Identifying the core transcriptional regulatory network initiating a tooth program

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PROJECT SUMMARY: Loss of teeth due to oral disease, trauma, aging or congenital defects is one of the most common organ failures. The first morphological sign of tooth development is the formation of the dental lamina (DL). How the DL—the critical structure for initiation of a tooth development program—is specified from naïve maxillary and mandibular ectoderm, remains poorly understood. We applied Laser Microdissection coupled spatiotemporal RNA-seq and single cell Multiome-seq to profile mouse mandibular ectoderm during DL specification and tooth initiation (E9.5-E12.5). In comparison to the non-DL epithelium (i.e., future skin), we identified a set of potential driver transcription factors (TFs) for DL specification including Pitx1, Pitx2, Sox2, and Foxe1. In contrast, another set of TFs was notably reduced in the DL, although enriched in aboral/ventral ectoderm, including Tfap2a, Tfap2b, Irx3, and Irx4. While single ectodermal knock-out (KO) of Tfap2a or Tfap2b resulted in normal DL and non-DL epithelial development we found that ectodermal double KO of both resulted in severe non-DL epithelial defects, a spatial expansion of DL specification, and an ectopic tooth. Similarly, we and other groups have found that the single KO of Pitx1, Pitx2 or Sox2 does not affect DL specification, despite expression of these genes prior to DL specification and tooth defects at later stages. Here, we propose that like the non-DL epithelium a redundant network exists to specify the DL epithelium. The long-term goal of this study is development of stem cell-based approaches for tooth repair and regeneration. The overall objective of the proposed research is to dissect the molecular and cellular mechanisms of tooth initiation, particularly the core transcriptional regulatory networks (TRNs) that drive specification of the DL within maxillary and mandibular ectoderm and initiation of a tooth development program. Our central hypothesis is that a few key TFs—including PITX1, PITX2, SOX2, FOXE1, etc.—redundantly drive the core TRNs that regulate DL formation and a tooth initiation program. Further, ectopic expression of a combination of these TFs could convert non-DL epithelium (e.g., skin) to DL epithelium and induce ectopic tooth development. In Aim 1, we will determine genetic redundancy and specificity of Pitx1, Pitx2 and Sox2 through double KO models and CUT&RUN-based TF target identification. In Aim 2, we will define the PITX1/PITX2-driven communication between DL epithelium and mesenchyme during tooth initiation using a Pitx1/Pitx2 KO mouse model. Finally, in Aim 3, we will determine the core set of TFs sufficient to drive DL lineage specification and non-DL epithelial reprogramming. Collectively, using a strong set of in vivo animal models, ex vivo explants, and genome-wide assays, these studies will fill a critical knowledge-gap in our current understanding of DL specification and its application to tooth regeneration. This experimental design and approach, along with PI’s/CoI’s with complementary expertise, and the strong environment outlined in this proposal provide the catalyst to improve oral and craniofacial health.