Organogenesis relies on complex and dynamic cellular processes that are mediated by genetic programs, governed by extrinsic and intrinsic signals, and are extensively coordinated through intercellular communication. During organ development, patterned gene expressions and biochemical signals regulate growth, proliferation, and mechanics to build complex tissues through tissue-level processing. Specific unit operations of tissue shaping include epithelial spreading, folding, and sculpting. These processes are essential for shaping two-dimensional epithelial sheets into complex three-dimensional structures. In addition, these morphogenetic modules are regulated throughout the development of an organism, from gastrulation to organogenesis. Dysregulation of these morphogenetic processes leads to various diseases, including congenital disabilities and cancer. However, the precise mechanisms through which the gene expression patterns regulate the coordinated cellular processes during organogenesis are poorly understood. A quantitative, mechanistic understanding of the biophysical mechanisms regulating the cellular processes is essential to understand the design principles regulating morphogenesis. These design principles would help develop effective therapeutics to repair defects during embryonic development and wound healing. Additionally, a systems-level understanding of combining multiple morphogenesis modules can guide advanced methods for regenerating tissues from basic building blocks.
In this dissertation, Drosophila melanogaster is used as a model system to investigate how critical biochemical signals coordinate collective cellular processes to drive epithelial morphogenesis. Epithelial tissues transduce key biochemical signals into specific cellular responses through second messenger dynamics such as calcium. Hence, it is crucial to understand how epithelial cells interpret diverse signals and transduce them into second messengers, such as calcium, to coordinate tissue-level processes. Calcium is one of the crucial second messengers regulating various physiological processes and is essential for regulating a diverse range of cellular functions such as growth, mechanics, apoptosis, and proliferation. Additionally, calcium signaling is also associated with a physiological signaling system, and its role in regulating neuronal and other physiological activity is well established. However, the mechanisms through which global patterning information integrates second messenger dynamics to regulate the key morphogenetic operations still need to be fully elucidated. Moreover, decoding the calcium signaling and its crosstalk with other signaling cascades during epithelial morphogenesis is highly relevant to wound healing and cancer. This is due to the high similarity of cell signaling pathways across the animal kingdom and the cross-hierarchical nature of biological systems. In fact, it is often possible to establish and leverage phenotypic analogies between genetic perturbations in a (slightly) simpler animal like the fruit fly and human diseases to identify promising drug targets and drug candidate systems.
This dissertation discusses studies investigating the mechanisms that integrate calcium signaling in regulating epithelial morphogenesis during development. A key motivation for this work is the long-term goal of inferring cell/tissue/organ states from dynamic measurements of a few ‘hubs’ within cell signaling networks that can capture a large percentage of the whole information processing network within cell systems. Such an integrated knowledge base contributes to defining over-arching design principles of organ development and homeostasis.
Chapter 1 of this thesis briefly introduces epithelial spreading, folding, the Drosophila model system, and key biochemical signals. In addition, it discusses Drosophila head involution, a poorly studied morphogenetic process that resembles key aspects of wound healing and is a focus of several chapters in this dissertation. Chapter 2 of this thesis extensively reviews the existing computational models, including mechanical and chemical models, to reverse engineer morphogenesis. This chapter discusses the types of mechanical and chemical signal modeling techniques that are currently in use. Additionally, it discusses coupled mechano-chemical models to elucidate the feedback mechanisms between morphogen signaling and cell mechanics. This chapter further discusses the future perspectives of these modeling techniques and the need for modeling to decouple the interplay between biochemical and mechanical signals. Chapter 3 of this thesis studies the significance of morphogen signaling, such as Hedgehog (Hh), during the late stages of Drosophila embryogenesis. This study generates quantitative maps of how Hh signaling is patterned during the late stages of embryogenesis. In addition, this study combines live imaging and advanced microscopic techniques to show how epithelial fold formation occurs at the segment boundaries. Additionally, this study shows that patterning of morphogen signaling is necessary for proper folding, and disruption to this pattern inhibits fold formation. Furthermore, this study proposes that folding occurs due to two coordinated and synchronous morphogenetic operations: pinching of segment groove cells and pushing of cells adjacent to the groove cells. This study validates this hypothesis by incorporating these processes into the computational biophysical model and simulating tissue folding. Of note, I have significantly contributed to the establishment and initial testing of a new light sheet microscope at the university during this study. This instrument can image thick tissues three-dimensionally, which is impossible with conventional confocal microscopes. Chapter 4 of this thesis investigates the role of calcium signaling during fold formation. This study shows that the calcium transients observed during the late stages of embryogenesis occur during the fold formation of epithelia at the segment boundaries. Surprisingly, this study discovered that the intercellular transients are absent during the epithelial spreading, which occurs prior to folding. Moreover, this study shows that these calcium transients are essential for proper segment positioning and shape formation, as inhibition of calcium signaling resulted in defects in segment shapes and widths. In addition, this study proposes that the transients are regulated by patterned Hh signaling, as the defects associated with Hh pattern disruption are similar to those associated with calcium signaling inhibition. In the next part of this study, I used wing discs as a model system to investigate the growth regulation mechanisms integrated with calcium signaling. Chapter 5 of this thesis presents a systematic screen of the family of G-Protein Coupled Receptors (GPCRs) during wing morphogenesis. GPCRs are key receptors that bind to diverse ligands and transduce external signals into the cytoplasm through second messenger dynamics such as Ca2+. In addition, GPCRs are targets for many drugs treating different pathological conditions. I, along with other graduate students, performed a genetic screening to identify potential GPCRs that play a role in wing morphogenesis. We identified several GPCRs, which, when inhibited, resulted in wing size and expansion defects. In Chapter 6 of this dissertation, I investigated the role of G-protein Galphaq (Gαq) during wing growth. This study demonstrates that the wing size is reduced when Gαq is inhibited or overexpressed. Moreover, it shows that overexpression of Gαq is sufficient to induce global calcium transients in the wing disc. Surprisingly, overexpression of Gαq reduces wing growth regardless of its stimulating global calcium waves. Furthermore, this study shows that the Gαq-mediated calcium signaling promotes growth, whereas growth inhibition occurs through its interaction with other downstream signaling cascades. Importantly, this study performs RNA sequencing to identify the downstream targets that function through Gαq to regulate growth. Based on RNA seq analysis, this study identifies that Gαq interacts with nuclear hormone signaling to regulate growth and developmental time. Further, it shows that Gαq disruption delays development through ecdysone signaling inhibition. In sum, this study demonstrates the significance of the Gαq-mediated calcium activity in regulating growth and development. Chapter 7 of this dissertation proposes future efforts to elucidate and leverage the underlying conserved molecular and mechanical mechanisms that govern epithelial morphogenesis.
Overall, these studies have shown the significance of calcium signaling in integrating morphogen and GPCR signaling to regulate growth and morphogenesis. Dysregulation of Hh signaling results in severe congenital disabilities in vertebrate systems, as Hh is a crucial regulator of tissue patterning during embryonic development. The results from the Hh and calcium studies would reveal novel insights into the crosstalk mechanisms between Hh and calcium signaling during embryonic development in other systems. They would aid the studies on the therapeutic development of Hh-mediated congenital disabilities. Further, these studies will serve as a basis for future studies investigating the role of Hedgehog and other signaling pathways in mediating the late stages of Drosophila embryogenesis. The Gαq study would provide novel perspectives on the mitogenic activity of G-proteins during organ development. Activating Gαq mutations are associated with uveal melanoma and cellular transformations. The results from our study would aid the studies investigating the downstream effectors of G-proteins in promoting growth during cancer-related pathologies. Additionally, this study would provide a basis for future studies investigating the crosstalk between Gαq and signaling cascades such as JAK/STAT and Toll signaling during organ development. A more quantitative understanding of the physical mechanisms behind epithelial morphogenesis advances and facilitates accelerated tissue repair capabilities after wounding, tissue rebuilding, and restoring tissue homeostasis.