Anoeska Van De Moosdijk

Summary The adult human body is built out of an estimated 30 trillion cells. All of these started The adult human body is built out of an estimated 30 trillion cells. All of these startedThe adult human body is built out of an estimated 30 trillion cells. All of these started out as a single cell, that had the potential to grow into a whole organism. This single out as a single cell, that had the potential to grow into a whole organism. This singleout as a single cell, that had the potential to grow into a whole organism. This single out as a single cell, that had the potential to grow into a whole cell, the fertilized oocyte, shows huge potential to turn into different cell types, but cell, the fertilized oocyte, shows huge potential to turn into different cell types, butcell, the fertilized oocyte, shows huge potential to turn into different cell types, but needs directions to know what to build. How does this work? This is where DNA needs directions to know what to build. How does this work? This is where DNAneeds directions to know what to build. How does this work? This is where DNA needs directions to know what to build. How does this work comes in. DNA is present in all cells, and it is the molecule on which the genetic code comes in. DNA is present in all cells, and it is the molecule on which the genetic codecomes in. DNA is present in all cells, and it is the molecule on which the genetic code comes in. DNA is present in all cells, and it is the molecule on wh is stored. It serves as a blueprint of the building plans for the body. is stored. It serves as a blueprint of the building plans for the body.is stored. It serves as a blueprint of the building plans for the body. is stored. It serves as a blueprint of the building plans for the body. Though just having a blueprint by itself is not enough: for cells to organize Though just having a blueprint by itself is not enough: for cells to organizeThough just having a blueprint by itself is not enough: for cells to organize themselves into a functional body with specialized cells and tissues, they need to be themselves into a functional body with specialized cells and tissues, they need to bethemselves into a functional body with specialized cells and tissues, they need to be able to work together. To achieve this, cells need to communicate. To this end, cells able to work together. To achieve this, cells need to communicate. To this end, cellsable to work together. To achieve this, cells need to communicate. To this end, cells have multiple networks of proteins inside them that can sense what is happening on have multiple networks of proteins inside them that can sense what is happening onhave multiple networks of proteins inside them that can sense what is happening on the outside and communicate these outside signals towards the inside of the cell. the outside and communicate these outside signals towards the inside of the cell.the outside and communicate these outside signals towards the inside of the cell. They do so via a chain of protein-protein interactions going from the cells They do so via a chain of protein-protein interactions going from the cellsThey do so via a chain of protein-protein interactions going from the cells They do so via a chain of protein-protein interactions surroundings towards the DNA in the centre of the cell, the nucleus. You can imagine surroundings towards the DNA in the centre of the cell, the nucleus. You can imaginesurroundings towards the DNA in the centre of the cell, the nucleus. You can imagine surroundings towards the DNA in the centre of the cell, the nucleus. Y this as a bucket of water passed down a line of people from a water source towards a this as a bucket of water passed down a line of people from a water source towards athis as a bucket of water passed down a line of people from a water source towards a this as a bucket of water passed down a line of people from a water source burning house, with the water being the signal, the people being the proteins, and burning house, with the water being the signal, the people being the proteins, andburning house, with the water being the signal, the people being the proteins, and burning house the burning house representing the nucleus of the cell where the DNA is located. So, the burning house representing the nucleus of the cell where the DNA is located. So,the burning house representing the nucleus of the cell where the DNA is located. So, an outside signal ultimately reaches the DNA. In response, the cell typically reacts an outside signal ultimately reaches the DNA. In response, the cell typically reactsan outside signal ultimately reaches the DNA. In response, the cell typically reacts by activating or inactivating specific genes, which are specific segments of DNA. One by activating or inactivating specific genes, which are specific segments of DNA. Oneby activating or inactivating specific genes, which are specific segments of DNA. One by activating or gene contains the instructions (a sort of recipe) to make one specific protein. Thus, gene contains the instructions (a sort of recipe) to make one specific protein. Thus,gene contains the instructions (a sort of recipe) to make one specific protein. Thus, when genes get activated, novel proteins can be made, and the cell can adjust its when genes get activated, novel proteins can be made, and the cell can adjust itswhen genes get activated, novel proteins can be made, and the cell can adjust its when genes get activated, novel proteins can be made, behaviour. We call the protein networks that are responsible for passing down the behaviour. We call the protein networks that are responsible for passing down thebehaviour. We call the protein networks that are responsible for passing down the behaviour. We call signals “signalling pathways”. signals “signalling pathways”.signals “signalling pathways”. signals “signalling pathways”. One signalling pathway that is essential for communication during embryonic One signalling pathway that is essential for communication during embryonicOne signalling pathway that is essential for communication during embryonic development and later in keeping the body balanced and healthy (a state termed development and later in keeping the body balanced and healthy (a state termeddevelopment and later in keeping the body balanced and healthy (a state termed “homeostasis”), is called the WNT signalling pathway. It is found and studied “homeostasis”), is called the WNT signalling pathway. It is found and studied“homeostasis”), is called the WNT signalling pathway. It is found and studied “home throughout the animal kingdom: from fly to frog, and from fish to mice and humans. throughout the animal kingdom: from fly to frog, and from fish to mice and humans.throughout the animal kingdom: from fly to frog, and from fish to mice and humans. One of the functions of the WNT signalling pathway is to balance two important One of the functions of the WNT signalling pathway is to balance two importantOne of the functions of the WNT signalling pathway is to balance two important One of the functions of the WNT signalling pathway is to balance processes in cells: cell division (“proliferation”) and cell specialization processesprocesses in cells: cell processes cellcells: (“proliferation”)(“proliferation”) andand cellcell specializationspecialization divisiondivision c in cells: in (“differentiation”). By correctly balancing these, the body will have enough cells – (“differentiation”). By correctly balancing these, the body will have enough cells –(“differentiation”). By correctly balancing these, the body will have enough cells – not too few or too many – and those cells will be of the right type, doing their rightnot too few or too many – and those cells will be of the right type, doing their rightnot too few or too many – and those cells will be of the right type, doing their rightnot too few or too many – & & function. If any of these processes get disrupted, this can lead to diseases. The most function. If any of these processes get disrupted, this can lead to diseases. The mostfunction. If any of these processes get disrupted, this can lead to diseases. The most function. If any of these pro prominent example is cancer, in which cell division gets out of control. prominent example is cancer, in which cell division gets out of control.prominent example is cancer, in which cell division gets out of control. prominent example is cancer, in which cell division gets out of control. This WNT signalling pathway is very complex: both the mouse and human DNA This WNT signalling pathway is very complex: both the mouse and human DNAThis WNT signalling pathway is very complex: both the mouse and human DNA contains 19 different genes encoding 19 different WNT proteins. Different cell types contains 19 different genes encoding 19 different WNT proteins. Different cell typescontains 19 different genes encoding 19 different WNT proteins. Different cell types in the body express different sets of WNT genes. To add to that, dozens of other in the body express different sets of WNT genes. To add to that, dozens of otherin the body express different sets of WNT genes. To add to that, dozens of other in the body express different sets of WNT proteins are involved in the WNT signalling pathway. They interact with the WNT proteins are involved in the WNT signalling pathway. They interact with the WNTproteins are involved in the WNT signalling pathway. They interact with the WNT Addendum proteins during the receiving or transmission of signals in the cell. Because of this complexity, after nearly 40 years of research on this pathway, there are still major gaps in our knowledge of the exact interactions happening in the cell when a WNT protein comes by on the outside. In this thesis, we have developed novel experimental tools to help visualize and track how WNT signalling controls development and homeostasis, and what mechanisms and interactions happen in cells that receive WNT signals. Tissues are complex 3D environments, consisting of different cell types that all have their specialized function. Breast tissue is no exception: it contains fat cells, cells forming the milk ducts, the milk glands and all kinds of support cell types as well. We know that WNT signalling is important for the development and homeostasis of breast tissue, but we also know that changes in WNT gene expression are difficult to measure in all of these different cell types, since they are often subtle. Therefore, very sensitive techniques are needed to detect these subtle changes. Since we cannot directly study these processes in humans, we make use of the mouse as mammalian model organism. In chapter 2, we use a technique called qRT-PCR for studying subtle gene expression changes during different developmental stages of the breast (also called mammary gland). To accurately quantify the subtle gene expression changes that occur during tissue development, normalization of the data is required using so-called reference genes. However, it turned out that the traditionally used ones were not sufficient. We therefore used large published gene expression datasets to identify novel reference genes and tested and validated these for qRT-PCR studies of the developing mouse mammary gland. Using the new reference genes Prdx1, Phf7 and Ctbp1, we are able to more reliably detect subtle WNT gene changes between different mammary gland developmental timepoints. The mouse mammary gland grows out during puberty, but in the adult the tissue remains remarkably dynamic. Like in every other tissue, maintenance is an active process where new cells replace old or damaged cells. On top of that, in the mammary gland, every reproductive cycle, the ducts meant to transport milk prepare themselves by building extra side ducts in the case a pregnancy happens. When that doesn’t occur, they regress again. The same thing happens during the menstrual cycle in humans, but in mice this cycle is only 4-5 days, so you can imagine this needs a lot of controlled cell growth. Specialized cells, called mammary stem cells, are responsible for producing the cells forming these ducts. We want to know where these stem cells are located and how exactly they contribute to cell turnover and homeostasis of the mammary gland tissue. In chapter 3 we describe a method to carry out such an investigation in intact mouse mammary glands. The method is called lineage tracing, and it allows us to label cells and follow exactly where they are and what their line of progeny is (hence the term lineage). We discuss the pros and cons of two different genetic labelling techniques to perform lineage tracing of mammary stem cells: one using Cre ERT2 /LoxP, the other rtTA/TeO-Cre/LoxP and point out the critical steps in designing and executing such experiments. Lineage tracing is possible via the use of genetically engineered mouse models. One mouse must carry a so-called “driver gene” as well as a “reporter gene” in the genome. The driver consists of a gene expressed in a specific cell-type (such as a stem cell) and the resulting protein encoded by that gene can in turn switch on the reporter gene, but only when a drug is given to the mouse, allowing us to control at with timepoint we switch on the reporter gene in a stem cell. This process is similar to flicking on a light switch in a dark room, with the difference that once this reporter is switched on, it will be switched on forever and permanently label the cell. Moreover, when the cell divides all daughter cells inherit it. In this way, we can track which cells are the progeny of the stem cell that was initially labelled. The label often used is a fluorescent protein. With the help of microscopes, we can literally make the cells carrying fluorescent proteins light up and visualize where they are in a tissue. Several lineage tracing mouse models exist. However, we noticed that the current driver lines were not perfect for studying stem cells in the mammary gland. Therefore, in chapter 4 we describe the generation of a novel genetic mouse model (its full name being Axin2 P2A-rtTA3-T2A-3xNLS-SGFP2 ) that has a double function: it serves as a driver line for lineage tracing, but also as a live fluorescent reporter for WNT- responsive stem cells (most stem cells found in adult tissues are receiving WNT signals, we call those cells “WNT-responsive”). This double function allows us to visualize the current stem cells (they will be labelled with a live, bright green fluorescent reporter) and at the same time visualize their lineage (once we cross this driver to a suitable reporter mouse line). This double function was not available yet for the WNT signalling target gene Axin2, which is expressed in most adult stem cells responding to WNT. We show that this novel mouse model faithfully reports WNT signalling activity during development and in different tissues, and that we can use it as a proper driver line for lineage tracing as well. To further improve our lineage tracing toolbox, in chapter 5 we describe the generation and analysis of a novel lineage tracing reporter (named Rosa26 PRIME ). This reporter cannot express just one, but four different fluorescent proteins, & & allowing more resolution when tracing the lineage of cells because we can distinguish cell lineages carrying different colours (red, cyan and yellow). It also expresses a far- red fluorescent protein in all non-labelled cells, allowing easy visualization of whole tissues. This novel reporter aims to be better and brighter than previously existing mouse models, although we did not yet manage to test this in a living organism (in vivo). Addendum Creating genetically engineered mouse models is a long and challenging process. But recently, the emergence of the CRISPR/Cas9 genome editing technique promised that this could become easier. In chapter 6 we therefore set out to create a toolbox for CRISPR/Cas9 genome editing, first starting in human and mouse cell lines and ultimately in mice. We focused on a very important protein in the WNT signalling pathway that is central to most of the signalling events: beta-catenin (with the gene name being Ctnnb1). We show that we can successfully edit the Ctnnb1 gene in cells: we can not only cut it up (an destroy it), but we can also modify it with high precision, so that it becomes labelled with a fluorescent protein. After that, we optimized CRISPR/Cas9 genome editing to specifically insert a gene encoding for a fluorescent protein into the Ctnnb1 gene in mouse embryos, creating precision engineered knock-in mice. The resulting mice carry a fusion of the fluorescent protein with beta- catenin. This allows us to image the localization of this protein in cells and tissues and study what it is doing in live cells for the first time. Overall, this thesis describes the generation of several novel tools to study WNT signalling at physiological levels in mice. Together they provide a strong basis for future generations of scientists to unravel the mechanisms of WNT signalling and its impact on development, tissue homeostasis and disease.