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
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.