Simmer Hu Lab

Enamel matrix proteins, amelogenin, enamelin, and ameloblastin, from developing porcine first permanent molars are isolated, purified and characterized. Roles of each enamel matric protein and its cleavage products in enamel ribbon initiation, elongation, and shaping are investigated.

To understand genetic regulation of tooth formation, we use routine histology, immunohistochemistry, and RNAScope in situ hybridization to determine the spatial and temporal expression pattern of selected genes during mouse odontogenesis.

Establishing and characterizing mouse models with dentin sialophosphoprotein (Dspp) gene mutations has allowed us to investigate the underlying pathophysiology of Dentinogenesis imperfecta, an autosomal dominant trait affecting 1:8,000 individuals in the general population.

Using optimized tissue preparation techniques, we are able to preserve and evaluate the intricate enamel architecture using back-scattered SEM and focused-ion beam SCM.

We provide consultations to families with members affected with tooth size, shape, number, and/or structure defects. Families diagnosed with inherited dental defects including but not limited to amelogenesis imperfecta, dentinogenesis imperfecta, oligodontia, supernumerary teeth, short root anomaly, etc. may join the genetic study conducted at the Dental Research Lab. Pending availability of the research funding, research-based genetic testing can be conducted on eligible participants.

Dentin dysplasia type II (DD-II), dentinogenesis imperfecta type II and type III (DGI-II and DGI-III) are autosomal dominant inherited conditions that occur at a frequency of 1 in 8,000 and are caused by specific types of mutations in one allele of the dentin sialophosphoprotein (DSPP) gene. More than half of the reported DSPP mutations that cause DD-II, DGI-II and DGI-III are -1 frameshifts in the C-terminal repetitive domain of DSPP. We generated Dspp+/-1fs mice using CRISPR/cas9 gene editing to introduce a FLAG-tag insert (DYKDDDDK) followed by a single nucleotide deletion that translated a frameshifted protein with 493 missense amino acids analogous to the genetic cause of dentin malformations in humans. Using Focused Ion Beam-Scanning Electron Microscopy, we assessed early stage dentinogenesis of the Dspp+/+, Dspp+/-1fs mice and Dspp-1fs/-1fs mice. Mandibular incisors of the heterozygous mice exhibited non-polarized odontoblasts with autophagosomes, enlarged rER, and fewer mitochondria. Adjacent to the abnormally thin predentin deposition, there were apoptotic cells and macrophages. We hypothesize that the aberrant DSPP produced by Dspp-1 frameshift cannot be folded properly, thus resulted in ER retention, which subsequently triggered an ER stress response and activated apoptotic cell death. Objective: to determine the proteinopathy and Endoplasmic-reticulum-associated protein degradation (ERAD) of Dspp -1 frameshift mutations in order to gain insights leading to clinical interventions. Approach: Dentin formation of postnatal day 5 mouse mandibular incisors will be characterized for unfolded protein response and assessed for molecular markers of ER stress and ERAD.

The objectives of this research are to advance our understanding of enamel formation and mineralization and to develop well-characterized mouse models to study secretory and maturation stage amelogenesis. Achieving these objectives will afford the research field valuable mouse models to study enamel mineralization. This research will also lead to a better understanding of the disease process in persons with Amelogenesis Imperfecta and provide insights into intervention of this group of disorders.

Protocols

• Materials and Supplies for LM/EM Histology
• Mouse Perfusion
• Decalcification Protocol
• Immunohistochemistry Protocol
• RNA Isolation
• In Situ Hybridization Protocol
• Odaph Gene Targeting, Genotyping, and Validation
• Slc13a5 Gene Targeting, Genotyping, and Validation

Description:

Ameloblasts of Slc13a5^R337*/R337* mice, in the early stage of enamel formation, contain a large number of secondary lysosomes (orange) likely due to a significant amount of proteins being redirected for degradation. The Slc13a5^R337* mouse model was generated using CRISPRcas9 gene-targeting to introduce a premature stop codon at Slc13a5^R337 homologous to human mutation SLC13A5^R333* that resulted in developmental and epileptic encephalopathy 25 (DEE25) and amelogenesis imperfecta (MIM#615915).
Yellow – primary lysosomes; Orange – secondary lysosomes; Cyan – a type of mostly large, dispersed mitochondria seen in the nuclear and supranuclear compartments; Green – the classic small, denser and more tightly packed mitochondria seen in the infranuclear compartment; Magenta – nuclei; Sky blue – gap junctions; Red – enamel proteins seen in a basolateral or abnormal location.