Cell-cell interactions in the regulation of neural progenitor fate: the role of PCDH19

The brain is a complex, yet highly ordered structure serving as the control centre for all vertebrates and some invertebrates. In mammals, one region of the brain, designated the cerebral neocortex, is particularly important for elaborate processes such as memory, attention, thoughts, perception and language. Neurons in the mammalian neocortex are distributed in specific layers and have distinct morphological and functional properties. These neurons are generated during development by specific cells, called neural progenitors. These progenitors need to produce the correct amount of neurons for each of the layers, and they do it sequentially. They first produce neurons for the deepest layer, then for the layer on top of that, and so on until all layers are generated. This means that progenitors need to maintain a balance between giving rise to neurons and maintaining their own population, to avoid getting depleted before all neurons are produced. Progenitors can divide in two different ways: symmetrically and asymmetrically. Symmetric divisions produce two identical daughter cells, either two progenitors or two neurons. Asymmetric divisions give rise to two different cells: one progenitor and one neuron. At very early stages in the formation of the cortex, progenitors divide symmetrically to increase their numbers. But at some point they need to change to asymmetric divisions to start producing neurons. How progenitors make this decision is currently not understood.
My laboratory is investigating the mechanisms that control development of the cerebral cortex. We have recently found that one cell-cell adhesion protein, protocadherin 19 (PCDH19), is involved in the regulation of neural progenitor behaviour. PCDH19 is mutated in a human disorder leading to epilepsy and varying degrees of cognitive impairment in very young girls. This molecule is present in neural progenitors around the time of the symmetric to asymmetric division switch. We have found that progenitors with PCDH19 produce neurons at different rates when they are alone than when they coexist with progenitors that lack this protein. The same is true for the PCDH19-deficient progenitors. This suggests that communication between cells is important to regulate progenitor behaviour and that we can use PCDH19 to investigate this process. We will first determine if the changes that we have seen at embryonic day E11.5 are maintained in time and how they affect overall neuronal production. We will then analyse the dividing progenitor cells to find out what makes the progenitors with PCDH19 different from the ones without this protein. Finally, we will use both direct and unbiased approaches to figure out which molecules and signalling pathways are responsible for the differential behaviour between the two progenitor types. We will investigate particular molecular pathways that have been shown to play a role in the maintenance of progenitor cells. At the same time, we will carry out an analysis of all genes expressed by PCDH19-positive and -negative progenitors to assess other potential differences in an unbiased way.
These experiments will provide valuable information to explain how progenitors decide to start producing neurons, but they will also reveal how cell-cell communication influences this process. This information is crucial to understand how brains are formed correctly, an essential knowledge when studying neurodevelopmental disorders.

Protocadherin 19 (Pcdh19) expressing and non-expressing neural progenitors in E11.5 heterozygous (het) mice brains behave differently from each other and from control brains: progenitors that lack Pcdh19 show higher mitotic index and quitting fraction, producing more neurons at the expense of radial glial cells. This is reversed in Pcdh19-expressing progenitors, which display both lower mitotic index and quitting fraction with reduced neurogenesis. We will use this unique system of Pcdh19 het and control brains to investigate the cellular and molecular mechanisms regulating the switch from symmetric, proliferative divisions to asymmetric, neurogenic divisions during corticogenesis, a crucial but poorly understood step during early brain development. We will first analyse cell cycle parameters at later stages to determine degree of change and consequences on neurogenesis. To investigate the cellular basis of this different symmetric to asymmetric switch (STAS), we will determine cleavage plane angles, cell cycle length, mode of neurogenesis (direct vs. indirect), and endfeet behaviour combining immunohistochemistry, injection of base analogues, live imaging and use of reporter mice. Finally, to reveal molecular determinants of the STAS, we will employ candidate and unbiased approaches. We will investigate the Notch signalling pathway, known to regulate progenitor identity and neurogenesis. This will be achieved by in utero electroporation of reporter plasmids and use of reporter mice strains. RNA sequencing analysis will complement the candidate approach, allowing us to compare the transcriptome of Pcdh19+ and Pcdh19- progenitors in wild type, ko and het animals. The project will benefit from our expertise in cortical development and in utero electroporation. Our results will shed light on how neural progenitors switch their division mode to start neurogenesis and will be relevant for the fields of neurodevelopment, cadherins, and regulation of cell division.

The beneficiaries from this project will be:

1. Wider public, particularly school pupils
2. Researchers and students directly involved in the project
3. Clinicians working on neurodevelopmental disorders
4. Patients suffering from neurodevelopmental disorders and their families

How will they benefit from it?

1. Wider public and pupils
Policy makers have identified a greater understanding of science and an increased interest in STEM subjects as key factors for the UK's prosperity by (see the policy paper "2010 to 2015 government policy: public understanding of science and engineering"). Neuroscience is an appealing topic to engage with a wider public and to attract students to scientific careers, since it can be approached from a variety of perspectives: medical, biological, engineering, etc. However, scientific results are generally complex and difficult to understand. We will disseminate our research results to the wider public in an easy to understand, visual way, and incorporate them into children-oriented engagement activities to increase the public understanding of science and to raise scientific interest in school-aged children.

2. Researchers and students directly involved in the project
In this project, we will train one postdoctoral researcher, who will acquire specialized technical skills in neuroscience, such as in utero electroporation and RNAseq analysis. In addition, (s)he will also acquire transferable skills like problem solving abilities, time and resource management, presentation of results, networking, etc. This training will increase employability and the chances for the development of a successful career. We also expect MRes students and Final Year students to get involved at different stages of the project. These students will benefit in a similar way as the postdoctoral researcher in terms of acquiring transferable skills that will increase their employability.

3. Clinicians working on neurodevelopmental disorders
Clinicians working on neurodevelopmental disorders will also benefit from our results. Even though the project addresses basic biological questions, the new knowledge that we will generate can be relevant to understand the pathophysiology of EIEE9, a disorder that presents with seizures and cognitive impairment due to mutations in PCDH19. This information should place clinicians in a better position to inform patients about their condition and to develop new treatment strategies for this disease. In addition, a better understanding of progenitor behaviour during corticogenesis will also benefit clinicians working on microcephaly and other disorders in which neuronal numbers are altered.

4. Patients suffering from neurodevelopmental disorders, their families and support groups
Our results will add to the existing knowledge about cortical development and about the function of PCDH19, and they will have potential to identify new candidate genes involved in neurodevelopmental disorders.
PCDH19 has become the second most clinically relevant gene in epilepsy after SCN1A and several groups have been created to support research into PCDH19 epilepsy:
- Pcdh19 Alliance (https://www.pcdh19info.org)
- Pcdh19 France (http://www.pcdh19france.fr/index.html)
- Asociación epilepsia rosa (http://www.pcdh19.net)
- Insieme per la Ricerca PCDH19 - ONLUS (http://www.pcdh19research.org/index.html)
The first way in which we expect our research to benefit patients, their families and patient support groups is by providing them with information to better understand the disease and how mutations in Pcdh19 lead to the different symptoms of the disorder.
In the longer term, our results should contribute to enhance the quality of life of patients and their families, by aiding in the design of better treatment options, in the form of drugs and other types of therapies.