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

Lead Research Organisation: University of Edinburgh
Department Name: Biomedical Sciences


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 Finally, our unpublished data show that the Inpp5e mutant cerebral cortex is elongated and forms several folds suggesting that Inpp5e coordinates the proliferation and differentiation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.

Technical Summary

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.

Planned Impact

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 ( 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 ( of which the principal applicant is a member. There are also several websites that provide links (; 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.


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Bas-Orth C (2020) The mitochondrial calcium uniporter is crucial for the generation of fast cortical network rhythms. in Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

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Hasenpusch-Theil K (2021) The Multifaceted Roles of Primary Cilia in the Development of the Cerebral Cortex. in Frontiers in cell and developmental biology

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Ratié L (2020) Loss of Dmrt5 Affects the Formation of the Subplate and Early Corticogenesis. in Cerebral cortex (New York, N.Y. : 1991)

Description The cerebral cortex is required for all higher cognitive functions unique to humans. In order to function properly, the cortex requires a large number of nerve cells (neurons). Too few and also too many neurons interfere with the cortical function and can lead to neurodevelopmental disorders such as autism, microcephaly and macrocephaly. Crucial for producing the correct numbers of neurons are neural stem cells and controlling when and how these cells divide to produce more stem cells (proliferation) or neurons (differentiation). This control requires stem cells and neurons to communicate with each other via cell signalling. The primary cilium acts as an antenna and constitutes a hub to coordinate the cell's response to signals and we have hypothesized that altered function of the primary cilium leads to defects in cortical stem cell proliferation/differentiation and to cortical growth defects. We characterize cortical stem cell development in a mouse mutant for the Inpp5e gene which leads to defective cilia signalling. Our characterisation showed that the formation of neurons is increased at the expense of a specific cortical stem cell type, basal progenitors, i.e. neurons form prematurely and there are not sufficient stem cells available to produce later born neurons. Using a conditional mouse mutant we could also show that this defect arises early in cortical development at patterning stages. We have also identified signalling pathway(s) defects in which underlie the abnormal neurogenesis. We could show that processing of the Gli3 transcription factor is affected by the Inpp5e mutation and that re-introducing Gli3 rescues the neurogenesis phenotype. In addition, we observed an up-regulation of mTOR signalling and we are currently testing the role of this alteration in pharmacological rescue experiments.
Exploitation Route Future replacement therapies for neurodegenerative diseases such as Parkinson's and Alzheimer will require the generation of neurons in vitro from stem cell culture. Our findings might provide instructions how the formation of neurons in such cultures can become more effective.
Sectors Healthcare,Manufacturing, including Industrial Biotechology

Description Modelling the role of the primary cilia gene CEP41 in autism spectrum disorder using human cortical organoids
Amount £127,671 (GBP)
Organisation Simons Foundation 
Sector Charity/Non Profit
Country United States
Start 11/2019 
End 07/2021
Description RS MacDonald Seedcorn Fund
Amount £5,000 (GBP)
Organisation RS Macdonald Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2017 
End 01/2018
Title mRNAseq dataset on Inpp5e mutants 
Description An mRNAseq experiment was performed comparing the gene expression profiles in the developing cerebral cortex of control and Inpp5e mutant embryos. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact We have only just generated this data set which will be made available to other researchers once we have evaluated the data set. 
Description Investigating the role of primary cilia genes in human brain development using brain organoids 
Organisation University of Edinburgh
Department Edinburgh Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution We characterized the brain organoids generated by the Chandran lab (see below).
Collaborator Contribution Prof Siddharthan Chandran's lab helped to generate a point mutation in the INPP5E gene in human iPS cells and to grow brain organoids from these mutant iPSCs.
Impact There have been no output or outcomes yet.
Start Year 2018
Description Primary cilia in Joubert Syndrome 
Organisation Radboud University Nijmegen Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution We are generating brain organoids from CEP41 mutant human iPSC lines.
Collaborator Contribution Prof Roepman provided us with a list of Cep41 interaction partners which will help us enormously to characterise Cep41's roles in neural development.
Impact No outcome yet.
Start Year 2019
Description Role of Dmrt genes in corticogenesis 
Organisation University Libre Bruxelles (Université Libre de Bruxelles ULB)
Department Neuroscience Institute
Country Belgium 
Sector Academic/University 
PI Contribution We performed part of the experiments and edited two manuscripts which were published in the last couple of years.
Collaborator Contribution Partner designed the study, performed most of the experiments and wrote manuscript.
Impact Published papers: 1) Loss of Dmrt5 Affects the Formation of the Subplate and Early Corticogenesis. Ratié L, Desmaris E, García-Moreno F, Hoerder-Suabedissen A, Kelman A, Theil T, Bellefroid EJ, Molnár Z. Cereb Cortex. 2019 Dec 16. pii: bhz310. doi: 10.1093/cercor/bhz310 2) DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium-Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors. Desmaris E, Keruzore M, Saulnier A, Ratié L, Assimacopoulos S, De Clercq S, Nan X, Roychoudhury K, Qin S, Kricha S, Chevalier C, Lingner T, Henningfeld KA, Zarkower D, Mallamaci A, Theil T, Campbell K, Pieler T, Li M, Grove EA, Bellefroid EJ. J Neurosci. 2018 Oct 17;38(42):9105-9121. doi: 10.1523/JNEUROSCI.0375-18.2018.
Start Year 2017
Description Role of primary cilia in cortical development 
Organisation Sorbonne University
Country France 
Sector Academic/University 
PI Contribution This is an ongoing collaboration with Prof Sylvie Schneider Maunoury. We have mainly analyzed the phenotype of Inpp5e mutant embryos in cortical development and have written the manuscript.
Collaborator Contribution Prof Schneider Maunoury's lab has performed an electron microscopy study to reveal the structure of primary cilia in the developing cerebral cortex of Inpp5e mutant embryos.
Impact Manuscript under review with Elife
Start Year 2016
Description CIP visit of PCD families 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact 2 Families affected by Primary Cilia Dyskinesia visited my lab which sparked questions and discussions. I will attend a future meeting of the Scottish PCD group.
Year(s) Of Engagement Activity 2017
Description German Circle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 15 members of the public attended a talk/presentation on Developmental Biology which sparked questions and a lively discussion.
Year(s) Of Engagement Activity 2019
Description PCD Awareness Day 2018 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact Ca. 15 PCD patients and their families and 15 scientists attended this event, where scientists gave brief presentations of their work followed by lively discussions.
Year(s) Of Engagement Activity 2018
Description University of Edinburgh Biomedical Sciences Student Association 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Ca 50 undergraduate students attended my general talk on how genes control the development of the brain given to the University of Edinburgh Biomedical Sciences Student Association
Year(s) Of Engagement Activity 2019