Patronin/CAMSAP promotes reactivation and regeneration of Drosophila quiescent neural stem cells.

The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. Drosophila quiescent neural stem cells (qNSCs) extend a primary protrusion that is enriched in acentrosomal microtubules and can be regenerated upon injury. Arf1 promotes microtubule growth, reactivation (exit from quiescence), and regeneration of qNSC protrusions upon injury. However, how Arf1 is regulated in qNSCs remains elusive. Here, we show that the microtubule minus-end binding protein Patronin/CAMSAP promotes acentrosomal microtubule growth and quiescent NSC reactivation. Patronin is important for the localization of Arf1 at Golgi and physically associates with Arf1, preferentially with its GDP-bound form. Patronin is also required for the regeneration of qNSC protrusion, likely via the regulation of microtubule growth. Finally, Patronin functions upstream of Arf1 and its effector Msps/XMAP215 to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. Our findings reveal a novel link between Patronin/CAMSAP and Arf1 in the regulation of microtubule growth and NSC reactivation. A similar mechanism might apply to various microtubule-dependent systems in mammals.

Histone lysine methyltransferase Pr-set7/SETD8 promotes neural stem cell reactivation.

The ability of neural stem cells (NSCs) to switch between quiescence and proliferation is crucial for brain development and homeostasis. Increasing evidence suggests that variants of histone lysine methyltransferases including KMT5A are associated with neurodevelopmental disorders. However, the function of KMT5A/Pr-set7/SETD8 in the central nervous system is not well established. Here, we show that Drosophila Pr-Set7 is a novel regulator of NSC reactivation. Loss of function of pr-set7 causes a delay in NSC reactivation and loss of H4K20 monomethylation in the brain. Through NSC-specific in vivo profiling, we demonstrate that Pr-set7 binds to the promoter region of cyclin-dependent kinase 1 (cdk1) and Wnt pathway transcriptional co-activator earthbound1/jerky (ebd1). Further validation indicates that Pr-set7 is required for the expression of cdk1 and ebd1 in the brain. Similar to Pr-set7, Cdk1 and Ebd1 promote NSC reactivation. Finally, overexpression of Cdk1 and Ebd1 significantly suppressed NSC reactivation defects observed in pr-set7-depleted brains. Therefore, Pr-set7 promotes NSC reactivation by regulating Wnt signaling and cell cycle progression. Our findings may contribute to the understanding of mammalian KMT5A/PR-SET7/SETD8 during brain development.

RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in Caenorhabditis elegans.

UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions are consistent with RHO-1 and RHGF-1 acting with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.

Flavin monooxygenases regulate Caenorhabditis elegans axon guidance and growth cone protrusion with UNC-6/Netrin signaling and Rac GTPases.

The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5:UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5:UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein αq subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

Correction to: A fly's eye view of quiescent neural stem cells.

[This corrects the article DOI: 10.1093/oons/kvac001.].

Golgi-dependent reactivation and regeneration of Drosophila quiescent neural stem cells.

The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. In Drosophila, quiescent neural stem cells (qNSCs) extend a primary protrusion, a hallmark of qNSCs. Here, we have found that qNSC protrusions can be regenerated upon injury. This regeneration process relies on the Golgi apparatus that acts as the major acentrosomal microtubule-organizing center in qNSCs. A Golgi-resident GTPase Arf1 and its guanine nucleotide exchange factor Sec71 promote NSC reactivation and regeneration via the regulation of microtubule growth. Arf1 physically associates with its new effector mini spindles (Msps)/XMAP215, a microtubule polymerase. Finally, Arf1 functions upstream of Msps to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. Our findings have established Drosophila qNSCs as a regeneration model and identified Arf1/Sec71-Msps pathway in the regulation of microtubule growth and NSC reactivation.

A fly's eye view of quiescent neural stem cells.

The balance between proliferation and quiescence of stem cells is crucial in maintaining tissue homeostasis. Neural stem cells (NSCs) in the brain have the ability to be reactivated from a reversible quiescent state to generate new neurons. However, how NSCs transit between quiescence and reactivation remains largely elusive. Drosophila larval brain NSCs, also known as neuroblasts, have emerged as an excellent in vivo model to study molecular mechanisms underlying NSC quiescence and reactivation. Here, we discuss our current understanding of the molecular mechanisms underlying the reactivation of quiescent NSCs in Drosophila. We review the most recent advances on epigenetic regulations and microtubule cytoskeleton in Drosophila quiescent NSCs and their cross-talk with signaling pathways that are required in regulating NSC reactivation.

Control of Growth Cone Polarity, Microtubule Accumulation, and Protrusion by UNC-6/Netrin and Its Receptors in Caenorhabditis elegans.

UNC-6/Netrin has a conserved role in dorsal-ventral axon guidance, but the cellular events in the growth cone regulated by UNC-6/Netrin signaling during outgrowth are incompletely understood. Previous studies showed that, in growth cones migrating away from UNC-6/Netrin, the receptor UNC-5 regulates growth cone polarity, as observed by polarized F-actin, and limits the extent of growth cone protrusion. It is unclear how UNC-5 inhibits protrusion, and how UNC-40 acts in concert with UNC-5 to regulate polarity and protrusion. New results reported here indicate that UNC-5 normally restricts microtubule (MT) + end accumulation in the growth cone. Tubulin mutant analysis and colchicine treatment suggest that stable MTs are necessary for robust growth cone protrusion. Thus, UNC-5 might inhibit protrusion in part by restricting growth cone MT accumulation. Previous studies showed that the UNC-73/Trio Rac GEF and UNC-33/CRMP act downstream of UNC-5 in protrusion. Here, we show that UNC-33/CRMP regulates both growth cone dorsal asymmetric F-actin accumulation and MT accumulation, whereas UNC-73/Trio Rac GEF activity only affects F-actin accumulation. This suggests an MT-independent mechanism used by UNC-5 to inhibit protrusion, possibly by regulating lamellipodial and filopodial actin. Furthermore, we show that UNC-6/Netrin and the receptor UNC-40/DCC are required for excess protrusion in unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of, or in parallel to, F-actin asymmetry and MT + end entry. F-actin accumulation might represent a polarity mark in the growth cone where protrusion will occur, and not protrusive lamellipodial and filopodial actin per se Our data suggest a model in which UNC-6/Netrin first polarizes the growth cone via UNC-5, and then regulates protrusion based upon this polarity (the polarity/protrusion model). UNC-6/Netrin inhibits protrusion ventrally via UNC-5, and stimulates protrusion dorsally via UNC-40, resulting in dorsally-directed migration. The polarity/protrusion model represents a novel conceptual paradigm in which to understand axon guidance and growth cone migration away from UNC-6/Netrin.