Using the jawed yet toothless Trp63 mouse mutant to understand the morphogenetic relationship between developing lower teeth and mandibles
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Across vertebrates, the coordinated evolution and synchronous development of teeth and the mandible must require specific timing and positioning of gene expression. While debate persists about whether teeth have evolved before or after mandible, currently, the consensus is that these systems evolved at separate times and thus have discreet origins. This raises an important question of whether tooth and mandibular tissues have over the course of their evolution become developmentally co-dependent or, as separate evolutionary origins would imply, remain developmentally autonomous of each other. The molecular signaling that patterns the genesis of upper versus lower jaw skeletons, as well as specifies tooth type (i.e., molar vs. incisor) is relatively well understood. To date, the distinct genetic processes that drive tooth development distinct from jaw skeletal development has been little-studied, in no small part due to the technical complexity of this task. The main hypothesis of thesis is that a collection of genes acting within a gene regulatory network (GRN) drives odontogenesis with neither input from, nor influence on, jaw morphogenesis. The Transformation Related Protein (TRP63) is a master transcription factor that is vital to odontogenesis because TRP63 maintains the competence and proliferation of the epithelial layer of the tooth organ. Thus the “toothless” TRP63 homozygote mouse mutant (Brdm2 mutant) fails to develop teeth even though it develops a virtually unperturbed mandible. This combination of lower jaw morphogenesis in the absence of odontogenesis presents a rare model to study the genetic changes that occur when teeth but not jaws fail to form. A previous microarray gene expression analysis (Boughner laboratory, unpublished data) of mandibular prominences (MdPs) derived from embryonic day (E) 10-13 revealed that, compared to heterozygote (Trp63+/-) MdPs, in Brdm2 mutant MdPs, transcript levels of cerebellin 1 (Cbln1); keratin 2-8 (Krt2-8); phospholipid transfer protein (Pltp) and fermitin 1 (Fermt1) were altered in at least some of the four embryonic stages. Specifically Cbln1 and Krt2-8 were up-regulated while Pltp and Fermt1 were down-regulated. None of these four genes have previously been linked to odontogenesis yet all are potential candidates for a “tooth-specific” GRN. Using RT-qPCR analysis, I aimed to test the validity of the microarray work and confirmed its veracity by showing that, generally, Cbln1 and Krt2-8 mRNA were up-regulated, while Fermt1 (but not Trp63 or Pltp) mRNA was significantly down-regulated in the MdPs of Brdm2 mutant mice relative to Trp63+/- mice. Conversely, western blotting protein expression analysis showed little-to-no change among Brdm2 MdPs relative to either wild type (Trp63 +/+) or Trp63+/- embryos, making it difficult to tease out the precise relationship between CBLN1, FERMT1, KRT2-8, and PLTP and TRP63. These results show a lack of strong correlation between mRNA and protein expression. Because the mRNA analyses showed disturbances in the expression level in a few of these five genes within the MdPs during the earliest stages of tooth development, these genes remain candidates for an odonto-specific GRN. In complement to the genetic work, to characterize the tandem developmental morphology of tooth and jaw skeleton tissues, my work included developing a new tissue staining protocol. Using Protargol, a silver-based compound, to enhance in uncut mouse embryos contrast among tiny, soft oral tissues and visualize their organization in 3D and microscopic detail across several embryonic stages. This novel protocol offers a simple, easy-to-follow, and relatively inexpensive way to effectively stain whole embryos aged E10-15 for X-ray based micro-computed tomography (μ-CT) imaging using synchrotron and desktop scanning systems. Because the scan data are digital, this new method also allows more precise, accurate and rapid empirical studies of the sizes, shapes and positions of teeth as they form within the jaw to clarify how these tissues are integrated as they develop. The work presented in this thesis investigated tooth development exclusive of mandible development from complementary molecular and morphological points of view. Driven by the lack of understanding of the genetic mechanisms that orchestrate tooth with jaw skeletal development, this study has for the first time isolated a set of genes that are potential candidates for tooth formation only. These results set the stage for next steps in testing the developmental-genetics that enable teeth and jaws to “fit” together as they develop.
DegreeMaster of Science (M.Sc.)
DepartmentAnatomy and Cell Biology
ProgramAnatomy and Cell Biology
CommitteeCooper, David; Kulyk, William; Nazarali, Adil
Copyright DateAugust 2015