Actin-dependent astrocytic infiltration is a key step for axon defasciculation during remodeling.

Astrocytes are essential for synapse formation, maturation, and plasticity; however, their function during developmental neuronal remodeling is largely unknown. To identify astrocytic molecules required for axon pruning of mushroom body (MB) γ neurons in Drosophila, we profiled astrocytes before (larva) and after (adult) remodeling. Focusing on genes enriched in larval astrocytes, we identified 12 astrocytic genes that are required for axon pruning, including the F-actin regulators Actin-related protein 2/3 complex, subunit 1 (Arpc1) and formin3 (form3). Interestingly, perturbing astrocytic actin dynamics does not affect their gross morphology, migration, or transforming growth factor ß (TGF-ß) secretion. In contrast, actin dynamics is required for astrocyte infiltration into the axon bundle at the onset of pruning. Remarkably, decreasing axonal adhesion facilitates infiltration by Arpc1 knockdown (KD) astrocytes and promotes axon pruning. Conversely, increased axonal adhesion reduces lobe infiltration by wild-type (WT) astrocytes. Together, our findings suggest that actin-dependent astrocytic infiltration is a key step in axon pruning, thus promoting our understanding of neuron-glia interactions during remodeling.

Glial Derived TGF-ß Instructs Axon Midline Stopping.

A fundamental question that underlies the proper wiring and function of the nervous system is how axon extension stops during development. However, our mechanistic understanding of axon stopping is currently poor. The stereotypic development of the Drosophila mushroom body (MB) provides a unique system in which three types of anatomically distinct neurons (γ, α'/ß', and α/ß) develop and interact to form a complex neuronal structure. All three neuronal types innervate the ipsi-lateral side and do not cross the midline. Here we find that Plum, an immunoglobulin (Ig) superfamily protein that we have previously shown to function as a TGF-ß accessory receptor, is required within MB α/ß neurons for their midline stopping. Overexpression of Plum within MB neurons is sufficient to induce retraction of α/ß axons. As expected, rescue experiments revealed that Plum likely functions in α/ß neurons and mediates midline stopping via the downstream effector RhoGEF2. Finally, we have identified glial-derived Myoglianin (Myo) as the major TGF-ß ligand that instructs midline stopping of MB neurons. Taken together, our study strongly suggests that TGF-ß signals originating from the midline facilitate midline stopping of α/ß neuron in a Plum dependent manner.

Combining Developmental and Perturbation-Seq Uncovers Transcriptional Modules Orchestrating Neuronal Remodeling.

Developmental neuronal remodeling is an evolutionarily conserved mechanism required for precise wiring of nervous systems. Despite its fundamental role in neurodevelopment and proposed contribution to various neuropsychiatric disorders, the underlying mechanisms are largely unknown. Here, we uncover the fine temporal transcriptional landscape of Drosophila mushroom body γ neurons undergoing stereotypical remodeling. Our data reveal rapid and dramatic changes in the transcriptional landscape during development. Focusing on DNA binding proteins, we identify eleven that are required for remodeling. Furthermore, we sequence developing γ neurons perturbed for three key transcription factors required for pruning. We describe a hierarchical network featuring positive and negative feedback loops. Superimposing the perturbation-seq on the developmental expression atlas highlights a framework of transcriptional modules that together drive remodeling. Overall, this study provides a broad and detailed molecular insight into the complex regulatory dynamics of developmental remodeling and thus offers a pipeline to dissect developmental processes via RNA profiling.

Contrasting developmental axon regrowth and neurite sprouting of Drosophila mushroom body neurons reveals shared and unique molecular mechanisms.

The molecular mechanisms regulating intrinsic axon growth potential during development or following injury remain largely unknown despite their vast importance. Here, we have established a neurite sprouting assay of primary cultured mushroom body (MB) neurons. We used the MARCM technique to both mark and manipulate MB neurons, enabling us to quantify the sprouting abilities of single WT and mutant neurons originating from flies at different developmental stages. Sprouting of dissociated MB neurons was dependent on wnd, the DLK ortholog, a conserved gene that is required for axon regeneration. Next, and as expected, we found that the sprouting ability of adult MB neurons was significantly decreased. In contrast, and to our surprise, we found that pupal-derived neurons exhibit increased sprouting compared with neurons derived from larvae, suggesting the existence of an elevated growth potential state. We then contrasted the molecular requirements of neurite sprouting to developmental axon regrowth of MB ɣ neurons, a process that we have previously shown requires the nuclear receptor UNF acting via the target of rapamycin (TOR) pathway. Strikingly, we found that while TOR was required for neurite sprouting, UNF was not. In contrast, we found that PTEN inhibits sprouting in adult neurons, suggesting that TOR is regulated by the PI3K/PTEN pathway during sprouting and by UNF during developmental regrowth. Interestingly, the PI3K pathway as well as Wnd were not required for developmental regrowth nor for initial axon outgrowth suggesting that axon growth during circuit formation, remodeling, and regeneration share some molecular components but differ in others.