Control of stem cell proliferation and differentiation by primary cilia in the developing cerebral cortex

Our proposed research aims to understand the formation of a part of the brain, the cerebral cortex, which is the seat of all higher cognitive functions unique to humans. Fulfilling these functions requires a huge diversity of nerve cells (neurons), it is thought that the cortex contains dozens of different types of neurons. During embryonic development, all these different neurons are generated from stem cells which can either proliferate to produce more of the stem cells or differentiate into neurons (neurogenesis). Tightly controlling the balance between proliferation and differentiation is required to produce neurons in sufficient numbers and at the correct time and place. If stem cells start to form neurons too early, too few neurons are produced affecting the functioning of the cortex. In contrast, continued divisions of progenitors may lead to the overproduction of some neuron types at the expense of others, again resulting in cortical malfunctioning.
Coordinating the proliferation and differentiation of cortical stem cells requires communication between cortical stem cells and their environment and involves a subcellular structure called the primary cilium. The primary cilium is a small protrusion from the cell surface that acts as an antenna for signals between cortical stem cells. Defects in the function and/or structure of primary cilia can have profound effects on brain development, however, it remains largely unknown how cilial defects could perturb the formation of cortical stem cells and the balance between stem cell division and neurogenesis.
Here, we will investigate roles of primary cilia in cortical stem cells using mice mutant for the Inpp5e gene which has dual roles in cilial signalling and in controlling cilia stability. Inpp5e encodes a phosphatase, an enzyme which removes phosphate groups from phospholipid molecules. Inpp5e inactivation perturbs several signalling systems important for stem cell function, including Akt and Sonic hedgehog signalling. Inpp5e also controls the stability of the cilium which becomes more labile in the absence of Inpp5e. Finally, our unpublished data show that the Inpp5e mutant cerebral cortex is elongated and forms several folds suggesting that Inpp5e coordinates the proliferation and differentiation of cortical stem cells. This hypothesis is also supported by a severe depletion of one type of cortical progenitor cells, namely basal progenitors. These findings emphasize Inpp5e's importance for cortical development and taken together with Inpp5e's dual roles in cilia stability and cell signalling make Inpp5e mutant mice an excellent tool to study roles of primary cilia in cortical stem cell development.
In this proposal, we will test the hypothesis that primary cilia control the signalling required to determine the balance between stem cell proliferation and neurogenesis. We will systematically analyse the formation and differentiation of cortical stem cells in Inpp5e mutants. Using high resolution live imaging of cilia, we will also investigate whether the Inpp5e mutation affects the asymmetric inheritance of cell fate determinants, an important characteristic of stem cells. Finally, using cell biology and genetic approaches, we will examine alterations in signalling mechanisms which underlie the stem cell defects in Inpp5e mutants. These analyses will be a vital step towards gaining a comprehensive understanding of how cilia can coordinate the proliferation and differentiation of cortical stem cells. This knowledge will not only have implications for our understanding of the role of primary cilia during the development of the cerebral cortex but also for regenerative medicine as differentiating stem cells into neurons is widely considered to be an attractive long-term approach for the replacement of nervous tissues that have been lost or damaged by disease or injury.

The cerebral cortex confers humans with their unique cognitive capabilities and relies on a striking neuronal diversity to fulfil its highly complex neural tasks. Generating these diverse neurons in sufficient numbers constitutes a major challenge in Biology and requires several signals to coordinate the proliferation and differentiation rates of cortical stem cells. The primary cilium acts as a signalling hub integrating signalling pathways during embryonic development and tissue homeostasis. Due to their prominent role in cell signalling, cilia are ideal candidates to control the development of cortical stem cells but surprisingly little is known about these roles. Here, we investigate cilia functions in cortical stem cells using mice mutant for Inpp5e that is essential for cilial signalling and cilia stability. Inpp5e mutants display growth defects and a severely reduced pool of basal progenitor cells, one of the major progenitor cell types in the developing cerebral cortex. We will systematically characterize the formation of cortical stem and progenitor cells and their proliferation and differentiation in Inpp5e mutants. Using high-resolution live imaging we will determine how defects in cilia affect asymmetric inheritance in cortical stem cells and thereby the cell fate of their daughter cells. Cell biological assays and a genetic rescue experiment will identify cilia mediated signalling mechanisms that lead to the severe reduction of basal progenitors. Elucidating the mechanisms and signals that primary cilia use to control cortical stem cell development will help us to understand how cortical stem and progenitor cells are maintained but are also able to produce neurons in sufficient numbers. A better understanding of this process may also be relevant to regenerative medicine in the context of replacement therapies of neurons which have been lost or damaged by disease or injury.

Impact Summary

This proposal outlines a plan of fundamental scientific research that addresses a challenging question in biological sciences, the answers to which will be complex. In the immediate future, we expect them to improve our understanding of normal brain development. In the long term, we consider these answers to have a significant impact on our understanding of disease in humans and will eventually contribute to the development of new therapies in regenerative medicine.

A. List of potential beneficiaries of this research

1. Academics
2. Clinicians/regenerative medical scientists
3. Patients and patient support groups
4. General public

B. Ways in which potential beneficiaries will benefit from this research:

1. Academics

As described in the academic beneficiaries section of the proposal, this work will have a significant impact on academic scientists working on stem cells, primary cilia, cell signalling and cortical development. Benefits include the additional biological knowledge gained and elucidating the molecular mechanisms which control stem cell characteristics. It will also provide these scientists with an improved framework of knowledge for interpreting their own results.

2. Clinicians/regenerative medical scientists

We expect this project to have a strong impact on clinicians working on syndromes which affect INPP5E (Joubert Syndrome) or cilia function (ciliopathies) and on intellectual disability in general. In the long term, an improved understanding of the biological basis of these syndromes could contribute to designing new treatments using a more evidence-based approach. Regenerative medical scientists interested in transplantation based regenerative therapies will also benefit from our findings in the long term. Investigating the mechanisms underlying neurogenesis in the cerebral cortex will provide more informed and efficient ways of developing methods for directing the generation of cortical neurons from stem cells.

3. Patients and patient support groups

Our research will allow patient support groups to provide informed advice to patients suffering from Joubert Syndrome (mutations in the INPP5E gene) or from other ciliopathies caused by defects in the structure or function of the primary cilium. The principal applicant and the co-applicant have participated in the "Cilia Conferences 2012 and 2014" organized by the Ciliopathy Alliance (http://www.ciliopathyallicance.org/). From these meetings, it was very clear that a greater understanding of the basis of the components of the syndrome is very much looked for. Direct communication and engagement with patient groups will be at a future "Cilia Conference 2016" or fundraising events organized by the Patrick-Wild Centre for research into Autism, Fragile X-syndrome and Intellectual Disability (http://patrickwildcentre.com/) of which the principal applicant is a member. There are also several websites that provide links (http://www.alstrom.org.uk; http://www.lmbbs.org.uk/) between sufferers, for whom understanding their condition and its implications for their and their families' livelihoods and prospects is of paramount importance. At future such meetings, we hope to be able to provide patients with continuously improving explanations.

4. General public

Stem cell biology is a particularly attractive topic to engage with the public in general and with pupils in science in particular. Research on stem cells is routinely disseminated via the popular media. We will participate in Science and Career Fair activities at local primary and secondary schools and use work from the current grant to underscore the importance of stem cell biology and the responsible use of animals in research.