Defining the Role of MEMO1 in Craniodental Mineralization

Abstract

Defects in the mineralization of craniofacial tissues are an underlying pathology in a variety of human congenital disorders. Our understanding of these developmental processes is confounded by the complex intertwinement of different tissue types (e.g. ectoderm, neural crest and mesoderm) and mineralization methods (e.g. endochondral and intramembranous ossification). Understanding the unique cellular and molecular mechanisms driving the development and mineralization of craniodental tissues (e.g. bone and teeth) is critical to explain, prevent, and treat associated genetic disorders. Our lab has identified the protein Mediator of Cell Motility 1 (MEMO1)—initially found to associate with breast cancer metastasis—is critical for bone and enamel mineralization. However, MEMO1’s biological and molecular function in these processes is unknown.We are using in vivo mouse-based and in vitro cell culture-based models to address this knowledge gap. With our in vivo models, we have utilized immunofluorescence, scanning electron microscopy, µCT, single cell RNA-sequencing, and basic histological examination to define the craniodental defects upon loss of Memo1 in the ectoderm or oral epithelium. With our in vitro models, we have utilized an oral epithelial cell line, along with CRISPR/Cas9 genome editing, Western Blot analysis, bulk RNA-sequencing and immunofluorescence to gain insight into the cellular and molecular function of MEMO1 within oral epithelial cells. Analyses of the in vivo Memo1 ectoderm or oral epithelial mutants, relative to controls, identifies an important role for MEMO1 in enamel development. Conditional mutants display chalky enamel associated with a significant decrease in enamel density (p-value < 0.03). Compromised enamel in conditional mutants is further supported by alterations in gene and protein expression within ameloblasts and may include defects in the cytoskeleton. Supporting this observation, our oral epithelial in vitro Memo1 loss-of-function model displays changes in cytoskeletal dynamics, including increased cellular contractility.These findings provide novel insight into how MEMO1 contributes to mineralization, potentially deploying its breast cancer metastatic function (i.e., cytoskeletal regulation), within a novel biological setting—amelogenesis. The oral epithelial specific role of MEMO1 expands our understanding of craniodental mineralization and MEMO1’s molecular control of these processes. Furthermore, this provides novel insight into genetic diseases of mineralization, including those impacting the craniodental complex. Additional investigation into our Memo1 loss-of-function models will progress both the specific and general knowledge of tissue mineralization—providing novel opportunities to exploit this protein, or its interacting partners, in a clinical or therapeutic setting.