm and ectoderm. At the ectoderm found in the oral cavity, there is the development of enamel and other tissues also come from associated mesenchyme. All teeth don’t develop at the same time, but rather in stages (Eckhardt, Jágr, Pataridis, & Mikšík, 2014). First buds are always observed in the region of anterior mandible, followed by anterior maxillary area then finally posteriorly in both jaws. This continuous development of teeth occurs in stages that include the bud stage, cap stage and the bell stage (Jernvall & Thesleff, 2012). Bud stage is also referred to as the proliferation stage forms the initial stage during teeth formation and the enamel organ usually looks like a small bud. At this stage, the enamel organ has centrally placed polygonal cells low columnar cells that are peripherally located. There is the proliferation of mesenchymal cells resulting in the condensation. Dental laminae are always seen after the sixth week as oral epithelium thickenings. For the cap stage, there is an invagination of ectodermal tooth buds by the mesenchyme referred to as dental papilla, giving rise to dental pulp and dentin. The ectodermal which is seen over the papilla as cap-shaped is known as enamel organ because it is responsible for the production of the future enamel. Finally, at the bell stage, the tooth acquires a bell shape due to continuous uneven growth in the enamel organ (Jernvall & Thesleff, 2012)
Knowledge in the chemical differentiation of teeth during its growth is essential for the study of experimental procedures in teeth and biochemical analyses of the teeth composition, changes induced by certain teeth diseases and the general study of chemical development. The article that has been used to describe tooth development and to illustrate one of the proteins involved in the process is about the analysis of amelogenin mRNA during bovine tooth development (Yuan et al. 1996). Amelogenins are enamel matrix proteins that have been highly conserved and are essential for a proper mineral formation. These tissues that are highly mineralized; the dental enamel, are secreted by ameloblast cells (Yuan et al. 1996).
Materials and method
Biochemical analyses involve different assays, methods, and procedures used to analyze certain substances of interest in living organisms. To realize a comprehensive analysis dealing with a biomolecule; a strategy has to be designed to detect the biomolecule, isolate it, characterize and then its functions analyzed. In this article, the biochemical method that was used for the analysis is the agarose gel electrophoresis with the help of northern blot.
Agarose gel electrophoresis is used to separate proteins in a medium of agarose or a mixed DNA. The proteins are separated based on charge, size and even on the length of the fragment in the case of RNA and DNA. The separation of these biomolecules is achieved by the application of an electric field that is responsible for moving the charged molecules through the agarose matrix and then separation of the molecules is realized by size in the matrix of agarose gel (Jin & Gassmann, 2012). Electrophoresis involves a set of procedures ranging from; casting of gel, loading of samples, to staining and visualization (Jin & Gassmann, 2012). During gel casting, the gel preparation is achieved by dissolving the agarose powder in a suitable buffer like TBE and TAE to be used during the electrophoresis. The agarose is then dispersed into the buffer before being heated to almost boiling point. The agarose that has melted is then allowed to cool sufficiently before the solution is poured into the cast to avoid cracking in case the gel is too hot. Wells are then created by placing the comb in the cast so that the sample can be loaded. The comb is then removed once the gel sets leaving the wells used to load the DNA samples. Before the loading is done, the DNA sample is mixed with the buffer containing a dense compound such as sucrose, or glycerol. The purpose of this dense compound is to raise the sample density to allow the DNA sink at the bottom of the well. In staining and visualization, the DNA and RNA are stained with ethidium bromide. The dye enters the DNA grooves and fluoresces in the presence of ultraviolet light. Other available staining methods include GelRed, SYBR Green, methylene blue, crystal violet and Nile blue. For northern blot, the separation of RNA sample is based on the size, and this is achieved with the help of gel electrophoresis. The RNA fragments that have been separated are then transferred to a membrane that serves as a support and then after that treated with a DNA probe that has been labeled. In case the sample has a complementary RNA sequence, then the probe binds to a complementary membrane and after which visualized. The principle of gel electrophoresis is based on the separation and migration of molecules under the influence of electric field. When these charged molecules are put in an electric field, the movement is achieved either toward a positive or negative pole depending on their charges. The migration of shorter molecules across the gel is easy and faster as compared to longer molecules, a process called sieving.
The stages involved in this analysis included RNA isolation, reverse transcriptase- polymerase chain reaction and DNA sequence analysis. At the stage of RNA isolation, bovine fetuses were measured from crown to rump for age approximation and unerupted mandibular molars were removed and placed in liquid nitrogen. In the laboratory, enamel organs were dissected, and RNA was separately extracted for each age, using the guanidinium isothiocyanate procedure. RNA quality was then checked by denaturing agarose gel electrophoresis. Reverse transcriptase – polymerase chain reaction ( RT-PCR) was carried out according to the procedure in the Gene-Amp RNA PCR kit from Perkin Elmer Corp. cDNA synthesis was then carried for one cycle at 42oC for 42 minutes, 99oC for 5 minutes, 5oC for 5 minutes in a Perkin Elmer DNA thermocycler 480. PCR amplification was done immediately after cDNA synthesis in a total volume of 100µ containing 10mM tri-HCL pH 8.3, 50mM KCl, 4nM MgCl2, 2.5 units of Taq DNA polymerase and 35 pmol of each primer. The amplification was done for 35 cycles of 95 oC for 1 min and 60 oC for 1 min, with an additional extension time 0f 7 min at 60 oC.RT-PCR products were then analyzed by agarose gel electrophoresis using Metaphor agarose as described in the principle of agarose gel above. DNA sequence was then obtained by the dideoxynucleotide chain termination procedure using the PCR amplification technique with fluorescent dye-labeled terminators on an ABI instrument, according to the manufacturer's recommendations. The sequence was assembled with sequencer software and was analyzed using the Genetics Computer group’s sequence analysis software package.
Results
A series of oligonucleotides corresponding to bovine amelogenin cDNA or genomic sequences were synthesized.
X and Y chromosomal amelogenin mRNAs
RNA was extracted from the enamel organs, and duplicate Nothern blots were made containing enamel-organ RNA from male and female animals of various ages, which were hybridized under the identical conditions using the X-chromosomes or Y-chromosomes amelogenin-specific probe, radiolabelled to the same specific gravity. Relatively wide bands at approximately 850 and 450 bases were evident in the Northern blot probed with the X-specific oligomer. When the Y-specific probe was used, there was no hybridization to samples from female animals, and the 450 bases band was not apparent as expected. The Northern blot hybridized with the Y-specific probe was then rehybridized with the X-specific probe to indicate the presence of amelogenin RNA in all lanes. The densitometric measurement showed at least six-fold more X-chromosomal transcripts at these stages of development.
LRAP mRNA during bovine tooth development
Densitometric comparison of large (850 nucleotides) to small (4500 nucleotides) alternatively spliced mRNAs, detected with the X-chromosomal amelogenin probe, revealed more than nine times more large mRNAs than small amelogenin mRNAs. To determine whether LRAP mRNA levels vary during tooth development; oligomer primers were used to amplify the alternatively spliced mRNA from animals at different stages of development, using RT-PCR. RNA samples from male and female fetuses between 142 days and 184 days of gestation were subjected to RT-CR. For each sample, the expected 200-bp products were seen, with no significant differences in the amount between ages. Control for this experiment was β-actin which indicated that each sample contained a similar amount of RNA. As quantification may prove difficult when a large number of alternative splice product are expected, a Northern blot was used to confirm the LRAP abundance during the development. By densitometry, it was determined that the ratio of LRAP to X-chromosomal amelogenin mRNA did not change appreciably during these stages of development
Discussion
References
Eckhardt, A., Jágr, M., Pataridis, S., & Mikšík, I. (2014). Proteomic Analysis of Human Tooth Pulp: Proteomics of Human Tooth. Journal Of Endodontics, 40(12), 1961-1966. http://dx.doi.org/10.1016/j.joen.2014.07.001
Jernvall, J. & Thesleff, I. (2012). Tooth shape formation and tooth renewal: evolving with the same signals. Development, 139(19), 3487-3497. http://dx.doi.org/10.1242/dev.085084
Jin, H. & Gassmann, W. (2012). RNA abundance analysis. New York: Humana Press.
Matalova, E., Janeckova, E., Buchtova, M., Smarda, J., & Misek, I. (2009). 13-P125 c-Myb expression correlated with apoptosis and proliferation in prenatal molar tooth germ development. Mechanisms Of Development, 126, S232-S233. http://dx.doi.org/10.1016/j.mod.2009.06.598
Roberts, G., McDonald, F., Andiappan, M., & Lucas, V. (2015). Dental Age Estimation (DAE): Data management for tooth development stages including the third molar. Appropriate censoring of Stage H, the final stage of tooth development. Journal Of Forensic And Legal Medicine, 36, 177-184. http://dx.doi.org/10.1016/j.jflm.2015.08.013
Teaford, M., Smith, M., & Ferguson, M. (2000). Development, function and evolution of teeth. New York: Cambridge University Press.
Wise, S. (2007). Bone morphogenetic proteins in teleost tooth development and evolution.
Yuan, Z., Collier, P., Rosenbloom, J., & Gibson, C. (1996). Analysis of amelogenin mRNA during bovine tooth development. Archives Of Oral Biology, 41(2), 205-213. http://dx.doi.org/10.1016/0003-9969(95)00119-0
