Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 100-111).

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Master of Science / Department of Chemistry / Daniel A. Higgins / Lithography techniques have been widely used to fabricate optical, electronic and optoelectronic devices with sub-micron scale spatial resolution. In the most common lithographic procedures, a light sensitive polymer, called a photoresist, is exposed and developed to form a binary relief pattern on a substrate. The finest features are produced by X-ray or electron-beam methods, both of which are very expensive to employ. Less expensive methods use ultraviolet (UV) light to expose the photoresist through a photomask. The resolution in these methods is somewhat lower and is governed by diffraction of light by the photomask, the quality of the photomask, and by any chemical/physical development steps subsequently employed. Due to the above limitations, we have been investigating direct-write, ablative multiphoton lithography as an alternative method for preparing high-resolution patterns. With this method, near-IR light from an ultrafast pulsed laser source is focused into a polymer film, leading to depolymerization and vaporization of the polymer. Arbitrary binary patterns can be produced by raster scanning the sample while controlling exposure of the film to the laser. Importantly, high-resolution etching of the polymer film is achieved without the use of a photomask and without chemical development steps. While arbitrary patters are easily prepared, it is also possible to prepare three-dimensional (i.e. grayscale) surface relief structures. In this study, ablative multiphoton photolithography is used to prepare binary and grayscale structures in thin films of PEDOT:PSS, an electrically conductive organic polymer blend. A simple kinetic model is proposed to explain the etching process. Data on the power-dependence of polymer etching can be fit to this model and is used to determine the order of the nonlinear optical process involved. The etch depth as a function of laser focus is also investigated and shown to follow the same kinetic model. The results show that three-dimensional (grayscale) patterns can be prepared by modulating either the laser power or the laser focus. Images of several binary and grayscale structures prepared by this method are presented.

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.

Cell behavior is influenced to a large extent by the surrounding microenvironment. Therefore, in the body, the cellular microenvironment is highly controlled with cells growing within well-defined tissue architectures. However, traditional culture techniques allow only for the random placement of cells onto culture dishes and biomaterials. Cell micropatterning strategies aim to control the spatial localization of cells on their underlying material and in relation to other cells. Developing such strategies provides us with tools necessary to eventually fabricate the highly-controlled microenvironments found in multicellular organisms. Employing natural extracellular matrix (ECM) materials in patterning techniques can increase biocompatibility. In the future, with such technologies, we can hope to conduct novel studies in cell biology or optimize cell behavior and function towards the development of new cell-based devices and tissue engineering constructs. Herein, a novel cell patterning platform was developed on a hydrogel base of crosslinked hyaluronic acid (HA). Hydrogels are often employed in tissue engineering due to their ability to mimic the physicochemical properties of natural tissues. HA is a polymer present in all connective tissues. Cell-adhesive regions on the hydrogel were created using the RGDS peptide sequence, found within the cell-adhesive ECM protein, fibronectin. The peptide was bound to a 2-nitrobenzyl “caging group” via a photolabile bond to render the peptide light-responsive. Finally, this “caged” peptide was covalently bound to the hydrogel to form a novel HA hydrogel with a cell non-adhesive surface which could be activated with near-UV light to become adhesive. In this way, we successfully formed chemically patterned cell-adhesive regions on a HA hydrogel using light as a stimulus to form controlled cell patterns. While the majority of cell patterning strategies to date are limited to patterning one cell population and cannot be changed with time, our strategy was novel in using small, adhesive, caged peptides combined with multiple, aligned light exposure steps to allow for dynamic chemical cell patterning on a hydrogel. Multiple cell populations, even held apart from one another, were successfully patterned on the same hydrogel. Furthermore, cell patterns were deliberately modified with time to direct cell growth and/or migration on the hydrogel base.

As the internet-of-things hardware integration continues to develop and the requirements for electronics keep diversifying and expanding, the necessity for specialized properties other than the classical semiconductor performance becomes apparent. The success of emerging semiconductor materials depends on the manufacturability and cost as much as on the properties and performance they offer. Solution-based semiconductors are an emerging concept that offers the advantage of being compatible with large-scale manufacturing techniques and have the potential to yield high-quality electronic devices at a lower cost than currently available solutions. In this work, patterns of high-quality MAPbBr3 perovskite single crystals in specific locations are achieved through the modification of the substrate properties and solvent engineering. The fabrication of the substrates involved modifying the surface adhesion forces through functionalization with self-assembled monolayers and patterning them by photolithography processes. Spin coating and blade coating were used to deposit the perovskite solution on the modified silicon substrates. While single crystal perovskites were obtained with the modification of substrates alone, solvent engineering helped with improving the Marangoni flows in the deposited droplets by increasing the contact angle and lowering the evaporation rate, therefore controlling and improving the shape of the grown perovskite crystals. The methodology is extended to other types of perovskites such as the transparent MAPbCl3 and the lead-free MABi2I9, demonstrating the adaptability of the process. Adapting the process to electrode arrays opened up the path towards the fabrication of optoelectronic devices including photodetectors and field-effect transistors, for which the first iterations are demonstrated. Overall, manufacturing and integration techniques permitting the fabrication of single crystalline devices, such as the method in this thesis work, are fundamental in pushing hybrid perovskites towards commercialization.

Stomata are cellular valves in plants that play an essential role in the regulation of gas exchange and are distributed in the epidermis of aerial organs. In Arabidopsis thaliana, stomatal production and development are coordinated by the mitogen-activated protein kinase (MAPK) signalling pathway, which modulates a variety of other processes, including cell proliferation, regulation of cytokinesis, programed cell death, and response to abiotic and biotic stress. The environment also plays a role in stomatal development, by influencing the frequency at which stomata develop in leaves. This thesis presents an analysis of stomatal development in Arabidopsis mutants in two MAPK pathways: MEKK1-MKK1/MKK2-MPK4, and MAP3K17/18-MKK3. Obtained results demonstrate the effect of stress conditions on stomatal development and specify the involvement of analysed MAPK in stomatal patterning. First, both analysed pathways modulate stomatal patterning in Arabidopsis cotyledons. Second, plant growth-promoting bacteria tested enhance stomatal density and affect guard cell morphology. Third, the sucrose or mannitol treatment increases defects in stomatal patterning. Finally, salt stress or high temperature can suppress stomatal defects in mutants of the MEKK1-MKK1/MKK2-MPK4 pathway.

<p>Here presents the studies of polymerization kinetics and crosslinking efficiency of nm-resolution striped phases on surface, which depends on lengths of alkyl segments and headgroup chemistry. While fluorescence readouts offer the overall efficiencies of polymerization and crosslinking transfer, SPM measurements reveal molecular details accounting for reactivity differences. Additionally, this research also demonstrates the utilization of primary amines striped phases on soft materials, achieving post-functionalization and specific  adsorption of nanocrystals, highlighting the versatile applications of this nm-scale chemistry boundary.</p>

Multi-tissue pattern formation during development is a complex process in which extracellular communication events specify distinct cell types and regulate exquisite embryonic morphogenesis. The sea urchin embryo provides an excellent model for studying pattern formation due to relative genetic and morphological simplicity. The larval skeleton is secreted via biomineralization by the skeletogenic primary mesenchyme cells (PMCs). The PMCs undergo an epithelial-mesenchymal transition and migrate as individual cells within the blastocoel into stereotypic positions; this regulated PMC migration and positioning is required for normal skeletal patterning. Elegant PMC transplant experiments have demonstrated that PMC positioning, and thus skeletal patterning, is directed by ectodermal cues, and not by cues internal to the PMCs. In recent years, new efforts have been made to identify the ectodermal gene products that regulate skeletal development. The transcription factors Otp, Pax2/5/8 and Strim1, signaling by FGF, VEGF, and Wnt5a ligands have all been implicated in skeletal development in the sea urchin embryo; however, loss-of-function analysis for most of these gene products results in inhibition of skeletogenesis, suggesting that they regulate biomineralization and not PMC positioning and patterning. Notably, no skeletal patterning genes have previously been identified that pattern specific parts of the larval skeleton. This dissertation takes candidate-based and systems-level approaches to identify novel skeletal patterning genes that pattern distinct parts of the larval skeleton. We find that activation of the Alk4/5/7 receptor is required during gastrulation for anterior PMC positioning and skeletal patterning. We next test the function of the TGF-ß ligand Univin and find that it is necessary and sufficient for secondary skeletal patterning, indicating that Univin is a likely signaling ligand in anterior skeletal patterning. We also report a ventral accumulation of sulfated proteoglycans that requires function of the sulfate transporter, SLC26a2/7. This SLC-dependent sulfated proteoglycan accumulation is necessary and sufficient to attract PMCs to the ventral territory, and thereby pattern the ventral transverse skeletal elements. Finally, BMP5-8 function is required for left-side skeletal and serotonergic neuron development. Together, these studies reveal novel ectodermal genes that specifically regulate skeletal patterning across the anterior-posterior, dorsal-ventral, and left-right axes in Lytechinus variegatus embryos. / 2017-01-01T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Loss of the finite cochlear hair cells of the inner ear results in sensorineural deafness. Human cochlear hair cells do not regenerate, and there is no cure for deafness. Our laboratory has established a three-dimensional culture system for deriving functional sensory hair cells from human pluripotent stem cells. A major limitation of this approach is that derived hair cells exhibit a morphological and gene expression phenotype reflective of native vestibular hair cells. Previous studies have shown that establishment of localized domains of gene expression along the dorso-ventral axis of the developing otic vesicle is necessary for proper morphogenesis of both auditory and vestibular inner ear structures. Sonic hedgehog (SHH) signaling has been shown to play a key role in specification of the ventral otic vesicle and subsequent cochlear development. Here, SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro. Single-cell RNAsequencing analysis revealed that while treatment with the SHH pathway agonist Purmorphamine reduced expression of markers for the vestibular-yielding dorsal otic vesicle, upregulation of ventral otic marker genes was modest. More strikingly, the number of otic progenitors exhibiting a neuroprogenitor phenotype increased in response to Purmorphamine treatment. These results suggest that SHH pathway modulation in early-stage inner ear organoids may bias their differentiation toward a neural lineage at the expense of an epithelial lineage. The present study is the first to evaluate the patterning phenotype of human stem cell derived otic progenitors, and sheds light on the transcriptomic profile at this critical point of inner ear development. This study may also cultivate future efforts to derive cochlear cell types as well as inner ear neural cell types from human pluripotent stem cells, and contribute to the establishment of a more complete in vitro model of inner ear development. / 2021-08-21