Browsing by Author "Di Talia, Stefano"

Regeneration occurs throughout the animal kingdom and is a well-studied

feature of many model organisms, yet the field lacks a fundamental understanding of

the real-time dynamics of cell behavior during regeneration. I discuss how existing

knowledge of regeneration may be used to inform efforts to translate these remarkable

feats of animals to human regeneration and present research that uses live imaging to

improve understanding of cell origins and diversification during regeneration in the

scale, focusing specifically on osteoblasts the matrix-depositing cells that divide and heal

bone injuries. I developed an imaging platform to monitor and quantify individual and

collective behaviors of osteoblasts in adult zebrafish scales. I show that a founder pool

of osteoblasts emerges through de novo differentiation within one day of scale plucking,

then diversifies across the primordium by two days after injury, with region-specific

changes in proliferation, cell shape, and cell death rates coincident with acquisition of

mature scale morphology. I also demonstrate a role for Fgf signaling in scale

regeneration and present tools for high resolution imaging studies of basal epidermal

cells during skin and scale injury. These findings demonstrate the value of live imaging

in revealing novel biology and gaining a more complete picture of the many complex

processes that must be elegantly choreographed to achieve tissue regeneration.

Coordination of biochemical signals across long distances is a ubiquitous feature of biological systems; however, the molecular and mechanical mechanisms which allow such signaling are still largely unknown. I discuss general classes of long-range signaling mechanisms as well as the regulatory pathways involved in creating and maintaining cellular coordination in different size scales. In particular, I present a thorough mathematical analysis of a reaction-diffusion model of Erk activity waves which control osteoblast regeneration in the zebrafish scale and use live and fixed imaging of Drosophila melanogaster embryos to elucidate the mechanisms of synchronized cell cycle control. In the regenerating zebrafish scale, I show that a simple three-component model consisting of Erk, an Erk activator, and an Erk inhibitor is sufficient to generate Erk activity waves which propagate across the millimeter sized region. The properties of these waves agree with chemical wave theory and are structured to enable proper timing of hypertrophy to enable precise regulation of size and shape of the regenerated tissue. In the early Drosophila embryo, I show that the ubiquitin ligase Cullin-5 acts to regulate the actin cytoskeleton. Using novel mutants, I show that mutations in the cullin-5 gene leads to a disruption in signaling across the embryo and an eventual mistiming of the mid-blastula transition. Furthermore, I show that the cell cycle is not controlled globally across the embryo or very locally but rather is coordinated across distances of ~100µm. This work highlights the different mechanisms and regulation which exists in different contexts to transmit signals across a domain and control the proper development and regeneration of large tissues.

Self-organization refers to the emergence of an overall order in time and space that results from collective interactions of its individual components. Cells and tissues in biological systems achieve dynamic organizations through self-organization processes. In this dissertation, I show two different organization principles observed from the developing embryos of the fruit fly Drosophila melanogaster: the Histone Locus Body size control and cortical migration of nuclei. The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. We show that Drosophila HLBs form through phase separation. During embryogenesis, the size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic histone gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which facilitates phase separation, and the nuclear concentration of the scaffold protein multi-sex combs (Mxc), which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Thus, our experiments identify a mechanism linking nuclear body growth and size with gene expression. The early syncytial Drosophila melanogaster embryo goes through 13 rapid cleavage divisions. Followed by axial expansion during nuclear cycles 4-6, the nuclei collectively migrate to the cortex through cycles 7-9, known as cortical migration. In this study, using light sheet microscopy and quantitative analysis, we track individual nuclei in 3D over the migrating stage and demonstrate that cortical migration is essentially a nuclear packing problem resulting from uniformly expanding nuclear shell or the energids. The division orientation distribution can accurately predict how many nuclei will make it to the next shell, suggesting that the division orientation controls the nuclear fate and overall energids organization. Changes in the cell cycle affects cortical migration phenotype by changing the shell expansion duration rather than speed, which leads to changes in the internuclear distance. The nuclei self-organize in a shell in which inter-nuclear distance potentially matches aster size. Our experimental data and mathematical modeling suggest an additional important role of aster-aster interactions, which is to orient the centrosomes in a way that minimizes crowding. Overall, our data highlights that cortical migration is a self-organized process driven by geometric packing of dividing nuclei.

Early development in most metazoans is characterized by remarkably fast and coordinated cell cycles. Nonetheless, it is unclear what organizational principles underlie cell cycle synchronization across a large developing embryo. We found that cell cycle synchronization in Drosophila arises through the self-organized positioning of nuclei, which is regulated by the spatiotemporal dynamics of the cell cycle, cortical contractions, and cytoplasmic streaming. First, local Cdk1 downregulation at mitotic exit initiates the damped spreading of PP1 activity, which is responsible for recruiting myosin II to cortical regions that surround the nuclei, where gradients of contractility are generated. These gradients drive cortical and cytoplasmic flows that properly position the nuclei across the embryo. Uniform positioning of nuclei across the embryo is required for the emergence of synchronous cell cycles. Once at the surface of the embryo, nuclei undergo four metachronous cell cycles, which spread in a wave-like manner with remarkable speed across the large distance of the egg. Using a Cdk1 biosensor, we found that travelling waves of Cdk1 activity propagate through the embryo and synchronize the cell cycle during S-phase through an active mechanism, while mitotic events simply follow S-phase synchronization with a delay. Taken together, a self-organized mechanism that spreads nuclei uniformly is required early on in development to give rise to synchronous divisions. Cell cycle synchrony is then maintained by waves of Cdk1 activity, ensuring that all nuclei initiate the mid-blastula transition simultaneously. This work highlights the importance of chemical waves and cytoplasmic flows in the spatiotemporal regulation of the cell cycle of large embryos.

Understanding the molecular mechanisms controlling tissue growth and patterning remains a fundamental open question in developmental biology. To investigate the dynamic biochemical signals regulating tissue growth, we have developed a system for live imaging zebrafish scales during development. Zebrafish scales are bony skin appendages which are positioned on the exterior of the fish and have a flat, circular shape that makes them conducive for live imaging. Scales develop sequentially in a spreading wave along the anterior-posterior and dorsal-ventral axes until the entire fish is covered in a hexagonal array. While some of the molecular pathways regulating this process have been identified, exactly how this wave of scale development is regulated remains unsolved. We have found that scale development requires communication between cells and thus likely proceeds through an active wave mechanism of positive feedback loops. Introducing a cut in the skin to interrupt cell-cell communication caused a delay in scale development until the cut healed. This indicates that new scales only form in response to a signal from neighboring cells. These cuts also block NF-κB activity, which acts downstream of Ectodysplasin A signaling to activate pathways required for scale development, including Fgf20a. A wave of NF-κB activity precedes scale initiation and is required for proper scale development. Experiments decoupling the propagation of the wave from dermal placode formation and osteoblast differentiation demonstrate that the Eda/NF-kB activity wavefront times the sequential patterning of scales. Moreover, this decoupling resulted in defects in scale size and significant deviations in the hexagonal patterning of scales. Thus, our results demonstrate that a biochemical traveling wave coordinates scale initiation and proper hexagonal patterning across the fish body.

Cell proliferation is one of the elementary operations involved in building and maintaining the bodies of organisms, and animal development employs diverse regulatory strategies to ensure that it happens in the correct spatial and temporal arrangements. This dissertation is a study of some of the mechanisms involved in timing the early cell cycles of the embryo of Drosophila melanogaster. In chapter 1, we introduce many of the important concepts and provide the reader with background on developmental regulation of the cell cycle. In chapter 2, we turn our focus to the problems associated with the cell-cycle transitions that accompany the maternal-to-zygotic transition. Specifically, it had been shown that slowing of the cell cycle following the initial rapid cleavage divisions is linked to the downregulation of protein phosphatase Cdc25/Twine activity. We pursue this problem with a structure-function analysis of Cdc25/Twine. In chapter 3, we turn our attention to the fourteenth round of cell divisions, which form exquisite spatio-temporal patterns called mitotic domains. Six heterochronic genes (btd, ems, kni, slp1, h, and hkb) had been identified that have dosage-sensitive effects on the timing of cell division in mitotic domain 2 (MD2). We tag two of these factors with GFPs using BAC trangenesis and measure their dynamics in MD2 and other head domains. We find that btd is expressed in a gradient that anticipates the mitotic schedule of MD2, and that slp1 is a powerful repressor of mitosis in the head domains. We conclude that these two factors contribute to the timing of MD2 via a mixed hourglass model that involves both activator-accumulation and repressor-depletion.