Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. This study shows that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, it was shown that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. These results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized (Brock, 2012).

The traditional model of the actin cytoskeleton in cell migration, based on in vitro cell culture and biochemical assays, provides a useful framework for the mechanics of how cell migration is regulated. However, there is need for in vivo studies in order to answer important questions that are not addressed by the current model: 1. Is there a role for Profilin-mediated recycling during wound-induced migration of differentiated cells in vivo? 2. Is there a role for transcriptional regulation of actin regulators during such migrations? This latter question emerges because the basic model generally assumes that migratory cells have an intact actin-regulatory apparatus that needs only to be activated to initiate and sustain migration. While this assumption may be correct for migrating cells in developmental contexts one could imagine that initially non-migratory differentiated cells may need more than their resting complement of actin regulators in order to effect long-distance migration (Brock, 2012).

Unwounded larval epidermal cells have an even distribution of actin and Profilin throughout the cytoplasm and are thought to be non-migratory. These fully differentiated epithelial cells secrete an apical cuticle and a basal lamina. They respond to the physiological signal of tissue damage by partially dedifferentiating and becoming migratory. This study shows that the leading edge cells form multiple actin-based structures including a discontinuous cable, filopodia, and lamellipodia. A working model is proposed where the basal levels of Profilin are sufficient to make actin-based structures, but wound-induced transcription of chic is required for the cells to efficiently migrate. The lack of actin-based structures at the wound edge in cells lacking Profilin would indicate that if Formin-mediated actin nucleation is involved in wound healing, it likely requires Profilin. An epidermal sheet lacking detectable Profilin fails to close wounds or form any actin-based structures at the wound edge whereas a sheet containing only a basal level of Profilin (i.e., one that is lacking proteins that transcriptionally regulate Profilin after wounding, such as JNK, Fos, or Jun) forms actin structures at the wound edge, but is ultimately unable to efficiently migrate and close the wound. This model is complicated by the fact that cells lacking JNK, Fos, or Jun also have defects in dedifferentiation, as these cells do not stop secreting cuticle following wounding. Thus, the possibility cannot completely excluded that the lack of wound closure is due to defects in dedifferentiation. However, it is entirely possible that upregulation of actin-binding regulators is an important component of the dedifferentiation process, as this involves returning to a state during which these cells were competent to migrate (Brock, 2012).

Current wound closure models have identified two signaling pathways that are important for healing. One is Pvr signaling, where the secreted VEGF-like ligand Pvf1 activates the Pvr receptor. Currently, only a few proteins are suspected of being downstream of Pvr signaling, but Profilin is not among them. Given that epidermal cells lacking Pvr are unable to mobilize actin to the wound edge, Pvr is likely upstream of actin regulatory proteins that initiate actin polymerization at the leading edge of migrating cells. The second pathway is JNK signaling, which is required for closure but not for actin polymerization at the wound edge. Naively, it was anticipated that wound-induced chic expression would be regulated by Pvr since epidermal expression of UAS-chicRNAi also blocks actin accumulation at the wound edge. Surprisingly, this is not the case. chiclacZ expression is instead regulated by JNK signaling, as it is in the developing embryo during DC . This data reveals that although the JNK signaling pathway is not required for actin nucleation at the wound edge it contributes to actin dynamics through regulating expression of chic and perhaps other genes important for migration (Brock, 2012).

How does JNK signaling activate chic transcription after wounding? Although the upstream signal for the JNK signaling pathway is still unknown, the kinase cascade is well-defined and is thought to culminate with the phosphorylation of the transcription factors, DJun and DFos. These two proteins are commonly thought to act as a dimer (AP-1) to mediate transcriptional activation of target genes. In the early DC studies chickadee expression was shown to depend on the JNK signaling pathway. This study did not address the roles of DJun and DFos in particular, although these transcription factors are required for DC. In wound healing contexts, however, it appears that DFos can act without DJun to activate a ddc-wound reporter and a msn-lacZ wound reporter. This study found that both DJun and DFos are required to activate chic. Additionally, two consensus binding sequences for the AP-1 transcription factor (TGANTCA) are located upstream of the chic start codon (depending on the message isoform the sites are located in the 5’UTR, the first intron, or the promoter region), indicating that it is at least possible that the upregulation of chic transcription is directly accomplished by Jun and Fos. The consensus sequence is also located upstream of the human Pfn1, indicating that there is potential for this regulation to be conserved. This suggests that in the migrating cells at the wound edge, DFos can act either as a homodimer, with unidentified binding partners, or with DJun to regulate the necessary transcriptional targets (Brock, 2012).

In Drosophila embryonic models of wound closure both the contractile actin cable and filopodial processes are important for wound closure, but their relative contributions are still unclear. There has been debate over whether the cable mediates closure through contraction, through serving as a platform for extension of processes into the wound gap, or through a combinaton of these functions. From the data shown in this study it seems that actin-based contraction is not a major contributor to larval wound closure. First, the actin concentrations that appear at larval wound edges are discontinuous. Second they do not appear to be locally contractile given that the cells behind prominent concentrations do not obviously taper toward the wound. This is similar to what has been observed in the embryonic Xenopus epithelium where actin cables form but differently shaped wounds do not round up as would be expected from cable contraction. Thus it would appear that in larvae the actin concentrated at the wound edge primarily facilitates process extension into the wound gap (Brock, 2012).

This study has establish a connection between a known wound-induced signaling pathway, JNK signaling, and Profilin-mediated regulation of the actin cytoskeleton. It is speculated that transcriptional induction of actin-regulators may be a general feature of cell migration in differentiated cells as suggested by a recent study of cells undergoing EMT. By connecting upstream signaling pathways to downstream actin dynamics, this work begins to unravel the logic of how the cellular movements required for wound closure are orchestrated (Brock, 2012).

Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) γ neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches. This study identified the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting. Whereas Imp is dispensable for the initial growth of MB γ neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, it was demonstrated that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. This study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation (Medroni, 2014).

In cultured vertebrate neurons, ZBP1 mediates the transport of β-actin mRNA to axons, a process required for the chemiotropic response of growth cones to guidance cues. Whether these observations reflect a general requirement for ZBP1 and axonal mRNA transport during brain development has remained unclear. This study found that Imp, the Drosophila ZBP1 ortholog, accumulates in the cell bodies of a large number of neural cells in adult brain. Strikingly, Imp was additionally observed in the axonal compartment of a subpopulation of mushroom body (MB) neurons. MBs are composed of three main neuronal types (αβ, α'β', and γ) with specific axonal projection patterns and developmental programs. α'β' and αβ neurons are generated during late larval stage and early metamorphosis and are maintained until adulthood. γ neurons are born during late embryogenesis and early larval stages and undergo extensive remodeling during metamorphosis. Imp was found to be enriched in adult γ neuron axons where it colocalized with FasciclinII, but it was not detected in the axons of nonremodeling MB neurons (αβ and α'β' neurons). To test whether Imp is expressed in αβ and α'β' neurons, brains expressing GFP in γ and αβ-core neurons were labelled with antibodies against Imp and Trio, a protein specifically expressed in adult α'β' and γ neurons. Imp was not detected in αβ-core neurons but accumulated in the cell bodies of both α'β' and γ neurons. Thus, both the expression and subcellular distribution of Imp are tightly regulated in Drosophila MB neurons (Medroni, 2014).

To investigate whether Imp axonal translocation is developmentally regulated, the distribution of Imp within γ neurons was examined at different stages. In third-instar larvae, Imp accumulated exclusively in the cell bodies and was not observed in axons. During metamorphosis (pupariation), MB γ neurons first prune the distal part of their axons and then re-extend a medial branch to establish adult-specific projections. Six hours after puparium formation (APF), Imp was weakly detected in γ neuron axons. Such an axonal accumulation of Imp was visible at the time larval γ neurons have completed the pruning of their axonal processes (18 hr APF). During the subsequent intensive growth phase, Imp was enriched at the tip of axons, where it accumulated in particles. Thus, the translocation of Imp to axons is developmentally controlled, and correlates with axonal remodeling (Medroni, 2014).

To test whether Imp is required for γ axon developmental remodeling, the morphology was analyzed of adult homozygous mutant neurons generated using the MARCM (mosaic analysis with a repressible cell marker) system. Clones in which the entire progeny of a neuroblast was mutant exhibited a reduced number of cells and an altered morphology. Although wild-type adult γ axons typically span the entire medial lobe, a mixture of elongated and nonelongated axons was observed upon imp inactivation. To better visualize the morphology of mutant neurons, single labeled neurons were analyzed. Wild-type adult γ neurons extend one main axonal process that reaches the extremity of the MB medial lobe. Several secondary branches typically form along this main axonal process. In contrast, about 50% of imp γ axons failed to reach the end of the medial lobe. These defects did not result from axon retraction, as the proportion of defective axons did not increase with age. Interestingly, mutant axons of normal length but lost directionality were observed, suggesting that imp may be required for the response of γ axons to guidance cues during metamorphosis. imp mutant neurons also exhibited an overall decrease in the complexity of axonal arborization patterns characterized by a reduced number of terminal branches. Both phenotypes were significantly suppressed upon expression of a wild-type copy of Imp in γ neurons, revealing that imp acts cell autonomously to control axonal regrowth and branching (Medroni, 2014).

To determine whether Imp function in axonal growth correlates with its accumulation in axons, the requirement for Imp was investigated in two neuronal cell types where it is exclusively detected in cell bodies: larval γ neurons and α'β' neurons. Both single larval γ neurons and single adult α'β' neurons mutant for imp projected their axons normally. Furthermore, larval γ neuron neuroblast clones exhibited a normal morphology, confirming that imp is not necessary for initial axon growth. These results show that Imp is specifically required for the growth and branching of remodeling γ axons and suggest that its translocation to axons is critical for this function (Medroni, 2014).

To address whether Imp is transported actively to the axons of regrowing γ neurons, a live-imaging protocol was developed using cultured pupal brains expressing functional GFP-Imp fusions specifically in γ neurons). The culture conditions supported efficient axonal growth, as MB neurons from cultured brains grew similarly to their counterparts developing inside the pupa. Fast confocal imaging of axon bundles revealed that GFP-Imp fusions accumulated in particles undergoing bidirectional movement. In contrast, no particles could be detected upon expression of GFP alone. Motile GFP-Imp particles were distributed into three classes: particles with a strong net anterograde (56%) or retrograde (36%) movement and particles with little net bias (8%). Individually tracked particle trajectories were broken into segments to calculate velocities. Segmental velocities distributed over a wide range, with mean anterograde and retrograde segmental velocities of 0.98 ± 0.05 microm/s and 0.73 ± 0.03 microm/s, respectively. Furthermore, curves matching the graph of a quadratic function were obtained upon plotting of the mean square displacement (MSD) values over time, indicating that GFP-Imp particles undergo directed transport rather than diffusion. To assess the role of microtubules (MTs) in this process, brains were treated with colchicine. This treatment abolished MT dynamics, as revealed by the loss of EB1-GFP comets characteristic of growing MT plus ends. Strikingly, motile GFP-Imp particles were no longer observed under these conditions. These results demonstrate that Imp is a component of particles undergoing active MT-dependent transport during midpupariation, consistent with a role of Imp in the transport of selected mRNAs to regrowing γ axons (Medroni, 2014).

Previous in vitro studies have revealed that the axons of immature neurons are enriched in mRNAs encoding regulators of the actin cytoskeleton that play critical roles in axonal growth and guidance. To identify Imp mRNA targets, an immunoprecipitation RT-PCR-based screen was performed for mRNAs encoding actin regulators. Imp was found to selectively associate with chickadee (chic) mRNA, which encodes the G-actin binding protein Profilin. As revealed by affinity pull-down assays, endogenous Imp associated with the chic 3' untranslated region (UTR), but not with the chic coding sequence. To test whether Imp can interact with chic mRNA directly, the binding of recombinant MBP-Imp to the chic 3' UTR was analyzed in electrophoretic mobility shift assays. Retarded complexes formed in the presence of the chic 3' UTR, but not in the presence of a nonrelated RNA (y14). Furthermore, no significant interaction was observed when other MBP-tagged proteins were used. Notably, two discrete complexes were detected in the presence of low amounts of Imp, whereas higher-order complexes were formed with increasing amounts of Imp. Formation of these complexes was outcompeted by the addition of nonlabeled RNAs corresponding to the chic 3' UTR, but not to the chic coding sequence. Altogether, these results show that Imp associates with chic mRNA in vivo and that it can bind directly and specifically to the chic 3' UTR (Medroni, 2014).

To test whether chic mRNA localizes to the neurites of regrowing γ neurons, in situ hybridization was performed on pupal and adult brains. The poor signal-to-noise ratio obtained with this method at these stages, combined with the relatively low levels of axonally localized mRNAs, did not allow chic transcripts or reporters to be unambiguously detected in axons. Thus chic reporter constructs expressed under the control of the γ-specific 201Y-Gal4 driver was used and fluorescent in situ hybridizations was used on dissociated neurons extracted from 24 hr APF pupae and cultured for 3-4 days. chic reporter mRNAs could be observed in the neurites of γ neurons at a significantly higher frequency than control gfp mRNAs. Furthermore, chic mRNA and Imp colocalized in developing neurites, consistent with their association within mRNA transport complexes (Medroni, 2014).

To test whether the region of chic bound by Imp is required for chic mRNA localization to developing neurites, the distribution of reporters containing both the chic coding sequence and 3' UTR was compared with that of reporters lacking the chic 3' UTR. Transcripts with the chic 3' UTR localized more efficiently than transcripts lacking it, suggesting that Imp binding to the 3' UTR promotes chic axonal targeting. To exclude an effect of Imp on chic mRNA stability, the levels of chic transcripts were analyzed in cultured S2R+ cells. No significant differences in chic mRNA and Chic protein levels could be observed upon imp inactivation in these conditions (Medroni, 2014).

To functionally test the importance of chic regulation in vivo, the phenotypes associated with chic downregulation were examined. Consistent with described roles of Profilin in regulating F-actin polymerization and axonal pathfinding, it was observed that chic mutant γ neurons fail to properly extend their axons. More importantly, overexpression of chic significantly rescued the axonal growth defects observed in imp mutant neurons. Similar results were obtained with two independent UAS-chic transgenes, but not with overexpression of another regulator of F-actin polymerization (enabled). These results suggest that imp controls axonal remodeling by regulating chic expression in vivo and reveal that forced accumulation of Chic protein in axons can partially compensate for the loss of imp function (Medroni, 2014).

In conclusion, the finding that Drosophila Imp is required for γ axon regrowth but is dispensable for initial axonal growth suggests a novel and specific function of axonal mRNA targeting in developmental remodeling of the brain. Furthermore, these results highlight mechanistic similarities between developmental axonal regrowth and postinjury axonal regeneration, a process known to depend on axonal mRNA transport. Finally, this study uncovers that the translocation of Imp to γ axons is tightly linked to their developmental remodeling program. This reveals that mRNA transport machineries are subject to precise spatiotemporal regulation and may be specifically recruited in the context of developmental rewiring of the brain. It will now be interesting to identify the signals controlling the localization and the activity of mRNA transport machineries during this process (Medroni, 2014).