Periodic Reporting for period 2 - SynPromiscuity (Synaptic Promiscuity in Brain Development)
Reporting period: 2023-04-01 to 2024-09-30
Genetically encoded development robustly produces brains, but how the genome ensures functional brain connectivity has remained a major scientific riddle. Sequence variations in the genome underlying the developmental processes can increase the probability for neurodevelopmental disorders like autism and schizophrenia. Yet, it has been difficult to pinpoint the contribution of single gene products and how they fit into the developmental programs that lead to brain wiring.
The overarching goal of SynPromiscuity (101019191) is to understand how the genetically encoded developmental programs unfold the daunting specificity and robustness of functional connectivity of the brain. The core hypothesis of 'Synaptic Promiscuity in Brain Development' posits that successive developmental programs restrict what potential synaptic partners encounter each other at the moment of partner choice; the precision and robustness observed in the outcome can only be understood as the product of such a series of successive developmental steps. In the limiting case, the developmental program prior to synapse formation ensures that only correct synaptic partners 'get to see each other', thereby allowing for promiscuity at the moment of synapse formation without losing specificity. The notion that synapses can form at least somewhat promiscuously is supported by a plethora of observations revealing that neurons are capable of making synapses with incorrect partners when given the opportunity. On the other hand, a number of quantitative contributors may further specify partners at the moment of choice, including interaction dynamics, competence to assemble the required pre- and postsynaptic protein machinery, and pre-post cell adhesion. The objective of SynPromiscuity is thus to test (1) to what extent development prior to the moment of choice specifies 'who gets to see each other', and (2) the quantitative contributions of each of the three mechanisms (interaction kinetics, synaptic competency, molecular recognition). To test the SynPromiscuity hypothesis and measure the contributions to synaptic partner selection in a quantitative fashion, we combine genetics and live imaging of Drosophila brain development prior to and at the moment of choice. We have recently published a summary and context of the overall objective of the project in a review article bearing the title of the ERC Project Title 'Synaptic Promiscuity in Brain Development' (Current Biology 2024, doi: 10.1016/j.cub.2023.12.037)
The overarching goal of SynPromiscuity (101019191) is to understand how the genetically encoded developmental programs unfold the daunting specificity and robustness of functional connectivity of the brain. The core hypothesis of 'Synaptic Promiscuity in Brain Development' posits that successive developmental programs restrict what potential synaptic partners encounter each other at the moment of partner choice; the precision and robustness observed in the outcome can only be understood as the product of such a series of successive developmental steps. In the limiting case, the developmental program prior to synapse formation ensures that only correct synaptic partners 'get to see each other', thereby allowing for promiscuity at the moment of synapse formation without losing specificity. The notion that synapses can form at least somewhat promiscuously is supported by a plethora of observations revealing that neurons are capable of making synapses with incorrect partners when given the opportunity. On the other hand, a number of quantitative contributors may further specify partners at the moment of choice, including interaction dynamics, competence to assemble the required pre- and postsynaptic protein machinery, and pre-post cell adhesion. The objective of SynPromiscuity is thus to test (1) to what extent development prior to the moment of choice specifies 'who gets to see each other', and (2) the quantitative contributions of each of the three mechanisms (interaction kinetics, synaptic competency, molecular recognition). To test the SynPromiscuity hypothesis and measure the contributions to synaptic partner selection in a quantitative fashion, we combine genetics and live imaging of Drosophila brain development prior to and at the moment of choice. We have recently published a summary and context of the overall objective of the project in a review article bearing the title of the ERC Project Title 'Synaptic Promiscuity in Brain Development' (Current Biology 2024, doi: 10.1016/j.cub.2023.12.037)
During the first half of the project, we have developed and applied methods to live observe developmental choices of individual neurons during the development of the intact fly brain. The key methodological approach has been intravital and ex vivo brain imaging using 2-photon microscopy. In order to live observe single neurons at the level of synapse formation during normal brain development, we have developed and used an increasing toolkit of cell-specific driver lines as well as reporter lines devised to stochastically and sparsely label neurons and their filopodial interactions. These tools and methods were applied to the developmental choices prior to the moment of choice (i.e. during developmental choices that restrict subsequently available partners), as well as at the moment of choice itself for more than 10 different neuron types so far.
Our first major result was recently published in the journal Science in March 2024 (PMID: doi: 10.1126/science.adk3043). Here, we applied our method of intravital live imaging of the filopodial interactions to six types of photoreceptor neurons and studied how these neurons pre-specify synaptic partnerships through a self-organization process. Remarkably, this developmental process does not require the target cells, yet ensures that the right pre- and postsynaptic neurons are sorted together prior to the time of synaptic partner choice. In this study, we combined live imaging with genetic manipulation (target cell ablation) and computational modeling to provide a comprehensive model for the developmental program that ensures the correct sorting of synaptic partners prior to the moment of choice. Here, the specificity of preceding development is in fact such, that only correct partners are available for synapse formation at the time and place when synapse formation occurs. Hence, this work supports the SynPromiscuity hypothesis and further suggests that genetically encoded synaptic specificity may develop without explicitly 'tagging' the pre- and post-synaptic partners, but instead through target-independent self-organization prior to synapse formation.
We have so far developed live imaging in the intact fly pupa or in ex vivo brain culture for a number of optic lobe interneurons, including L3, Mi1, M4, Dm4 and Dm8. The roles of the observed dynamics of these neurons for the developmental programs leading to synapse-specific brain wiring are ongoing.
Our first major result was recently published in the journal Science in March 2024 (PMID: doi: 10.1126/science.adk3043). Here, we applied our method of intravital live imaging of the filopodial interactions to six types of photoreceptor neurons and studied how these neurons pre-specify synaptic partnerships through a self-organization process. Remarkably, this developmental process does not require the target cells, yet ensures that the right pre- and postsynaptic neurons are sorted together prior to the time of synaptic partner choice. In this study, we combined live imaging with genetic manipulation (target cell ablation) and computational modeling to provide a comprehensive model for the developmental program that ensures the correct sorting of synaptic partners prior to the moment of choice. Here, the specificity of preceding development is in fact such, that only correct partners are available for synapse formation at the time and place when synapse formation occurs. Hence, this work supports the SynPromiscuity hypothesis and further suggests that genetically encoded synaptic specificity may develop without explicitly 'tagging' the pre- and post-synaptic partners, but instead through target-independent self-organization prior to synapse formation.
We have so far developed live imaging in the intact fly pupa or in ex vivo brain culture for a number of optic lobe interneurons, including L3, Mi1, M4, Dm4 and Dm8. The roles of the observed dynamics of these neurons for the developmental programs leading to synapse-specific brain wiring are ongoing.
Live observation of a single neuron's choices prior to and at the moment of synaptic partner choice in an intact, normally developing brain has remained a challenge in the field. As a consequence, many live imaging approaches have been restricted to cultured neurons. Furthermore, most genetic perturbation approaches have been conducted without identifying or live observing the actual live moment of partner choice; instead, it remains common in the field to focus on fixed preparations of the adult outcome. By contrast, our approach is to live observe - both in wild type as well as under condition of targeted genetic perturbation - how neurons choose their partners. This approach is currently beyond state of the art for most in vivo systems; in flies, our work is made possible by recent advanced in genetics (driver and reporter lines) and the size and time frame of Drosophila brain development.
Our recent publication of a comprehensive model for Drosophila visual map formation based on live imaging data represents an example of the power of this approach. In this work, we made better progress than anticipated at the beginning of SynPromiscuity project. The findings support a limiting case of the SynPromiscuity hypothesis, namely a case where development prior to synapse formation ensures correct partnerships to such an extend that synapse formation can, in theory, occur with almost complete promiscuity at the actual moment of choice. This prediction remains to be tested.
Our subsequent live imaging efforts of the moment of choice are yielding similarly exciting results, but are ongoing. The key goal is to quantitatively establish the contributions of interaction dynamics, synaptic competency and molecular target adhesion for at least three synaptic pairs in the developing fly brain. We expect to provide such quantitative assessments by the end of the project, as planned.
Our recent publication of a comprehensive model for Drosophila visual map formation based on live imaging data represents an example of the power of this approach. In this work, we made better progress than anticipated at the beginning of SynPromiscuity project. The findings support a limiting case of the SynPromiscuity hypothesis, namely a case where development prior to synapse formation ensures correct partnerships to such an extend that synapse formation can, in theory, occur with almost complete promiscuity at the actual moment of choice. This prediction remains to be tested.
Our subsequent live imaging efforts of the moment of choice are yielding similarly exciting results, but are ongoing. The key goal is to quantitatively establish the contributions of interaction dynamics, synaptic competency and molecular target adhesion for at least three synaptic pairs in the developing fly brain. We expect to provide such quantitative assessments by the end of the project, as planned.