Primary cilia are ubiquitous microtubule-based organelles that coordinate multiple signaling pathways critical for craniofacial development including Hedgehog, Wnt, and PDGF. Ciliary dysfunction causes a range of human disorders, collectively referred to as ciliopathies, many of which display craniofacial defects such as cleft lip/palate, micrognathia, midface dysplasia, and craniosynostosis. This dissertation explores how defects in the transition zone complex- a ciliary gatekeeper- results in a narrowing or collapse of the midface utilizing transgenic and knockout mouse models. Developmental analysis uncovered that the first molecular defects occur in the prechordal plate, a central organizing center in the developing midface. These early prechordal plate defects were transmitted to the adjacent developing forebrain resulting in a massive increase in cell death and culminating in collapse of the midface. Surprisingly, we could fully rescue the midface defects in multiple transition zone mutants by decreasing Ptch1 gene dosage. These results have uncovered the molecular underpinnings underlying craniofacial dysmorphology in a group of poorly characterized ciliopathies and hold powerful clinical implications for future strategies aimed at treatment and prevention of these defects.

Purpose: To determine if genetically deficient DAP12 mice demonstrate impaired fracture healing responses.

Methods: All studies were performed under IACUC approval. Genetically deficient DAP12 and age-matched C57BL/(6) (B6) control mice were used. The right tibia was cleaned and prepped for surgery and a closed, mid-diaphysis fracture was created. The fractures remained unstabilized to promote healing through the formation of a cartilage intermediate. Mice were sacrificed at 7, 10, 14, 21, and 28 days post fracture and processed for histology by decalcifying the tibia and embedding the samples into paraffin wax. Serial sections were collected through the callus and every tenth slide was stained with Milligan’s Trichrome, which stains for bone tissue. Using stereology, the volume of the fracture callus, cartilage, bone, bone marrow, and fibrous tissue was determined.

Results: Histological and stereological analysis demonstrated less trabecular bone percentage and volume, increased and delayed cartilage resorption, decreased bone marrow volume, and decreased overall callus volume in the DAP12-/-. Furthermore, DAP12-/- mice displayed elevated levels of fibrous tissue within the callus. Statistical significance was not observed in the callus volume between the DAP12-/- and control B6 mice.

Conclusion: Our data indicates that DAP12 deficiency disrupts the fracture healing process. Enhancing the understanding behind the mechanism of fracture repair can lead to improve healing strategies and potential therapeutics.

Background: Periodontal disease is an inflammatory disease that increases in prevalence with increasing age. The elderly demonstrate an elevated and dysregulated inflammatory response. Macrophages act as a key regulator of inflammation. The study aim was to evaluate the extent to which age-related changes and chemical inhibition in the macrophage affect periodontal disease in a mouse model.

Methods: Old (24 month) and young (3 month) mice were utilized for this study. Periodontal status was examined in mice at baseline. Periodontal disease was induced via Porphyromonas gingivalis (P. gingivalis) inoculated ligatures in old and young mice for 7 days. A second cohort had disease induced for 7 days, followed by ligature removal, and recovery for 7 additional days. Half the mice in each induction or recovery group were treated with Pexidartinib (PLX), which inhibits macrophage recruitment. Linear bone loss, alveolar bone volume, and macrophage quantification were examined by t test.

Results: PLX successfully inhibited macrophage numbers in all treatment groups. The young periodontal disease group had significantly more vertical bone loss (0.234±0.024mm) compared to the young periodontal disease PLX group (0.140±0.023mm)(p≤0.001) and less alveolar bone volume (0.460±0.016BV/TV) compared to the young periodontal disease PLX group (0.586±0.004BV/TV)(p≤0.001). The old periodontal disease group exhibited no difference in vertical bone loss (0.242±0.025mm) compared to the old periodontal disease PLX group (0.238±0.032mm)(p=0.825) and significantly less alveolar bone volume (0.536±0.030BV/TV) compared to the old periodontal disease PLX group (0.614±0.030BV/TV)(p≤0.01). The young recovery control group trended towards less vertical bone loss (0.170±0.020mm) compared to the young recovery + PLX group (0.190±0.011mm)(p=0.055) and had no difference in alveolar bone volume (0.557±0.015 BV/TV) compared to the young recovery PLX group (0.545±0.011 BV/TV)(p=0.131). The old recovery control group exhibited no difference in vertical bone loss (0.241±0.019mm) compared to the old recovery PLX group (0.218±0.035mm)(p=0.187) and had significantly less alveolar bone volume (0.567±0.021BV/TV) compared to the old recovery PLX group (0.617±0.023BV/TV)(p≤0.01).

Conclusion:

PLX prevents periodontal disease induction in old and young groups and improves recovery in older age groups. The difference in response during recovery could suggest that there are age related changes in the macrophage that affect disease progression.

Aging is characterized by physiologic changes leading to a predisposition to a myriad of age-related diseases. Accompanying these age-related diseases is a process of systemic inflammatory dysregulation known as inflamm-aging. This body of work focuses on fracture healing and periodontal disease, as both involve dysregulation of inflammation within bone and both demonstrate increased complications or prevalence with age. We chose to investigate the macrophage and its contribution to age-related pathologies affecting bone. Macrophages are important regulators of inflammation during fracture healing and periodontal disease. An improved understanding of the age-related changes to macrophages will advance our understanding of the biology of aging and lead to enhanced healthcare for the aging population.

Mouse models of fracture healing and periodontal disease using young and old mice were employed in this work. Macrophages from young and old mice were characterized via RNA-seq. Macrophage were depleted pharmacologically during disease or fracture healing to elucidate the contribution of macrophages within the given models.

The results demonstrated that macrophages from old mice present an aged-macrophage phenotype with increased pro-inflammatory and M1 gene expression. By eliminating the influence of the aged macrophage phenotype via macrophage depletion, periodontal disease severity was significantly reduced and fracture healing was significantly improved. Further, we have demonstrated that TREM2 expression on macrophages is decreased with age which drove increased inflammatory cytokine expression and poorer fracture healing outcomes. Taken together, the aged-related changes that occur to the macrophage are likely involved in numerous disease pathologies, and this work presents potential therapeutic targets to address the macrophage-driven inflammatory dysregulation in the elderly population.

For many diseases, the patient population exhibits a wide range of severity, but the cause of this variation is largely unknown. This is the case for many structural craniofacial diseases, including holoprosencephaly (HPE). In HPE patients, the forebrain fails to develop into two hemispheres and the midline of the face does not fully form; however, the disease manifests in a broad spectrum of severity, with phenotypes ranging from mild midfacial narrowing to cyclopia. Although mutations in Sonic hedgehog (SHH) and in other members of the SHH signaling pathway have been linked to HPE, the source of the extreme phenotypic variability characterizing the disease is not understood. Intriguingly, research suggests that development largely buffers against variation in morphological form occurring, but that nonlinearities in development are a rich source of such buffering. Previous research in an exogenously manipulated chick model suggests that there is a non-linear relationship between SHH-signaling and midfacial shape, but it has not been tested whether such a non-linear relationship exists in a genetic model. This dissertation utilizes an allelic series of embryonic mice with discrete, genetic reductions in Shh and in low-density lipoprotein receptor-related protein (Lrp) 2 to test whether nonlinearities in SHH signaling underlie the HPE spectrum. LRP2 is an endocytic receptor required to activate SHH-signaling in the developing forebrain, and Lrp2-/- mice exhibit mild HPE. Geometric morphometric (GM) analysis uncovered no statistically significant difference in craniofacial shape between wild-type, Lrp2+/-, Shh+/-, and Lrp2+/-;Shh+/- embryos. However, the upper jaw is significantly more narrow relative to the lower jaw in Lrp2-/- and Lrp2-/-;Shh+/- embryos in comparison to other genotypes, and there is significantly higher face shape variance in Lrp2-/- and Lrp2-/-;Shh+/- embryo groups than in other genotypes. Surprisingly, SHH pathway activation (as defined by Shh, Ptch1, Gli1 mRNA levels) did not predict face shape or face shape variance. Interestingly, however, GM and gene expression analysis suggest that that growth is delayed in embryos missing functional LRP2 protein, and that this delay may underlie the HPE phenotype—but that a threshold of such delay is required for HPE to manifest.

Teeth, with their remarkable preservation in the fossil record, have long been instrumental in uncovering insights into human history. Similar to the way tree rings record growth patterns, dental tissues capture and preserve evidence of environmental disruptions at the time of their formation. Deciduous (primary) teeth, forming from Week 6 in utero into early childhood, capture prenatal and postnatal time windows that overlap with brain and nervous system development. Unlike bone or neural tissues, primary teeth do not actively remodel and thus provide a lasting and faithful record of an individual's early life, making them potential predictors neurodevelopmental outcomes. However, further research is needed to fully understand how primary teeth develop in altered environments before they may be used as diagnostic tools of neurodevelopment. Given strong evidence linking early life stress to neurodevelopmental and mental health conditions, we investigated whether environmental stress leave imprints in primary teeth. Microcomputed tomographic (microCT) analysis of kindergarten children’s lower central incisor revealed measurable changes in the enamel, dentin-pulp complex, and eruption patterns in children with elevated cortisol levels. Furthermore, our empirical investigations in a mouse model of Early Life Adversity (ELA) showed that stressed pups exhibited distinct changes in tooth enamel mineralization and gene expression of key regulatory proteins (Amelx, Enam, Dlx3, Igfbp2, Per1, Nrd1). Identifying and understanding how these early life stress biomarkers are produced, is a crucial step forward towards using primary teeth as diagnostic tools for children at risk of stress-related mental health conditions.

The fossil record is widely informative about evolution, but fossils have not been systematically used to study the evolution of stem cells. Moreover, while the mechanisms underlying tooth development have been widely studied in model organisms, the role of genetic regulatory elements in patterning the different elements of the occlusal surface and crown height across species is not well understood. Here, I examined evolution of the rodent adult dental stem cell niche, which enables continuous growth (hypselodonty) of molar teeth. Moreover, I compared the variation in dental morphology across nine taxa of rodents to the variation in sequences of non-coding evolutionary conserved regions (ECRs) of Fgf3, 4, 8, 9, and 10 and the function of Fgf10 signaling on retention of molar stem cell niches. I studied the occurrences of 3500 North American fossil rodent specimens, ranging from 50 million years ago (mya) to 2 mya. I examined evolutionary changes in molar height to determine if evolution of continuously growing molars shows distinct patterns in the fossil record, and we found that hypselodont taxa emerged through intermediate forms of increasing crown height. Next, we designed a two-parameter Markov simulation model, which correctly accounted for molar height increases throughout the Cenozoic, and, moreover, evolution of hypselodonty. Finally, I correlated the variation in molar tooth cusp shape and the evolution of high molar crowns (hypsodonty) to the patterns of sequence variation in two ECRs, Fgf10ECR3 and Fgf9ECR1, respectively. By conducting luciferase and electrophoretic mobility shift assays, we determined that these ECRs could function as enhancers. Thus, by extension, the retention of the adult stem-cell niche appears to be a predictable quantitative rather than a stochastic qualitative process. Our analyses predict that hypselodonty will eventually become the dominant rodent phenotype. Mammalian dental morphology is under strong evolutionary pressure because of its importance for mastication and diet. My data suggest that emergence of hypsodonty and occlusal cusp patterning may have happened through the evolutionary changes in enhancers, such as Fgf9ECR1 and Fgf10ECR3, which affected the expression of major signaling molecules involved in tooth development.

Craniofacial anomalies are disfiguring, debilitating birth defects that afflict a large portion of the world's population. These malformations can be caused by genetic and/or environmental factors, and one such factor, prenatal hypoxia, is of particular interest in our research on craniofacial dysmorphology. There is experimental and clinical evidence for a correlation between hypoxia and craniofacial malformations, however, the mechanisms underlying such defects are not yet understood. The goal of our research has been to understand the mechanisms whereby hypoxia causes abnormal craniofacial morphology in early embryonic development.

Chick embryos were incubated in either normoxic (21% O2) or hypoxic (7%-19% O2) conditions and collected on days 2-6 for morphological and cellular analyses. Embryos were photographed for morphological analyses, and facial shape variation was quantified via two-dimensional geometric morphometrics. 13-day embryos were cleared and stained for analysis of skeletal and cartilaginous development. Cell proliferation was assessed via BrdU staining, and apoptosis was assessed by whole-mount and section TUNEL and caspase 3 immunoassay. Embryos were examined for oxidative stress using a phospho-AMPK immunoassay.

In morphometric analyses of normal growth, older embryos (HH22-28) showed greater shape variation among embryos than younger embryos (HH16-21). All the embryos fell along a well-defined nonlinear curve of normal facial growth in relation to size, and displayed variation in relation to chronological age and discrete morphological stage. Within stages, older embryos displayed greater variation within stages than younger embryos. In studies of hypoxic embryos, their survival was reduced in a step-wise manner in comparison to normoxic control embryos. Hypoxic embryos showed a wide range of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. They also displayed delayed skull bone development, with some skeletal defects. Abnormal facial shape variation occurred in relation to centroid size and age among individuals in hypoxic groups versus the normoxic population. Hypoxia disrupted cell proliferation and caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed increased metabolic stress response.

Objectives: Craniofacial malformations are among the most common birth defects, affecting ~1 in every 700 live births. However, our understanding of the mechanisms that result in these diseases is limited. Many cases have been associated with genetic predispositions that have allowed us to attempt to investigate the underlying mechanisms for these diseases. Sonic Hedgehog (SHH) signaling pathway has undergone significant investigation due to the involvement of Shh in the development of the midface. In humans, deletion of a single copy of Shh is associated with a spectrum of phenotypes comprising Holoprosencephaly (HPE), ranging from mild hypotelorism and midfacial hypoplasia to cyclopia indicates that diseases like HPE are multifactorial. Although our long-term goal is to discover the underlying mechanisms that cause these diseases, we must first understand the normal progression of development before trying to understand the abnormal. Therefore, the goal of this study was to determine how Shh expression and face shape relate with each other during normal development.

Methods: We collected wild-type chicken embryos at 72 hrs, 96 hrs, and 120 hours of incubation. Chickens infected with RCAS-wnt3a at 72 hrs of incubation were also included in our sample. We used in situ hybridization to identify Shh expression domains and 2D geometric morphometrics to quantify changes in shape in Shh expression domains and face shape. We performed Principal Components Analysis (PCA) as well as multivariable regression analyses of Shh expression shape and face shape on somite number and centroid size. We used Partial Least Squares (PLS) to evaluate covariation in shape between Shh expression domains and facial shape.

Results: Changes in Shh expression shape and face shape are dependent on developmental time. As the embryos progressed in development, there were significant changes in both Shh expression shape and face shape. While the overall size of the embryo grew, both the Shh expression shape and face shape constricted. More specifically, Shh expression shape tapered into a narrow ¬V-shaped band in the ectoderm of the stomodeum while face shape constricted as a result of the nasal pits growing closer together while the mouth became smaller. Our PLS regression identified that the changes in Shh expression and face shape are correlative in which Shh expression shape is associated with face shape at specific timepoints during development.

Conclusion: Changes in Shh expression shape correlate with changes in face shape. This suggests that Shh expression shape may serve as a predictor for face shape during embryonic growth. Although we are unable to determine if Shh is directly responsible for the observed changes in face shape, this potential predictive relationship could be valuable for future studies to identify when and how disease progression initiates.