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SummaryDuring animal development, neurons often form exuberant or incorrect axons and dendrites at early stages, followed by the refinement of neuronal circuits at late stages. Neural circuit refinement leads to the production of large amounts of neuronal debris in the form of neuronal cell corpses, fragmented axons and dendrites, and pruned synapses requiring disposal. In particular, the predominant phagocytes acting during the neuronal remodeling and degeneration are glial cells and critical signaling pathways between neurons and glia leading to phagocytosis are required. Chemokine-like mushroom body neuron secreted Orion ligand was shown to be essential to the astrocyte infiltration into the γ axon bundle leading to γ axon pruning and clearance of debris left from axon fragmentation. Here we show a role oforionalso in debris engulfment and phagocytosis. Interestingly, we show thatorionis also involved in the overall transformation of astrocytes into phagocytes. In addition, analysis of several neuronal paradigms demonstrates the role oforionin the elimination of both peptidergic vCrz+and PDF-Tri neuronsviaadditional phagocytic glial cells as cortex and/or ensheathing glia. Our results suggest that Orion is essential for phagocytic activation of three different types of glial cells: astrocytes, cortex and ensheathing glia and point to Orion as a trigger not only of glial infiltration but also engulfment and phagocytosis.

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 CX

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 inDrosophila. However, how PS is recognized by Drpr-expressing phagocytesin vivoremains poorly understood. Using multiple models of dendrite degeneration, we show that theDrosophilachemokine-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 inDrosophilaand imply conserved mechanisms of phagocytosis of neurons.SIGNIFICANCE STATEMENTPhagocytes efficiently clear sick or damaged neuronal branches by engulfing them, while leaving healthy branches untouched. How phagocytes recognize degenerating neurites remains poorly understood. Here, we identified a key role for the secreted protein Orion in the detection and engulfment of degenerating neurites inDrosophila. Using multiple models of dendrite degeneration, we found that Orion acts as a bridging molecule between the neuronal “eat-me” signal phosphatidylserine and the engulfment receptor Draper on phagocytes, enabling phagocytosis. Our study reveals a missing link in phosphatidylserine-mediated phagocytosisin vivo, sheds light on factors determining the sensitivity of phagocytes, and implies the potential for manipulating the detection of neuronal “eat-me” signals in neurodegenerative diseases.

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., Astrocytes can engulf axonal debris in the developing brain. However, the mechanisms regulating astrocyte recruitment to the proper axons is unclear. Here, the authors identify Orion as a signal for astrocyte infiltration and engulfment to the mushroom bodies in the Drosophila developing brain.

SummaryThe remodeling of neurons is a conserved fundamental mechanism underlying nervous system maturation and function. Glial cells are known to clear neuronal debris but also to have an active role in the remodeling process. Developmental axon pruning ofDrosophilamemory center neurons occurs by a degenerative process mediated by infiltrating astrocytes. However, how these glial processes are recruited by the axons is unknown. In an unbiased screen, we identified a new gene (orion) which is necessary for both the pruning of some axons and removal of the resulting debris. Orion is secreted from the neurons and bears some features common to the chemokines, a family of chemoattractant cytokines. Thus, chemokine involvement in neuron/glial cell interaction is an evolutionarily ancient mechanism. We propose that Orion is the neuronal signal that elicits astrocyte infiltration required for developmental neuronal remodeling.

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 αβ neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αβ 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., Author summary Understanding the normal physiological roles of proteins involved in neurodegenerative diseases can provide significant insight into disease mechanisms. Drosophila offers a powerful system in which to ask these fundamental questions. Both Htt, related to Huntington’s disease, and Appl, related to Alzheimer’s disease, have well-conserved single orthologs in the fly genome. Appl has been shown to be a conserved modulator of a Wnt-PCP signaling pathway required for axon outgrowth in the mushroom body (MB) in the Drosophila brain. However, roles for Htt in fly brain development have not been reported. Unexpectedly, we found that htt mutations suppress the axon outgrowth defects of Appl mutants in the MB, indicating a link between these two neurodegenerative proteins and a cryptic role of Htt during development. Abl tyrosine kinase is a downstream effector of the Appl receptor, and we show here that Abl is also required for MB axon outgrowth. Importantly, Abl activity must be tightly regulated as evidenced by our observations that both under and overexpression of Abl result in similar axonal defects. We demonstrate that Htt is an inhibitor of Abl activity and provide evidence that the phenotypic rescue of αβ axons in Appl mutants by reducing htt is mediated by the restoration of proper levels of Abl signaling. These data, therefore, suggest that Appl and Htt act antagonistically to maintain an optimal balance of activation and inhibition of Abl, and thereby promote the growth of MB αβ axons.

International audience; 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.

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 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.

SummaryIt 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 αβ MB neurons; (2) STM information is not transferred from γ neurons to αβ 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 αβ neurons to form LTM. Taken together, these results demonstrate that appetitive short- and long-term memories are formed and processed in parallel.

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.

Summary 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 β 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.

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.

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.

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.

P element insertion is essentially random at the scale of the genome. However, P elements containing regulatory sequences from Drosophila engrailed and polyhomeotic genes and from the Bithorax and Antennapedia complexes show some insertional specificity by frequently inserting near the parent gene (homing) and/or near genes containing Polycomb group response elements (preferential insertion). This phenomenon is thought to be mediated by Polycomb group proteins. In this report, we describe a case of homing of P elements containing regulatory sequences of the linotte gene. This homing occurs with high frequency (up to 20% of the lines) and high precision (inserted into a region of Polycomb group proteins but by a new, as yet unknown, mechanism. We also suggest that P element homing could be a more frequent phenomenon than generally assumed and that it could become a powerful tool of Drosophila reverse genetics, for which there is no other described gene targeting technique.

The linotte (lio) mutant was first isolated as a memory mutant. The lio gene encodes a putative receptor tyrosine kinase (RTK), homologous to the human protein RYK. This gene has been independently identified in a screen for embryonic nervous system axonal guidance defects and called derailed (drl). Here, we report that linotte mutants present structural brain defects in the adult central complex (CX) and mushroom bodies (MB). linotte and derailed are allelic for this phenotype, which can be rescued by a drl+ transgene. The Lio RTK is expressed preferentially in the adult CX and MB. Our results suggest that, analogous to its role within the embryonic nervous system, the Lio RTK is involved in neuronal pathway selection during adult brain development.

We used the white gene as an enhancer trap and reporter of chromatin structure. We collected white+ transgene insertions presenting a peculiar pigmentation pattern in the eye: white expression is restricted to the dorsal half of the eye, with a clear-cut dorsal/ventral (D/V) border. This D/V pattern is stable and heritable, indicating that phenotypic expression of the white reporter reflects positional information in the developing eye. Localization of these transgenes led us to identify a unique genomic region encompassing 140 kb in 69D1–3 subject to this D/V effect. This region contains at least three closely related homeobox-containing genes that are constituents of the iroquois complex (IRO-C). IRO-C genes are coordinately regulated and implicated in similar developmental processes. Expression of these genes in the eye is regulated by the products of the Polycomb -group (Pc-G) and trithorax-group (trx-G) genes but is not modified by classical modifiers of position-effect variegation. Our results, together with the report of a Pc -G binding site in 69D, suggest that we have identified a novel cluster of target genes for the Pc-G and trx-G products. We thus propose that ventral silencing of the whole IRO-C in the eye occurs at the level of chromatin structure in a manner similar to that of the homeotic gene complexes, perhaps by local compaction of the region into a heterochromatin-like structure involving the Pc-G products.

The Drosophila homolog of the Alzheimer's disease protein APP, known as APPL, regulates axon growth during brain development., 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 αβ neurons and, in particular, is required cell-autonomously for the β-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., Author Summary Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but in neurons it regulates axon outgrowth, 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. In the present work we investigate the role of the Drosophila neuron-specific APP homologue, called APPL, during brain development. We find that APPL is required for the development of αβ neurons in the mushroom body, a structure critical for learning and memory. We find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not for cell polarity. Molecularly, both human APP and fly APPL are found in membrane complexes with PCP receptors. Moreover, we show that APPL regulates PCP pathway activation through its downstream effector Abelson kinase (Abl), which modulates the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) and the subsequent activation of Wnt-PCP signaling. Taken together our data suggest that APPL is the first example of a neuron-specific modulator of the Wnt-PCP pathway.

The polyhomeotic (ph) gene of Drosophila is a member of the Polycomb group of genes and encodes a chromatin protein required for negative regulation of homeotic genes and other loci, in particular the ph locus itself. We have studied the genetic control of ph transcription during development. Early ph expression is under the control of bicoid and engrailed as activators and of oskar as an inhibitor. The negative autoregulation of ph starts at the blastoderm stage and is partly mediated by a transvection effect. As the number of functional copies of ph increases in the same genome, a concomitant reduction of the transcription of each copy is observed. This regulation is ensured, likely at the chromatin level, positively by the trithorax group and negatively by the Polycomb group gene products like a homeotic gene, but it occurs in the same cells. We propose that an equilibrium between these two states of chromatin activity ensures an accurate level of ph transcription.

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and in normal development of posterior compartments. One of its regulatory targets appears to be polyhomeotic (ph), a Polycomb group gene. We observed, by immunostaining, that Engrailed protein binds to the site of the polyhomeotic locus in region 2D of polytene chromosomes. The same analysis carried out on a transgenic line containing one copy of a P(ph-lacZ) construct shows an additional Engrailed-binding site at the location of the insert. In vivo, polyhomeotic depends on engrailed function in germ-band-elongated embryos, when engrailed and polyhomeotic genes are expressed in similar patterns. By in vitro immunoprecipitations and gel shift assays, we identified two classes of high affinity Engrailed-binding sites upstream of each of the two polyhomeotic transcription units. DNA fragments containing these sites were also immunoprecipitated from embryonic UV crosslinked chromatin in presence of anti-Engrailed antibody. These results suggest that polyhomeotic activation in germ-band-elongated embryos could be mediated by Engrailed-binding to these sites.

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 β-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-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. We propose that a signal from a TGF-β 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.

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 TGFβ signaling pathway. We found that the ftz-f1 and Hr39 pathway apparently acts independently of TGFβ signaling, suggesting that EcR-B1 is the target of two parallel molecular pathways that act during γ neuron remodeling.

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.

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 a screen for Drosophila genes that interfere with transcriptional repression mediated by the Polycomb group of genes, we identified a dominant mutation affecting the Alhambra (Alh) gene, the fly homologue of the human AF10 gene. AF10 has been identified as a fusion partner of both MLL and CALM in infant leukemias. Both fusion proteins retain the leucine zipper domain of AF10 but not its PHD domain. We show here that, while the full-length ALH protein has no activity on Polycomb group-responsive elements (PREs), overexpression of the isolated ALH leucine zipper domain activates several PREs. Within the ALH full-length protein, the PHD domain inhibits the PRE deregulation mediated by the leucine zipper domain. This deregulation is conserved in the human AF10 leucine zipper domain, which confers the same activity on an oncogenic MLL-AF10 fusion protein expressed in Drosophila melanogaster. These data reveal new properties for the leucine zipper domain and thus might provide new clues to understanding the mechanisms by which AF10 fusion proteins in which the PHD domain is lost might trigger leukemias in humans.

The Drosophila linotte 1 mutation was isolated from a genetic screen designed to identify learning and memory genes. For some authors, this mutation affects a novel gene specifically involved in adult learning and memory, whereas for others, it is an allele of the derailed receptor tyrosine kinase gene (the linotte/derailed gene) involved in nervous system development. Here, we show that the original derailed mutation induces a memory phenotype. We also report that a new null mutation, lio exc21 , affecting specifically the linotte/derailed gene causes behavioral defects, which can be partially rescued by expression of a lio + /drl + transgene. The data presented here suggest that the memory phenotype of linotte and derailed mutants is a consequence of abnormal brain development due to loss of function of the linotte/derailed encoded receptor tyrosine kinase.

Members of the Polycomb group (Pc-G) and trithorax group (trx-G) of genes, as well as the enhancers of trx-G and Pc-G (ETP), function together to maintain segment identity during Drosophila development. In order to obtain new marked P mutations in these genes, we screened for dominant modifiers of the extra-sex-combs phenotype displayed by males mutant for the polyhomeotic (ph) gene, a member of the Pc-G group. Five P{lacW} insertions in four different genes were found to stably suppress ph: two are allelic to trithorax, one is the first allele specific to the Minute(2)21C gene, and the remaining two define new trx-G genes, toutatis (tou) in 48A and taranis (tara) in 89B10-13. tou is predicted to encode a 3109 amino acid sequence protein (TOU), which contains a TAM DNA-binding domain, a WAKZ motif, two PHD zinc fingers and a C-terminal bromodomain, and as such is likely to be involved in regulation of chromatin structure as a subunit of a novel chromatin remodelling complex. In a previous study, we found that insertion of a P{ph} transposable element containing ph regulatory sequences creates a high frequency of mutations modifying ph homeotic phenotypes. One such insertion enhanced the ph phenotype and we show that it is a new allele of UbcD1/eff, a gene encoding a ubiquitin-conjugating enzyme that is involved in telomere association and potentially in chromatin remodelling.

Gene silencing by heterochromatin is a well-known phenomenon that, in Drosophila, is called position effect variegation (PEV). The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct support only recently. Another gene-silencing phenomenon in Drosophila, although similar in its phenotype of variegation, has been shown to be associated with euchromatic sequences and is dependent on developmental regulators of the Polycomb group (Pc-G) of gene products. One model proposes that the Pc-G products may cause a local heterochromatinization that maintains a repressed state of transcription of their target genes. Here, we test these models by measuring the accessibility of white or miniwhite sequences, in different contexts, to the Escherichia coli dam DNA methyltransferase in vivo. We present evidence that PEV and Pc-G-mediated repression mechanisms, although based on different protein factors, may indeed involve similar higher-order chromatin structure.

Variegation of the miniwhite gene is observed in a euchromatic context in transformant lines that contain a P transposon including regulatory sequences of the polyhomeotic (ph) gene upstream of the resident miniwhite gene (P[ph]). This variegated phenotype is not affected by most of the genetic modifiers of heterochromatic position-effect variegation (PEV) nor by removal of the Y chromosome. Interestingly, it is sensitive to ph and Polycomb (Pc) mutations, which are known to affect homeotic gene regulation. Regulatory DNA of ph can also mediate transvection of the miniwhite gene. This transvection is abolished in a ph but not in a zeste mutant background. In addition, P[ph] inserts preferentially in sites corresponding to PH/PC protein-binding sites as defined at the polytene chromosome level. These insertions induce an unusually high proportion of mutations in genes affecting homeotic gene regulation. In particular, one insertion is located within the tramtrack locus, which is thought to regulate fushi tarazu, an Ultrabithorax activator. We suggest that a multimeric complex containing PH and PC proteins, at a minimum, causes a local and clonally inherited heterochromatinization, which maintains the repressed state of transcription of the homeotic genes.

A heat shock applied at the blastoderm stage to Drosophila embryos produces phenocopies of dominant mutants of the bithorax-complex (BX-C). We have studied the effect of different genetic variations upon the frequency of Ultraabdominal phenocopies. The results indicate that heat shock acts upon regulation rather than expression of the BX-C. They are also in accordance with the possible role of Polycomb and a gene included in Deficiency (3) red in the regulation of BX-C. We postulate that the actual repression of the Ultraabdominal locus is due to at least two factors, one governed by the wild-type allele of the Polycomb locus and one governed by the BX-C itself. The results suggest that the heat shock interferes with the BX-C factor and not with the product of Polycomb.

Two mutagenic events are required to make null mutations of polyhomeotic (ph), which suggests that the locus is complex. Amorphic mutations (ph degrees) die in mid-embryogenesis and completely lack ventral thoracic and abdominal epidermal derivatives, whereas single-event mutations lead to transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes. After a chromosomal walk, the ph gene was localized using deficiencies and ph mutations that result from DNA rearrangements. Hybridization analyses show that there are two large, duplicated sequences in the ph region, and DNA lesions affecting either one of these repeats alter the function of the ph locus. We propose a model that may account for this unusual functional organization.

A new locus in Drosophila melanogaster that is required for the correct expression of segmental identity has been discovered. The new locus, termed polyhomeotic (ph), is X-linked and maps cytologically to bands 2D2-3. Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANT-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb (Pc), Polycomblike (Pcl), and extra sex comb (esc), and acts as a dominant enhancer of Pc. The expression of ph depends on the ANT-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph2 show that the ph+ product is required during embryogenesis and larval development. We propose that ph mutants in some way disrupt the normal expression of the ANT-C and BX-C, and, therefore, that ph+ is needed for maintenance of segmental identity.

The synthesis of heat shock proteins (hsp) has been examined during the early embryogenesis of Drosophila melanogaster. Normal protein synthesis stops after heat shock at all developmental stages, while hsp synthesis is induced only after treatment at blastoderm and later stages. The small hsps continue to be synthesised after heat shock for a longer period than the larger ones. Heat shocks at 35 degrees C, 37 degrees C and 40 degrees C were compared for their effect on hsp synthesis and the effect of heat shock on the normal course of development was analysed.

Three Ultrabithorax (Ubx) alleles and three different deficiencies of the bithorax complex (BX-C) of Drosophila melanogaster have been tested for maternal effects in the germ line. The dominant female sterile technique was used. The Ubx alleles and a deletion of the abdominal region of the BX-C are homozygous viable in germ line clones and show no maternal effects. Two deletions which lack the proximal portion of the BX-C are lethal in the female germ line indicating either that these deficiencies lack genes apart from the BX-C that are necessary for fertility or that there are BX-C genes that are essential for normal maternal germ line function. The significance of the bias in the isolation of only zygotic mutations at the BX-C are discussed with respect to these results.

I-R hybrid dysgenesis in Drosophila melanogaster occurs in female progeny of crosses between reactive strain females and inducer strain males, and is controlled by transposable elements called I-factors. These are 5.4 kb elements that are structurally similar to mammalian LINE elements and other retroposons. We have tested the activity of an I-factor directly, by introducing it into the genome of a reactive strain, using P-element mediated transformation. It confers the complete inducer phenotype on the reactive strain, and can stimulate dysgenesis when transformed males are mated with reactive females. It has transposed in the transformed lines, and we have cloned one of the transposed copies. This is the first time that it has been possible to demonstrate that a particular retroposon is transposition proficient, and to compare donor and transposed elements. We propose a mechanism for I-factor transposition based on these results, and the coding capacity of these elements. We have been unable to detect either autonomous transposition of a complete I-factor from a plasmid injected into reactive strain embryos, or transposition of a marked I-factor when co-injected with a complete element.

The distributions of the products of the homeotic genes Sex combs reduced (Scr) and Ultrabithorax (Ubx) and of the segmentation genes, fushi tarazu (ftz), even skipped (eve) and engrailed (en) have been monitored in polyhomeotic (ph) mutant embryos. None of the genes monitored show abnormal expression at the blastoderm stage in the absence of zygotic ph expression. Both Scr and Ubx are ectopically expressed in the epidermis of ph embryos, confirming the earlier proposal, based on genetic analysis, that ph+ acts as a negative regulator of Antennapedia (ANT-C) and bithorax (BX-C) complex genes. At the shortened germ band stage, en is also ectopically expressed, mainly in the anterior region of each segment. In contrast to these effects in the epidermis, the expression of en, Ubx, Scr and ftz is largely or completely suppressed in the central nervous system, whereas eve becomes ectopically expressed in most neurones.

The complex genetic locuspolyhomeotic (ph) is a member of thePolycomb (Pc)-group of genes and as such is required for the normal expression of ANT-C and BX-C genes. It also has probably other functions since amorphicph alleles display a cell death phenotype in the ventral epidermis of 12-h-old embryos. Here it is shown that lethal alleles ofph (amorph and strong hypomorph) show transformation of most of their segments towards AB8. Theph + product is required autonomously in imaginal cells. The total lack ofph + function prevents viability of the cuticular derivatives of these cells.ph has a strong maternal effect on segmental identity and epidermal development that can not be rescued by one paternally supplied dose ofph + in the zygote. These phenotypes differ substantially from those of previously describedPc-group genes. AmongPc-group genes,ph seems to be the only one that is strongly required both maternally and zygotically for normal embryonic development.

In the drosophila brain, mushroom bodies are involved in olfactory memory and learning. This structure is composed of different types of / neurons. These neurons form an orthogonal structure, with the branch projecting dorsally and the branch projecting medially. The aim of this study is to understand mechanisms and pathways involved during the development of these neurons.The drosophila APPL protein (Amyloïd Precursor Protein-Like) is the homologue of the human APP, known to be involved in Alzheimer’s disease. This pathology is characterized by neuronal degeneration inducing cognitive and memory defects. In spite of the numerous studies focused on the pathological function of APP during the last decades, few things are known on the physiological functions of this protein and more particularly during the development. This is from this perspective that we studied the APPL function and its interaction with proteins during the mushroom bodies development.The APPL protein was identified as a co-receptor of the PCP pathway (Planar Cell Polarity), involved in the axonal growth regulation. During the development, APPL allows the recruitment and the activation of the ABL protein (Abelson Tyrosine Kinase), which phosphorylates DSH (dishevelled) and so activates the axonal growth pathway.The first part investigates the regulation of ABL activity during the / neuron development. If it’s already established that APPL regulates positively the kinase activity of ABL, I show here that the HTT protein (Huntingtin) allows a negative regulation of ABL activity. In human, HTT is involved in the Huntington’s disease, another neurodegenerative disorder. This thesis work shows that HTT regulates the phosphorylation level of ABL, and therefore its activity.The second part investigates the interaction between APPL and ARM (armadillo), the homologue of the human -catenin, during the development of the / neurons. I show that this interaction is independent of the APPL function in the PCP pathway. Moreover, this interaction between APPL and ARM involves the actin cytoskeleton dynamic function of ARM, and not its Wnt pathway function.The third and last part presents new mutant alleles of APPL obtained with the CRISPR-CAS9 technique. The creation and analysis of these new alleles lead us to propose that vnd (ventral nervous system defective), neighbor gene of Appl, is also involved in / neurons development, and can interact genetically with Appl.; Le cerveau de drosophile est constitué entre autres des mushroom body, siège de la mémoire et de l’apprentissage. Cette structure est composée de différents types de neurones, parmi lesquels les neurones /. Ces neurones se présentent sous une forme orthogonale, avec l’axone qui se divise en une branche dorsale : la branche et une branche médiale : la branche . Le but de cette étude est de comprendre les mécanismes et voies de signalisation mis en jeu lors du développement de ces neurones.Chez la drosophile, la protéine APPL (Amyloïd Precursor Protein-Like) est l’homologue de la protéine APP humaine, connue pour son implication dans la maladie d’Alzheimer chez l’homme. Cette pathologie est caractérisée par une dégénérescence neuronale entraînant des défauts cognitifs et mnésiques. Malgré les nombreuses études focalisées sur la fonction pathologique d’APP durant les dernières décennies, peu de choses sont actuellement connues sur les fonctions physiologiques de cette protéine et notamment pendant le développement. C’est dans cette optique que nous avons étudié la fonction d’APPL et son interaction avec différentes protéines lors du développement des mushroom body. La protéine APPL a été identifiée comme étant un co-récepteur de la voie PCP (Planar Cell Polarity), permettant la régulation de la croissance axonale. Lors du développement, APPL permet le recrutement et l’activation de la protéine ABL (Abelson Tyrosine Kinase), qui phosphoryle DSH (dishevelled) et ainsi active la voie de signalisation permettant la croissance axonale.Le premier volet de cette thèse porte sur la régulation de l’activité ABL lors du développement des neurones /. S’il est établi qu’APPL permet une régulation positive de l’activité kinase d’ABL, je montre ici que la protéine HTT (huntingtine) permet de réguler négativement cette activité. Cette protéine HTT est impliquée dans la maladie de Huntington chez l’homme, une autre pathologie neurodégénérative. Ces travaux démontrent qu’HTT régule le niveau de phosphorylation d’ABL et par conséquent son activité. Le deuxième volet de cette thèse porte sur l’interaction d’APPL avec la protéine ARM (armadillo), homologue de la -caténine, lors du développement des neurones /. Je démontre que cette interaction est indépendante du rôle d’APPL dans la voie PCP. Je démontre aussi que cette interaction entre APPL et ARM est dépendante uniquement de la fonction d’ARM dans la dynamique du cytosquelette d’actine.Enfin le troisième volet de cette thèse porte sur la création de nouveaux allèles mutants pour Appl grâce à la technique du CRISPR-CAS9. La production de ces allèles permet d’avancer d’une part un possible rôle du gène voisin vnd (ventral nervous system defective) dans le développement des mushroom body, et d’autre part une interaction génétique entre Appl et vnd.