UC Berkeley

To the observer it is immediately visible that an elephant and an ant are very

different animals. However, when observed at a molecular level, we realize that

they share a comparable basic machinery of development. This concept is

essentially true for all the animals, either vertebrates or invertebrates.

Over the years, different biologists tried to explain how phenotypic diversity is

created, to conclude that living beings are made of similar genetic material.

However, the instructions that drive their development may vary considerably.

To make a simple comparison, we can combine the same ingredients to form

multiple types of food. For example, we can mix flour, salt, water and a bit of

yeast to make pizza dough. The same ingredients can be used to make bread or

savory cookies. The final products depend on the quantity ratio among these

ingredients, the preparation, cooking strategy and the time we allow for each of

these products to develop consistency and flavor.

Similarly, the same genes may lead to the formation of various cell fates just

because they are expressed (or not) at different levels or different time in that

specific cell. But what does regulate when, where and how much a particular

gene is expressed? In other words, where can we find the manual that instructs

on how organism form and develop?

When in the sixties, Jacob and Monod described that in bacteria the Lac operon

transcription is controlled by non-coding elements positioned right upstream the

gene (Jacob & Monod 1961), no one would imagine that a similar process would

regulate gene expression in eukaryotic cells. It took about two extra decades to

discover that also in eukaryotes some non-coding DNA elements were able to

enhance the activity of a gene (for a complete review on enhancer discovery, see

Schaffner 2015). Contrarily to bacteria, these elements, called enhancers, were

functioning in any orientation and also on a distance. At the same time, it was

discovered that enhancers contain binding sites for activator and repressor

proteins (Lewis 1978). Thus, a gene could be either active or inactive depending

on whether activators (or repressors) were bound to its enhancer. When all the

genes of a multicellular organism are expressed at the right place, time and in the

right quantity, development proceeds normally.

Regulation of gene expression is far from a static process; on the contrary it is

characterized by a series of dynamic events. The amount of regulatory proteins

changes constantly among cells and even within the same cell. To complicate

things further, enhancers can be found everywhere in the genome, often, far

away from the gene they regulate. More over, DNA is a very mobile molecule and

acquires multiple conformations that potentially could prevent, or favor, protein

accessibility to regulatory domains and interaction between enhancers and

genes.

Until recently, most of the information about gene expression derived from

experimental analysis in fixed biological samples, using for example in situ

hybridization assays that allow detection of nuclear and cytoplasmic mRNA. This

technique has been useful to understand the spatial information of gene

expression. Yet, in fixed samples it is very challenging to deduce the dynamic

changes that occur in a developing embryo.

The continuous advancement in the molecular biology field, improvements on the

visualization and computational techniques, allow now seeing and analyzing

biological processes occurring in real time at increasingly higher resolution. This,

together with collaborations among people with different expertise, represents a

multi-disciplinary task force to understand the rules that govern gene regulation

and, to a large extent, how multicellular organisms are produced.

In this thesis I intend to discuss how newly developed imaging techniques allow

to push our knowledge forward about gene regulation dynamics. I will present

three instances in which we reveal subtle mechanisms that fine tune mRNA

production in Drosophila melanogaster embryos. Gene regulation is a pervasive

feature that underlies the formation of all living beings and I believe that by being

able to observe and analyze how biological processes occur in real-time in vivo

we will, to some extent, be able to comprehend how life is generated.

This thesis consists of four chapters. In the first introductory chapter I present the

background on gene regulation and imaging techniques. In particular I focus on

the bacteriophage MCP-MS2 system since this has been my elective way to

visualize transcription. In the following chapters I discuss my work done in

collaboration with several bright people to visualize transcriptional dynamics

during the hours that precede gastrulation in the Drosophila embryo. I present

how transcription occurs in burst of expression in a pair-rule gene (chapter 2),

describe the existence of post-mitotic transcriptional memory (chapter 3) and, in

the forth chapter, I report the analysis done to understand how transcriptional

repression regulates gene activity during the formation of the mesoderm ectoderm

boundary. In this work we provide evidence that mitotic silencing

facilitates repression and we suggest a model whereby repressor exploit pauses

in transcriptional activity to ensure rapid gene inactivation.