Recent advances in live imaging technologies as well as non-equilibrium models are revealing new insights into the dynamic process of transcriptional regulation. The works described here all utilize MS2 imaging of transcription in live, developing embryo of fruit flies and zebrafish to investigate transcriptional dynamics in these model organisms. We utilize the resulting high resolution data to develop and test physics-based models of transcription.

We investigate two different regulatory enhancers in fruit fly embryos in three different projects. Through the first of these projects, we illuminate new information about the timing of transcriptional onset and the role of the pioneer-like factor Zelda. We connect newly discovered Zelda-dependent “hubs” of an activating transcription factor to transcriptionaloutput for the first time. Next, we undertake a systematic dissection of transcriptional regulation in eukaryotes. Through this project, we challenge the standard equilibrium model of transcriptional regulation and demonstrate the need for non-equilibrium models. We propose a model of transcription that incorporates transient transcription factor dynamics to drive DNA accessibility. The third project extends the study of transcriptional dynamics beyond initiation. We devise a novel experimental and theoretical foundation for the study of the interconnected regulation of the entire transcription cycle. In the last project we describe here, we move beyond the Drosophila field and expand our MS2 technology to zebrafish and established a transgenic MS2 reporter line of fish for the first time.

The imaging technology and modeling used in these projects have already proven to be incredibly useful for studying the dynamic process of transcription, revealing new mechanisms of regulation as well as enabling the development and testing of new theoretical models of transcription. This is just the beginning and, in the coming years, further improvements in imaging technologies in combination with continued efforts on the theoretical modeling frontwill surely continue to bring about new discoveries in the field of transcriptional regulation.

The main objective of this thesis is to advance a quantitative understanding of the developmental genetic network. Three projects are presented toward this goal. The first project involves the quantitative dissection of transcription factor dynamics and gene regulation focusing on optogenetics to examine and manipulate transcription factors within a multicellular organism. This project provides a precise understanding of how transcription factors regulate target gene expression.

The second project explores the concept of bistability in autoactivation network motifs, focusing on how feedback loops contribute to binary cell fate decisions. Using a combination of mathematical modeling and experimental validation, this work elaborates on the dynamics governing cellular behavior and its implications for better understanding medical therapies related to cell development.

The final project investigates the flexible design principles governing the binding-site organization within enhancers. This entails unraveling the enhancer sequence's essential role in gene regulation, an insight that could inform the design of synthetic regulatory sequences for biotechnological applications and the development of novel therapeutic strategies.

In conclusion, this thesis provides insights into the developmental genetic network, contributing toward predicting complex biological processes based on specific genetic network parameters. This work may ultimately improve our understanding of organismal development and inform therapeutic strategies for developmental disorders.

Predicting how interactions between transcription factors and regulatory DNA sequence dictate rates of transcription and, ultimately, drive developmental outcomes remains an open challenge in quantitative biology. Indeed, despite decades of biochemical and genetic studies that have established a reasonably complete "parts list" of the molecular components driving eukaryotic transcription, the field nonetheless lacks a satisfactory understanding of how interactions between these molecular components unfold across space and time to give rise to gene regulatory logic. Recently, technical advancements have begun to provide glimpses into this molecular black box, dramatically improving our ability to trace how molecular pieces move, interact, and assemble. However, if we are to fully realize the immense potential of these exciting new technologies, then our ideas need to catch up with our experiments.

In this thesis, we argue that quantitative models have a central role to play in synthesizing the ever-increasing array of cutting-edge experimental measurements into a coherent theory for the molecular basis of transcriptional control. To this end, we seek to develop conceptual, theoretical and computational frameworks for dissecting how molecular reactions at individual gene loci give rise to the formation of dynamic patterns of gene expression and facilitate cellular decision-making. Chapters 2 and 3 describe previously published works that combine live imaging, statistical inference, and simple quantitative models to probe how transcription factor proteins regulate the dynamics of transcriptional bursting at target gene loci to give rise to stripes of gene expression early on in fruit fly development. Chapter 4 describes a series of analyses following-up on various results from these works. We also use this chapter to break new ground, however, examining how 2 spot experiments, which track the output of two identical gene loci in each cell, can be used to estimate rates of information transmission at individual gene loci from live imaging data.

In Chapters 5 and 6, we connect phenomenological models of transcriptional bursting employed in the preceding sections to truly molecular models that seek to understand how key transcriptional behaviors emerge from molecular interactions at the gene locus. Chapter 5 examines a puzzling gap revealed by recent live imaging studies between the rapid timescale (seconds) of transcription factor binding and the slow timescale (minutes to hours) of transcriptional bursts, and proposes two simple theoretical frameworks for bridging this gap. Chapter 6 investigates how the presence of energy-dissipating processes within the eukaryotic transcriptional cycle can open the door to new kinds of gene regulatory logic that increase the rate at which gene loci transmit information. Finally, Chapter 7 describes ongoing work to develop a novel Bayesian framework, burstMCMC, that uses efficient inference techniques to examine how transcription factor proteins regulate transcriptional burst dynamics.

Enhancers drive the transcription of genes in defined spatio-temporal patterns of gene expression that orchestrate the development of animal body plans. The spatial aspects of enhancer function and gene expression pattern formation have been elucidated with elegant experiments in ​Drosophila melanogaster and other organisms. However, our knowledge regarding the temporal dynamics of pattern formation is still incomplete.

In this work, I coupled the MS2 reporter system, which enables real-time, single-locus analysis of transcription, with the regulatory sequences of the gene ​even-skipped (eve)​, a fine model of enhancer function active in seven antero-posterior stripes that ring the fruit fly embryo before gastrulation.

Our results show highly dynamic expression patterns that emerge from stochastic bursts of transcription in single loci. We found that the dynamic expression of ​eve stripes stems from shared transcriptional kinetics. Nuclei at the center of all stripes increase their frequency of transcriptional bursting. Nuclei in the inter-stripe boundaries remain quiescent and do not show any detectable bursting. All the while stripes shift anteriorly, propelled by expression flow. The bursting kinetics in the ectopic patterns of eve expression that result from lifting the repression of ​eve enhancers are similar to the kinetics in the endogenous stripes. These observations show that the various ​eve enhancers generate similar bursting kinetics regardless of the myriad of distinct inputs that shape their activity.

This dissertation consists of four chapters. Chapter 1 describes the construction of our MS2-based ​eve reporter and the generation of the transgenic fruit flies carrying it. Chapter 2 goes through our imaging methods and their optimization. It ends with an account of our data analysis to turn movies of transcription in embryonic development into meaningful and quantifiable data. Chapter 3 summarizes our findings regarding the conserved kinetics of ​even-skipped transcriptional bursting. Finally, Chapter 4 explores the activity of ​eve​ enhancers in body regions where they are not normally active.

Due to their apparent passive behavior, plants have long been considered something in between inanimate objects and ”proper” living things, that is, animals. When an animal is incapable of moving or communicating we refer to it as being in a vegetative state. Research over the last three decades has shown that, when it comes to molecules, plants cells are bustling with activity. It was our task to just go and take a look.These dynamic molecular events operating at the cellular level include a myriad of signaling pathways that can sense environmental inputs such as light intensity and quality, temperature, osmotic pressure, the axis of gravity, mineral nutrients and several hormones to name but a few. The output of these signaling pathways often involves changes in the mRNA abundance of responsive genes. Plant growth and development depends on quantitative aspects of this regulation -when, where and how much of each mRNA species each cell makes in response to a specific stimulus. In turn, human societies ultimately depend on plants, which makes these problems not only fascinating from a basic science point of view, they are also pressing from a very practical perspective. In large part, we owe our current understanding of how this information transfer occurs to studies in other eukaryotic model organisms, in particular the fruit fly Drosophila melanogaster (Drosophila). From the first experimental description of morphogens to the recognition of transcriptional enhancers, foundational discoveries related to gene regulation in higher eukaryotes have been made using the fruit fly embryo as a model system. The fact that this inter-kingdom comparisons are possible and useful highlights one of the most important lessons of the molecular biology revolution, the notion that all life forms share fundamental principles because they are built largely of the same type of molecules. Jacques Monod captured this view when he famously claimed that ’what is true for E. coli is true for the Elephant’. While Monod’s quote nicely justifies the logic behind a specific research program, it also challenges us to question whether paradigms drawn in one model organism bias and limit our understanding when applied uncritically to other biological systems. Monod certainly was not expecting us to believe that the specific details of how bacterial genes are turned on and off in response to sugar sources are conserved all the way from bacteria to animals. However, the physical principles that nature exploits to bring about a bacterium and an elephant are ultimately the same. The genomic era has nothing but confirmed the underlying unity across all life kingdoms by revealing that the way these principles are encoded in DNA derives from one common ancestor. One of the challenges we face as biologists is finding what these shared principles are. In this effort, by distinguishing what is shared from what is different, drawing from different experimental systems can be illuminating. There are more practical reasons why a trans-kingdom perspective can be beneficial. Engaging at a deep level with the technical details of an experimental method developed in one model organism allows one to more easily transfer that knowledge to other contexts. A substantial part of this dissertation builds on this premise. It should be possible to express these biophysical principles as models that can be applied across biological contexts. Since the dawn of molecular biology, transcriptional regulation has had a central role as a test bed to combine theory and experiments to un- cover generalizable biological principles. A good example of this is the Monod-Wyman-Changeux (MWC) model of allostery, which captures such seemingly different behaviors as sugar regulation of gene expression in bacteria, oxygen transport in the blood by hemoglobin and the effect of drugs on cell surface receptors. During the summer of 2016 I had the privilege of attending the Physiology Course at the Marine Biological Laboratory where, under the guidance of Professor Rob Phillips, I used statistical mechanics to derive and test the MWC model in the context of sugar sensing in E. coli. This type of simple and intuitive mechanistic model is of a fundamentally different nature to the statistical regression-based models that are becoming increasingly popular in biology. Because they are conceived as arbitrarily complex black box ’fits’ these models can certainly ’recapitulate the data’, but, for that very same reason, cannot be used to test molecular or biophysical hypotheses. We should, however, recognize the relative failure of using theory and experiments to develop predictive models of transcription in eukaryotes. Compared to the resounding successes in phage λ and the E. coli Lac operon, we are far from achieving this type of understanding in plants or in any other higher eukaryote. Recent calls to advance a unified model of transcription in eukaryotes advocate for making an inventory of moving parts and building tools to measure their dynamics in single cells. Many of the results presented here align with the goal of closing technical gaps to make it possible to measure the phenomena under study. It may well be that we have reached a technical wall beyond which the dialog between theory and experiments will come naturally. For example, there is mounting evidence that the formation of biomolecular condensates of RNA Polymerase and transcription factors is a fundamental property of transcriptional regulation. The theoretical frame- work for these processes is an active area of research and it is thus still unclear what experiments could be designed to falsify these models . Theoretical explorations, even if frustrating, can sharpen our thinking of what experimental tools we are still missing. For example, as I argue in the following chapters, there is a still a need for live cell technologies to measure post-translational modifications of histones and other proteins at a specific locus. Beyond this quest for building unifying models based on physical principles, we should not forget that transcriptional regulation lies at the core of a myriad of diverse biological phenomena that we have only scratched the surface of. Simply taking a quantitative, single-cell view of gene regulation can offer unprecedented insights about the way a particular biological process works and make us revisit the way we think about it. As I have argued here, toggling between model systems offers several advantages, how- ever, it is does not come without risk. The practice of citing faulty research in animals models to buttress claims about plants goes back to the very origins of plant biology. Theophrastus, considered the father of botany, claimed that spontaneous generation in plants should not be taken with suspicion since it had already been demonstrated in animals: If some [plants] are generated in both ways, spontaneously as well as by seed, there is no absurdity: so some animals similarly come from two sources, both from other animals and from earth. It would be presumptuous for me to pretend that I have avoided the same mistake, but that did not stop me from trying. Chapter 1 consists on a comparative overview of novel insights about transcriptional regulation in Arabidopsis thaliana (Arabidopsis) and Drosophila acquired in the course of my research. In it, I try to find unifying themes and put results in the context of the field at large. Chapter 1 is also intended to give a relatively detailed overview of this dissertation and so many of its figures are found in chapters that deal with specific topics. The rest of the chapters are as follows. In chapter 2, I describe the motivations to establish optogenetic tools to manipulate transcription factors in Drosophila embryos and the results from our efforts. Chapter 3 contains a manuscript that is currently in preparation that describes the development of a minimal synthetic transcriptional system in Drosophila and the insights we have gained from it. Chapter 4 contains a manuscript currently in bioRxiv and under peer review that describes how transcription operates at the single cell level in plants. Chapter 5 builds upon the live imaging tools presented in Chapter 4 and describes experiments to measure the dynamics of light signaling during a dark to light transition in Arabidopsis seedlings. Finally, in Chapter 6, I outline experiments that I was unable to finish that point to potential future directions.

Crustaceans are a group of organisms with tremendous diversity in the organization of limbs alongtheir body, also known as the bauplan, or body plan. This diversity is hypothesized to arise from evolutionary changes to the expression domains of the Hox genes, an ancient gene family conserved to most animals. Previous researchers have used the amphipod crustacean Parhyale hawaiensis as a genetic model organism to examine the expression and function of Hox genes within crustaceans. This work revealed that each of the Parhyale Hox genes is expressed in a unique domain of expression that prefigures the ultimate arrangement of different limb types within the Parhyale body plan. Moreover, RNA interference, CRISPR-Cas9 mutagenesis, and heat shock overexpression experiments revealed that each of the Hox genes in Parhyale is required – or sufficient – for the development of different types of limbs. Taken in the context of changing Hox expression patterns, these results suggest that crustacean body plan evolution may be driven by changes to Hox gene regulation between different crustacean groups. In this thesis, I present several avenues of investigation into the genetic and genomic basis of crustacean body plan evolution. First, I describe the generation of a developmental functional genomic database for the amphipod crustacean Parhyale hawaiensis. Using Omni-ATAC-Seq and short- and long-read RNA-Seq, I generate an improved genome annotation for Parhyale, discover hundreds of thousands of candidate cis-regulatory elements across the Parhyale genome, and infer additional features of the chromatin landscape of Parhyale development. Using this dataset, I demonstrate the potential to generate novel reporter genes. This dataset will serve as a platform for future researchers to investigate cis-regulation across the genome during Parhyale development, and will also enable the discovery of Hox cis-regulatory mechanisms. Second, I describe experiments that reveal the role of two families of general transcriptional regulators, the Polycomb Group and Trithorax Group proteins, in regulation of the Parhyale Hox genes. Loss of function of Polycomb Group genes in particular results in homeotic transformations and changes to Hox expression. By examining the timing of these changes and synthesizing these data with other work, 2 I demonstrate that Parhyale Hox regulation occurs via at least three distinct mechanisms: early PcG-independent boundary establishment, PcG-dependent boundary maintenance, and Hox cross-regulatory interactions. I propose that crustacean Hox genes have evolved decoupled regulation at the level of developmental timing, tissue type, and specific PcG gene function. These results provide some of the first insights into the genetic mechanisms of Parhyale Hox regulation. Moreover, these data provide new mechanistic explanations for how crustaceans may have evolved their tremendous body plan diversity. Finally, I describe efforts to establish animal husbandry, injection, and dissection techniques for the isopod crustacean Asellus aquaticus and the decapod crustacean Neocaridina davidi. I provide a developmental staging guide for each crustacean. I also describe the methods I have used to isolate early embryos for injection and the various types of troubleshooting I have performed in an attempt to establish these organisms as new genetic model systems. The work presented here will prove useful in future efforts to establish new crustacean genetic model systems.