Visualization of transcriptional dynamics in the early Drosophila embryo

To the observer it is immediately visible that an elephant and an ant are very/ndifferent animals. However, when observed at a molecular level, we realize that/nthey share a comparable basic machinery of development. This concept is/nessentially true for all the animals, either vertebrates or invertebrates./nOver the years, different biologists tried to explain how phenotypic diversity is/ncreated, to conclude that living beings are made of similar genetic material./nHowever, the instructions that drive their development may vary considerably./nTo make a simple comparison, we can combine the same ingredients to form/nmultiple types of food. For example, we can mix flour, salt, water and a bit of/nyeast to make pizza dough. The same ingredients can be used to make bread or/nsavory cookies. The final products depend on the quantity ratio among these/ningredients, the preparation, cooking strategy and the time we allow for each of/nthese products to develop consistency and flavor./nSimilarly, the same genes may lead to the formation of various cell fates just/nbecause they are expressed (or not) at different levels or different time in that/nspecific cell. But what does regulate when, where and how much a particular/ngene is expressed? In other words, where can we find the manual that instructs/non how organism form and develop?/nWhen in the sixties, Jacob and Monod described that in bacteria the Lac operon/ntranscription is controlled by non-coding elements positioned right upstream the/ngene (Jacob & Monod 1961), no one would imagine that a similar process would/nregulate gene expression in eukaryotic cells. It took about two extra decades to/ndiscover that also in eukaryotes some non-coding DNA elements were able to/nenhance the activity of a gene (for a complete review on enhancer discovery, see/nSchaffner 2015). Contrarily to bacteria, these elements, called enhancers, were/nfunctioning in any orientation and also on a distance. At the same time, it was/ndiscovered that enhancers contain binding sites for activator and repressor/nproteins (Lewis 1978). Thus, a gene could be either active or inactive depending/non whether activators (or repressors) were bound to its enhancer. When all the/ngenes of a multicellular organism are expressed at the right place, time and in the/nright quantity, development proceeds normally./nRegulation of gene expression is far from a static process; on the contrary it is/ncharacterized by a series of dynamic events. The amount of regulatory proteins/nchanges constantly among cells and even within the same cell. To complicate/nthings further, enhancers can be found everywhere in the genome, often, far/naway from the gene they regulate. More over, DNA is a very mobile molecule and/nacquires multiple conformations that potentially could prevent, or favor, protein/naccessibility to regulatory domains and interaction between enhancers and/ngenes./nUntil recently, most of the information about gene expression derived from/nexperimental analysis in fixed biological samples, using for example in situ/nhybridization assays that allow detection of nuclear and cytoplasmic mRNA. This/ntechnique has been useful to understand the spatial information of gene/nexpression. Yet, in fixed samples it is very challenging to deduce the dynamic/nchanges that occur in a developing embryo./nThe continuous advancement in the molecular biology field, improvements on the/nvisualization and computational techniques, allow now seeing and analyzing/nbiological processes occurring in real time at increasingly higher resolution. This,/ntogether with collaborations among people with different expertise, represents a/nmulti-disciplinary task force to understand the rules that govern gene regulation/nand, to a large extent, how multicellular organisms are produced./nIn this thesis I intend to discuss how newly developed imaging techniques allow/nto push our knowledge forward about gene regulation dynamics. I will present/nthree instances in which we reveal subtle mechanisms that fine tune mRNA/nproduction in Drosophila melanogaster embryos. Gene regulation is a pervasive/nfeature that underlies the formation of all living beings and I believe that by being/nable to observe and analyze how biological processes occur in real-time in vivo/nwe will, to some extent, be able to comprehend how life is generated./nThis thesis consists of four chapters. In the first introductory chapter I present the/nbackground on gene regulation and imaging techniques. In particular I focus on/nthe bacteriophage MCP-MS2 system since this has been my elective way to/nvisualize transcription. In the following chapters I discuss my work done in/ncollaboration with several bright people to visualize transcriptional dynamics/nduring the hours that precede gastrulation in the Drosophila embryo. I present/nhow transcription occurs in burst of expression in a pair-rule gene (chapter 2),/ndescribe the existence of post-mitotic transcriptional memory (chapter 3) and, in/nthe forth chapter, I report the analysis done to understand how transcriptional/nrepression regulates gene activity during the formation of the mesoderm ectoderm/nboundary. In this work we provide evidence that mitotic silencing/nfacilitates repression and we suggest a model whereby repressor exploit pauses/nin transcriptional activity to ensure rapid gene inactivation.

Advisor: Levine, Michael S