Chemokine-like Orion is involved in the transformation of glial cells into phagocytes in different developmental neuronal remodeling paradigms.

During animal development, neurons often form exuberant or inappropriate axons and dendrites at early stages, followed by the refinement of neuronal circuits at late stages. Neural circuit refinement leads to the production of neuronal debris in the form of neuronal cell corpses, fragmented axons and dendrites, and pruned synapses requiring disposal. Glial cells act as predominant phagocytes during neuronal remodeling and degeneration, and crucial signaling pathways between neurons and glia are necessary for the execution of phagocytosis. Chemokine-like mushroom body neuron-secreted Orion is essential for astrocyte infiltration into the γ axon bundle leading to γ axon pruning. Here, we show a role of Orion in debris engulfment and phagocytosis in Drosophila. Interestingly, Orion is involved in the overall transformation of astrocytes into phagocytes. In addition, analysis of several neuronal paradigms demonstrates the role of Orion in eliminating both peptidergic vCrz+ and PDF-Tri neurons via additional phagocytic glial cells like cortex and/or ensheathing glia. Our results suggest that Orion is essential for phagocytic activation of astrocytes, cortex and ensheathing glia, and point to Orion as a trigger of glial infiltration, engulfment and phagocytosis.

The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons.

Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, we show that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.

Neuron-glia crosstalk in neuronal remodeling and degeneration: Neuronal signals inducing glial cell phagocytic transformation in Drosophila.

Neuronal remodeling is a conserved mechanism that eliminates unwanted neurites and can include the loss of cell bodies. In these processes, a key role for glial cells in events from synaptic pruning to neuron elimination has been clearly identified in the last decades. Signals sent from dying neurons or neurites to be removed are received by appropriate glial cells. After receiving these signals, glial cells infiltrate degenerating sites and then, engulf and clear neuronal debris through phagocytic mechanisms. There are few identified or proposed signals and receptors involved in neuron-glia crosstalk, which induces the transformation of glial cells to phagocytes during neuronal remodeling in Drosophila. Many of these signaling pathways are conserved in mammals. Here, we particularly emphasize the role of Orion, a recently identified neuronal CX3 C chemokine-like secreted protein, which induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling. Although, chemokine signaling was not described previously in insects we propose that chemokine-like involvement in neuron/glial cell interaction is an evolutionarily ancient mechanism.

Axonal chemokine-like Orion induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling.

The remodeling of neurons is a conserved fundamental mechanism underlying nervous system maturation and function. Astrocytes can clear neuronal debris and they have an active role in neuronal remodeling. Developmental axon pruning of Drosophila memory center neurons occurs via a degenerative process mediated by infiltrating astrocytes. However, how astrocytes are recruited to the axons during brain development is unclear. Using an unbiased screen, we identify the gene requirement of orion, encoding for a chemokine-like protein, in the developing mushroom bodies. Functional analysis shows that Orion is necessary for both axonal pruning and removal of axonal debris. Orion performs its functions extracellularly and bears some features common to chemokines, a family of chemoattractant cytokines. We propose that Orion is a neuronal signal that elicits astrocyte infiltration and astrocyte-driven axonal engulfment required during neuronal remodeling in the Drosophila developing brain.

Htt is a repressor of Abl activity required for APP-induced axonal growth.

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αß neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αß neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.

Guidance of Drosophila Mushroom Body Axons Depends upon DRL-Wnt Receptor Cleavage in the Brain Dorsomedial Lineage Precursors.

In vivo axon pathfinding mechanisms in the neuron-dense brain remain relatively poorly characterized. We study the Drosophila mushroom body (MB) axons, whose α and ß branches connect to different brain areas. We show that the Ryk family WNT5 receptor, DRL (derailed), which is expressed in the dorsomedial lineages, brain structure precursors adjacent to the MBs, is required for MB α branch axon guidance. DRL acts to capture and present WNT5 to MB axons rather than transduce a WNT5 signal. DRL's ectodomain must be cleaved and shed to guide α axons. DRL-2, another Ryk, is expressed within MB axons and functions as a repulsive WNT5 signaling receptor. Finally, our biochemical data support the existence of a ternary complex composed of the cleaved DRL ectodomain, WNT5, and DRL-2. Thus, the interaction of MB-extrinsic and -intrinsic Ryks via their common ligand acts to guide MB α axons.

Nuclear receptors and Drosophila neuronal remodeling.

During the development of both vertebrates and invertebrates, neurons undergo a crucial remodeling process that is necessary for their new function. Neuronal remodeling is composed of two stages: first, axons and dendrites are pruned without the loss of the cell body; later, this process is most commonly followed by a regrowth step. Holometabolous insects like the fruitfly Drosophila exhibit striking differences between their larval and adult stages. These neuronal remodeling processes occur during metamorphosis, the period of transformation from a larva to an adult. All axon and dendrite pruning events ultimately depend on the EcR nuclear receptor. Its ligand, the steroid molting hormone ecdysone, binds to heteromeric receptors comprising the nuclear receptor ECR and USP, and this complex regulates target genes involved in neuronal remodeling. Here we review the nuclear receptor-mediated genetic control of the main neuronal remodeling events described so far in Drosophila. These events consist of neurite degeneration in the mushroom bodies (MBs: the brain memory center) and in the dendritic arborizing sensory neurons, of neurite retraction or small scale elimination in the thoracic ventral neurosecretory cells, in the olfactory circuits and in the neuromuscular junction. MB axon regrowth after pruning and the role of MB neuron remodeling in memory formation are also reviewed. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles.

One of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of our understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. Here, we analyze the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24h of metamorphosis. We find that ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, we show that the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Finally, we show that unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis.

The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αß neurons and, in particular, is required cell-autonomously for the ß-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-ß/BMP signaling and orphan nuclear receptors.

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of ß-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-ß signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-ß signaling and nuclear receptor) triggers axon retraction. We propose that a signal from a TGF-ß family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.

Mushroom body neuronal remodelling is necessary for short-term but not for long-term courtship memory in Drosophila.

The remodelling of neurons during their development is considered necessary for their normal function. One fundamental mechanism involved in this remodelling process in both vertebrates and invertebrates is axon pruning. A well-documented case of such neuronal remodelling is the developmental axon pruning of mushroom body γ neurons that occurs during metamorphosis in Drosophila. The γ neurons undergo pruning of larval-specific dendrites and axons at metamorphosis, followed by their regrowth as adult-specific dendrites and axons. We recently revealed a molecular cascade required for this pruning. The nuclear receptor ftz-f1 activates the expression of the steroid hormone receptor EcR-B1, a key component for γ remodelling, and represses expression of Hr39, an ftz-f1 homologous gene. If ectopically expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. The mushroom bodies are a bilaterally symmetric structure in the larval and adult brain and are involved in the processing of different types of olfactory memory. How memory is affected in pruning-deficient adult flies that possess larval-stage neuronal circuitry will help to explain the functional role of neuron remodelling. Flies overexpressing Hr39 are viable as adults and make it possible to assess the requirement for wild-type mushroom body pruning in memory. While blocking mushroom body neuron remodelling impaired memory after short-term courtship conditioning, long-term memory was normal. These results show that larval pruning is necessary for adult memory and that expression of courtship short-term memory and long-term memory may be parallel and independent.

Parallel processing of appetitive short- and long-term memories in Drosophila.

It is broadly accepted that long-term memory (LTM) is formed sequentially after learning and short-term memory (STM) formation, but the nature of the relationship between early and late memory traces remains heavily debated [1-5]. To shed light on this issue, we used an olfactory appetitive conditioning in Drosophila, wherein starved flies learned to associate an odor with the presence of sugar [6]. We took advantage of the fact that both STM and LTM are generated after a unique conditioning cycle [7, 8] to demonstrate that appetitive LTM is able to form independently of STM. More specifically, we show that (1) STM retrieval involves output from γ neurons of the mushroom body (MB), i.e., the olfactory memory center [9, 10], whereas LTM retrieval involves output from αß MB neurons; (2) STM information is not transferred from γ neurons to αß neurons for LTM formation; and (3) the adenylyl cyclase RUT, which is thought to operate as a coincidence detector between the olfactory stimulus and the sugar stimulus [11-14], is required independently in γ neurons to form appetitive STM and in αß neurons to form LTM. Taken together, these results demonstrate that appetitive short- and long-term memories are formed and processed in parallel.

ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling.

Developmental axon pruning is a general mechanism that is required for maturation of neural circuits. During Drosophila metamorphosis, the larval-specific dendrites and axons of early γ neurons of the mushroom bodies are pruned and replaced by adult-specific processes. We found that the nuclear receptor ftz-f1 is required for this pruning, activates expression of the steroid hormone receptor EcR-B1, whose activity is essential for γ remodeling, and represses expression of Hr39, an ftz-f1 homologous gene. If inappropriately expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. EcR-B1 was previously identified as a target of the TGFß signaling pathway. We found that the ftz-f1 and Hr39 pathway apparently acts independently of TGFß signaling, suggesting that EcR-B1 is the target of two parallel molecular pathways that act during γ neuron remodeling.

Ryks: new partners for Wnts in the developing and regenerating nervous system.

Conserved Ryk transmembrane proteins, tyrosine kinase-related Wnt receptors, are important during neurogenesis, axon guidance and synaptogenesis. Here, we review the increasingly complex biology of the Wnt/Ryk pathway, emphasizing the mechanisms by which Ryks transduce or sometimes block the Wnt signal. Recent studies reveal that Wnts signal through Ryk via multiple mechanisms, including nuclear translocation of their intracellular domains and pathways employing Src Family Kinases and members of the canonical Wnt pathway. We also discuss reports indicating that Wnt/Ryk axon guidance roles are evolutionarily conserved and Wnt/Ryk interactions are required for motoneuron target selection and synaptogenesis at the neuromuscular junction. Recent findings that injury-induced Wnt/Ryk pathway activation inhibits axon regeneration underscore the importance of further understanding this novel pathway.

Identification of linotte, a new gene affecting learning and memory in Drosophila melanogaster.

We describe the identification of linotte, a new autosomal gene in Drosophila involved with learning and memory. The linotte(1) mutant was derived from a PlacW transposan mutagenesis and was screened for three-hour memory deficits after classical conditioning of an olfactory avoidance response. Sensory and motor systems (olfactory acuity and shock reactivity) required for the classical conditioning experiments were normal in mutant linotte(1) files--indicating that the mutation disrupts learning/memory specifically. A chromosomal deficiency of the 37D region, where the linotte(1)P insert was localized in situ, failed to complement linotte(1)'s memory defect, and files from two lines homozygous for independent PlacW excisions show normal memory--indicating that the P insertion is responsible for the mutant phenotype. Additional behavior-genetic data suggest that linotte gene is non-vital.

Respective roles of the DRL receptor and its ligand WNT5 in Drosophila mushroom body development.

In recent decades, Drosophila mushroom bodies (MBs) have become a powerful model for elucidating the molecular mechanisms underlying brain development and function. We have previously characterized the derailed (drl; also known as linotte) receptor tyrosine kinase as an essential component of adult MB development. Here we show, using MARCM clones, a non-cell-autonomous requirement for the DRL receptor in MB development. This result is in accordance with the pattern of DRL expression, which occurs throughout development close to, but not inside, MB cells. While DRL expression can be detected within both interhemispheric glial and commissural neuronal cells, rescue of the drl MB defects appears to involve the latter cellular type. The WNT5 protein has been shown to act as a repulsive ligand for the DRL receptor in the embryonic central nervous system. We show here that WNT5 is required intrinsically within MB neurons for proper MB axonal growth and probably interacts with the extrinsic DRL receptor in order to stop axonal growth. We therefore propose that the neuronal requirement for both proteins defines an interacting network acting during MB development.

In vivo evidence for a regulatory role of the kinase activity of the linotte/derailed receptor tyrosine kinase, a Drosophila Ryk ortholog.

The RYK subfamily of receptor tyrosine kinases is characterised by unusual, but highly conserved, amino acid substitutions in the kinase domain. The linotte/derailed gene encodes a Drosophila RYK subfamily member involved in embryonic and adult central nervous system development. Previous studies have shown that the kinase activity of this receptor is not required in vivo for its embryonic function. In this study, we have investigated the role of the cytoplasmic domain and the kinase activity of the linotte/derailed receptor tyrosine kinase in adult brain development. Our results indicate that these domains are not essential for adult brain development but they are required for the proper regulation of the activity of this receptor. This sheds light on a regulatory role for the kinase activity of a RYK subfamily member.

Gain-of-function screen identifies a role of the Src64 oncogene in Drosophila mushroom body development.

Mushroom bodies (MB) are substructures in the Drosophila brain that are essential for memory. At present, MB anatomy is rather well described when compared to other brain areas, and elucidation of the genetic control of the development and projection patterns of MB neurons will be important to the understanding of their functions. We have performed a gain-of-function screen in order to identify genes that are involved in MB development. We drove expression of genes in MB neurons by crossing 2407 GAL4-driven UY element lines to lines containing an MB GAL4 source and UAS-GFP elements, and looked for defects in the MB structure. We have molecularly identified the genomic regions adjacent to the 26 positive UY insertions and found 18 potential genes that exhibit adult MB gain-of-function phenotypes. The proteins encoded by these candidate genes include, as well as genes with yet unknown function, transcription factors (e.g., tramtrack), nanos RNA-binding protein, microtubule-severing protein, vesicle trafficking proteins, axon guidance receptor, and the Src64 cytoplasmic protein tyrosine kinase. These genes are involved in key features of neuron cell biology. In three cases, tramtrack, nanos, and Src64, we show that the open reading frame located directly downstream of the UY P element is indeed the expressed target gene. Loss-of-function mutations of both ttk and Src64 lead to MB phenotypes proving that these genes are involved in the genetic control of MB development. Moreover, Src64 is shown here to act in a cell-autonomous fashion and is likely to interact with the previously-identified linotte/derailed receptor tyrosine kinase in MB development.

Genome-wide prediction of Polycomb/Trithorax response elements in Drosophila melanogaster.

Polycomb/Trithorax response elements (PRE/TREs) maintain transcriptional decisions to ensure correct cell identity during development and differentiation. There are thought to be over 100 PRE/TREs in the Drosophila genome, but only very few have been identified due to the lack of a defining consensus sequence. Here we report the definition of sequence criteria that distinguish PRE/TREs from non-PRE/TREs. Using this approach for genome-wide PRE/TRE prediction, we identify 167 candidate PRE/TREs, which map to genes involved in development and cell proliferation. We show that candidate PRE/TREs are bound and regulated by Polycomb proteins in vivo, thus demonstrating the validity of PRE/TRE prediction. Using the larger data set thus generated, we identify three sequence motifs that are conserved in PRE/TRE sequences.

Identification and characterization of polyhomeotic PREs and TREs.

The polyhomeotic (ph) gene is a member of the Polycomb group of genes (Pc-G), which are required for the maintenance of the spatial expression pattern of homeotic genes. In contrast to homeotic genes, ph is ubiquitously expressed and it is quantitatively regulated. ph is negatively regulated by the Pc-G genes, except Psc, and positively regulated by the antagonist trithorax group of genes (trx-G), suggesting that Pc-G and trx-G response elements (PREs and TREs) exist at the ph locus. In this study, we have functionally characterized PREs and TREs at the ph locus that function in transgenic constructs. We have identified a strong PRE and TRE in the ph proximal unit as well as a weak one in the ph distal unit. The PRE/TRE of both ph units appear atypical compared with the well-defined homeotic maintenance elements because the minimal ph proximal response element activity requires at least 2 kb of sequence and does not work at long range. We have used chromatin immunoprecipitation experiments on cultured cells and embryos to show that Pc-G proteins are located in restricted regions, close to the ph promoters that overlap functionally defined PRE/TREs. Our data suggest that ph PRE/TREs are cis-acting DNA elements that modulate rather than silence Pc-G- and trx-G-mediated regulation, enlarging the role of these two groups of genes in transcriptional regulation.