Doctor of Philosophy in Craniofacial Science (PhD)
The role of small Rho GTPases in normal and abnormal lip fusion
The role of non-canonical WNT signaling in facial and limb development in the chicken embryo
Rare diseases - validation of disease-causing human variants in animal models
The molecular basis of the ever-renewing reptilian dentition using the gecko model
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The mechanisms of embryonic facial morphogenesis are poorly understood because direct visualization of the growing embryo is challenging. We refined an organ culture method to visualize the individual cells within the frontonasal mass at high resolution. The fate of the frontonasal mass is to form the premaxilla, nasal septum and facial midline. During normal development the frontonasal mass begins as wide prominence but then the nasal pits relocate to the midline, causing dramatic medio-lateral narrowing. We confirmed that in vitro, the frontonasal mass narrowed over a 48h period, similar to in vivo. Removing the eyes, brain and most of the surrounding face did not impede facial narrowing suggesting intrinsic rather than extrinsic mechanisms were involved. Indeed, blocking the cytoskeletal rearrangements with the Rho GTPase inhibitor, Y27632 (ROCKi) completely inhibited narrowing. Organ cultures were stained with Hoechst dye and imaged using confocal microscopy. Nuclei were imaged for 4-6 hours at 10 minute intervals. Manual cell tracking was carried out across the frontonasal mass. At 10X magnification, striking patterns of order, disorder and then order in vectors of movement were observed. In ROCKi treated cultures there was disorder for the entire culture period. Clustering the vectors according to similarity of the angle revealed large groups of cells were moving in a similar manner in the controls but in the ROCKi treated cultures, clusters were poorly defined and smaller in size. In order to assess symmetry and patterns of divergence and convergence the vector data was interpolated over an evenly spaced grid. The controls had a high degree of right-left symmetry whereas the ROCKi-treated cultures lacked symmetry. We also examined the data to look for sources or sinks in the cell movements. Divergence and convergence bands were located in the mediolateral axis with branches at the lateral edges. The change in direction from convergence to divergence and then back to convergence was rapid, often switching within 20 minutes. This rapid cycling is on the same scale as GTPase switching. Our data suggest that orchestrated mesenchymal cell behaviors, mediated by Rho GTPases are involved in convergent-extension in the face.
The study of rare genetic diseases provides valuable insights into human gene function. The chicken embryo was used as a model to investigate the role of WNT signaling in skeletogenesis and to elucidate the functional consequences of mutations in dominant Robinow Syndrome (RS). RS mutations affect non-canonical WNT signaling that controls a variety of developmental events to regulate convergent extension, cell polarity, and cytoskeletal rearrangement. RS is characterized by short stature, mesomelic limb shortening, hypertelorism, and mandibular hypoplasia. Mutations in dominant RS occur in several components of the non-canonical WNT signaling pathway, and this study is focusing on two mutations in WNT5A ligand (WNT family member) and three mutations in Dishevelled1 (DVL1), a protein that relays WNT signals intracellularly. We delivered the human genes to the chicken embryo using replication competent retroviruses (RCAS) and analyzed morphologic, cellular, and molecular effects in the forelimbs and mandible. Misexpression of mutants in dominant RS led to a shortening of the forelimb and mandible and caused polarity disruptions in the chondrocytes that were not seen in the GFP virus controls. The variants were unable to activate canonical WNT signaling and over-activated non-canonical WNT signaling, demonstrating the importance of non-canonical WNT signaling in skeletogenesis. Dominant RS mutations have dominant neomorphic effects on chondrogenesis that interfere with the function of the wild-type protein. This work establishes that the dominant effect of the mutations leads to elevated non-canonical WNT signaling and randomizes the distribution of planar cell polarity molecules of which produces shortened skeletal elements.
Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis and morphogenesis is unknown. The aim of this thesis is to study functions of WNT5A during mandibular development in chicken embryos.We initially determined that WNT5A is expressed in developing Meckel's cartilage but in mature cartilage expression was decreased to background. This pattern suggested that WNT5A is regulating chondrogenesis so to determine whether initiation, differentiation or maintenance of matrix was affected I used primary cultures of mandibular mesenchyme. I found that Wnt5a conditioned media allowed normal initiation and differentiation of cartilage but the matrix was subsequently lost. Collagen II and aggrecan, two matrix markers, were decreased in treated cultures. Degradation of matrix was due to the induction of metalloproteinases, MMP1, MMP13, and ADAMTS5 and was rescued by an MMP antagonist. The effects of Wnt5a on cartilage were mainly due to stimulation of the non-canonical JNK/PCP pathway as opposed to antagonism of the canonical Wnt pathway.To increase the clinical relevance of my work I studied the functional consequences of two human WNT5A mutations (C182R and C83S) causing human Robinow syndrome. Retroviruses containing mutant and wild-type versions of WNT5A caused shortening of beaks and limbs; however, the phenotypes were more frequent and severe with mutations. Mechanisms responsible for micrognathia were assessed. Decreased cell proliferation and impaired chondrocyte organization and intercalation were seen with all constructs. The effects of mutant proteins on the migration of mesenchymal cells were tested in organ cultures of the mandible. The C83S and to a lesser extent C182R forms of WNT5A inhibited the normal migration of dye-labeled mesenchymal cells. The lack of cell migration was similar to that reported in Wnt5a null mice and therefore suggested that the WNT5A mutations are causing a loss-of-function.We conclude that WNT5A is required during early chondrogenesis to block canonical signaling thereby allowing cartilage to form. In addition, WNT5A is required for cells to migrate within the mandible and perhaps to form the elongated shape of the lower jaw. Finally WNT5A in conditions of excess has detrimental effects on cartilage integrity.
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Objectives: Secular change refers the increased rate of growth and development to maturity that children are undergoing over successive generations. Secular change has been postulated to be the result of non-genetic changes in the environment that are associated with an improvement in the overall standard of living, such as improved quality of nutrition and lessening of infectious diseases. Evidence of secular change has been described across a multitude of populations, including Canada, and has included changes in height, craniofacial morphology, fetal growth and birth weight, and age of menarche. Though some secular changes in dental maturation and tooth emergence have been demonstrated, results have been mixed and limited and no studies on secular changes in dental maturation have been conducted in Canada. We sought to determine if a secular change in dental maturation has occurred in Vancouver children ages 6-12.99 over a 30-year period. Methods: Two cohorts of age and gender matched children age 6-12.99 were identified from a retrospective chart review of Orthodontic/Pediatric patients treated in the Vancouver, BC area. The historical group was born between 1960-1980 (n= 220) and the recent group between 2004-2010 (n=220). Stages of tooth mineralization were scored from panoramic radiographs according to the Demirjian method of assessing crown and root development (Demirjian et al., 1973, Hum. Biol. 45, 211-227). Seven mandibular teeth on the left were used were assigned a maturity score (A-H). To assess changes in the timing of dental maturation, the median age of attainment for each stage of maturation was calculated, using logistic regression. Differences in median age of attainment between the two time periods and between males and females will be obtained by logistic regression. Results: A significant positive secular change in dental maturation was observed. Advancement in median age-of-attainment in the recent sample relative to the historic sample ranged between 0.06 and 1.43 years (mean = 0.61years) in boys and between 0.05 and 1.43 years in girls (mean = 0.90 years). Conclusions: Positive secular changes have taken place in British Columbia during the last 30-years and that tooth maturation is a sensitive way in which to measure childhood development.
The leopard gecko is an emerging reptilian model for the molecular basis of indefinite tooth replacement. Here we characterize the tooth replacement frequency and pattern of tooth loss in the normal adult gecko. We chose to perturb the system of tooth replacement by activating the Wingless signaling pathway (Wnt). Misregulation of Wnt leads to supernumerary teeth in mice and humans. We hypothesized by activating Wnt signaling with LiCl, tooth replacement frequency would increase. To measure the rate of tooth loss and replacement, weekly dental wax bites of 3 leopard geckos were taken over a 35-week period. The present/absent tooth positions were recorded. During the experimental period, the palate was injected bilaterally with NaCl (control) and then with LiCl. The geckos were to be biological replicates. Symmetry was analyzed with parametric tests (repeated measures ANOVA, Tukey’s post-hoc), while time for emergence and total absent teeth per week were analyzed with non-parametric tests (Kruskal-Wallis ANOVA, Mann-Whitney U post-hoc and Bonferroni Correction). The average replacement frequency was 6-7 weeks and posterior-to-anterior waves of replacement were formed. Right to left symmetry between individual tooth positions was high (>80%) when all teeth were included but dropped to 50% when only absent teeth were included. Two animals were followed for 14 weeks after NaCl injections and 14 weeks after LiCl injections. NaCl did not affect the replacement dentition but LiCl delayed and disrupted the pattern of replacement. The phenotypes were more severe for one animal including 1) increased time before emergence, 2) increased total number of absent teeth per week, 3) a greater effect on anterior teeth and 4) disruption of symmetry. The most affected period began 7 weeks post LiCl injection. At the end of the study, in vitro CT scans of both animals revealed normal patterns of unerupted teeth however there was bone loss in one animal. Gecko tooth replacement is rapid enough to be useful for longitudinal studies. Between-animal variation is high when studying individual teeth therefore each animal should be used as its own control. Future work includes increasing the biological replicates and detailed molecular studies to confirm the effect of LiCl.
Objectives: In order to study abnormal facial development, reference standards of normal development are required. It is challenging to obtain 3D data on early embryos, since they are comprised of non-differentiated tissue. We used optical projection tomography (OPT) (Bioptonics, UK), which images transparent specimens with UV light. Here we used carefully staged chicken embryos to measure facial morphogenesis over time.Methods: Chicken eggs (n=32) were incubated for 3.5-6 days (stage 20, 24, 28, 29). Embryo heads were fixed in formaldehyde, embedded in agarose, dehydrated in methanol, and then cleared in Benzyl Alcohol Benzyl Benzoate. Embryos were scanned with the OPT, images were reconstructed, and then the head was digitally resliced in the frontal plane using NRecon and CTan. Resliced files were imported into Amira, facial prominences were outlined, and isosurfaces were created. Volumetric measurements were assessed using Amira. Landmarks were applied to the surface of each prominence using Landmark. These landmarks were then superimposed from different embryos using MorphoJ, whereby they underwent Procrustes superimposition, Principal Component Analysis, Canonical Variate Analysis, and Discriminant Function Analysis.Results: Traditional morphometrics revealed that the greatest amount of growth was a 24-fold difference in volume of the lateral nasal prominence between stages 20 and 29, followed by the maxillary, mandibular, and frontonasal mass. Geometric morphometrics revealed that embryonic facial prominences had minimal changes in shape between stages 20 and 24, however, after this time, there was more separation of the data in morphospace. Strikingly, the greatest morphological change was between stages 28 and 29, which was only 12 hours apart. This rapid change suggests that other mechanisms in addition to cell proliferation are involved. In addition, the data show that major morphological changes precede lip fusion. Therefore, we can pinpoint our studies to stage 28, when critical events in the mesenchyme are taking place. Conclusion: Embryonic chicken facial prominences undergo major shape changes. Each prominence varies in morphology with respective stage, with the frontonasal mass and mandibular prominence having the most dynamic shape changes.
Though most dentate vertebrates replace their teeth at least once in the course of their lives, the process of tooth replacement is poorly understood. This is mainly because the major tooth development model is the mouse which only has one generation of teeth. Our previous work suggested that tooth renewal in geckos might involve dental epithelial stem cells and that these putative stem cells become transit- amplifying cells when exposed to canonical WNTs. Here we further investigate this idea using adult leopard geckos (Eublepharis macularius). To further previous findings from our lab that the dental apparatus is a WNT responsive tissue we perturbed the WNT pathway by agonist and antagonist organ cultures of oral tissue explants. BIO stimulated proliferation at an intermediate concentration of 20 μM but not at higher or lower concentrations. This suggests that in vivo, cells are responding to gradients of WNT activity. We also looked at associated BMP and FGF pathways via in situ histology and organ culture manipulation respectively and found alternating patterns of gene expression. We then mapped areas of high canonical WNT signaling and found that nuclear staining for phospho beta catenin was principally found in the outer enamel epithelium and successional lamina. We moved to an in vivo strategy to allow for better tissue survival. Palatal injections of LiCl or the control reagent NaCl were delivered to the base of the maxillary teeth. We found that LiCl increased proliferation in the successional lamina and cervical loops, areas that normally have higher proliferation. We conclude that certain regions of the dental epithelium are sensitive to change in canonical WNT signaling and that this signaling is potentially kept to a localized region via BMP inhibition of the WNT pathway. Regions of the dental lamina that contain putative stem cells may require signals in addition to WNTs to stimulate the formation of transit amplifying cells. Future work will further elucidate the many signaling cascades required for tooth succession to occur.
Objectives: The human secondary palate forms between 6-12 weeks of gestation. There has been controversy as to whether palatal shelves in the soft palate join by fusion similar to the hard palate, or whether merging and proliferation of the mesenchyme at the posterior edge of the developing hard palate is the mechanism. The purpose of this study is to examine the mode of soft palate closure in a more representative sample than was used in the single previous study on which all textbooks are based.Methods: Serial sections of secondary palates from 13 human fetuses from 54-74-days of development post conception were stained, photographed and imported into WinSurf 3D software. Anatomical structures were traced including the palatal shelves, midline epithelial seam and palatine aponeurosis, the images aligned and then stacked to create a 3D representation.Results: We analyzed the following numbers of specimens: 54-days-2; 57-days-4; 59 days -2; 64-days-1; 67-days-1; 70-days–2; 74-days-1. At 54-days, a midline seam is present close to the hard palate but more posteriorly the soft palate is open. Between 57 and 59 days a thick midline seam is observed throughout the soft palate. There is some variability between specimens such that the soft palate was closed early in one 59 day specimen and open in a 67-day specimen. One 70-day specimen had no seam whereas the other retained the seam. By 74-days the specimen had complete soft palate union with the presence of a continuous palatine aponeurosis. Overall, our sample included a total of 7 fetuses with a midline seam in the soft palate. Conclusions: The formation of a bilayered epithelial seam followed by breakdown of the seam and mesenchymal fusion is the primary mode of soft palate formation in humans. Epithelial seam removal is rapid and could explain why a seam was not observed in earlier studies.
Vertebrate craniofacial development and speciation has been studied in great detail, with major emphasis placed on mammalian species and highly derived archosaurs (birds). However, less is known about reptiles and in particular turtles. Turtles are speculated as to have retained many ancestral features of amniotes. Therefore, studying the Testudine (turtle) order not only helps to better understand amniote head development, but also the derivation of modern form. This thesis will investigate the formation of the hard palate in a representative turtle species, E. subglobosa, not only because of its evolutionary significance but also because this region is frequently affected in orofacial clefting. Origins of the palatine bones were first examined since other amniotes form these bones within outgrowths of the maxillary prominence, or the palatal shelves. Surprisingly no palatal shelves were found at the position or time when they should have been forming. Instead palatine bones condensed directly in the mesenchyme beneath the nasal cavity Furthermore there was no evidence from cell proliferation or apoptosis analysis of the maxillary prominences that vestigial shelves were ever present. The hypothesis following was that gene expression in the maxillary prominences might be different in turtles compared to the chicken or mouse in which shelves do form. I found no major differences but interestingly several of the genes I studied were also markers of the primitive stomodeum. Results show the turtle retains gene expression patterns of the chicken stomodeum, the primitive oral roof before palatal shelf formation, suggesting the turtle oral roof is still primitive in nature rather than advanced in other amniotes. This unfamiliar mechanism of hard palate development with no vestigial traits of palatal shelf formation supports arguments for a more basal placement of the turtle in the phylogenetic tree. Contrary to these findings, the similarity in gene expression and sequence to the chicken argues for a more derived placement closer to the archosaurs. While these present results do not allow for confident placement of the turtle as more basal or derived in the amniote tree, the data collected shows that ontological studies can help shed light on evolutionary debates.
The aim of this thesis is to study the effects of reducing retinoic acid (RA) levels in the embryonic face on jaw morphogenesis. One member of the Cytochrome P26 class of enzymes, CYP26A1, which degrades retinol products, was locally overexpressed in chicken embryos. I hypothesized that lowering RA levels would either affect jaw patterning, cell survival and/or cytodifferentiation. Chicken embryos at stage 15 and 20 (E2.5, 3.5) were injected with RCAS
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