<p> Caenorhabditis elegans (C. elegans) is a model organism which is known for its transparent body, small body size, high reproductivity and short lifecycle. Several important genes and signal transduction pathways are well conserved in C. elegans. lin//, a LIM homeobox family member, plays a crucial role in the development of the vulva in C. elegans. LIM homeobox genes are a subgroup of Homeobox family that play fundemental role in animal development. In C. elegans lin-If mutant animals fail to form a functional vulva and vulval-uterine connection and consequently exhibit egg-laying defective phenotype. The cell lineage and marker gene expression studies have shown that lin-// is required for the patterning of all primary and secondary lineage vulval cells. lin- II also functions in the nervous system. </p> <p> lin-// expression is mainly observed in the developing vulval cells and in the pi cells which are involved in the formation of vulval-uterine connection. lin-If expression is also seen in VCs and in some of the head and tail neurons. The completed genome sequences of closely related species in Caenorhabditis genus serve as a power tool to do systematic comparative studies. The lin-If regulatory sequences from these species have been compared along with the expression patterns. </p> <p> We looked at the regulation of lin-// in closely related nematode species like C. elegans, C. briggsae, C. remanei and Caenorhabditis n species. </p> <p> Consistent with this. expression of lin-11 is observed in the developing vulval cells. We are interested in understanding evolutionary changes in the regulation and function of lin-II in reproductive system </p> <p> /in-11 is a LIM homeodomain family member which IS involved in several developmental events. lin-11 role is documented in the thermoregulatory circuit specifying AIY interneuron, in chemosensory neurons like A W A and olfactory neurons A WS. During vulval development lin-II expression is dynamically expressed in subset of secondary lineage cells and is broadly expressed in all the cells indicating its role in cell identity and cell fusion of the vulval cells. lin-II is also required for the formation of vulval uterine connection which is the passage to lay eggs in the hermaphrodite. linllloss of function hermaphrodites have change in the axis of the secondary lineage cells during vulval development, uterine Jt cell migration defect, defects in the AIY, A W A and A WS interneurons resulting in egg-laying defect and protruding vulva and neuronal defects and reduced mating efficiency. </p> <p> The expression pattern of lin-If in closely related species is highly similar but not identical. From the sequence comparison of lin-If regulatory sequences a 1 kb conserved block of sequences have been identified which includes the regulatory sequences responsible for the expression of lin-If in vulva and Jt cells. We propose that cisregulatory elements controlling lin-If gene expression are slowly evolving though there is no change in the function which indicates that lin-If plays critical role during the development of the vulva and other tissues. </p> / Thesis / Master of Science (MSc)

Fungi of the Crepidotaceae are characterized by saprotrophic habit, filamentous cuticle, and brown-pigmented basidiospores that lack either a germ pore or plage. The majority of species belong to Crepidotus, distinguished by their pleurotoid basidiomata. Because of their diverse morphology, the presence of several conflicting classifications, and lack of data regarding the biology, phenotypic plasticity, or phylogeny of these fungi, the present study sought first to determine phylogenetic relationships among the different taxonomic groups as a basis for addressing other aspects of Crepidotus biology and evolution. Sequencing analyses show the Crepidotaceae is not monophyletic, and the family concept is revised. Crepidotus and its sister genus Simocybe are found to be monophyletic. At least nine phylogenetic lineages within Crepidotus were uncovered, although relationships between them could not be resolved. However, none of the previously proposed infrageneric classifications are reflective of phylogeny. Morphological, biological, and phylogenetic species concepts were compared within a single phylogenetic unit, termed the Sphaerula group, showing an unusual amount of phenotypic plasticity exists within species, and a taxonomic revision of these species proposed. Also reported are several unique or unusual aspects of Crepidotus biology, including presence of a prolonged latent period prior to basidiospore germination; spontaneous reversion of differentiated hymenial cells to vegetative growth; and the revelation that structures previously termed pleurocystidia are actually the expression of secondary growth from basidia. Results from mating system, culture, and type studies, reassessment of morphological characters traditionally applied to agaric taxonomy, and a revised life cycle for the Crepidoti are presented. / Ph. D.

The characteristic loops of the small intestine arise during embryonic development from differential growth as the intestinal tube elongates against the constraint of its attached dorsal mesentery, which compresses the tube until it buckles into loops. The number and shape of loops are conserved for a given species and are predictable from tube and mesentery geometries and stiffnesses. Importantly, the mesentery readily accommodates a certain amount of stretch from the elongating tube before stiffening by several orders of magnitude and resisting further extension—thereby dictating how differential growth translates into buckling forces. While such constitutive nonlinearity is well appreciated in adult soft tissues, its determinants and consequences remain largely unexplored in buckling morphogenesis and embryogenesis as a whole. In this work, we undertake to establish a mechanistic link between molecular control of cell behaviors and organ-scale buckling morphogenesis of the small intestine. Using pharmacological treatments, mechanical testing, image analysis of tissue microstructure, and computational modeling, we test the hypothesis that actomyosin contractility regulates mesentery constitutive nonlinearity and thereby organ-scale buckling of the small intestine through its effects on extracellular matrix recruitment. Our findings suggest that highly contractile cells could act as a mechanical ‘clutch’, modulating the stiffening transition of the mesentery by compacting stiff matrix fibers that must be decompressed by applied forces before contributing to stretch resistance. However, we also find that low levels of contractility control the initial soft response of the mesentery through a mechanism largely independent of matrix fiber straightness and alignment. Despite the apparent simplicity of buckling from a mechanical standpoint, its underlying biological determinants are evidently quite complex. The present study begins to unpack those intricate links between the molecular and biophysical aspects of buckling morphogenesis by revealing how cell forces and matrix organization interactively dictate tissue-scale mechanics during development in sometimes counterintuitive ways.

Embryo development is a highly coordinated process where genetic regulation and mechanical forces interplay to drive the transformation of a single cell into a complex, multicellular organism. It involves many fundamental processes such as cell division, cell differentiation, and morphogenesis. Morphogenesis, the shape changes of tissue, results from collective cell movement, growth, proliferation, and shape changes, guided by genetic and mechanical cues. Despite the comprehensive data obtained from experimental measurements and advanced imaging, the physical mechanisms underlying morphogenesis are poorly understood, a quantitative cell shape pattern that describes morphogenesis has yet to be discovered, and the coupling between cytoskeleton that generates stress and shape changes has not been quantitatively demonstrated. To address these unsolved questions, we utilized a powerful combination of first-principles modeling and empirical, data-driven approaches. Chapter 1 presents our mathematical model of Drosophila ventral furrow formation, which incorporates actomyosin contractile stress and viscous tissue responses. With all model parameters fitted from experiment, our model quantitatively explained numerous experimental observations in wild-type and genetically perturbed embryos, which were not fully explained by other models assuming elastic tissue responses. Our model revealed that the tissue-scale contraction in ventral furrow formation is driven by the curvature of the multicellular myosin profile. We also demonstrated that the pulsatile time-dependence of myosin acts as a protective mechanism for tissue contraction, suppressing cell-to-cell myosin fluctuations through a low-pass filter effect. This is crucial because tissue contraction is highly sensitive to even small myosin fluctuations, which would otherwise lead to significant inhomogeneous contractions. Chapter 2 details our data-driven approach to studying Drosophila ventral furrow formation, utilizing time-lapse 3D data from light sheet microscopy. We developed computational algorithms to systematically parameterize over 28,000 cell shapes, designed interpretable cell shape features, and employed unsupervised learning to classify cell shape evolution trajectories. By mapping these classes onto the embryo, we extracted the first quantitative cell shape pattern in the Drosophila embryo. This pattern unveiled key physical mechanisms underlying embryo development, including how mechanical stresses propagate, how cell packing is influenced by embryo curvature, and the stochastic nature of apical constriction during tissue contraction. Chapter 3 explores the coupling between actomyosin density and shape changes. We developed a mathematical model of the actomyosin cortex, using partial differential equations to describe the evolution of actomyosin density on a deformable surface, which is represented through differential geometry. Our model revealed that although under physiological conditions, the cell cortex is observed to maintain a homogeneous density and shape, this stability is challenged by two factors: increased cortical tension, which is mechanical in nature, and an elongated aspect ratio, which is a geometric feature. Higher cortical tension disrupts this homogeneity, leading to patterned actomyosin density and multiply furrowed shape. In contrast, an elongated aspect ratio drives constriction through a mechanism we named active Rayleigh instability, a modified form of the Plateau-Rayleigh instability. Furthermore, friction plays a crucial role in protecting the homogeneous state by preserving a large region of homogeneity in the state diagram of the cortex. When friction is reduced, this homogeneous region shrinks significantly, making the cortex more vulnerable to destabilization caused by increased tension and an elongated aspect ratio.

No description available.

No / Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (TfamEKO), which induces loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that TfamEKO mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. TfamEKO mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, TfamEKO mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1, and β-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial–mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in TfamEKO mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial–mesenchymal interactions that drive overall development of the skin and its appendages.

Mechanical forces are largely responsible for shaping sheets of epithelial cells into tissues and organs. During epithelial morphogenesis, cells dynamically tune their mechanical forces to locally promote or resist shape changes. Our current understanding of how these mechanical forces coordinate and drive tissue shape changes is lacking. In particular, it is not well understood why some epithelial cell sheets remodel and flow like fluids, while others stretch and bend like elastic solids, or at what length scale mechanical behaviors of individual cells give rise to tissue-level behaviors. In this work, I develop a framework to analyze tissue structure and dynamics through quantification of cell packings and protein localization patterns. I then combine this framework with optogenetic perturbations and nanoengineering techniques to measure and manipulate the mechanical properties and forces of cultured cells with high spatiotemporal precision. In chapter 2, I address these challenges by studying how cells respond to different mechanical cues in the fruit fly, Drosophila melanogaster, during early development. Specifically, I quantify global system disorder arising from internal and external stresses by using order parameters that describe the area and stretch distortion between the center of cells. Using these order parameters, we find that structural disorder is synchronized with internal and external stresses imposed on the germband. Furthermore, we dissect the contribution of each stress on the embryo using different mutants that inhibit one or more of these stresses. In chapter 3, I refine this analysis to identify the cellular and supracellular myosin networks that tune cellular mechanics and forces, connect them to local cell packings, and determine their overall impact on tissue movement. Using cell geometries to infer mechanical properties, I analyze the cell packings during the active processes of fluid-like germband extension (GBE) and the solid-like bending during ventral furrow formation (VFF). We find that the local cell packings match the global cell packings in the ventral furrow, while the local cell packings match the regional behavior in the germband when averaging over N = 3 nearest neighbors. In chapter 4, I investigate how the mechanical forces of epithelial tissue sheets can be altered using an in vitro culture system. Using optogenetic tools, I manipulate myosin activity in cultured cells and quantify the induced forces by measuring the deflection of an array of micropillars on which the tissue is grown. I show that the traction forces resulting from activation of the optogenetic tool are increased up to 3-fold higher than baseline during activation and decrease to near-baseline levels 30 minutes after activation is stopped. These studies improve our understanding of the mechanisms and mechanics underlying cellular self-organization and clarify how perturbations of these mechanisms can lead to disease states like congenital anomalies and cancer.

The lung is an organ that contains a vast system of airways carefully constructed to achieve maximal surface area in a confined space, requiring guidance from a multitude of developmental factors. The Shh pathway is one such signaling mechanism that is critical to proper lung formation, guiding branching morphogenesis and cellular proliferation through its downstream Gli transcription factors. Additionally, Foxf1 has been shown to be a key developmental factor required for proper lung formation during embryogenesis. Although theorized that the Gli transcription factors are responsible for regulating foxf1 levels, their exact relationship has yet to be revealed. Using five different models for Shh signaling (gli2 null, gli2 over-expressor [hVER-Gli2], gli3 null, Gli3 constitutive repressor [Gli3Δ699] and cyclopamine treated lung explants), I compared and contrasted the role of Gli2 and Gli3 in terms of their effect on cell cycle regulation, and on the expression levels of foxf1 and its potential downstream target genes tbx4, tbx5 and fgf10. I found that ectopic over-expression of gli2 resulted in increased Shh pathway activation, and increased expression of G1/S phase cyclins, which was associated with increased cellular proliferation and lung growth. However, no change in the levels of G1/S phase cyclins due to altered Gli3 signaling was observed. Foxf1 levels positively correlate with the levels of gli2, and appear to be independent of Gli3 activity. The amount of tbx4, tbx5, and fgf10 transcripts were observed to follow the levels of gli2 in the different gli2 mouse models, however, there was no significant change in gli3 null or Gli3Δ699 mice. Finally, by analyzing gene expression at different time points during gestation, I found that while gli2 levels affect foxf1 throughout gestation, the relationship to tbx4, tbx5 and fgf10, occurs only during the latter stages of lung development. I conclude, that Gli2 and not Gli3 appears to be the primary transducer of Shh signaling influencing cyclin regulation, leading to changes in embryonic lung growth. Furthermore, that Gli2 and not Gli3 appears to regulate foxf1 expression levels, and that this may extend downstream to influence tbx4, tbx5 and fgf10 expression.

The lung is an organ that contains a vast system of airways carefully constructed to achieve maximal surface area in a confined space, requiring guidance from a multitude of developmental factors. The Shh pathway is one such signaling mechanism that is critical to proper lung formation, guiding branching morphogenesis and cellular proliferation through its downstream Gli transcription factors. Additionally, Foxf1 has been shown to be a key developmental factor required for proper lung formation during embryogenesis. Although theorized that the Gli transcription factors are responsible for regulating foxf1 levels, their exact relationship has yet to be revealed. Using five different models for Shh signaling (gli2 null, gli2 over-expressor [hVER-Gli2], gli3 null, Gli3 constitutive repressor [Gli3Δ699] and cyclopamine treated lung explants), I compared and contrasted the role of Gli2 and Gli3 in terms of their effect on cell cycle regulation, and on the expression levels of foxf1 and its potential downstream target genes tbx4, tbx5 and fgf10. I found that ectopic over-expression of gli2 resulted in increased Shh pathway activation, and increased expression of G1/S phase cyclins, which was associated with increased cellular proliferation and lung growth. However, no change in the levels of G1/S phase cyclins due to altered Gli3 signaling was observed. Foxf1 levels positively correlate with the levels of gli2, and appear to be independent of Gli3 activity. The amount of tbx4, tbx5, and fgf10 transcripts were observed to follow the levels of gli2 in the different gli2 mouse models, however, there was no significant change in gli3 null or Gli3Δ699 mice. Finally, by analyzing gene expression at different time points during gestation, I found that while gli2 levels affect foxf1 throughout gestation, the relationship to tbx4, tbx5 and fgf10, occurs only during the latter stages of lung development. I conclude, that Gli2 and not Gli3 appears to be the primary transducer of Shh signaling influencing cyclin regulation, leading to changes in embryonic lung growth. Furthermore, that Gli2 and not Gli3 appears to regulate foxf1 expression levels, and that this may extend downstream to influence tbx4, tbx5 and fgf10 expression.

Organ formation is an important step during development of an organism that combines different scales from the molecular to the tissue level. Many organogenesis phenomena involve epithelial morphogenesis, where sheets of cells undergo rearrangements to form complex architectures – organ precursors, which subsequently develop into mature organs. Timely development of the characteristic architectures of the organ precursors is crucial for successful organogenesis and is determined by the choice of epithelial rearrangements that organise the constituent cells in space and time. However, for many organogenesis events the cellular dynamics underlying such epithelial rearrangements remain elusive. In the work presented here, I investigated the morphogenesis of the hemispherical retinal neuroepithelium (RNE), that serves as an organ precursor of the neural retina. Formation of RNE is an important event in vertebrates that shapes the optic cup and sets the stage for subsequent eye development. I investigated RNE morphogenesis in the developing zebrafish embryo by visualising and investigating the cellular dynamics of the process in vivo. My findings show that the zebrafish RNE is shaped by the combined action of two different epithelial rearrangements – basal shrinkage of the neuroepithelial cells and involution of cells at the rim of the developing optic cup. The basal shrinkage of the neuroepithelial cells bends the neuroepithelial sheet and starts the process of invagination. However, my results show that the major player in RNE morphogenesis is rim involution. Rim involution translocates prospective RNE cells to their designated location in the invaginating layer and contributes to RNE invagination. My work unravelled the so far unknown mechanism of rim involution. I show that the rim cells involute by collective epithelial migration using directed membrane protrusions and dynamic cell-matrix contacts. If rim migration is perturbed, the prospective RNE cells cannot reach the invaginating layer. As a result, these migration-defective cells attain the RNE fate at an ectopic location and disrupt the tissue architecture. Therefore, rim migration coordinates the cellular location with the timing of RNE fate determination and orchestrates RNE morphogenesis in space and time. Overall, my work highlights how morphogenetic processes shape the organ precursor architecture and ensure timely organ formation. These findings provide important insights not only for eye development but also for epithelial morphogenesis and organogenesis in many other systems. / Für die Entwicklung eines Organismus ist die Bildung von Organen (Organogenese) von zentraler Bedeutung. Organogenese umfasst Prozesse auf allen Ebenen der Längenskala: von der molekularen Ebene, der Gewebeebene, bis hin zur Ebene des ganzen Organismus. Viele Phänomene der Organogenese beinhalten dabei Veränderungen von Epithelien, bei der sich Schichten von Zellen zu komplexen Strukturen - Organvorläufern - umwandeln. Diese entwickeln sich später zu vollständigen Organen. Die rechtzeitige Entwicklung der charakteristischen Architektur der Organvorläufer ist entscheidend für eine erfolgreiche Organogenese und wird durch die Wahl der epithelialen Umwandlungsprozessen bestimmt, welche die Zellen in Raum und Zeit koordinieren müssen. Für viele dieser Prozesse ist jedoch genau diese zugrundeliegende Zelldynamik unklar. In der hier vorgestellten Arbeit untersuchte ich die Bildung des hemisphärischen retinalen Neuropepithels (RNE). Das RNE ist der Organvorläufer der neuralen Retina, weshalb dessen korrekte Bildung die Voraussetzung für die korrekte Entwicklung der Augen ist. Ich untersuchte die RNE-Morphogenese in sich entwickelnden Zebrafisch-Embryos durch Visualisierung und Untersuchung der zellulären Dynamik der beteiligten Prozesse in vivo. Meine Ergebnisse zeigen, dass das RNE in Zebrafischen durch die kombinierte Umwandlung von zwei verschiedenen Epithelien geformt wird. Zum einen findet eine Verkleinerung des basalen Prozesses der neuroepithelialen Zellen statt, zum anderen die Involution von Randzellen. Die basale Verkleinerung der neuroepithelialen Zellen verbiegt die neuroepitheliale Schicht und führt zur Einstülpung des RNE. Meine Ergebnisse zeigten allerdings, dass Involution von Randzellen noch bedeutsamer für die RNE-Morphogenese ist. Die involution von Randzellen transportiert potenzielle RNE-Zellen in das Neuroepithel und trägt zur RNE-Einstülpung bei. Die Bedeutung meiner Arbeit liegt darin, den bisher unbekannten Mechanismus der Randzell-Involution entdeckt zu haben. Ich zeigte, dass die Randzellen sich aktiv durch kollektive epitheliale Migration bewegen indem sie gerichtete Membranforsätze und dynamische Zell zu Matrix Kontakte etablieren. Wird die Migration der Randzellen inhibiert, so führt dies dazu, dass diese Zellen die eingestülpte RNE Schicht nicht erreichen. Sie landen dann an den falschen Positionen, wo sie die Gewerbearchitektur stören können. Daher koordiniert die Randzellmigration die Position der Zellen und orchestriert die RNE-Morphogenese in Raum und Zeit. Insgesamt zeigt meine Arbeit, wie morphogenetische Prozesse die Organvorläuferarchitektur prägen und eine rechtzeitige Organbildung sicherstellen. Diese Erkenntnisse sind sowohl für das Verständnis der Augenentwicklung, als auch für das der epithelialen Morphogenese und Organogenese in anderen Systemen von großer Bedeutung.