Deciphering how the dazzling diversity of neuronal types of the nervous system (CNS) emerges from seemingly identical neural progenitors during development constitutes a long-standing question in Neuroscience. We proposed to address this question in the neural retina, a network of >70 types of neurons providing easy experimental access compared to the brain. The project will combine state-of-the art technologies developed by two research teams at Institut de la Vision for cell lineage tracing with combinatorial labels and functional neuron characterization with multiphoton Calcium imaging and optogenetics. Using these approaches, we will mark clones of retinal neurons originating from individual retinal progenitor cells during development, and functionally characterize their cell-type composition. This will enable us to understand the logic of neuronal type production by retinal progenitors and probe how the clonal origin of retinal neurons influences their connectivity.

The central nervous system (CNS) comprises a dazzling diversity of neuronal types whose specific morphology, connectivity and functional properties are key to proper circuit operation. Deciphering the origin of this diversity constitutes a long-standing question in Neuroscience, whose answer will have profound fundamental and translational implications. The neural retina is a model of choice to address this question: it comprises a vast diversity of neurons which can be grouped in 6 main classes occupying specific retinal layers, and >70 neuronal types interspaced across its surface according to semi-regular mosaic patterns. Embryologically, the retina derives from the diencephalon and is thus part of the CNS. As in the brain, all retinal neurons are generated during development from seemingly identical neural progenitors. Lineage tracing studies conducted in vivo and in vitro have demonstrated that early retinal progenitor cells (RPCs) are multipotent and produce the 6 classes of retinal neurons in sequential yet partially overlapping waves: ganglion cells (RGCs), followed by cone photoreceptors (PRs), horizontal cells (HCs), amacrine cells (ACs), followed by rod PRs and bipolar cells (BCs). Clones generated by individual RPCs typically form columns comprising cells of all retinal classes.

According to the prevalent model, RPCs may transition through different states of competence to generate these different classes of neurons. However, this model does not explain how the full diversity of retinal neuron types is generated. An intriguing possibility is that cell lineage could play a role in the process of cell type determination in the retina. Indeed, certain types of amacrine cells have been shown to be already specified around their birth, and direction-selective RGCs may originate from a molecularly defined RPC subpopulation. Testing whether retinal type identity may be encoded in their lineage requires tracing back their developmental origin with both single-cell precision and high throughput. Yet, clonal analysis techniques based on monochrome markers require very sparse labeling to achieve clonal resolution. The Brainbow strategy efficiently resolves cells in a population using combinations of fluorescent protein (FPs) which are faithfully transmitted to their progeny through cell division. Based on this idea, the Livet team at Institut de la Vision (IDV) has developed optimized Brainbow transgenes and mouse lines for multiplex clonal tracking in the developing nervous system, using FP color labels activated by Cre/lox recombination [1, 2]. These transgenes efficiently delineate the columns of retinal neurons generated by individual RPCs. The team has also introduced a breakthrough scheme termed “iOn” to experimentally manipulate neural stem cells and their output using exogenous DNA transgenes activated by genomic integration, thanks to a novel type of genetic switch [3]. iOn vectors open the way to complex genetic manipulations and provide a new avenue to rapidly probe gene function in the intact retina through simple electroporation of RPCs. The Emiliani and Marre teams (also at IDV), who will collaborate on the project, have established methods to finely characterize retinal neuron types by recording their response to different light stimuli using Calcium imaging [4].

The proposed project will aim at probing the relation between neuronal identity and linage in the retina, by applying the above methodologies to characterize the cell type composition of clones of retinal neurons originating from individual mouse RPCs, in normal and experimentally perturbed contexts.
The first objective of the PhD will be to establish the conditions for combined functional and lineage characterization of mouse retinal neurons. The student will generate retinas with multicolor labeling of retinal clones by triggering Brainbow clonal labels in RPCs at the beginning of retinal neurogenesis. Functional analysis will take place in mature retinas ex vivo, using a genetically encoded fluorescent Calcium sensor expressed by AAV vectors enabling restricted labeling of neuronal classes (BCs, ACs or RGCs). Calcium activity will be recorded by two-photon imaging in response to an array of light stimulation patterns, enabling to functionally classify cell types according to their response. Following this, retinal clones will be mapped at high resolution with multichannel confocal microscopy.
Based on this approach, the second objective of the project will be to probe the cell-type composition of retinal clones. We will first seek to characterize biases in the composition of retinal clones within individual classes of neurons, starting with RGCs and followed by ACs and BCs. For instance, we will test whether clonally-related RGCs more frequently comprise (or exclude) certain cell types or associations of cell types (such as OFF, ON-OFF, fast-ON…). We may then try to extend our analysis to clonal biases between distinct classes of retinal neurons, such as RGCs and BCs. Results from this part of the project will provide unprecedented data on the links between the clonal and functional organization of the retina.
In a third objective, we will test to which extent clonally-related neurons are functionally connected. Using the approach established in Objective 1, we aim at defining maps of functional connections between distinct classes of retinal neurons clonally-related in a column. We will employ two-photons optogenetics to activate presynaptic cells and to monitor post-synaptic cells activity with Calcium imaging. We will use AAVs to genetically encode a protein sensitive to light (opsin) in a presynaptic class of neurons, i.e. BCs, and a Calcium indicator in a different class of neuron, i.e. RGCs [4]. Emiliani and Marre teams demonstrated previously that it is possible to locally map local connections between neurons in the retina and in cortical layer2/3, by activating one or more pre-synaptic cells with targeted two-photons illumination and by simultaneously recording the activity in post synaptic cells with Calcium imaging or single cell electrophysiological recordings [4,5].

The project will take place at IDV in the center of Paris. The student will be co-mentored by Jean Livet (neurodevelopment, genetic engineering) and Valeria Zampini (functional imaging and optogenetics), with the collaboration of Olivier Marre (neurophysiology of the retina). Overall, the project will provide a quantitative exploration at the single-cell level of the links between cell lineage and functional organization in the retina. It will enable to tackle major questions, such as the composition of retinal clonal units. It will also provide a rich multidisciplinary training for the PhD student concerning state-of-the art techniques to analyze and link neuronal function and development. The approaches generated during this project will have general applicability in Neuroscience.

References :
1. K. Loulier et al. Neuron 2014
2. S. Clavreul et al. Nat Commun 2019
3. T. Kumamoto et al. Neuron 2020
4. G. Spampinato et al., Cell Rep Methods, 2022
5. Shemesh et al., Nat Neurosci. 2017