Focus on tobacco-related disease concerns has shifted from cigarettes to other forms of tobacco use over the last 20 years. Due to their perception as “safer than a cigarette” by the general public, harm-reduction tobacco products (HRTPs) offer an appealing alternative for women struggling with nicotine addiction who find themselves pregnant. Some studies, however, suggest that HRTPs may increase risk of adverse pregnancy outcomes and hinder fetal skeletal development. To date, the mechanistic etiology of HRTP embryotoxicity is unreported. This thesis aims to address this knowledge gap by answering some of the questions surrounding how HRTPs molecularly and biochemically cause changes in the developing skeleton.

Early in vivo studies reported herein indicated that HRTP exposure directly targets early osteogenesis of the skull following in utero exposure of mouse embryos. To explore the molecular etiology of this outcome, the embryonic stem cell test (EST) protocol was adapted to an in vitro model of developmental osteogenesis using human pluripotent stem cells (hPSCs). Cultures were concurrently exposed to conventional sidestream cigarette smoke (CSC), harm-reduction sidestream cigarette smoke (HSC), or harm-reduction Snus smokeless tobacco extract (STE). While conventional and harm-reduction extracts both inhibited in vitro osteogenesis, only the HSC and STE harm-reduction extracts did so at sub-cytotoxic doses. Furthermore, inhibitory doses increased cellular levels of reactive oxygen species and reduced endogenous antioxidant enzyme activity. Molecular analysis found that CSC exposure incurred both DNA damage and a concurrent apoptotic response that was absent in cultures exposed to either HRTP extract. Biochemical exploration of HRTP impact on developing cultures found exclusive activation of survival kinase AKT and reduction of stress rescue kinase JNK in STE-exposed cultures. Concurrent treatment with an isoform-specific inhibitor of AKT or JNK activator rescued osteogenesis in STE cultures, implicating the specific misregulation of these kinases in poor osteogenic outcomes. Global proteomic analysis of AKT signaling targets also identified exclusive hyperphosphorylation of FOXO transcription factors—required for oxidative stress defense and adult bone homeostasis—in STE-treated cultures, marking FOXOs for nuclear exclusion. Collectively, our data suggest that HRTPs inhibit normal osteogenesis by disrupting the balance between embryonic osteogenesis, survival, and redox equilibrium mechanisms.

Bone is a complex mineralized tissue that functions as a structural support for the body, as well as in mineral storage and homeostasis. Its development is as complex as it’s function, with multiple molecular cues governing several aspects of its development and maintenance. Multiple previous studies over several decades have shown that failure to maintain tight regulation of these cues results in varying degrees of bone malformation, most frequently of the craniofacial variety. Various reports have cited a fundamental importance of both non-canonical and canonical Wnt signaling in regulating osteogenesis. Multiple studies have also revealed the importance of temporal-spatial regulation of their expression in promoting proper bone development.

MicroRNAs (miRNAs) are small non-coding RNAs that are able to negatively regulate protein expression, serving as a regulatory mechanism in a variety of developmental processes and diseases. They function by binding to the 3’ Untranslated Region of target mRNAs to induce mRNA degradation or translational inhibition. They are crucial for early embryonic differentiation, as studies have shown that embryonic stem cells that cannot generate miRNA exhibit impaired differentiation.

The work presented in this dissertation focuses on two miRNAs that have not been previously characterized in osteogenesis: miR-690 and miR-361. These miRNAs were selected from a screen based on their differential patterns of accumulation in response to osteogenic factors, and their ability to positively regulate osteogenesis. Using embryonic stem cells as a model for early embryonic development, we observed that both miRNAs were capable of affecting Wnt signaling. We also noted a correlation of miRNA expression with accelerated and/or sustained expression of genes that are associated with neural crest specification or differentiation. These studies have established (i) that miR-690 supports osteogenesis by directly regulating Ctnnb1 during differentiation from the mesenchyme; and (ii) that miR-361 promotes osteogenesis by directly regulating the non- canonical Wnt inhibitor Prickle during mesenchymal commitment. This research presents novel possibilities in miRNA-mediated regulation of early osteogenic development, specifically with regard to regulation of Ctnnb1.

Cigarette smoke is a mixture of over 7000 toxic components with at least 69 smoke chemicals producing cancers, suggesting that smoking is harmful to smokers. More importantly, the World Health Organization has estimated that 10% of the world’s population is involuntarily exposed to environmental tobacco smoke, a number that does not include developing embryos. However, tobacco exposure during embryonic development is one of the risk factors for developing a congenital birth defect. An understudied, yet important abnormality caused by maternal smoking is the improper development of the embryonic skeleton, which can result in long-term burdens on the affected child and their family, creating a need for investigation that is addressed in this thesis.

Due to the difficulty in quitting smoking while pregnant, women often look to use harm reduction tobacco products (HRTPs) because they are perceived as a safer alternative in comparison to conventional cigarettes. These products include ultra-filtered cigarettes with reduced carcinogen content, and chewing tobacco, which omits exposure to harmful combustion products. However, there is a lack of investigation to reveal the molecular mechanisms involved in skeletal embryotoxicity induced by conventional and harm-reduction tobacco, the unraveling of which is another objective of this thesis.

In the process of this investigation, an in vitro model based on human embryonic stem cells was developed. These cells are the only currently available cell culture system, which mimics the development of the human embryo. As this thesis shows, their differentiation into osteoblasts in vitro can be used to study chemical toxicity and the molecular mechanisms thereof.

Regulation of cell fate decisions, such as the decision to self-renew or to differentiate along a certain lineage, is critical during embryonic development and adult life. During embryogenesis, these decisions are critical and are very tightly controlled processes that involve input from a variety of signals, from the extracellular environment to the intracellular nutrient availability. This review will focus on AKT and CTNNB1 and the roles they play in promoting proliferation, differentiation, survival, and energy production. Due to the complexity of these processes, a number of other signaling families will also be discussed, including the Forkhead Box O (FOXO) protein, the mammalian Target of Rapamycin (mTOR) complexes, and the AMP-regulated kinase (AMPK).

Traumatic brain injury (TBI) often results in damage to the cerebral vasculature which leads to hypoperfusion, edema, and hemorrhage. Repairing the injured vasculature after TBI is critical for neuroprotection and improving outcomes. While numerous studies have shown that the cerebral vessels are damaged after TBI, there are scant studies looking at repair of the vessel network following brain injury. Furthermore, there is a paucity of studies that have examined the molecular mechanisms underlying vascular repair after TBI. One possible signaling factor is β-catenin, which promotes blood vessel formation during embryonic development. To address this gap in knowledge, we developed a novel method to stain, visualize, and analyze the cerebral vasculature in the entire rodent brain. This technique, referred to as Vessel Painting (VP), effectively stains pial, penetrating, and parenchymal vessels and cerebral vessels can be imaged by wide-field fluorescent microscopy to acquire whole brain images. We introduce two complimentary methods to analyze vessel morphology and complexity in the whole brain. Our novel VP and analysis protocol was used to study the vascular alterations after TBI. Adult male mice received a moderate controlled cortical impact followed by VP at 1 and 7 days post injury (dpi). We assessed β-catenin inside blood vessels around the injury site and utilized a Wnt reporter mouse line (TCF/LEF:H2B-GFP) to monitor Wnt gene expression. We report that TBI results in vascular loss at 1 dpi followed by an increase in new vessels at 7 dpi. We observed an acute increase in β-catenin expression and increased Wnt reporter activity in cerebral vessels after TBI. To assess the role of β-catenin in vascular repair, we utilized Lithium to increase β-catenin expression and JW74 to reduce β-catenin expression. Lithium treatment after TBI enhanced vascular repair and lead to elongated vessel segments at 7 dpi while JW74 treatment after TBI reduced vascular repair and lead to fragmented vessels. Overall, these findings suggest that β-catenin becomes activated after TBI to initiate vascular repair. Treatment strategies to enhance β-catenin appear to contribute to vascular repair after TBI and represents a potential target for future therapeutics.

A class of small, non-coding RNAs, microRNAs (miRNAs), may play a pivotal role in the development of the neural crest (NC)- and mesoderm-derived skeleton. They are involved in a variety of biological processes that contribute to skeletal development, such as cell migration, Wnt signaling and specification of cellular fate towards osteogenesis. Identifying miRNAs involved with regulating genes that drive the two lineages that give rise to osteoblasts during development can provide distinct gene regulatory mechanisms that give rise to craniofacial deformities. Potential microRNA-to-gene target interaction (MTIs) pairs can then potentially be used as biomarkers for in utero diagnostic or treatments for skeletal deformities in the face or limbs. The aim of this study was to develop a set of miRNA biomarkers that correlate with NC and mesoderm cell differentiation into osteoblasts, and to identify miRNAs specific to these pathways. Our study was conducted in vitro using human embryonic stem cells (hESCs) that were differentiated into osteoblasts. Toxicants known to repress NC and mesoderm induction were supplied to the in vitro cultures and the RNA sequenced to obtain both small RNAs and mRNAs. To facilitate this analysis, an R Shiny web app was developed to implement the sequencing results of both microRNA and RNA. The app provides tools to separately display and visualize the results of the differential regulation of microRNAs and mRNAs. Gene ontology (GO) analysis revealed that the identified miRNA-target interactions were indeed involved in select GO pathways related to osteogenic, NC and mesoderm differentiation. The app narrowed down significantly correlated MTIs specific to each differentiation lineage. Within the NC lineage miR-30a, miR-148a and miR-195 were correlated with cadherin2 (CDH2), DNA methyltransferase 3 beta (DNMT3B) and transcription factor AP-2 alpha (TFAP2A), respectively. Within the mesoderm lineage miR-148a, miR-7 and miR-20a were correlated with DNMT3B, JARID2 and VANGL1, respectively. In the future, these miRNAs could be further validated as potential biomarkers in the context of neurocristopathies.

Developmental toxicology is an ever-changing field dedicated to ensuring the safety and efficacy of foods, drugs and chemicals available to the public, specifically concerned with effects on a developing embryo. While many studies have uncovered the detrimental health effects of tobacco use, a concern to the health problems associated with harm reduction tobacco products (HRTPs) grows as the usage increases, specifically in women who are, or may become pregnant. Using a novel approach analyzing mitochondrial network morphology called Mitochondrial Network Analysis (MiNA), significant changes in mitochondrial network morphology were found in human embryonic stem cells (hESCs) exposed to both Camel and Camel Blue cigarettes, conventional and HRTP respectively. Supplementation with ascorbic acid led to a general improvement of network morphology for both products suggesting ROS as one mechanism of toxicity. To determine if prenatal exposure to Snus, a non-combustible HRTP, causes any increased risk of neurological damage such as anxiety and motor control, pregnant mice were injected with a Snus tobacco extract, and allowed to age into adulthood. The adult mice were then tested for such neurological issues using the elevated plus maze (EPM) and the Suok test. Results suggest female mice are more susceptible to increased behavioral issues such as anxiety and reduced motor control when exposed in utero to Snus tobacco extract than male mice compared to the untreated. This suggest that these neurological concerns may be linked in some way to the X-chromosome, and environmental toxicants such as tobacco may increase chances of occurrence. These studies together suggest that embryonic exposure to HRTPs may lead to other increased risks, not typically evaluated, that may be equal or more detrimental than conventional tobacco and must be taken into consideration when making claims of reduced harm.

Because of their pluripotent nature, as defined by their self-renewal and differentiation capacity, mouse embryonic stem cells (mESCs) have become an invaluable resource for unraveling the underlying mechanisms that control developmental programs. Understanding the formation and maintenance of bone tissue will undoubtedly facilitate the creation of new therapeutic approaches that address bone-related pathologies. Numerous reports have demonstrated the importance of the Wnt signaling pathway in regulating osteogenesis, however, given the revealed complexity of this regulation further elucidation of the time- and cell-specific response is needed. The in vitro culture of mESCs allows investigators to examine multiple stages of differentiation while simultaneously manipulating this signaling pathway in a time-dependent manner. Through the use of known modulators and activity reporters, studying beta-catenin (CTNNB1) (i.e. the central effector of canonical Wnt signaling) will provide invaluable insights towards a greater understanding of osteogenic processes. For instance, we have found that supplementation of the non-canonical Wnt ligand Wnt5a, during a narrow time window of osteoblast precursor formation, was able to improve osteogenic differentiation at later stages. This supplementation resulted in increased activation of multiple downstream effectors and was also correlated with a decrease in CTNNB1-dependent LEF/TCF transcriptional activity. Interestingly, when studying the impact of high concentrations of glucose within culture media, we found a decrease in osteogenic differentiation, which corresponded to an upregulation of CTNNB1-dependent LEF/TCF reporter activity within the same time period. This change in transcriptional activity was linked to differential activation of the AKT/FOXO signaling pathway. Thus, our work continues to reinforce the dynamic role of CTNNB1 in regulating in vitro osteogenesis. With this knowledge we employed multi-stage supplementation of either canonical or non-canonical Wnt ligands to direct osteogenic differentiation of mESCs in serum-free cultures. We found that these ligands, or corresponding chemical modulators, could yield measures of osteogenic output that was comparable to cultures containing fetal bovine serum. With these advancements in our understanding of the role of CTNNB1 in both developmental osteogenesis and the dysregulated processes underlying bone-related pathologies, novel strategies can be developed that improve differentiation protocols and therapeutic applications.

Bone formation is a complex, yet key process in the early development of all vertebrates. Therefore, understanding bone development, and more specifically the early stages of bone development, is crucial to further enhance our overall understanding of embryo development as well as finding ways to ameliorate skeletal birth defects. During development bones are derived from mesenchymal cells that either form from mesoderm (axial, paraxial, and lateral) or neural crest origins. These different origins and their derivatives contribute to specific bones in vertebrate organisms.

Origin determination and the subsequent bone formation are tightly controlled processes regulated by signaling pathways, microRNAs (miRNAs), transcription factors and environmental factors. All four elements have been the subject of many studies. However, our understanding of the precise role that miRNAs play in the early stages of bone development, and specifically in early cell fate decisions is lacking. This thesis aims to improve the understanding of miRNAs and their role in origin determination and osteoblast potentiation.

Furthermore, birth defects may arise when normal development is interrupted, delayed or otherwise negatively affects as is the case with exposure to environmental toxicants during embryonic development. The most common exposures, cigarette smoke and various cigarette smoke components, have been shown to reduce bone density and inhibit osteoblast formation. But as with miRNA studies, our understanding of the role that these toxicants play in early development faulters. Therefore, the second aim of this thesis is to elucidate the extent of damage caused by different types of cigarette smoke and tobacco products on early bone development.

Our work shows that miR-690 enhances osteogenesis and alters cell fate through CTNNB1 and FOXO3A inhibition. In addition, we showed that cigarette smoke inhibits early cartilage and bone formation consistent with earlier work from our lab showing cigarette smoke inhibition of FOXO3A and in vitro osteogenesis.

Purpose of review

Recent findings