The people behind the papers – Rana Fetit and David Price Free

Rana Fetit and David Price

David, can you give us your scientific biography and the questions your lab is trying to answer?

DP: I've always been fascinated by brains – particularly ours – and even more so by how they're made. When I was a medical student at Edinburgh, I discovered a love of neuroanatomy and neurodevelopment. I also met my future PhD supervisor, Colin Blakemore, and his work on the effects of early experience on development of the brain inspired me to move into research rather than clinical practice. Towards the end of my PhD at Oxford University I decided that I wanted to get deeper into the mechanisms that generate the brain. That was the mid-1980s, and the homeobox had just been discovered. Molecular biology was revolutionising our ability to investigate the mechanisms of brain development, and I decided to go to learn the methods. I went to work with David Weisblat and his extremely patient and knowledgeable postdoc Cathy Wedeen on homeobox genes in leech at the University of California at Berkeley. After returning to Edinburgh University to set up my own lab, I used the knowledge I had gained to start investigating the molecular genetics of brain development in mice. This has been the main focus of my lab ever since. Much of our effort has been devoted to research on the actions of transcription factors. It sometimes feels like I've had a 30-year love affair with one of them in particular: Pax6! I am pleased to say that this affair has borne fruit, and we now have a clear idea of what Pax6 does during normal brain development, and we published a long paper on this last year (Manuel et al., 2022). Increasingly, we have been working towards a better understanding of how brain development can become disordered in humans, and that's where Rana's fantastic work comes in (and just to be clear, the idea for this project was all hers).

Rana, how did you come to work on this project and what drives your research today?

RF: When I joined the lab, my colleagues were using in-silico approaches and human foetal tissue to investigate the expression of 16p11.2 genes, together with murine models of the deletion. We had just received the induced pluripotent stem cell (iPSC) lines from our collaborators, but the project had not been fully outlined. I knew I wanted to work with stem cells and organoids in a neurodevelopmental context, so I took on the project! I consider myself very lucky that I was in a programme that allowed me to design my own study and propose a research project tailored to my own interests. Having worked with organoids, I understand the great potential this tool offers. As such, I am now exploring the use of 3D-organoids to model other human diseases, primarily cancers.

What was known about the effects of 16p11.2 microdeletions in autism spectrum disorder (ASD) before your work?

RF: The genetic link between 16p11.2 microdeletions and ASD has been clearly demonstrated by several studies, but how this genetic deletion brings about the patient phenotype was, and still is, largely unknown. Before I started this project, most of the research on 16p11.2 deletion either focused on investigating the roles of individual genes within the 16p11.2 locus or used animal models, such as zebrafish, Drosophila or mouse, to recapitulate the human deletion. Studying single candidate genes within the region highlighted their roles in a number of neurodevelopmental processes like cell cycle regulation, neuronal migration and cortical lamination, but it is more likely that multiple genes within the 16p11.2 region interact with each other through common pathways to contribute to ASD and the variable clinical symptoms observed in patients with the deletion. Mouse models of this deletion recapitulated many of the behavioural symptoms of ASD and revealed synaptic abnormalities, perturbations in cortical cytoarchitecture and perturbations in brain size, all of which are observed in post-mortem ASD tissue. Moreover, this large genetic region has been associated with transcriptional and synaptic pathways that are implicated in several hypotheses of ASD pathogenesis.

Recently, iPSC technology has allowed researchers to specifically investigate early disruptions in foetal development, which is of great relevance to the neurodevelopmental mechanisms leading to ASD manifestations. These studies used 2D-cultures, with a focus on excitatory, cortical progenitors and neurons, revealing aberrant neuronal morphology, and implicated genes outside the 16p11.2 locus that were related to psychiatric disorders, including ASD. There were only a handful of publications investigating the effects of 16p11.2 deletion using 3D-whole brain cortical organoids, recapitulating the macrocephalic patient phenotype with an excess in neuron numbers. However, there were no reports of using region-specific ventral organoids to specifically address the effects of this deletion on interneuron development.

Can you give us the key results of the paper in a paragraph?

RF: We generated ventral organoids, which mimic only the region in the brain where inhibitory neurons are produced, from cells that were genetically modified to harbour this deletion and others that kept the two copies intact. Our work showed that, at early developmental stages, ventral organoids exhibit greater variations in size, with more potential to form circular, radial arrangements of progenitor cells, commonly referred to as ‘rosettes’. Moreover, the time it took the progenitor cells to undergo a cycle of cell division was remarkably prolonged in organoids with the deletion. This was primarily due to an elongated G1 phase, the duration of which also varied more than normal. At later stages, deletion organoids exhibited increased production of inhibitory interneurons, suggesting that this deletion may contribute to ASD aetiology by lengthening the cell cycle of ventral progenitors and promoting premature differentiation into interneurons.

In your study you use multiple different control and deletion lines, why was this so important?

RF: 16p11.2 microdeletion has a very variable clinical presentation. In addition to ASD, the patients with the deletion may present with seizures, developmental and language delay and cognitive impairment to varying extents. This variability could be due to a number of reasons: the length of the deletion fragment, the patient's genetic background and their familial cognitive, social and motor performance levels.

iPSC lines are equally variable because they acquire additional mutations as they're maintained. Consequently, the organoids derived from these lines ought to exhibit variability, especially given the additional factors that come into play when generating organoids. These include variations in reagents from suppliers and variability due to culturing environment, which contribute to the batch-batch variability consistently observed in organoids. When designing the experiment, these sources of variability had to be taken into consideration and accounted for in our statistical analysis. Therefore, using as many lines as possible allows us to generate statistical models that account for the different sources of variability introduced by cell lines and organoid batches, and focus specifically on the differences observed due to the genetic deletion.

Are there any candidate genes in the 16p11.2 region that might affect the regulation of the cell cycle during neurogenesis?

RF: Many 16p11.2 genes, such as KIF22, ALDOA, HIRIP3, PAGR1 and MAZ, were found to be expressed in neural progenitors and their deletion could, therefore, influence neurogenesis. The region also encompasses the gene MAPK3, which converges on ERK signalling, a crucial pathway that affects cell growth and proliferation. Impaired MAPK signalling also contributes to many cancers, and recent studies have demonstrated the association of 16p11.2 microdeletion with neuroblastoma.

What implications will your study have on understanding or treating ASD?

RF: I believe our work provides an insight into one potential mechanism that contributes to the manifestations of 16p11.2 deletion, including ASD. The dysregulation of the cell cycle inevitably renders the brain vulnerable during development and may individually, or in synergy with additional perturbations in other tightly controlled spatiotemporal developmental processes, contribute to the pathogenesis of ASD. This study is yet another piece of evidence implicating the excitatory/inhibitory imbalance in ASD pathology. However, to fully understand the molecular mechanisms underlying ASD and attempt to translate that into treatment options, we really need to adopt an integrated approach in investigating ASD. As such, we need to use the variety of research tools and methodologies from iPSC models, clinical and post-mortem studies to offer complementary insights on perturbations that may occur during the different stages of brain development including proliferation, neurogenesis, migration, neuronal morphogenesis and synaptogenesis, as well as gliogenesis and myelination. The integration of additional layers of analysis from genetic studies, animal models and functional neuroimaging studies in living individuals will give us a more comprehensive understanding of the neuropathology of ASD.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

RF: Initially, this project started as an exploratory study. I ran a small pilot study with few organoids from one control line and one mutant line. I observed potential differences, but the sample size was too small to derive any conclusions with confidence. Nonetheless, it gave me a thread to follow. The optimisation of antibody combinations for imaging took a while. Some stains worked, but a lot didn't. I remember after spending hours in the dark at the microscope, not observing much, I finally stumbled upon a staining combination that worked! The rosette looked like the ‘Eye of Sauron’ from Lord of the Rings and is now printed on my favourite jumper!

And what about the flipside: any moments of frustration or despair?

RF: Oh plenty! For starters, the 16p11.2 deletion cell lines were not the easiest to maintain. We constantly had to tweak and optimise the protocols to keep them happy. They needed special care, otherwise they died, and I had to start all over again. Then, there was the pandemic, which meant shortages of media and delays in supplies. I remember having to discard tens of organoids, cutting my experimental sample size by half because the deliveries were delayed or cancelled. Also, I became a mother of two while working on this project. Unfortunately, being an international student, I didn't get maternity leave. This meant going to the lab and running experiments at night, having Zoom meetings in the park, and sometimes even attending lab meetings with my children! It was challenging, but thankfully everyone was supportive.

Rana, what is next for you after this paper?

RF: Currently, I am working on modelling human tumours using 3D-organoid systems. Specifically, I am looking into the interaction of cancer organoids with different immune cell populations in vitro to better understand the roles of tumour-infiltrating immune cells and characterise their subtypes both in primary cancer and in metastases. Tumour-derived organoids and immune cell co-cultures offer a dynamic way to test tumour-specific therapies and, hopefully, establish a more personalised approach to immunotherapy and cancer treatment. As I explore the great potential of organoid models in research, I hope that one day my research path takes me to the field of neuro-oncology.

David, where will this story take your lab next?

DP: In my opinion, one of Rana's most intriguing findings is that the 16p11.2 deletion made some aspects of the organoid development much more variable than normal. It emphasises that a mutation's main phenotype might be increased variability, even if there is no effect on the average of whatever is being measured. I find this interesting because it fits with the idea that mutations such as this do not inevitably lead to abnormalities, but they increase the chances that things will deviate. We see a similar thing with other genes, such as Pax6. This might be part of the reason why some mutations have such variable penetrance; they increase the risk of things going awry, but that doesn't mean they necessarily will do. Many genes might be important because they somehow provide belt-and-braces for mechanisms that, in an ideal setting, don't need those genes. I'd like to explore this idea a bit more.

Finally, let's move outside the lab – what do you like to do in your spare time?

RF: If I am not pipetting in the lab, then I am mixing colours as I paint or sketch. In fact, I have recently illustrated a children's scientific book – a project I worked on with my partner for our little ones that we eventually published. Hopefully the first of many. I also play the fiddle, guitar and keyboards; not in any way professionally, but good enough to unleash the emotions within.

DP: I grow vegetables and love cooking, eating and drinking with friends and family. I love cycling, particularly touring, and other outdoor things like climbing the Scottish hills. I too like drawing and painting and enjoy music of all sorts.