Across vertebrates, the coordinated evolution and synchronous development of teeth and the mandible must require specific timing and positioning of gene expression. While debate persists about whether teeth have evolved before or after mandible, currently, the consensus is that these systems evolved at separate times and thus have discreet origins. This raises an important question of whether tooth and mandibular tissues have over the course of their evolution become developmentally co-dependent or, as separate evolutionary origins would imply, remain developmentally autonomous of each other. The molecular signaling that patterns the genesis of upper versus lower jaw skeletons, as well as specifies tooth type (i.e., molar vs. incisor) is relatively well understood. To date, the distinct genetic processes that drive tooth development distinct from jaw skeletal development has been little-studied, in no small part due to the technical complexity of this task. The main hypothesis of thesis is that a collection of genes acting within a gene regulatory network (GRN) drives odontogenesis with neither input from, nor influence on, jaw morphogenesis. The Transformation Related Protein (TRP63) is a master transcription factor that is vital to odontogenesis because TRP63 maintains the competence and proliferation of the epithelial layer of the tooth organ. Thus the “toothless” TRP63 homozygote mouse mutant (Brdm2 mutant) fails to develop teeth even though it develops a virtually unperturbed mandible. This combination of lower jaw morphogenesis in the absence of odontogenesis presents a rare model to study the genetic changes that occur when teeth but not jaws fail to form. A previous microarray gene expression analysis (Boughner laboratory, unpublished data) of mandibular prominences (MdPs) derived from embryonic day (E) 10-13 revealed that, compared to heterozygote (Trp63+/-) MdPs, in Brdm2 mutant MdPs, transcript levels of cerebellin 1 (Cbln1); keratin 2-8 (Krt2-8); phospholipid transfer protein (Pltp) and fermitin 1 (Fermt1) were altered in at least some of the four embryonic stages. Specifically Cbln1 and Krt2-8 were up-regulated while Pltp and Fermt1 were down-regulated. None of these four genes have previously been linked to odontogenesis yet all are potential candidates for a “tooth-specific” GRN. Using RT-qPCR analysis, I aimed to test the validity of the microarray work and confirmed its veracity by showing that, generally, Cbln1 and Krt2-8 mRNA were up-regulated, while Fermt1 (but not Trp63 or Pltp) mRNA was significantly down-regulated in the MdPs of Brdm2 mutant mice relative to Trp63+/- mice. Conversely, western blotting protein expression analysis showed little-to-no change among Brdm2 MdPs relative to either wild type (Trp63 +/+) or Trp63+/- embryos, making it difficult to tease out the precise relationship between CBLN1, FERMT1, KRT2-8, and PLTP and TRP63. These results show a lack of strong correlation between mRNA and protein expression. Because the mRNA analyses showed disturbances in the expression level in a few of these five genes within the MdPs during the earliest stages of tooth development, these genes remain candidates for an odonto-specific GRN. In complement to the genetic work, to characterize the tandem developmental morphology of tooth and jaw skeleton tissues, my work included developing a new tissue staining protocol. Using Protargol, a silver-based compound, to enhance in uncut mouse embryos contrast among tiny, soft oral tissues and visualize their organization in 3D and microscopic detail across several embryonic stages. This novel protocol offers a simple, easy-to-follow, and relatively inexpensive way to effectively stain whole embryos aged E10-15 for X-ray based micro-computed tomography (μ-CT) imaging using synchrotron and desktop scanning systems. Because the scan data are digital, this new method also allows more precise, accurate and rapid empirical studies of the sizes, shapes and positions of teeth as they form within the jaw to clarify how these tissues are integrated as they develop. The work presented in this thesis investigated tooth development exclusive of mandible development from complementary molecular and morphological points of view. Driven by the lack of understanding of the genetic mechanisms that orchestrate tooth with jaw skeletal development, this study has for the first time isolated a set of genes that are potential candidates for tooth formation only. These results set the stage for next steps in testing the developmental-genetics that enable teeth and jaws to “fit” together as they develop.

published_or_final_version / Botany / Master / Master of Philosophy

Acute injury of major epithelial organ systems (kidney, liver, lung, etc.) is collectively a principal cause of death worldwide. Regenerative medicine promises to meet these human health challenges by harnessing intrinsic cellular processes to repair or replace damaged tissues. Epithelial morphogenesis is a hard-wired, multicellular differentiation program that dynamically integrates microenvironmental cues to coordinate cell fate processes including adhesion, migration, proliferation, and polarization. Thus, epithelial morphogenesis is an instructive mode of tissue assembly, maintenance, and repair. Three-dimensional epithelial cell cultures in natural basement membrane (BM) extracts produce hollow, spherical cyst structures and have indicated that the BM provides the critical cell adhesion ligands to facilitate cell survival, stimulate proliferation, and promote polarization and lumen formation. However, the utility of natural BMs for detailed studies is generally limited by lot-to-lot variations, uncontrolled cell adhesive interactions, or growth factor contamination. The goal of this thesis was to engineer bioartificial extracellular matrices (ECM) that would support and modulate epithelial cyst morphogenesis. We have engineered hydrogels, based on a multi-arm maleimide-terminated poly (ethylene glycol) (PEG-4MAL), that present cell adhesive molecules and enzymatic degradation substrates and promote polarized epithelial cyst differentiation in vitro. To investigate the influence of matrix physical and biochemical signals on cyst morphogenesis, we independently varied the polymer weight percentage (wt%), the density of a cell adhesion ligand (RGD), and crosslink degradation rates of the hydrogels. Then, we evaluated functional outcomes including Madin-Darby canine kidney (MDCK II) epithelial cell survival, proliferation, cyst polarization, and lumen formation. We found that cell proliferation, but not cell survival, was sensitive to the polymer wt%, which is related to elastic modulus and crosslink density. This result defined a working range of PEG-4MAL concentration (3.5% - 4.5%) that promotes robust proliferation. Analysis of mature cysts indicated that 4.0% and 4.5% gels produced cysts resembling those typically grown in type I collagen gels while 3.5% gels produced cysts with higher incidence of inverted polarity and multiple lumens. Perturbation of matrix degradability using a slow-degrading crosslink peptide or matrix metalloproteinase inhibitors showed that the rate of matrix degradation exerts major influence on cyst growth in PEG-4MAL gels. We employed 4.0% PEG-4MAL hydrogels with RGD ligand density ranging over 0 – 2000 uM to discover that (1) lumen formation was eliminated in the absence of RGD, (2) extent of lumen formation increased with increasing RGD concentration, and (3) cyst polarity was inverted below a threshold of integrin binding to RGD. Together, these results show that the biochemical and physical properties of the matrix, particularly integrin binding and matrix degradability, effectively modulate establishment of apico-basal polarity and lumen phenotypes in MDCK II epithelial cyst structures. Furthermore, these studies validate PEG-4MAL hydrogels as a powerful culture platform to enable detailed investigation of matrix-directed modulation of epithelial morphogenesis.

No description available.

The bony fin rays of the zebrafish caudal fin are a convenient system for studying bone morphogenesis and patterning. Joints and bifurcations in fin rays follow predictable spatial patterns, though the mechanisms underlying these patterns are not well understood. We developed simulation models to explore ray pattern formation mechanisms in growing fins. In all models, the fin ray growth rates are based on quantitative experimental data. The different models simulate ray joint formation and bifurcation formation using different hypothetical mechanisms. In the most plausible model, ray joint and bifurcation formation result from the accumulation of two substances, arbitrarily named J and B. Model parameters were optimized to find the best fit between model output and quantitative experimental data on fin ray patterns. The model will be tested in the future by evaluating how well it can predict fin ray patterns in different fin shapes, mutant zebrafish fins, and other fish species.

The development of spatial structure and form on vertebrate skin is a complex and poorly understood phenomenon. We consider here a new mechanochemical tissue interaction model for generating vertebrate skin patterns. Tissue interaction, which plays a crucial role in vertebrate skin morphogenesis, is modelled by reacting and diffusing signal morphogens. The model consists of seven coupled partial differential equations, one each for dermal and epidermal cell densities, four for the signal morphogen concentrations and one for describing epithelial mechanics. Because of its complexity, we reduce the full model to a small strain quasi-steady-state model, by making several simplifying assumptions. A steady state analysis demonstrates that our reduced system possesses stable time-independent steady state solutions on one-dimensional spatial domains. A linear analysis combined with a multiple time-scale perturbation procedure and numerical simulations are used to examine the range of patterns that the model can exhibit on both one- and two-dimensions domains. Spatial patterns, such as rolls, squares, rhombi and hexagons, which are remarkably similar to those observed on vertebrate skin, are obtained. Although much of the work on pattern formation is concerned with synchronous spatial patterning, many structures on vertebrate skin are laid down in a sequential fashion. Our tissue interaction model can account for such sequential pattern formation. A linear analysis and a regular perturbation analysis is used to examine propagating epithelial contraction waves coupled to dermal cell invasion waves. The results compare favourably with those obtained from numerical simulations of the model. Furthermore, sequential pattern formation on one-dimensional domains is analysed; first by an asymptotic technique, and then by a new method involving the envelopes of the spatio-temporal propagating solutions. Both methods provide analytical estimates for the speeds of the wave of propagating pattern which are in close agreement with those obtained numerically. Finally, by numerical simulations, we show that our tissue interaction model can account for two-dimensional sequential pattern formation. In particular, we show that complex two-dimensional patterns can be determined by simple quasi-one-dimensional patterns.

The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. This study demonstrates that neuronal complexity controlled by p21-activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. Double knockout (DK) mice lacking both PAK1 and PAK3 were severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume at maturity. Remarkably, the reduced brain was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified dendritic arbors/axons and reduced synapse density. Surprisingly, the DK mice were elevated in basal synaptic responses due to enhanced individual synaptic potency, but severely impaired in bi-directional synaptic plasticity. The PAK1/3 action is likely mediated by cofilin-dependent actin regulation because the activity of cofilin and the properties of actin filaments were specifically altered in the DK mice.

Preplacodal ectoderm (PPE) is a contigous horse-shoe shaped domain that enwraps the anterior neural plate towards the end of gastrulation and eventally resolves into a number of focal epithelial thickenings called placodes. These placodes together with Neural Crest (NC), contributes to the peripheral nervous system in vertebrates. PPE and NC arise at the neural-non neural interface by distinct mechanisms during development. However, a general idea in the field was that a Bmp signaling gradient specifies different ectodermal fates: high Bmp levels specify epidermis, intermediate levels PPE and NC and no Bmp signaling is required for neural fate specification. We showed that while NC responds to intermediate levels of Bmp signals, PPE is specified by a distinct mechanism that involves a two step model for PPE specification. In the first step, Bmp is positively required to activate four competence factors, tfap2a, tfap2c, foxi1 and gata3 throughout the ventral ectoderm and renders this domain competent to respond to inductive factors. In the second step, inductive factors Fgf and Bmp antagonists act to completely block all Bmp signaling to specify PPE at neural-non neural interface. These Bmp-activated competence factors do not need Bmp for subsequent maintenance because they positively cross-regulate and autoregulate each other’s expression forming a gene regulatory network. This network is sufficient to rescue both PPE and NC in the complete absence of Bmp. The subsequent resolution of PPE into discerte placodal thickenings was hypothesized to involve localized migration of placodal progenitors and one of the molecules that could play an important role during cell migration was extracellular matrix binding molecule, integrin alpha 5 (itga5) because it was expressed at the right time and place. Knockdown of itga5 results in disorganised trigeminal, epibranchial ganglia and smaller otic placodes. Tracing the cell trajectories of placodal progenitors revealed that cells failed to migrate directionally. Additionally, we observed elevated levels of cell death in itga5 morphants which could be rescued by overexpression of Fgf ligands suggesting that Itga5 and Fgf pathways cooperate during placodal development. All together, this dissertation reveals novel genetic mechanisms that regulate placodal development from late-blastula to mid-somitogenesis stages.

Signaling molecules play essential roles in communication between cells. Wnt signaling molecules are critical for embryonic development of several organisms. I examined the involvement of Wnt signaling during two major developmental processes, namely embryo implantation and formation of the embryonic body axes. Using RT-PCR analysis, I showed that multiple Wnt genes are expressed in the blastocyst at the time of implantation. Moreover, expression of Wnt 11 requires both estrogen produced by the mother and the uterine environment. Using a transgenic approach, I showed that beta-catenin-regulated transcriptional activity, which is a major transducer of Wnt signaling, is activated in the uterus specifically at the site of implantation in an embryo-dependent manner. These results introduce Wnts as candidate signaling factors that may mediate the communication between the embryo and uterus that initiates implantation. / Wnt/beta-catenin signaling triggers axis formation in Xenopus and zebrafish embryos. I showed that, during embryonic development, beta-catenin-regulated transcriptional activity is first detected in the prospective primitive streak region prior to gastrulation. This demarcates the posterior region of the embryo. This activity then becomes restricted to the elongating primitive streak and to the node. In Xenopus embryos, beta-catenin participates in the formation of the organizer through the activation of the homeodomain transcription factors Siamois and Twin. I obtained evidence that a Siamois/Twin-like binding activity exists in mouse embryos and is localized in the node. These results strongly suggest that, as the case in Xenopus and zebrafish, the Wnt/beta-catenin pathway is involved in establishing embryonic body axes. / Furthermore, using the transgenic mouse line that I generated for these studies, I mapped the transcriptional activity of beta-catenin during mouse embryonic development. These results revealed when and where this activity, and presumably Wnt signaling, is active during the development of several organs and embryonic structures.

Thesis (Ph. D.)--University of Sydney, 1997. / Title from title screen (viewed 25 March 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Dept. of Linguistics, Faculty of Arts. Includes bibliographical references. Also available in print form.