Periodic Reporting for period 1 - NeuRemodelBehavior (Sculpting circuits and behavior by developmental neuronal remodeling)
Reporting period: 2022-05-01 to 2024-10-31
This proposal aims to understand the mechanisms of developmental neuronal remodeling: at the (Obj1) molecular level; at the (Obj2) neural circuits; and the (Obj3) function level of the circuit. The stereotypic developmental remodeling of the Drosophila mushroom body (MB) is a unique system for combining the molecular, circuit and behavioral aspects. This is due to the awesome genetic power of Drosophila, combined with our EM-resolution knowledge of the circuit in larva and in adult, as well as the extensive tools and knowledge of the role of the MB in learning and memory. We have made substantial progress in our work towards achieving our goals, and published, thus far, three erc-funded research articles in Curr Biol, Cell Rep and Open Biol, one preprint in bioRxiv, and two reviews in Front Neurosci and Learn Mem. Additionally, we are preparing four manuscripts for publication and making progress in all directions proposed.
Obj 1 focuses on the mechanisms that regulate remodeling, with a specific emphasis on cell-cell communication via cell surface proteins (CSPs).
In Obj1.1 we completed the single gene RNAi knockdown (KD) of Dprs/DIPs and Beats/Sides, among other CSPs. Unfortunately, we hit a setback on our research on two molecules originally proposed – DIP-α and DIP-β. However, we found other molecules – including DIP-γ, Beat-Ia and Beat-VII – to be required for axon pruning, and are currently exploring the mechanistic basis. In this aim we originally proposed a cis-inhibition mechanism underlying the DIP-α/β phenotypes. While these specific molecules may no longer be relevant, the proposed mechanism remains valid for other molecules. Furthermore, our hypothesis led us to collaborate with the Honig-Shapiro lab at Columbia University, and together we established cis inhibition among Dpr/DIPs in vitro, as well as potential in vivo occurrence in MB development: Morano et al, bioRxiv 2024.03.04.583391. As mentioned in the original proposal, we also expanded our study to glia-neuron interactions. We recently published that astroctyes are not passive bystanders but actually play instructive roles in reducing axon-axon adhesion by infiltrating the axon bundle in a process that depends on astrocytic actin dynamics (Marmor-Kollet et al and Schuldiner, Cell Rep, 42, 112117, 2023). In a parallel project, which is under preparation, we revealed the requirement of CSPs of the matrix metalloproteinase (MMP) family in both glia and neurons for pruning of MB axons.
In Obj1.2 we have made substantial progress and are preparing a manuscript for publication.
In Obj1.3 we completed single gene KD experiment as mentioned above, and due to the redundant interactions in the Dpr-DIP families, we embarked on double RNAi KD experiments, which are ongoing. We focus on DIP-ε, as well as double KD of DIP-β and DIP-λ, as key candidates for future mechanistic research since they display defects in MB zone formation. Additionally, we made the trasngenic flies required for the proximity labeling experiments aimed at uncovering the Dpr12 and DIP-δ signaling mechanism.
Obj 2 focuses on the circuit level of neuronal remodeling – from how different cells affect each other to coordinate pruning, to how remodeling affects the circuit.
In Obj2.1 we have made progress in applying new techniques for circuit mapping, as well as constantly screening Gal4 and LexA lines to identify drivers consistently expressed in the different neurons within the MB circuit throughout remodeling. Additionaly, we published a manuscript describing how the APL (a GABAergic inhibitory neuron that innervated the entire MB) and the MB neurons coordinate their pruning. In this study, we found that neuronal excitability, at least partly controled by APL mediated inhibition, is a regulator of circuit remodeling (Mayseless et al and Schuldiner, Curr Biol, 33, 981-989.e3 2023).
In Obj2.2 we screened and identified additional Gal4 drivers expressed in cells innervating the axonal branchpoint during the pruning timeframe. We began utilizing the drivers to kill these branchpoint cells and examine how it affects the location of pruning termination.
In Obj2.3 we have made substantial progress and found, using GRASP, that unpruned axons do not seem to maintain their larval connectivity. Rather, they ‘hitchhike’ on adult-specific connectivity and are innervated by MBONs and modulatory neurons (DANs) that normally innervate other adult axons (of other neuronal types) in their immediate environment.
In Obj2,4, we determined that upon loss of the γ4/5 zones in dpr12 mutants, the MBON that typically targets the γ5 zone is redirected to the γ3 zone (similar to the γ4 MBON). We are also implementing new tools for ultra-sparse labelling to resolve the fate of the mis-projected DANs in the dpr12 mutants. In addition, we began exploring the matching between the different members of the MB circuit.
Obj 3 explores the link between neuronal remodeling and organismal behavior, which is currently unknown in any system.
In Obj3.1 we are building upon our findings in Obj2.3 and exploring whether unpruned vertical axons can compensate for the loss/silencing of the adult vertical MB lobes in mediating long term memory (LTM). We have made progress by setting up a behavioral facility in the lab and establishing aversive olfactory conditioning assays. We already optimized short term memory (STM) and are currently calibrating the LTM assay (testing 24 hours after olfactory training), as required for the proposed experiments.
Obj3.2 was completed and we are currently preparing a manuscript for publication in collaboration with the Fiala lab in the University of Göttingen. Following calibration of larval LTM assays in the lab, we found that unpruned axons cannot maintain larval-acquired memories, as predicted by our connectivity experiments, and in contrast to a previous, controversial study that claimed memory retention throughout metamorphosis (Tully 1994).
In Obj3.3 we have made substantial progress are currently preparing a manuscript for publication.
In Obj1.1 we completed the single gene RNAi knockdown (KD) of Dprs/DIPs and Beats/Sides, among other CSPs. Unfortunately, we hit a setback on our research on two molecules originally proposed – DIP-α and DIP-β. However, we found other molecules – including DIP-γ, Beat-Ia and Beat-VII – to be required for axon pruning, and are currently exploring the mechanistic basis. In this aim we originally proposed a cis-inhibition mechanism underlying the DIP-α/β phenotypes. While these specific molecules may no longer be relevant, the proposed mechanism remains valid for other molecules. Furthermore, our hypothesis led us to collaborate with the Honig-Shapiro lab at Columbia University, and together we established cis inhibition among Dpr/DIPs in vitro, as well as potential in vivo occurrence in MB development: Morano et al, bioRxiv 2024.03.04.583391. As mentioned in the original proposal, we also expanded our study to glia-neuron interactions. We recently published that astroctyes are not passive bystanders but actually play instructive roles in reducing axon-axon adhesion by infiltrating the axon bundle in a process that depends on astrocytic actin dynamics (Marmor-Kollet et al and Schuldiner, Cell Rep, 42, 112117, 2023). In a parallel project, which is under preparation, we revealed the requirement of CSPs of the matrix metalloproteinase (MMP) family in both glia and neurons for pruning of MB axons.
In Obj1.2 we have made substantial progress and are preparing a manuscript for publication.
In Obj1.3 we completed single gene KD experiment as mentioned above, and due to the redundant interactions in the Dpr-DIP families, we embarked on double RNAi KD experiments, which are ongoing. We focus on DIP-ε, as well as double KD of DIP-β and DIP-λ, as key candidates for future mechanistic research since they display defects in MB zone formation. Additionally, we made the trasngenic flies required for the proximity labeling experiments aimed at uncovering the Dpr12 and DIP-δ signaling mechanism.
Obj 2 focuses on the circuit level of neuronal remodeling – from how different cells affect each other to coordinate pruning, to how remodeling affects the circuit.
In Obj2.1 we have made progress in applying new techniques for circuit mapping, as well as constantly screening Gal4 and LexA lines to identify drivers consistently expressed in the different neurons within the MB circuit throughout remodeling. Additionaly, we published a manuscript describing how the APL (a GABAergic inhibitory neuron that innervated the entire MB) and the MB neurons coordinate their pruning. In this study, we found that neuronal excitability, at least partly controled by APL mediated inhibition, is a regulator of circuit remodeling (Mayseless et al and Schuldiner, Curr Biol, 33, 981-989.e3 2023).
In Obj2.2 we screened and identified additional Gal4 drivers expressed in cells innervating the axonal branchpoint during the pruning timeframe. We began utilizing the drivers to kill these branchpoint cells and examine how it affects the location of pruning termination.
In Obj2.3 we have made substantial progress and found, using GRASP, that unpruned axons do not seem to maintain their larval connectivity. Rather, they ‘hitchhike’ on adult-specific connectivity and are innervated by MBONs and modulatory neurons (DANs) that normally innervate other adult axons (of other neuronal types) in their immediate environment.
In Obj2,4, we determined that upon loss of the γ4/5 zones in dpr12 mutants, the MBON that typically targets the γ5 zone is redirected to the γ3 zone (similar to the γ4 MBON). We are also implementing new tools for ultra-sparse labelling to resolve the fate of the mis-projected DANs in the dpr12 mutants. In addition, we began exploring the matching between the different members of the MB circuit.
Obj 3 explores the link between neuronal remodeling and organismal behavior, which is currently unknown in any system.
In Obj3.1 we are building upon our findings in Obj2.3 and exploring whether unpruned vertical axons can compensate for the loss/silencing of the adult vertical MB lobes in mediating long term memory (LTM). We have made progress by setting up a behavioral facility in the lab and establishing aversive olfactory conditioning assays. We already optimized short term memory (STM) and are currently calibrating the LTM assay (testing 24 hours after olfactory training), as required for the proposed experiments.
Obj3.2 was completed and we are currently preparing a manuscript for publication in collaboration with the Fiala lab in the University of Göttingen. Following calibration of larval LTM assays in the lab, we found that unpruned axons cannot maintain larval-acquired memories, as predicted by our connectivity experiments, and in contrast to a previous, controversial study that claimed memory retention throughout metamorphosis (Tully 1994).
In Obj3.3 we have made substantial progress are currently preparing a manuscript for publication.
I am proud that we established a behavioral assay in our lab. This will help us propel Obj. 3 forward, and has already allowed us to complete Obj. 3.2. This is a key step in achieving our goal to understand multiple aspects of cell-cell interactions during neuronal remodelling at the molecular, circuit and function levels.
Our work on the branch specific pruning (Obj. 1.2) has the potential to uncover how specific axonal parts are dismantled while others are preserved. While we are studying this in the context of neural development our findings will likely impact also ideas on how to prevent neurodegeneration.
Our ideas on cis-inhibition have resulted in a transatlantic collaboration with the Honig, Shapiro and Mann labs at Columbia University: Morano et al, bioRxiv 2024.03.04.583391. Here, the idea that expression of receptors and ligands in the same cell might provide an additional, post translational, level of regulation, is profound and generalizable.
Our published (Marmor-Kollet et al. 2023) and yet unpublished work on astro-neuron interactions is transforming our understanding on how profound the crosstalk is.
We pushed the field one step further in understanding how the circuit controls remodelling (Mayseless et al, 2023).We found, to our surprise, that Hebbian like mechanisms also apply to developmental processes in which neural activity is not apparent.
Our work on the branch specific pruning (Obj. 1.2) has the potential to uncover how specific axonal parts are dismantled while others are preserved. While we are studying this in the context of neural development our findings will likely impact also ideas on how to prevent neurodegeneration.
Our ideas on cis-inhibition have resulted in a transatlantic collaboration with the Honig, Shapiro and Mann labs at Columbia University: Morano et al, bioRxiv 2024.03.04.583391. Here, the idea that expression of receptors and ligands in the same cell might provide an additional, post translational, level of regulation, is profound and generalizable.
Our published (Marmor-Kollet et al. 2023) and yet unpublished work on astro-neuron interactions is transforming our understanding on how profound the crosstalk is.
We pushed the field one step further in understanding how the circuit controls remodelling (Mayseless et al, 2023).We found, to our surprise, that Hebbian like mechanisms also apply to developmental processes in which neural activity is not apparent.