The Interactive Fly

Genes involved in tissue and organ development

Axonogenesis


Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila
Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain
A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity
WldS prevents axon degeneration through increased mitochondrial flux and enhanced mitochondrial Ca2+ buffering
Drosophila growth cones advance by forward translocation of the neuronal cytoskeletal meshwork in vivo
The NAV2 homolog Sickie regulates F-actin-mediated axonal growth in Drosophila mushroom body neurons via the non-canonical Rac-Cofilin pathway
The Homeodomain Transcription Factor Hb9 Controls Axon Guidance in Drosophila through the Regulation of Robo Receptors
Drosophila Strip serves as a platform for early endosome organization during axon elongation
Robo-3-mediated repulsive interactions guide R8 axons during Drosophila visual system development
The guanine exchange factor Vav controls axon growth and guidance during Drosophila development
Pavarotti/MKLP1 regulates microtubule sliding and neurite outgrowth in Drosophila neurons
Regulation of axon regeneration by the RNA repair and splicing pathway
Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development
DISCO Interacting Protein 2 regulates axonal bifurcation and guidance of Drosophila mushroom body neurons
The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation
Drosophila Dunc-115 mediates axon projection through actin binding
MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2
Islet coordinately regulates motor axon guidance and dendrite targeting through the Frazzled/DCC receptor
Axon death pathways converge on Axundead to promote functional and structural axon disassembly
The large and small SPEN family proteins stimulate axon outgrowth during neurosecretory cell remodeling in Drosophila
Transcription factors
abrupt
motor axons and muscle

apterous
optic discs

asense
optic lobe

disconnected and disco-related
optic lobe

buttonless
transverse nerve outgrowth and bifurcation

castor
expressed in neuroblasts - effects commissures

cheerio
growth cone progression

drifter
midline glia

Dynactin 1, p150 subunit (common alternative name: p150Glued)
axonal transport

eagle
serotonin interneurons

extra machrochaete
motor neurons

fruitless
CNS axon tracts

huckebein
motor neurons

glass
eye

glial cells missing
glia

Hormone receptor 51
regulates mushroom body axon growth and pathfinding

islet
dopamine and serotonin interneurons of the CNS

jing
expressed in CNS midline - mutants show disrupted axon scaffold

Kinesin-like protein at 64D & Kinesin-like protein at 68D

Lim1
CNS, mutants have motor coordination problems but no obvious defects in axon guidance

longitudinals lacking
central nervous system (ventral cord)

nerfin-1
zinc finger transcription factor required for the proper development of CNS commissural and connective axon fascicles

nervy
regulates repulsive axon guidance by linking the cAMP-dependent Protein kinase (PKA) to the Semaphorin 1a (Sema-1a) receptor Plexin A

Nkx6
CNS motoneurons

prospero
central nervous system

reversed polarity
photoreceptor axons

orthodenticle
ventral midline

pointed
ventral midline

pou domain motif 3
odor receptor neurons

scribbler (alternative name: brakeless)
essential for R1-R6 growth cone targeting

sequoia
functions in PNS neurons, photoreceptors, and motoneurons in the CNS, functions in confering dendritic morphology in the PNS

shuttle craft
segmental and intersegmental nerves

single minded
ventral midline

tinman
mesodermal effect on exit glia

trachealess
sensory axons

X-box-binding protein 1


Cell surface and secreted

activin-β
optic lobe - TGFβ ligand of Baboon - required for tiling of photoreceptor R7 axon terminals

amalgam
CNS and PNS

beta amyloid protein precursor-like
neuromuscular junction

argos
eye

beaten path
motor axons, Bolwig's organ

borderless
optic lobe neurite-neurite interaction

breathless
midline glia

Cadherin-N
motor axon guidance

caskin
a neuronal adaptor protein required for axon growth and guidance -
interacts physically and genetically with Lar receptor protein tyrosine phosphatase

commissureless
axon guidance across the midline

capricious
LRR proteins participate in the selection appropriate synaptic targets for motor neurons

Connectin
a homophilic cell adhesion molecules expressed in muscles and the motoneurons that innervate them

derailed
axon guidance in the brain and CNS

Dlar
motor axon guidance

draper
required during metamorphosis for recognition and engulfment of degenerating axon branches by glia

Dscam
Bolwig's organ, brain and CNS

Dscam2
immunoglobulin superfamily - Dscam2 homophilic interactions mediate repulsion between neurites of L1 laminar cells in neighbouring columns in the optic lobe

Eph receptor tyrosine kinase
expressed by interneurons of the ventral cord - protein expression confined to axons

Ephrin
functions as a ligand for Eph receptor - expressed on the surface of neuron cell bodies - acts to confine Eph receptor bearing axons to longitudinal tracts by repulsion

faint sausage
central nervous system (ventral cord): effect is secondary to defects in cell positioning

Fasciclin1
commissural axons

Fasciclin2
motor neurons

Fasciclin3
muscle and nerve

frazzled
motor axon guidance and commissures

fizzled
controls mushroom body axon branching

Gliolectin
mutation disrupts the formation of commissural pathways and delays the completion of longitudinal pathfinding

Golden goal
regulates axon-axon and axon-target interactions in R8 photoreceptor axon -
acts as a functional partner of Flamingo for R8 photoreceptor axon targeting in the medulla

Guanylyl cyclase at 76C
cGMP production - receptor-type protein possessing a single transmembrane domain - required for Semaphorin-1a-Plexin A directed repulsive axon guidance of motor neurons

Histone H2B
regulates photoreceptor axon guidance in the optic lobe by effecting glial migration

kuzbanian
axon extention in CNS

Laminin A
CNS and Ocelli

leak (robo2)
transmembrane receptor that regulates axon guidance

Matrix metalloproteinase 1 and Matrix metalloproteinase 2
enzymes required for several stereotyped motor axon pathfinding decisions and essential for axon fasciculation

Multiplexin
homolog of Collagen XV/XVIII, a matrix protein that can be proteolytically cleaved to release Endostatin, an antiangiogenesis signaling factor -
required for normal calcium channel abundance, presynaptic calcium influx, and neurotransmitter release - functions in motor axon pathfinding

Netrin-A and Netrin-B
commissural axons, CNS neurons, and motor neurons

Neurotactin
ocellar pioneer axons and interneurons

Neurotrophin 1
secreted ligand that maintains neuronal survival - expression rescues
naturally occurring cell death and enables targeting by motor neurons

Notch
intersegmental nerves

off-track
CCK-4 family of 'dead' receptor tyrosine kinases, Ig-domains - required for lamina-specific targeting of R1-R6 axons -
associates with Plexin, the receptor for Semaphorin ligand

Olig family
HLH transcription factor expressed in the brain and CNS - motor neuron identify factor - regulates axon guidance

plexin A
CNS

plexin B
semaphorin domain protein, receptor for the secreted semaphorin Sema-2a -
regulates axon extension from the sensory neuron cell body in regions of direct contact with oenocytes

Protein tyrosine phosphatase 4E
broadly expressed type III receptor tyrosine phosphatase closely related to Ptp10D - shows synthetic phenotypes with other Pdps

Protein tyrosine phosphatase 10D
receptor tyrosine phosphatase - required for embryonic and larval axon guidance - along with Ptp69D regulates segregation
of the young axons into a single core bundle in the larval mushroom body - mutants are defective in long-term memory formation

Protein tyrosine phosphatase 69D
central nervous system and eye

Ptp52F
motor neuron axon guidance

rhomboid
ventral midline

roughest
eye

roundabout
expressed on all longitudinally projecting growth cones and axons

semaphorin I
CNS

Semaphorin-2a
expressed in muscle and inhibits the neuronal growth cone from forming a synaptic arborization -
secreted from oenocytes and acts represses axon extension from the sensory neuron cell body

Semaphorin-2b
attractive clue that promotes the initial assembly of a subset of CNS longitudinal projections and
subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives

sidestep
Ig domain cell adhesion molecule - ligand for Beaten path Ia - axon guidance

slit
ventral midline

Src oncogene at 42A
axons linking the larval eye (Bolwig's organ) to the optic lobe

Star
ventral midline

Syndecan
a heparan sulfate proteoglycan - a necessary component of Slit/Robo signaling required in Slit target cells

tartan
LRR proteins participate in the selection appropriate synaptic targets for motor neurons

Tenascin major transmembrane protein that acts together with the Filamin Cheerio to influence growth cone progression

Toll
motor neurons

Toll-6 & Toll-7
Toll-like receptors - Neurotrophin receptors - regulation of locomotion, motor axon targeting and neuronal survival - innate immunity

turtle
Ig superfamily - regulates the tiling pattern of R7 photoreceptor terminals

unc-5
multiple domain protein that functions as a repulsive netrin receptor

Wnt oncogene analog 5
required for the formation of the anterior of the two midline-crossing commissures present in each hemisegment


Others

Abelson Interacting Protein
plays an opposing role to Abl in Drosophila axonogenesis and synaptogenesis

Abl oncogene
neuronal growth cone pathfinding

abstrakt
ventral midline Bolwig's organ and CNS

Ack
targets the sorting nexin DSH3PX1 to regulate axonal guidance

APP-like protein interacting protein 1
JNK scaffolding scaffolding protein that is part of motor-cargo linkage complexes for both kinesin-1 and dynein - acts downstream of the JNK pathway
to affect axonal transport - binds APP family proteins

bifocal
functions downstream of misshapen to reorganize actin cytoskeleton in decelerating R cell growth cone motility

Calmodulin
axon guidance, including both defects in fasciculation and abnormal crossings of the midline

Calnexin 99A
misexpression causes embryonic axon guidance phenotype

chromosome bows
mediates the action of Slit and its receptors acting as a partner of the Abelson tyrosine kinase

COP9 complex homolog subunit 5
required for photoreceptor neurons (R cell) axon targeting in the optic lobe

crooked neck
mRNA splice factor - required in glial cells to control migration and axonal wrapping

cut up
homologue of the LC8 dynein light chain - binds proteasomes and functions specifically during their axonal transport

Cyclin-dependent kinase 5
axon patterning

Disabled
axon pathfinding in the central nervous system

dhshevelled
controls mushroom body axon branching

dreadlocks
axon pathfinding in the central nervous system and eye

Dynein heavy chain 64C
axon pathfinding and synaptogenesis

dunce
aberrant axons

embryonic lethal, abnormal vision (synonym: elav)
aberrant axons

enabled
central nervous system - axonal outgrowth and fasciculation

futsch
motor neurons

G protein oalpha 47A
motor neurons

kakapo
arborization and dendritic sprouting of motorneurons

kette (preferred name: HEM-protein)
a transmembrane protein that transduces information to the neuronal cytoskeleton affecting axon guidance

Liprin-α
scaffolding protein that physically interacts with LAR and is essential for R7 axon targeting

MICAL
flavoprotein monooxygenase - large, multidomain protein expressed in axons - interacts with the neuronal Plexin A -
enzymatic function required for Semaphorin 1a/PlexA-mediated repulsive axon guidance

mummy
acetylglucosamine diphosphorylase - functions in apical extracellular matrix formation by producing GlcNAc residues needed for
protein glycosylation - embryonic phenotypes in axon guidance are characteristic of defects in midline signaling

non-stop
ubiquitin-specific protease expressed in glia - involved in development of laminal glia

numb
motor neurons

Ornithine decarboxylase antizyme common alternative name: gutfeeling)
PNS growth cone guidance and fasciculation

PAK-kinase
photoreceptor R cell guidance

partner of drosha
mutation causes dendrite mistargeting in the antennal lobe and altered axonal terminations in higher brain centers

Ptpmeg
protein tyrosine phosphatase involved in neuronal circuit formation the central brain - regulates establishment
and the stabilization of axonal projection patterns

RacGAP50C
functions during brain development to limit axon overextension

RhoGAP
part of an axon retraction pathway from Rho to myosin in mature neurons -
inactivation of RhoGAP leads to axon retraction - functions in mushroom bodies

pod-1
WD repeat protein - crosslinks actin and microtubules - proper levels of Pod-1 must be maintained in the growth cone for correct axon guidance

prickle
controls mushroom body axon branching

SCAR
promotes actin polymerization via Arp2/3 protein complex

sec5
promotes neurite outgrowth

sec15
functions in polarized exocytosis/secretion - involved in targeting photoreceptor axons that involves localization of specific cell adhesion and signaling molecules

Son of sevenless
a GEF that is recruited to the plasma membrane, where it forms a ternary complex with Roundabout
and Dreadlocks to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand

spenito
stimulates axon outgrowth during neurosecretory cell remodeling

split ends
ventral midline

tau
vesicular axonal transport

Ter94
regulation of motor neuron degeneration

Tip60
mediates axonal growth of the Drosophila sleep regulating pacemaker cells and their production of the neuropeptide pigment dispersing factor
that functions to stabilize Drosophila sleep-wake cycle

trio
motorneuron, mushroom body and photoreceptor axon pathfinding

twinstar
encodes Drosophila actin-depolymerizing and actin-severing protein cofilin - function in axon growth is inhibited by LIM kinase and activated by Slingshot phosphatase in convergent and divergent pathways from Rho GTPases to result in different developmental outcomes

unc-104 (common alternative name, immaculate connections or imac)
a Kinesin-3 family member that is essential for transporting synaptic vesicle precursors

UV-resistance associated gene (common alternative name: Vps38)
subunit of the PI3K-cIII complex - autophagy - endosomal-lysosomal pathway - axon pruning tumor suppressor -
receptor downregulation through endolysosomal degradation - the establishment of proper cell polarity
in the developing wing - UV-induced DNA damage repair

Vav ortholog
prevents longitudinal axons from crossing the midline, regulates photoreceptor axon targeting to the optic lobe

wallenda
>MapKKK member of the JNK pathway - influences axonal transport by functioning as a kinesin-cargo dissociation factor -
suppresses autophagy-induced neuromuscular junction overgrowth - required for normal axon degeneration


Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila

The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, principles underlying the organisation of the motor system have been uncovered. Dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. These results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

The analysis began by correlating the positions of motor neuron dendrites with the distribution of their muscle targets in the periphery. Motor neurons were retrogradely labelled in a pairwise fashion and the positions of their dendritic arbors were mapped. Because of an interest in the mechanisms that underlie the assembly of the motor system, focus was placed on stages when each motor neuron first establishes a characteristic domain of arborisation within the neuropile (early stage 17, 15h after egg-laying [AEL]) (Landgraf, 2003).

Motor axons project into the muscle field via two main nerves, the intersegmental (ISN) and the segmental nerve (SN). The transverse nerve (TN) runs along the segment border and has few motor axons. Choice of nerve root is one of several features that divide the motor neurons into two principal sets, the ISN and SN. (1) The cell bodies of SN motor neurons are located in the same segment as the muscles that they innervate, whereas ISN motor neuron somata are located in the segment next anterior (with the exception of the RP2 and two neuromodulatory efferent ventral unpaired median [VUM] neurons. (2) ISN motor neurons innervate internal muscles, which span a segment from anterior to posterior, whereas SN (and the TN) motor neurons innervate external muscles. External muscles are distinct from the internal set in several respects: (1) they are generally transverse; (2) unlike internal muscles, they require wingless (wg) signalling for their specification; (3) external (but not internal) muscles and their innervating motor neurons express the cell adhesion molecule (CAM) Connectin, with the single exception of muscle ventral transverse 1 (VT1) (Landgraf, 2003 and references therein).

In addition, ISN and SN motor neurons elaborate their dendrites in distinct regions of the neuropile. Dendrites of ISN motor neurons occupy a domain extending posteriorly from the posterior part of one neuromere into the anterior part of the next. SN motor neuron dendrites occupy a domain that lies between the domains of ISN motor neuron arbors (Landgraf, 2003).

Thus, the organisation of the body wall muscles into internal and external sets is reflected centrally in patterns of motor neuron arborisations. The innervating motor neurons project their axons through different nerves and elaborate their dendritic fields in distinct regions of the neuropile. Although dendritic arbors become progressively more elaborate and extensive over developmental time, their separate domains remain clearly recognisable and appear to be maintained at least until the motor system is fully functional (18 h AEL) (Landgraf, 2003).

Having established that there is a central representation of the muscle field, the organisation of the motor neuron dendrites was analyzed in greater detail. (1) The set of external muscles and their innervating (SN) motor neurons were examined. Muscles of similar anteroposterior positions, such as the ventral acute muscle (VA3) and the segment border muscle (SBM), are innervated by motor neurons whose dendritic arbors lie in a common region of the neuropile. Conversely, motor neurons supplying the anterior (lateral transverse 1-2 [LT1-LT2]) versus the posterior (SBM) muscles have dendritic arbors that are correspondingly separated in the anteroposterior axis of the CNS (Landgraf, 2003).

To put the idea of a regular map to the test, focus was placed on an unusual external motor neuron-muscle pair. Muscle VT1 is innervated by a TN rather than an SN motor neuron. However, VT1 lies at the same place in the anteroposterior axis as the SBM, although VT1 is ventral and the SBM more dorsal. The VT1 motor neuron dendritic field is found to overlaps with that of the SBM motor neuron. For the external set, it is concluded that differences in target muscle location in the anteroposterior axis are mapped centrally as regular differences in dendritic position, but dorsoventral distinctions are not (Landgraf, 2003).

It was next asked whether there is a similarly regular representation of the internal muscles in the developing CNS. While most external muscles are transverse and have unique anteroposterior locations, the internal muscles span the width of a segment so that positional distinctions between them are solely in the dorsoventral axis. It was found that the set of internal muscles is represented centrally by three dendritic domains. Motor neurons innervating ventral internal muscles elaborate their dendritic arbors in the anterior half of the ISN dendritic domain. Motor neurons with dorsolateral internal muscle targets (lateral longitudinal [LL] 1, dorsal acute [DA] 3, dorsal oblique 3-5 [DO3-DO5]) put their arbors into the posterior part of the ISN dendritic domain. Finally, dorsal muscles are represented by a motor neuron dendritic domain that lies between those representing ventral (anterior) and dorsolateral (posterior) internal muscle groups. Thus, the internal muscles are represented in the neuropile by three domains of dendritic arborisation that reflect their different dorsoventral locations in the periphery. Once again, it is concluded that there is a regular mapping of muscle position in the neuropile: in this case, it is positions in the dorsoventral axis peripherally that are represented centrally as differences in the anteroposterior locations of dendrites (Landgraf, 2003).

To test the idea that dendritic arbor positions relate to the distribution of muscles, an atypical motor neuron-muscle pair was examined. The RP2 motor neuron is reported to innervate dorsal muscle DA2, yet its dendrites span the domains that represent both dorsal and dorsolateral internal muscles. However, on careful analysis it was found that DA2 is, in fact, specifically innervated by a U neuron whose dendrites lie in the dorsal internal domain, whereas the RP2 axon forms endings generally on all dorsolateral and dorsal muscles by 19 h AEL. These seem to correspond to the type 1s boutons found in late larvae. Thus, the RP2 neuron puts its dendrites into a region of the neuropile that does indeed represent its targets, namely the dorsolateral and dorsal internal muscles (Landgraf, 2003).

Like the muscle field itself, the map of motor neuron dendrites is metamerically repeated. However, the boundaries of these two units are out of register with one another, since the dendrites of the motor neurons innervating internal muscles lie in the next anterior neuromere. The anterior border of the dendritic map, as defined by the extent of these anterior dendrites, coincides with the anterior margin of engrailed (en) expression. Thus, while the muscles are segmental in their organisation, the domains occupied by the dendrites of their innervating motor neurons are parasegmental (Landgraf, 2003).

To test whether genes that implement the parasegmental pattern in the epidermis are also required for the formation of the parasegmental organisation of the neuromuscular system, the formation of SN and ISN dendritic fields was studied in embryos singly mutant for the following segment polarity genes: en/invected (Df(enE)), wg (wgCX4), naked (nkd2), patched (ptc9), hedgehog (hh21), and gooseberry (Df2R(gsb)). Every one of the six different mutants that were analysed has partially aberrant patterns of neuroblasts (NBs). Nevertheless, SN and ISN motor neurons still form and can be identified by their characteristic axonal projections into the periphery. In addition, it was found that the fundamental separation between SN and ISN dendritic domains is present despite often severe perturbations in CNS structure. For example, in gsb mutant embryos, both nerve roots are frequently fused so that the SN and ISN share a common CNS exit point. Nevertheless, SN and ISN axons as well as their dendritic fields do not intermingle but remain separate. These results suggest that the subdivision of the neuropile into the principal ISN and SN dendritic domains is a robust feature of the system, which appears to be specified early in development, since the embryo subdivides into parasegmental units (Landgraf, 2003).

It was next asked what mechanisms underlie the formation of the myotopic map. Because ISN and SN motor neurons lie at different positions in the CNS and their axons grow out into the muscle field through different nerves, it is reasonable to suppose that at least the major subdivision of dendritic arborisations into internal and external domains could be a byproduct of the locations at which the motor neurons are generated and the paths taken by their growing axons. This ‘passive mapping' explanation can be excluded by considering a single motor neuron-muscle pair, namely dorsal transverse 1 (DT1) and its innervating motor neuron. DT1 is an external muscle (by position, orientation, wg dependence, and Connectin expression), yet its motor neuron is clustered with the internal muscle innervating set and its axon (uniquely for the external muscles) grows out through the ISN. Despite its packing within the ‘internal motor neuron' set, the DT1 motor neuron makes a long posterior projection through the internal muscle domain of the myotopic map to reach the external domain, where it arborises appropriately, reflecting the orientation and external nature of its target muscle. In contrast, motor neurons derived from the same NB as DT1 innervate neighboring internal muscles DO3-DO5 and put their dendrites in a more anterior region characteristic of the dorsolateral muscles. These findings strongly suggest that the mapping of the muscle field within the CNS is an active process of growth and arborisation that partitions dendrites into subdomains of the neuropile that are appropriate to their function, rather than a passive subdivision of available space by position of origin or axon trajectory (Landgraf, 2003).

Since dendritic arbors form after motor axons have reached their targets, the muscles could be instrumental in dictating the organisation of the central map. To test this idea, the UAS/GAL4 system was used to misexpress an activated form of Notch (Kidd et al. 1998) in the developing mesoderm, suppressing the formation of muscle founder cells while leaving other tissues intact. In such muscleless embryos, the main nerve trunks, SN and ISN, still form and project into the periphery. Retrograde labellings of these nerves show that SN and ISN motor neurons form relatively normal dendritic arbors that consistently conform to the characteristic separation of SN and ISN dendrites. Thus, the neuropile is partitioned into distinct fields of dendritic arborisation independently of the muscles. It is concluded that the mapping process is likely to be an autonomous property of the motor neurons and their neighboring cells (Landgraf, 2003).

It was next asked whether motor neuron dendritic fields could be patterned by the substrates on which they grow. In the Drosophila ventral nerve cord (VNC), motor neuron dendrites form in the dorsal-most region of the neuropile, sandwiched between longitudinal glia above and the underlying scaffold of axons. Glial cells can act as substrates for supporting and guiding axonal growth. To test whether they might also be required for the growth and spatial patterning of dendritic fields, dendritic arbors were analysed in glial cells missing (gcm) mutant embryos, which are defective in glial cell differentiation. Although the structure of the nervous system is disrupted in gcm mutant embryos and the dendritic arbors are abnormal, they continue to form in their characteristic locations and the fundamental distinction between the ISN and SN motor neuron dendritic fields is maintained. Remarkably, even the long posterior dendritic projection of the DT1 motor neuron forms and reaches its target region, the SN external muscle dendritic domain. These results suggest that the patterning of the neuropile into distinct motor neuron dendritic domains is a process that appears to be intrinsic to the motor neurons and their neighboring neurons, but does not require proper glial cell differentiation (Landgraf, 2003).

One likely explanation for the division of dendrites into separate domains is that there is a process of mutual exclusion between the arborisations of neighboring cells. Such a process of dendritic ‘tiling' has so far only been documented between particular classes of sensory neurons, but could also occur in the motor system. The idea of tiling was tested by considering two groups of motor neurons whose axons have a common trajectory, but whose dendritic fields form in adjacent territories. The DO3-DO5 and DT1 motor neurons project their dendrites posteriorly, and at their most-anterior point, these dendrites meet the axons and dendrites of the anterior corner cell (aCC) and U/CQ neurons. To show whether the aCC and U/CQ axons and/or dendrites inhibit the growth of DO3-DO5 and DT1 dendrites anteriorly, these neurons (as well as RP2 and the posterior corner cell [pCC] interneuron) were selectively ablated. Using anti-Even-skipped (Eve) staining as a marker for aCC, RP2, and U/CQs (there are an additional two medially located eve-expressing interneurons, pCC and friend of pCC [fpCC], it was found that these neurons can be selectively ablated before they form dendrites (at approximately 11 h AEL): on average, by 10.5 h AEL all but 0.6 and by 12 h AEL all but 0.06 of the seven medially located eve-expressing neurons have been ablated per half-neuromere. In no instance was a concomitant anterior expansion of the DO3-DO5 and DT1 motor neuron dendrites into the regions vacated by the aCC and U/CQ dendrites observed. It is concluded that, at least in this instance, the initial dendritic territory of one set of motor neurons (DO3-DO5 and DT1) is not defined by a process of tiling, in which they are excluded by neighboring (aCC and U/CQ) dendritic arbors. However, it is possible that the elaboration of motor neuron dendritic arbors during later developmental stages may involve interactions between neighboring dendritic territories, activity-dependent processes, or both (Landgraf, 2003).

Thus, in summary, these results suggest that the mechanisms that subdivide the neuropile into distinct dendritc domains are very robust and refractory to perturbations. They further suggest that the cues that organise the map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

The patterning of the motor neuron dendritic arbors in the Drosophila embryo represents a first layer of organisation in the motor system. This is likely in part to be mirrored by the endings of higher-order neurons of central pattern generating circuits, which converge onto the myotopic map. While motor neuron cell body positions may, as has been proposed for vertebrate systems, relate to the ontogeny of target muscles, the operation of mature muscles is reflected by the allegiance of corresponding motor neuron dendrites to a particular territory in the neuropile. Thus, changes in muscle operation could be accommodated by a change of allegiance of the appropriate motor neuron dendrites from one domain to another (e.g., the DT1 motor neuron-muscle pair) without the need for rewiring the underlying higher-order circuitry. Such a model resolves the apparent discrepancy between the distributions of motor neuron cell bodies centrally and target muscles in the periphery. It also implies a considerable degree of flexibility, particularly at the level of motor output, yet suggests that elements of the underlying motor circuitry may have been highly conserved (Landgraf, 2003).

Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain

Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. This study used the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and to selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. The results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult-specific compartments to establish the role of glia. This study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development (Spindler, 2009).

Secondary neurons, which are born during the larval period, form SATs that have to extend over relatively long distances, finding their way amidst a complex array of (primary) axons, dendrites, and glia. The association of glia and SATs varies for different lineages. SATs either (1) remained wrapped within the neuropile or joined other tracts that were then ensheathed as a larger tract system, (2) encountered strands of glial condensations, or (3) had no association with glial sheaths. The association between individual SATs and glia was highly invariant. Thus, if SAT A joined SAT B to form a larger tract system that became wrapped by glia, the same densities of glia could be observed in other brains for the corresponding SATs (Spindler, 2009).

To address the role of glia during SAT fasciculation, growth, and guidance, cortex and neuropile glia, the two glial types in contact with growing SATs, were selectively eliminate. Expression of the pro-apoptotic proteins Hid and Rpr were effective in inducing apoptosis of most cortex and neuropile glia by the early or mid larval stage. It can be assumed that the primary axon tract formation is not disrupted, given that expression of the nrv2-GAL4 driver line does not set in prior to stage 12, primary axon tract (PAT) patterning is complete before glia invade the neuropile, and the first signs of apoptosis appear after hatching. In addition, the surface glia remain intact, providing general ensheathment around the brain, a functional blood–brain barrier, and potential signaling molecules used to control neuroblast proliferation (Spindler, 2009).

Upon the elimination of glia, frequent abnormalities are seen in the pattern of SATs. Importantly, the strength of an SAT phenotype appears to correlate with the degree of glial association of that SAT in a wild-type brain. Of all SATs analyzed, the mushroom body (associated with a complete glial sheath) exhibits the most severe defects, including complete SAT misguidance and aberrant fasciculation of neurites from adjacent SATs. In contrast, SATs that were less endowed with glia in the wild type typically had a normal projection pattern (Spindler, 2009).

This study does not differentiate between a role for glia in producing chemo-attractant signals or acting as a physical scaffold for guidance. In previous studies, ectopic expression of dominant-negative Drosophila E-cadherin in either cortex/neuropile glia or SATs themselves results in non-radial trajectories of SATs into the neuropile. This suggests a requirement for SAT-glia adhesion as secondary axons project toward the cortex-neuropile boundary. However, the direction of neuroblast division is also disrupted with DE-cadherin knock-down, thus aberrant SAT trajectories may be a secondary effect of abnormal cell body layering within the cortex. In support of a signaling role for glia, the midline guidance defect of the CP1 SAT (anteromedially, crossing the peduncle and entering the diagonal commissure) is reminiscent of the robo-slit phenotypes in the Drosophila ventral nerve cord. Whether robo/slit signaling is also used between SATs and glia in the brain is a question that warrants further investigation (Spindler, 2009).

The situation that SATs in part require glia for pathway guidance in the neuropile is different than the embryonic brain in which pioneer axons and PATs are formed in the absence of extensive glial processes. Why are SATs different? One can imagine the scenario in which the PATs generate the neuropile de novo, forcing cell body movement outwards as the central neuropile grows. By first instar, a full neuropile is established, and SATs must guide through a dense maze of neurites, glia, and trachea. It therefore appears that: (1) PATs establish the initial connectivity of the brain; (2) glia grow in around this initial scaffolding, and finally (3) SATs use both the PAT scaffolding and the glial boundaries for guidance into and around the neuropile (Spindler, 2009).

Insects have long been used to evaluate glial-neuronal interactions from embryonic to adult stages. An important focus of these studies was whether or not glia or axon tracts appear first, and in how far axonal pathfinding is disrupted if glia is ablated. In this regard, clear developmental differences have been found between distinct regions of the nervous system, as well as between insect species for homologous nervous system domains (Spindler, 2009).

In the Drosophila ventral nerve cord, two subpopulations of neuropile glia were studied in the context of axonal pathfinding: midline glia and longitudinal glia. Both types of glial cells appear around the same stage when pioneer neurons extend their axons. The proper number and positioning of midline glia is clearly required for the formation of commissural axon tracts. The loss of longitudinal glia (by ablation and in embryos mutant for the gcm gene) primarily affects the defasciculation and fasciculation events of longitudinal pioneer tracts, subsequently affecting the follower neuron trajectories. Note that gcm is required for all classes of glia, including surface glia; thus, in very late gcm mutant embryos, severe disruptions of the entire neuropile result from the fact that, with the onset of embryonic movement, the CNS lacking surface glia is literally 'shredded to pieces' (Spindler, 2009).

In the embryonic Drosophila peripheral nervous system, ablation of the peripheral glia (i.e. exit glia) via targeted overexpression of the cell death genes grim and ced-3 lead to aberrant pathfinding of both motor and sensory axons as they exited the CNS; although the motor neurons eventually overcame the absence of peripheral glia finding correct muscle targets, suggesting a limited role for the peripheral glia in the initial trajectory of motor neurons. In grasshopper, ablation of the cell-segment boundary glial guidepost cell lead to more severely aberrant axon trajectories. Ablation of glia surrounding the antennal lobe of adult Manduca generates olfactory axon de-fasciculation and misguidance. Finally, guidance phenotypes have also been observed with disruption of the Drosophila lamina glia; R1-R6 photoreceptor axons show aberrant guidance past the lamina into the medulla of the optic lobe (Spindler, 2009 and references therein).

An important aspect of the developmental role of glia added by this study is the focus on the correlation between closeness of axon tract/glia association, and axon tract abnormality in the absence of glia. In other words: the nervous system is formed by a large number of fascicles, and these fascicles vary in their degree of glial wrapping. It would be misleading when carrying out a genetic study to only focus on a single fascicle (or small subset of fascicles), and extrapolate from the phenotype observed for this fascicle onto the brain as a whole. In this analysis, SATs of several lineages, notably those that in normal brains have little glia covering them in the neuropile, show few abnormalities in glia-less brains. By contrast, other lineages were affected in the majority of cases, and typically, these lineages also were the ones whose SATs were associated more closely with glia (Spindler, 2009).

Neuropile compartments are formed by terminal branches of axons and dendrites and their synapses. For example, the antennal lobe of insects consists of the axonal terminals of sensory neurons located in the antenna, and dendritic terminals of antennal projection neurons and local interneurons located in the deuterocerebrum, aside from a relatively small number of other modulatory neurons. Sensory neurons expressing the same olfactory receptor all converge onto the dendrites of a small number of projection neurons to form an olfactory glomerulus. In many insects, notably Manduca, olfactory glomeruli are individually compartmentalized by neuropile glia, and it has been shown that glia plays a prominent role in establishing the glomeruli organization. According to the prevailing view, glomeruli are initially ordered by the specialized endings of sensory terminals into protoglomeruli; however, glial processes soon invade the space in between protoglomeruli and restrict the arborization of receptor axons. Therefore, in Manduca antennal lobes, glia is required for early maintenance of the glomerular map (Spindler, 2009 and references therein).

A recent analysis of the time course of glial development in the Drosophila antennal lobe suggested that in this species, glia plays an even lesser role, since glomeruli are only incompletely, and at a late time point, wrapped by glia. While glia were never ablated in those studies, later work found that elimination of Neuroglian from midline glia resulted in an inability of ORN axons to cross through the antennal commissural tract to the contralateral lobe. In this study, most adult brains lacking cortex and neuropile glia still form antennal lobes with glomeruli, however in some samples the glomeruli are poorly defined, and cannot be identified by their position and shape in the antennal lobe. The variability in phenotype penetrance likely stems from larvae containing the most glia surviving to eclosion; therefore, a relatively normal looking adult brain could stem from a weak glial apoptosis early in development. Whether the antennal lobe disorganization is a secondary defect due to a lack of definition normally provided by the presence of glia, a result of SATs that normally contribute to the AL misguiding or truncating early, or a maintenance defect in which axons begin inappropriately intermingling among glomeruli is not clear. Perhaps the Drosophila antennal lobe glia is required for glomeruli maintenance even after glomeruli organization is established, and future studies will hopefully address this possibility (Spindler, 2009).

In the post-embryonic midline of Drosophila, ablation of the transient interhemispheric fibrous ring (TIFR), a transient population of midline glia, by ectopic expression of the pro-death gene hid, generates defects in the adult central complex. What is unclear, however, is the cellular event that is effected to cause the defects. This study suggests that morphological abnormalities in adult compartments from glial manipulation are due to the misguidance of larval SATs to the correct neuropile compartment in the brain, affecting the formation of adult neuropile structures. This study presents evidence that glia is an important mediator of axon guidance in the Drosophila larval brain; the mechanism for glia-neuron communication during this process is an exciting area for future investigation (Spindler, 2009).

A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity

Axons damaged by acute injury, toxic insults, or during neurodegenerative diseases undergo Wallerian or Wallerian-like degeneration, which is an active and orderly cellular process, but the underlying mechanisms are poorly understood. Drosophila has been proven to be a successful system for modeling human neurodegenerative diseases. This study established a novel in vivo model of axon injury using the adult fly wing. The wing nerve highlighted by fluorescent protein markers can be directly visualized in living animals and be precisely severed by a simple wing cut, making it highly suitable for large-scale screening. Using this model, an axonal protective function of WldS and nicotinamide mononucleotide adenylyltransferase (Nmnat) was confirmed. It was further revealed that knockdown of endogenous Nmnat triggered spontaneous, dying-back axon degeneration in vivo. Intriguingly, axonal mitochondria were rapidly depleted upon axotomy or downregulation of Nmnat. The injury-induced mitochondrial loss was dramatically suppressed by upregulation of Nmnat, which also protected severed axons from degeneration. However, when mitochondria were genetically eliminated from axons, upregulation of Nmnat was no longer effective to suppress axon degeneration. Together, these findings demonstrate an essential role of endogenous Nmnat in maintaining axonal integrity that may rely on and function by stabilizing mitochondria (Fang, 2012).

This study presents a novel in vivo model for axon injury and degeneration based on the adult fly wing. Using this model, the following was uncovered: (1) endogenous dNmnat is required for axonal integrity, (2) axonal mitochondria are depleted rapidly upon axotomy or downregulation of dNmnat, (3) upregulation of dNmnat preserves mitochondria in injured axons and delays Wallerian degeneration, and (4) removal of mitochondria from axons abolishes the protective effect of WldS and Nmnat. The levels of mNmnat2 rapidly decline in mammalian neurite culture upon injury. Drosophila has only one gene encoding Nmnat, and axon degeneration was observed as early as 18 hr after axotomy, suggesting a rapid turnover of dNmnat. Hence, reduction of Nmnat levels, either by rapid turnover of Nmnat upon axotomy or genetic knockdown of dNmnat, may render mitochondria unstable and/or dysfunctional, thus triggering axon degeneration. The self-destructive mechanisms of axon degeneration in injury and loss of endogenous Nmnat appear to converge at axonal mitochondria. This may underlie the morphological similarity between Wallerian degeneration and spontaneous axon degeneration in dying-back diseases. As such, endogenous Nmnat and axonal mitochondria may be key to identifying additional downstream events and therefore providing exciting new targets for therapeutic interventions of both acute neural injury and chronic axonal disorders (Fang, 2012).

WldS prevents axon degeneration through increased mitochondrial flux and enhanced mitochondrial Ca2+ buffering

WldS (slow Wallerian degeneration) is a remarkable protein that can suppress Wallerian degeneration of axons and synapses, but how it exerts this effect remains unclear. Using Drosophila and mouse models, this study identified mitochondria as a key site of action for WldS neuroprotective function. Targeting the NAD(+) biosynthetic enzyme Nmnat to mitochondria was sufficient to fully phenocopy WldS, and WldS was specifically localized to mitochondria in synaptic preparations from mouse brain. Axotomy of live wild-type axons induced a dramatic spike in axoplasmic Ca(2+) and termination of mitochondrial movement-WldS potently suppressed both of these events. Surprisingly, WldS also promoted increased basal mitochondrial motility in axons before injury, and genetically suppressing mitochondrial motility in vivo dramatically reduced the protective effect of WldS. Intriguingly, purified mitochondria from WldS mice exhibited enhanced Ca(2+) buffering capacity. It is proposed that the enhanced Ca(2+) buffering capacity of WldS+ mitochondria leads to increased mitochondrial motility, suppression of axotomy-induced Ca(2+) elevation in axons, and thereby suppression of Wallerian degeneration (Avery, 2012).

The mechanistic action of WldS has remained controversial, but recent work has established a nonnuclear role for WldS after injury. This study shows that WldS is localized to mitochondria in vivo. It is important to note that protein localization studies with WldS must be interpreted cautiously -- the primarily nuclear localization of WldS suggested a nuclear role for WldS and initially misled the field. However, this study also found that WldS increases mitochondrial Ca2+ buffering capacity and results in maintained mitochondrial motility after axotomy. Taken together, these data argue strongly that the mitochondrial compartment is a key site of action for WldS in vivo (Avery, 2012).

This study has shown that axonal injury in live Drosophila preparations leads to a dramatic and transient rise in axonal Ca2+. Increased axonal Ca2+ has been observed in mammals after acute nerve crush and entry of extracellular Ca2+ is necessary and sufficient for Wallerian degeneration. Impressively, WldS expression resulted in a striking suppression of this axotomy-induced rise in axonal Ca2+. The most plausible explanation for this enhanced buffering is that increased ATP and energy production observed in WldS+ mitochondria -- presumably via increased mitochondrial NAD+ production, though essential roles cannot be formally excludes for other substrates of Nmnat -- is linked to increased mitochondrial membrane potential (ΔΨm), and thereby increased Ca2+ entry through the ΔΨm-regulated mitochondrial Ca2+ uniporter. This model is supported by the observation that WldS-expressing mitochondria isolated from mouse brain exhibit an enhanced ability to maintain their membrane potential and avoid PTP formation in the face of increasing extramitochondrial Ca2+. In the future, it will be important to confirm that such changes are also observed in Drosophila axonal mitochondrial physiology in vivo in WldS-expressing neurons (Avery, 2012).

Axonal Ca2+ spikes could result solely from entry of extracellular Ca2+ into the axon after injury. This would be consistent with the observation that blocking Ca2+ channels inhibits Wallerian degeneration. Mitochondria are a well-established sink for Ca2+ in axons and this study shows that WldS+ mitochondria exhibit enhanced Ca2+ buffering capacity and resistance to Ca2+-induced formation of the permeability transition pore (PTP). Indeed PTP formation appears to be a key final execution step in Wallerian degeneration. A model is therefore favored whereby extracellular Ca2+ enters the axon after axotomy and normally acts as a switch to activate Wallerian degeneration. In WldS axons, this Ca2+ is instead rapidly buffered by mitochondria, thereby blocking induction of axonal destruction. Consistent with this model, uncoupling mitochondria, which suppresses mitochondrial Ca2+ uptake, completely abrogates the protective effect of WldS in vitro (Avery, 2012).

WldS-expressing neurons exhibit a roughly 2-fold increase in the number of motile versus stationary mitochondria compared to WT controls, which could result from changes in mitochondrial Ca2+ buffering. Notably, genetic suppression of enhanced mitochondrial flux using mutations in miro also resulted in a remarkable suppression of WldS-mediated axonal protection in vivo. However, because this suppression was only partial, additional factors beyond increases in mitochondrial motility must also contribute to WldS-mediated axonal protection. For example, axonal energy supplies are likely closely intertwined with mitochondrial transport and bioenergetics. WldS+ mitochondria are known to exhibit an enhanced ability to generate ATP. This change in bioenergetics, coupled with increased mitochondrial motility in WldS+ axons, might enhance distribution of ATP or other mitochondrially-derived metabolites. At the same time, enhanced mitochondrial motility could also speed the removal of metabolic byproducts normally processed by mitochondria. Similarly, increased mitochondrial motility in axons could further enhance mitochondrial Ca2+ buffering in WldS+ axons because motile mitochondria would be predicted to traverse more 'axonal space' and perhaps be exposed to more Ca2+ than stationary mitochondria. Together, these could have the combined effect of increasing energy delivery, removing harmful byproducts, and increased buffering of Ca2+, a signal that can potently activate axonal degeneration (Avery, 2012).

A role for mitochondria in the WldS neuroprotective mechanism is intriguing because defects in mitochondria respiration and dynamics are emerging as critical underlying factors in a number of neurological disorders. For example, in mouse models of ALS (SOD1 transgenics), anterograde and retrograde mitochondrial transport is reduced, altered mitochondrial trafficking has been observed in models of Alzheimer's disease, and mutant, but not WT Huntington, protein blocks mitochondrial movement in cortical neurons. However, in the majority of models, whether these mitochondrial alterations are a cause or consequence of disease remains an open question. The current study shows, reciprocally, that enhanced mitochondrial flux is associated with and is required for maximal axon protection by WldS (Avery, 2012).

In vitro studies conducted in Aplysia and chick sensory neurons indicate that in addition to microtubule assembly, long microtubules in the C-domain of the growth cone move forward as a coherent bundle during axonal elongation. Nonetheless, whether this mode of microtubule translocation contributes to growth cone motility in vivo is unknown. This question was addressed in Drosophila. Using docked mitochondria as fiduciary markers for the translocation of long microtubules, motion along the axon was examined to test if the pattern of axonal elongation is conserved between Drosophila and other species in vitro. When Drosophila neurons were cultured on Drosophila extracellular matrix proteins collected from the Drosophila Kc167 cell line, docked mitochondria moved in a pattern indicative of bulk microtubule translocation, similar to that observed in chick sensory neurons grown on laminin. To investigate whether the C-domain is stationary or advances in vivo, the movement of mitochondria was tracked during elongation of the aCC motor neuron in stage 16 Drosophila embryos. Docked mitochondria were found to move forward along the axon shaft and in the growth cone C-domain. This work confirms that the physical mechanism of growth cone advance is similar between Drosophila and vertebrate neurons and suggests forward translocation of the microtubule meshwork in the axon underlies the advance of the growth cone C-domain in vivo. These results highlight the need for incorporating en masse microtubule translocation, in addition to assembly, into models of axonal elongation (Roossien, 2013).

The NAV2 homolog Sickie regulates F-actin-mediated axonal growth in Drosophila mushroom body neurons via the non-canonical Rac-Cofilin pathway

The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. This study demonstrates that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. Phenotypic similarities and genetic interactions were observed among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. It is proposed that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner (Abe, 2014).

During brain development, neurons undergo multiple morphological changes to form an elaborate neural network. The Drosophila mushroom body (MB), which forms bilaterally symmetric and dorsomedially bifurcated axonal lobe structures in the central brain, has been well studied as a model of neuronal development. Among various regulators of neuronal morphogenesis, ADF/Cofilin and Rac GTPase (Rac) are key molecules in controlling cytoskeletal remodeling in axonal development. Cofilin [Twinstar (Tsr) in Drosophila] plays an essential role as a regulator of axonal growth by severing and depolymerizing F-actin. Because Cofilin is activated by dephosphorylation by the Slingshot (Ssh) phosphatase and is inactivated by phosphorylation by LIMK, the loss of Ssh or excessive activation of LIMK results in an axonal growth defect. In Drosophila, Rac has been proposed to act as a bidirectional switch for signaling cascades. One signaling event is the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth. The overexpression of Pak, a downstream effector of Rac, induces axonal growth defects similar to those observed with LIMK overexpression. In addition, introducing one mutant copy of Rac or Pak suppresses the axonal defect induced by LIMK overexpression. Another signaling event is the Pak-independent non-canonical pathway to positively regulate axonal growth. Rac mutant animals show multiple MB axonal defects, but the axonal growth defect is alleviated by the exogenous expression of Rac1Y40C, which lacks the ability to activate Pak but does not affect lamellipodia formation. Furthermore, Rac1Y40C overexpression remarkably suppressed the LIMK overexpression phenotype (Abe, 2014).

Although several pieces of evidence have suggested the importance of the non-canonical pathway and predicted the existence of its mediator, whether this pathway finally converges upon the downstream Cofilin pathway and subsequent F-actin reorganization remains unclear. Moreover, many biochemical studies have assessed the regulation of Cofilin function and F-actin states using in vitro systems; the endogenous changes in F-actin and Cofilin phosphorylation appear not to have been analyzed simultaneously with an internal control in developing brain. To address these issues, a novel factor was sought that interacts with Rac-Cofilin signaling components and positively regulates MB axonal growth. It was observed that Sickie, which has a calponin homology (CH) actin-binding domain and shares structural similarities with the human microtubule-associated protein (MAP) neuron navigator 2 (NAV2), showed prominent expression in F-actin-rich newborn MB axons and genetically interacted with Rac-Cofilin signaling regulators. Although Sickie was originally identified by genome-wide RNAi screening in Drosophila S2 cells and the report proposed the involvement of Sickie in the innate immune response (Foley, 2004), in this report focus was placed on the function of Sickie in the regulation of Cofilin-mediated F-actin remodeling and propose an expanded model of regulatory mechanisms during axonal development (Abe, 2014).

By combining the MARCM technique with epistatic analysis, this study demonstrated that Sickie regulates the axonal growth of Drosophila MB neurons via the non-canonical Rac-Cofilin pathway. The following model is proposed. In wild type, Sickie relays the non-canonical pathway signal to Ssh to facilitate F-actin-mediated axonal growth by counteracting the canonical signal. In a sickie mutant, mediation of the non-canonical pathway is defective, which causes an imbalance in the regulation of Cofilin activity. Because neurons are morphologically polarized and the amount of actin is limited in each cell, the growing axons may efficiently control actin recycling by facilitating F-actin turnover by balancing between the non-canonical and canonical pathways. Consistently, a stronger axonal growth defect was found with increased P-Cofilin in the LIMKWTM6 ssh1-63 and sickieΔ LIMKKD double-mutant animals than in the single mutants ssh1-63, sickieΔ and LIMKKD. Cofilin activity might be decreased in the developing axons of these double mutants by the preponderance of the canonical pathway. If so, these results highlight an essential role of the non-canonical pathway to balance Cofilin activity in axonal growth (Abe, 2014).

Unlike the clear elevation of P-Cofilin levels in the ssh1-63 mutant, constitutive activation of LIMK did not result in a similar increase in P-Cofilin despite F-actin elevation. This apparent paradox might be explained by considering the positive regulation of Ssh by F-actin. The phosphatase activity of SSH-1L is F-actin dependent, and the addition of F-actin dramatically increases its phosphatase activity. In the LIMKKD mutant axons, endogenous Ssh may be activated by a large amount of F-actin and subsequently dephosphorylates Cofilin. Consistently, highly elevated signals of both F-actin and P-Cofilin were detected in the LIMKWTM6 ssh1-63 double-mutant clones. In this mutant, Cofilin activity was severely reduced by high phosphorylation levels due to constitutive LIMK activation and a lack of Ssh phosphatase activity, resulting in the posterior arrest severe axonal defect, similar to the cofilin knockdown mutant. In addition, relatively moderate increases in P-Cofilin signal were detected in the developing axons of the sickieΔ LIMKKD double mutant. These results also support the model that Sickie functions in the same pathway as Ssh to positively regulate Cofilin function by counteracting the canonical Rac-Pak-LIMK pathway. Ssh might be downregulated in the sickie mutant axons due to defects in the mediation of Pak-independent Rac1 function or in the interaction among Ssh and F-actin by the loss of Sickie. The similar increases in the P-Cofilin and F-actin signals and the similar posterior arrest phenotype in the LIMKWTM6 ssh1-63 double-mutant clone and those of the PakMyr mutant clone are also consistent with results of in vitro studies that showed that SSH-1L is inactivated by Pak4. Thus, in the current model, Pak concurrently inactivates Ssh and activates LIMK in axonal growth (Abe, 2014).

Whereas ssh or cofilin mutants are embryonic lethal and their mutant clones display developmental defects in non-neuronal tissues, sickie mutants are not embryonic lethal, and conspicuous phenotypes are found only in the substructures of the central brain, such as MB and EB, implying that more elaborate mechanisms involving Sickie function are required for ensuring their proper development. Given that MB neurons exhibit a densely bundled axonal morphology, the growing MB axons might require Sickie to smoothly extend their neurites within the lobe core region by coordinating the dynamics of actin and microtubules (MTs). Sickie and human neuron navigator proteins (NAVs) have conserved EB1-binding motifs, and Sickie shows a genetic interaction with MT components. Double RNAi of sickie and EB1 or β-tubulin both resulted in synergistic increases in the axonal defects. In addition, a recent cell biological study demonstrated a functional link between Cofilin and MTs. Through its interaction with EB1, Sickie might act as a navigator for the plus-end of MTs to link to the F-actin complex and thereby ensure elaborate neuronal wiring. To further elucidate the signaling mechanism, the relationships with other components of the Ssh-dependent Cofilin pathway need to be studied. Recent studies have revealed that PKD, 14-3-3 protein and Pak4 play key roles in suppressing Ssh function (Abe, 2014).

Finally, preliminary data suggest a post-developmental role for Sickie. Adult stage-specific knockdown of Sickie in MBs impairs olfactory memory. Moreover, recent mammalian studies have suggested the possible involvement of NAVs and Cofilin in neurodegenerative disease. In Alzheimer's disease brains, the NAV3 transcript level is elevated, and Cofilin-actin rod-shaped inclusions, which are formed by the hyperactivation of Cofilin, are enriched. Further studies are required to understand the wide variety of contributions of sickie and the general importance of this evolutionarily conserved gene in brain development and function (Abe, 2014).

The Homeodomain Transcription Factor Hb9 Controls Axon Guidance in Drosophila through the Regulation of Robo Receptors

Transcription factors establish neural diversity and wiring specificity; however, how they orchestrate changes in cell morphology remains poorly understood. The Drosophila Roundabout (Robo) receptors regulate connectivity in the CNS, but how their precise expression domains are established is unknown. This study shows that the homeodomain transcription factor Hb9 acts upstream of Robo2 and Robo3 to regulate axon guidance in the Drosophila embryo. In ventrally projecting motor neurons, hb9 is required for robo2 expression, and restoring Robo2 activity in hb9 mutants rescues motor axon defects. Hb9 requires its conserved repressor domain and functions in parallel with Nkx6 to regulate robo2. Moreover, hb9 can regulate the mediolateral position of axons through robo2 and robo3, and restoring robo3 expression in hb9 mutants rescues the lateral position defects of a subset of neurons. Altogether, these data identify Robo2 and Robo3 as key effectors of Hb9 in regulating nervous system development (Santiago, 2014).

Combinations of transcription factors specify the tremendous diversity of cell types in the nervous system. Many studies have identified requirements for transcription factors in regulating specific events in circuit formation as neurons migrate, form dendritic and axonal extensions, and select their final synaptic targets. In most cases, the downstream effectors through which transcription factors control changes in neuronal morphology and connectivity remain unknown, although several functional relationships have been demonstrated (Santiago, 2014).

Conserved homeodomain transcription factors regulate motor neuron development across phyla. Studies in vertebrates and invertebrates have shown that motor neurons that project to common target areas often express common sets of transcription factors, which act instructively to direct motor axon guidance. In mouse and chick, Nkx6.1/ Nkx6.2 and MNR2/Hb9 are required for the specification of spinal cord motor neurons, and for axon pathfinding and muscle targeting in specific motor nerves. In Drosophila, Nkx6 and Hb9 are expressed in embryonic motor neurons that project to ventral or lateral body wall muscles, and although they are not individually required for specification, they are essential for the pathfinding of ventrally projecting motor axons. Axons that project to dorsal muscles express the homeodomain transcription factor Even-skipped (Eve), which regulates guidance in part through the Netrin receptor Unc5. Eve exhibits cross-repressive interactions with hb9 and nkx6, which function in parallel to repress eve and promote islet and lim3. Hb9 and Nkx6 act as repressors to regulate transcription factors in the spinal cord; however, guidance receptors that act downstream of Hb9 and Nkx6 have not been characterized. Interestingly, in both flies and vertebrates, Hb9 and Nkx6 are also expressed in a subset of interneurons, and knockdown experiments in Drosophila have suggested a role for hb9 in regulating midline crossing (Santiago, 2014).

Roundabout (Robo) receptors regulate midline crossing and lateral position within the developing CNS of invertebrates and vertebrates. Two recent studies in mice have also identified a role for Robos in regulating motor axon guidance in specific motor neuron populations. The three Drosophila Robo receptors have diversified in their expression patterns and functions. Robo2 is initially expressed in many ipsilateral pioneers and also contributes to Slit-mediated repulsion. Subsequently, robo2 expression is more restricted, and it is required to specify the medio-lateral position of axons. Robo3 is expressed in a subset of CNS neurons and also regulates lateral position (Santiago, 2014).

Characterization of the expression domains of the Drosophila Robos revealed an intriguing pattern, in which Robo1 is expressed on axons throughout the width of the CNS, Robo3 is found on axons in intermediate and lateral zones, and Robo2 is enriched on the most lateral axons. These patterns are transcriptional in origin, as replacing any robo gene with the coding sequence of another Robo receptor results in a protein distribution that matches the endogenous expression of the replaced gene (Spitzweck, 2010). A phenotypic analysis of these gene-swap alleles revealed the importance of transcriptional regulation for the diversification of robo gene function (Spitzweck, 2010). Robo2 and robo3's roles in regulating lateral position are largely dependent on their expression patterns, although unique structures within the Robo2 receptor are also important for its function in lateral position (Evans, 2010; Spitzweck, 2010). In the peripheral nervous system, the Atonal transcription factor regulates robo3 in chordotonal sensory neurons, directing the position of their axon terminals. In the CNS, the transcription factors lola and midline contribute to the induction of robo1. However, how the expression patterns of robo2 and robo3 are established to direct axons to specific medio-lateral zones within the CNS remains unknown (Santiago, 2014).

This study identifies a functional relationship between Hb9 and the Robo2 and Robo3 receptors in multiple contexts. Hb9 acts through Robo2 to regulate motor axon guidance and can direct the medio-lateral position of axons in the nerve cord through its effects on robo2 and robo3. Furthermore, hb9 interacts genetically with nkx6 and requires its conserved repressor domain to regulate robo2. Together, these data establish a link between transcriptional regulators and cell surface guidance receptors, providing an example of how upstream factors act through specific guidance receptors to direct circuit formation (Santiago, 2014).

This study has demonstrated a functional relationship between Hb9 and the Robo2 and Robo3 receptors in multiple contexts in the Drosophila embryo. In the RP motor neurons, hb9 is required for robo2 expression, and genetic rescue experiments indicate that robo2 acts downstream of hb9. Hb9 requires its conserved repressor domain and acts in parallel with Nkx6 to regulate robo2 and motor axon guidance. Moreover, hb9 contributes to the endogenous expression patterns of robo2 and robo3 and the lateral position of a subset of axons in the CNS, and can redirect axons laterally when overexpressed via upregulation of robo2. Finally, restoring Robo3 rescues the medial shift of MP1 axons in hb9 mutants, indicating that hb9 acts through robo3 to regulate medio-lateral position in a defined subset of neurons (Santiago, 2014).

Hb9 and nkx6 are required for the expression of robo2 in motor neurons, and rescue experiments suggest that the loss of robo2 contributes to the phenotype of hb9 mutants. However, nkx6 mutants and hb9 mutants heterozygous for nkx6 have a stronger ISNb phenotype than robo2 mutants, implying the existence of additional downstream targets. One candidate is the cell adhesion molecule FasIII, which is normally expressed in the RP motor neurons and appears reduced in nkx6 mutant embryos. Identifying the constellation of effectors that function downstream of Hb9 and Nkx6 will be key to understanding how transcription factors expressed in specific neurons work together to drive the expression of the cell surface receptors that regulate axon guidance and target selection (Santiago, 2014).

Robo2's activity in motor axon guidance appears distinct from the previously described activities of the Drosophila Robo receptors. Although Robo1 can replace Robo2's repulsive activity at the midline (Spitzweck, 2010), Robo2's function in motor axon guidance is not shared by either Robo1 or Robo3. Moreover, Robo2's antirepulsive activity at the midline and its ability to shift axons laterally when overexpressed both map to Robo2's ectodomain, whereas this study has found that Robo2's activity in motor axon guidance maps to its cytodomain (Evans, 2010; Spitzweck, 2010). The signaling outputs of Robo2's cytodomain remain unknown, as it lacks the conserved motifs within Robo1 that engage downstream signaling partners. How does Robo2 function during motor axon guidance? In mice, Robo receptors are expressed in spinal motor neurons and prevent the defasciculation of a subset of motor axons (Jaworski, 2012). Does Drosophila Robo2 regulate motor axon fasciculation? The levels of adhesion between ISNb axons and other nerves must be precisely controlled during the different stages of motor axon growth and target selection, and several regulators of adhesion are required for ISNb guidance. Furthermore, whereas Slit can be detected on ventral muscles, it is not visibly enriched in a pattern that suggests directionality in guiding motor axons, making it difficult to envision how Robo2-mediated repulsive or attractive signaling might contribute to ISNb pathfinding. Future work will determine how Robo2's cytodomain mediates motor axon guidance, whether this activity is Slit dependent, and whether Robo2 signals attraction, repulsion, or modulates adhesion in Drosophila motor axons (Santiago, 2014).

Elegant gene-swap experiments revealed the importance of transcriptional regulation in establishing the different expression patterns and functions of the Drosophila Robo receptors (Spitzweck, 2010). By analyzing a previously uncharacterized subset of axon pathways, this study has uncovered a requirement for Hb9 in regulating lateral position in the CNS. Although Hb9 can act instructively to direct lateral position when overexpressed, its endogenous expression in a subset of medially projecting neurons suggests that its ability to shift axons laterally is context dependent. A complex picture emerges in which multiple factors act in different groups of neurons to regulate robo2 and robo3. In a subset of interneurons, hb9 is endogenously required for lateral position through the upregulation of robo3 and likely robo2. In other neurons, such as those that form the outer FasII tracts, the expression patterns of robo2 and robo3 rely on additional upstream factors. What might be the significance of a regulatory network in which multiple sets of transcription factors direct lateral position in different groups of neurons? One possibility is that hb9-expressing neurons may share specific functional properties, such as the expression of particular neurotransmitters or ion channels. Alternatively, hb9 may regulate other aspects of connectivity. Indeed, Robo receptors mediate dendritic targeting in the Drosophila CNS, raising the exciting possibility that hb9 regulates both axonal and dendritic guidance through its effects on guidance receptor expression (Santiago, 2014).

What is the mechanism by which Hb9 regulates the expression of robo2, robo3, and its other downstream effectors? This study has found that Hb9 requires its conserved putative repressor domain and acts in parallel with Nkx6 to regulate robo2 and motor axon guidance. It has previously been shown that hb9 and nkx6 function in parallel to regulate several transcription factors. Hb9, nkx6 double mutants show decreased expression of islet and lim3 and upregulation of eve and the Nkx2 ortholog vnd. Are Hb9 and Nkx6 regulating robo2 or robo3 through any of their previously identified targets? Hb9 and nkx6 single mutants show no change in islet, lim3, or vnd expression, arguing that hb9 and nkx6 do not act solely through these factors to regulate robo2 or robo3. Eve expression is unaffected in nkx6 mutants, and whereas it is ectopically expressed in two neurons per hemisegment in hb9 mutants, these do not correspond to RP3 or MP1, the identifiable cells in which changes can be detected in robo2 and robo3. Therefore, the data do not support the hypothesis that Hb9 and Nkx6 regulate robo2 or robo3 primarily through their previously identified targets islet, lim3, vnd, or eve (Santiago, 2014).

Gain-of-function experiments in vertebrates suggest that Hb9 and Nkx6 act as repressors to regulate gene expression in the spinal. The finding that Hb9's Eh domain is required for motor axon pathfinding and robo2 regulation suggests that Hb9 acts as a repressor in this context as well, most likely through a previously unidentified intermediate target. In contrast, the Eh domain is not required for Hb9's ability to regulate robo3 or lateral position in hb9GAL4+ neurons that project to intermediate zones of the CNS. The finding that Hb9;delta;Eh retains significant activity in rescuing lateral position and robo3 expression indicates that Hb9 may regulate robo2 and robo3 via distinct mechanisms, perhaps involving different transcriptional cofactors or intermediate targets. In support of this hypothesis, hb9 overexpression in the ap neurons can induce robo2, but not robo3. These data raise the intriguing possibility that Hb9's ability to regulate robo2 and robo3 via different mechanisms contributed to the diversification of their expression patterns in the CNS. Determining how Hb9 and Nkx6 regulate their effectors will be key to achieving a complete understanding of how these conserved transcription factors control changes in cell morphology and axon pathfinding during development. Of note, Hb9 mutant mice exhibit defects in a subset of motor nerves, including the phrenic and intercostal nerves, which are also affected in Robo mutants. It will be of great interest to determine if despite the vast divergence in the evolution of nervous system development between invertebrates and vertebrates, Hb9 or Nkx6 has retained a role for regulating Robo receptors across species (Santiago, 2014).

Drosophila Strip serves as a platform for early endosome organization during axon elongation

Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. This study showed that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis (Sakuma, 2014).

Robo-3-mediated repulsive interactions guide R8 axons during Drosophila visual system development

The formation of neuronal connections requires the precise guidance of developing axons toward their targets. In the Drosophila visual system, photoreceptor neurons (R cells) project from the eye into the brain. These cells are grouped into some 750 clusters comprised of eight photoreceptors or R cells each. R cells fall into three classes: R1 to R6, R7, and R8. Posterior R8 cells are the first to project axons into the brain.Using a microarray-based molecular screen as a starting point, this study identified the early and transient expression of Robo3 in R8 growth cones. Loss of Robo3 demonstrated a specific axon guidance choice point at an early stage of optic lobe innervation. In the absence of Robo3, posterior R8 growth cones inappropriately extend across Slit-expressing glial cells joining axon fascicles of the C+T lobula neurons, instead of remaining alongside the glial process as they extend into the lamina. This early repulsive function of Robo3 plays a crucial role in segregating axons and thereby contributes to the orderly assembly of columnar units comprising the fly visual system (Pappu, 2011).

The microarray data, coupled with antibody staining and the identification of a robo3-Gal4 enhancer trap, identified Robo3 as an R8-specific guidance receptor. Robo3 expression is transient in the R8 growth cone and prolonging Robo3 expression in R8 axons results in defects in R8 targeting. The microarray analysis suggests that restricted Robo3 expression during early stages of R8 differentiation occurs downstream of the transcription factors Sens and Run. However, the expression of both Sens and Run persists beyond the expression of Robo3. For example, during mid- to late-pupal development, Sens regulates both targeting of R8 axons to their final target layer in the medulla and the expression of R8-specific opsins. Therefore, other mechanisms must exist to control the expression of Robo3 in R8 (Pappu, 2011).

The importance of the tightly regulated expression of Robo receptors is emerging as a central theme in axon guidance. Indeed, previous studies have revealed a set of discrete posttranslational mechanisms controlling Robo functions both in vertebrates and invertebrates. For example, commissural axons in the fly embryo express Robo1 transcripts before crossing the midline, but Robo1 protein in these axons is sequestered into endosomes by the action of Commissureless protein, thereby preventing precocious repulsion from the midline and, thus, allowing these axons to cross. Subsequent up-regulation of Robo1 prevents them from recrossing. In vertebrates, alternative splicing of a divergent Robo receptor Rig1/Robo3, perhaps coupled with translational regulation, governs the switch from midline attraction to repulsion (Pappu, 2011).

The regulated expression of Robo3 in the R8 photoreceptors is similar to its expression in the chordotonal neurons in the embryonic peripheral nervous system. Sens is activated downstream of Ato in both chordotonal and in R8 neurons, suggesting that a conserved transcriptional program regulates Robo3 expression in these neurons. In a broader sense, these findings raise the possibility that conserved regulatory cassettes exist, which link specific transcriptional hierarchies controlling neuronal differentiation with specific constellations of downstream guidance receptors controlling wiring specificity (Pappu, 2011).

Posterior-most R8 neurons face at least three different guidance choices as they extend from the eye disk into the developing optic lobe. These early choices have a profound effect on later aspects of visual system assembly. First, R8 axons must navigate to the posterior of the eye disk and enter the optic stalk. This process is facilitated, in part, by retinal basal glial cells at the posterior edge of the eye disk. If glial cells are displaced anteriorly, R-cell axon fascicles project away from the optic stalk rather than toward it. Although it seems likely that this directional choice relies upon R8, it is not known whether posterior growth requires R8-specific functions or whether all retinal neurons are endowed with this function (Pappu, 2011).

Second, R8 axons from each ommatidium must possess molecular mechanisms to retain their individuality. As the R8 axons extend down the optic stalk they form a tight fascicle. Fasciculation is transient, however, because R8s defasciculate as they exit the optic stalk. R8 defasciculation relies on two cell surface receptors, Flamingo (Fmi) and Golden Goal (Gogo) that are expressed in the R8 growth cones as they exit the optic stalk. Fmi and Gogo mediate repulsive interactions between R8 axons, and thus play a key role ensuring that columns remain as separate modules. These repulsive interactions between R8 axons of adjacent columns also explain why axons from later-born (anterior) R8 neurons are not affected in robo33 mutant optic lobes; only the posterior R8 axons traverse through the optic lobe with access to the glial cell boundaries that separate them from the C+T lobular neuron axons (Pappu, 2011).

Third, in this article it was demonstrated that posterior R8 axons rely upon Robo3 to prevent inappropriate fasciculation with C+T lobula neurons. This process requires early, transient, and specific expression of Robo3 in R8 growth cones and is likely to require the reciprocal expression of Slit in the glial cells that these posterior R8 axons encounter when they enter the optic lobe. Thus, the posterior R8 axons are unique because they navigate a choice point that is not encountered by later arriving, more anterior R8 axons. The robo33 mutant phenotype described in this study is reminiscent of the loss of another Ig receptor encoded by the irregular chiasm/roughest (irre-C) locus. Whether IrreC acts in the same molecular pathway as Robo3 and, indeed, whether it acts in photoreceptor growth cones or lamina neurons is not known (Pappu, 2011).

In summary, Fmi, Gogo, and Robo3 play crucial roles in R8s in regulating fascicle organization, which provides the structural basis for columnar organization of the visual system. Although Fmi and Gogo mediate interactions between axons of the same class of cells (R8s), Robo3 prevents axons from one class of neurons (R8s) from inappropriately associating with a different class of axons (C+T lobular neurons) projecting into the same neuropil along a different pathway. Given the cellular complexity of columns (e.g., medulla columns comprise more than 50 axons from many different neuronal subclasses) and the stereotyped organization of axons and synaptic connections within them, it is speculated that many additional cell-surface proteins must act in a coordinated fashion in space and time to promote the orderly assembly of columnar units (Pappu, 2011).

Does Robo3 function in the R8 rely on Slit? This is indeed the most parsimonious model for Robo3 function in R8. Slit expression is detected around glial cells separating the posterior R8 growth cones from C+T lobula neurons. Although R8 projection defects seen in slit mutants are similar to those seen in robo3 mutants, they are more severe. In contrast to robo3, slit mutant optic lobes are extremely disorganized, arguing that Slit has a broader role in neuropil organization. Indeed, mutants deficient in all three Robo proteins exhibit cell migration defects, which largely phenocopy the loss of Slit. It has been proposed that Slit provides a repellent function in the optic lobe preventing cell migration between cell populations in the lamina and lobula. However, the residual Robo3 function in the robo31 hypomorphic allele available for study at that time masked the robo3 phenotypes uncovered in this study. Thus, although slit mutants uncover a broader role for Slit-Robo signaling in many aspects of optic lobe development and patterning, the unique robo3 mutants described in this study uncover a specific role for the Robo3 receptor in R8 axon guidance (Pappu, 2011).

The repulsive role of Robo3 in R8 neurons in response to locally secreted Slit is proposed in this study to be analogous to the role of Robo1 in the guidance of ipsilateral longitudinal pioneer axons in the ventral nerve cord. Robo1 is expressed on the growth cones of ipsilateral pioneer axons and prevents these axons from crossing the midline in response to Slit secreted from midline glia. In contrast, Robo3 is not expressed in the growth cone of commissural and longitudinal pioneer axons and is dispensable for the midline crossing during the development of the embryonic ventral nerve cord. Thus, the function of Robo3 in posterior R8s is analogous to the function of Robo1 in embryonic ipsilateral pioneers. However, analyses of knock-in mutants indicate that Robo1 and Robo3 must be sensitive to cell-type-specific regulatory functions. Robo1 has a unique role in midline repulsion of ipsilateral pioneers as it cannot be functionally replaced by Robo2 or Robo3. In contrast, as reported in this study, either Robo1 or Robo2 can functionally replace Robo3 in the R8s. Thus, repulsive signaling downstream of ipsilateral pioneers in the embryo is dependent on unique structural features of the Robo1 protein, but repulsion of posterior R8 axons does not depend on unique structural features of Robo3. Instead it is the unique and context-specific expression of Robo3 that allows it to determine R8 axon guidance and in a broader context function in the orderly assembly of a subset of columnar elements in the visual circuit (Pappu, 2011).

The guanine exchange factor Vav controls axon growth and guidance during Drosophila development

The Vav proteins are guanine exchange factors (GEFs) that trigger the activation of the Rho GTPases in general and the Rac family in particular. While the role of the mammalian vav genes has been extensively studied in the hematopoietic system and the immune response, there is little information regarding the role of vav outside of these systems. This study reports that the single Drosophila vav homolog is ubiquitously expressed during development, although it is enriched along the embryonic ventral midline and in the larval eye discs and brain. The role that vav plays during development was analyzed by generating Drosophila null mutant alleles. The results indicate that vav is required during embryogenesis to prevent longitudinal axons from crossing the midline. Later on, during larval development, vav is required within the axons to regulate photoreceptor axon targeting to the optic lobe. Finally, it was demonstrated that adult vav mutant escapers, which exhibit coordination problems, display axon growth defects in the ellipsoid body, a brain area associated with locomotion control. In addition, this study showed that vav interacts with other GEFs known to act downstream of guidance receptors. Thus, it is proposed that vav acts in coordination with other GEFs to regulate axon growth and guidance during development by linking guidance signals to the cytoskeleton via the modulation of Rac activity (Malartre, 2010).

Vav members are key regulators of the Rho GTPases and the Rac proteins in particular. However, although many studies have implicated the Rac proteins in controlling several aspects of axon growth and guidance during development, understanding of vav function in these processes is far more primitive. This is quite surprising given that all vav members are expressed in neural tissues in mammals. Recently, analysis of postnatal vav2/vav3 -deficient mice has revealed abnormal retinogeniculate projections. This study demonstrates that in Drosophila vav is required for axon growth and guidance at embryonic, larval, and pupal stages. Hence, these data strengthen the role of vav in multiple aspects of axogenesis during development (Malartre, 2010).

During the formation of the embryonic central nervous system of Drosophila, the neurons send out axons that project either ipsilaterally or contralaterally to form the complex axonal lattice. A small number of neurons project ipsilaterally as they receive repulsive signals from the midline glia and never cross the midline, while most neurons project contralaterally, cross the midline, and form the commissures. In vav2/vav3 mutant mouse brains, ipsilateral but not contralateral projections are affected. These results are consistent with the current data showing that Drosophila vav is required to regulate proper ipsilateral axon projection, as in vav mutant embryos the most medial longitudinal axons occasionally cross the midline when they should not. These fascicles are particularly sensitive to perturbations in axon guidance mechanism and cross the midline whenever repulsive signaling is altered, suggesting that Vav might participate in the regulation of repulsive signaling from the midline (Malartre, 2010).

This study also demonstrated that vav is required during subsequent larval development in regulating photoreceptor axon targeting to the optic lobe. This is again consistent with the finding that vav2/vav3 mutant mice display abnormal projections of axons connecting the retinal cells to the brain, suggesting that the role of vav in mediating axon guidance decisions is conserved between species. Interestingly, vav function in photoreceptors (R cells) seems to be more important than in the embryonic CNS. Indeed, this study found that in 100% of the larvae, R cell axons projected aberrantly to the lamina and the medulla target regions, while only 14% of vav mutant embryos displayed guidance defects. Finally, it was shown that later, during metamorphosis, vav is required once more for the correct formation of the ellipsoid body, one of the central brain structures. The ellipsoid body, in a majority of vav mutant adult brains, does not close properly and remains ventrally opened, most likely reflecting defects in the growth of the axons forming the ellipsoid body rather than guidance errors. Interestingly, the ellipsoid body has been involved in the control of locomotion, and vav mutant adults display strong locomotion defects. Opened ellipsoid bodies have also been found in ciboulot mutants. However these mutants do not display locomotion defects, suggesting that a disruption of the ellipsoid body alone is not sufficient to produce the locomotion phenotype observed in vav mutants. This implies that vav might also be required to regulate other aspects of axogenesis, in addition to the ones identified in this study (Malartre, 2010).

In summary, these results show that vav is required reiteratively throughout life to regulate different axogenesis events, including axon growth and guidance (Malartre, 2010).

During larval development, R cell axon targeting to the optic lobe is controlled, on the one hand, by some genes that are acting within the axons themselves, and on the other hand, by some genes that are sending signals to the axons from the glia to guide them. MARCM experiments clearly demonstrate a role for vav within the R cell axons to regulate their projections. This is also the case in mammals, where Vav2 is highly expressed in the growth cones of cultured neurons where it is required to control guidance (Malartre, 2010).

Interestingly, in Drosophila, the GEF Trio has been shown to activate Rac, which in turn activates Pak, which is recruited to the membrane by Dock. These proteins participate in a signal transduction pathway that plays an essential role during photoreceptor axon guidance. Vav also acts via Rac in photoreceptors, and vav and trio interact genetically. Thus, in this context, it is tempting to speculate that like Trio, Vav could also contribute to the precise spatial control of Pak activity. In this scenario, the combination of signals via Vav, Trio, and Dock would allow growth cones to integrate multiple guidance signals (Malartre, 2010).

Vav function in the axons could be to regulate the intracellular trafficking of guidance receptors through the activation of Rac. In mammals, for instance, vav2 has been proposed to be required in axons downstream of ephrin signaling for proper axon guidance. In this case, when ephrins bind their Eph receptors, Vav becomes transiently activated upon phosphorylation and promotes local Rac-dependent endocytosis of the ephrin/Eph complex, a key event in axonal repulsion. In Drosophila however, mutations in Eph surprisingly show no obvious axon guidance defects in the photoreceptor axons targeting to the optic lobe nor in the embryonic CNS. This suggests that in Drosophila, vav would need to be acting downstream of other guidance signals besides Eph (Malartre, 2010).

In conclusion, it is proposed that Vav, after being activated by signaling receptors, could be required to stimulate Rac proteins to participate in the regulation of axon growth and guidance during development (Malartre, 2010).

The Drosophila genome contains 22 GEFs. At least nine of them are expressed in the CNS, five of which are thought to be Rac activators. Why are there several Rac GEFs acting in the nervous system? A possible explanation is that the different GEFs might be activated in response to distinct guidance cues, thus triggering Rac-dependent specific cellular responses. For instance, beside its function in longitudinal axon growth, Trio has been involved in promoting commissure formation through its interaction with the attractive Netrin receptor Frazzled. Furthermore, another GEF, Sos, has been proposed to mediate Rac activation downstream of the Robo receptor to control axon repulsion at the midline. In this scenario, Vav, Sos, and Trio could coexist and be activated in response to different guidance molecules to control distinct aspects of axon guidance during the formation of the CNS (Malartre, 2010).

In another scenario, different set of GEFs could also act redundantly to activate Rac proteins to a certain level, or at precise time points or in specific subcellular locations, allowing a unique cellular response. In fact, this study has shown that the loss of both vav and sos function enhances dramatically the individual midline guidance phenotypes, suggesting that vav and sos can act redundantly in a common pathway. Similarly, the phenotype of the vav;trio double mutant in the nervous system, both at the midline and along the longitudinal axons, is more severe than the single mutants. In addition, while mutations in either vav or trio do not show any obvious defects outside the nervous system despite their widespread expression, elimination of both results in gross morphological defects. This indicates that both genes can act redundantly in vivo in different tissues and suggests that vav and trio are the main regulators of Rac activity (Malartre, 2010).

A final explanation for the existence of different rac GEFs is that they could preferentially activate a particular Rac. There are three highly related rac genes in Drosophila, rac1, rac2, and mtl, and it has been suggested that Rac1 and Rac2 are preferred substrates of Trio. By performing a similar epistasis analysis, this study has shown that in photoreceptor cells Vav activates preferentially Rac1 and Rac2. The fact that vav and trio show similar substrate specificities could explain why these two genes were found to be redundant during embryogenesis (Malartre, 2010).

In conclusion, although the vav family has been mainly implicated in the hematopoietic system and immune response, new roles are beginning to emerge for these genes. The fact that vav is required for axon growth and guidance at different stages of development suggests that it could be playing a multiplicity of functions in response to diverse signals. The existence of various protein-protein interaction domains in Vav represents a means of integrating Rac activities. These results also suggest that vav function must overlap with that of other Rac modulators. Having isolated mutations in the Drosophila vav gene will help elucidate not only the role of this GEF during neural development but also the molecular mechanisms underlying general remodeling of the embryonic and adult nervous systems (Malartre, 2010).

Pavarotti/MKLP1 regulates microtubule sliding and neurite outgrowth in Drosophila neurons

Kinesin-1 can slide microtubules against each other, providing the mechanical force required for initial neurite extension in Drosophila neurons. This sliding is only observed in young neurons actively forming neurites and is dramatically downregulated in older neurons. The downregulation is not caused by the global shutdown of kinesin-1, as the ability of kinesin-1 to transport membrane organelles is not diminished in mature neurons, suggesting that microtubule sliding is regulated by a dedicated mechanism. This study has identified the "mitotic" kinesin-6 Pavarotti (Pav-KLP) as an inhibitor of kinesin-1-driven microtubule sliding. Depletion of Pav-KLP in neurons strongly stimulated the sliding of long microtubules and neurite outgrowth, while its ectopic overexpression in the cytoplasm blocked both of these processes. Furthermore, postmitotic depletion of Pav-KLP in Drosophila neurons in vivo reduced embryonic and larval viability, with only a few animals surviving to the third instar larval stage. A detailed examination of motor neurons in the surviving larvae revealed the overextension of axons and mistargeting of neuromuscular junctions, resulting in uncoordinated locomotion. Taken together, these results identify a new role for Pav-KLP as a negative regulator of kinesin-1-driven neurite formation. These data suggest an important parallel between long microtubule-microtubule sliding in anaphase B and sliding of interphase microtubules during neurite formation (Del Castillo, 2014).

Previous work showed that microtubule sliding by kinesin-1 drives initial neurite outgrowth in Drosophila neurons and that sliding is downregulated as neurons mature. This paper, has demonstrated that the 'mitotic' kinesin Pav-KLP functions as a negative regulator of interphase microtubule sliding both in Drosophila S2 cells and in Drosophila neurons. Knockdown of Pav-KLP stimulated microtubule sliding, producing longer axons, while overexpression of Pav-KLP inhibited both sliding and neurite outgrowth. Increased length of axons after Pav-KLP depletion was also observed in vivo in Drosophila. Therefore, Pav-KLP attenuates neurite outgrowth by downregulation of kinesin-1-powered microtubule-microtubule sliding (Del Castillo, 2014).

Pav-KLP and its orthologs (members of the kinesin-6 family) were originally identified as essential components for central spindle assembly and cleavage furrow formation. Pav-KLP depletion induces defects in morphology of the mitotic spindle at telophase and failure to recruit contractile ring components. However, it has been demonstrated that CHO1/MKLP1, the mammalian ortholog of Pav-KLP, has an additional function in neurodevelopment. CHO1/MKLP1 plays a role in establishing dendrite identity in differentiated neurons. Depletion of CHO1/MKLP1 induces progressive loss of dendrites. It has concluded that CHO1/MKLP1 organizes microtubules in dendrites by transporting short minus-end-distal microtubule fragments into the dendrites. More recent work has revisited the role of CHO1/MKLP1 in developing neurons and suggested that CHO1/MKLP1 can regulate neurite outgrowth. Depletion of CHO1/MKLP1 increased transport of short microtubule fragments. The current data are in agreement with the idea that Pav-KLP regulates formation of neurites. However, the mechanisms reported in this study are clearly different from the results obtained by the mammalian studies in two significant aspects. First, this study has shown that the reorganization of microtubules required for neurite formation is driven by kinesin-1. Second, the current visualization technique clearly demonstrates that microtubules in developing Drosophila neurons are moved as long polymers. It is possible that the differences between the results and the mammalian study can be explained by different models (Drosophila versus mammalian neurons). A more attractive idea is that similar mechanisms work in both systems, but further work is required to understand the role of kinesin-1 in neurite outgrowth in mammalian neurons (Del Castillo, 2014).

Interestingly, work by several groups has shown that proteins that function together with kinesin-6 in the cytokinesis pathway could also regulate neuronal morphogenesis. For example, Tumbleweed or Ect2/Pebble/RhoGEF depletion increases the extent of neurite outgrowth, suggesting that Tumbleweed and RhoGEF control neurite outgrowth through actin reorganization. However, the current results demonstrate that the primary regulator of neurite outgrowth is kinesin-6 family member Pav-KLP, the essential partner of Pebble and Tumbleweed. Furthermore, the effect of Pav-KLP on process formation is independent of actin or small GTPases (although more subtle effects of Tumbleweed or Ect2 on the actin cytoskeleton in developing neurons cannot be completely excluded). Indeed, a recent work concluded that an actin-signaling pathway regulated by the Centralspindlin complex controls protrusive activity required for directional neuronal migration (Del Castillo, 2014).

The original idea that mitotic motors regulate cytoplasmic microtubules in neurons suggested that microtubule arrays in neurons are established by mechanisms that are analogous to those that organize the mitotic spindle. Supporting this idea, it was demonstrated that inhibition of other mitotic motors, e.g., kinesin-5, affected the axon length Advancing this concept, this paper proposes that Pav-KLP/kinesin-6 directly regulates cytoplasmic microtubule arrangement by crosslinking them. It has been shown that loss-of-function mutations on ZEN-4/MKLP1, the C. elegans form of Pav-KLP, produce longer spindles, suggesting that kinesin-6 motors inhibit sliding of microtubules against each other during anaphase B. If this is indeed the case, the current results suggest an important functional similarity between the molecular mechanisms of cell division and process formation in neurons. While anaphase B is driven in part by microtubule-microtubule sliding powered by bipolar kinesin-5 and negatively regulated by kinesin-6 (mammalian MKLP1/C. elegans Zen-4/Drosophila Pav-KLP), the initial formation of neurites requires microtubule-microtubule sliding by kinesin-1 and, similar to anaphase B, is negatively regulated by kinesin-6. Thus, kinesin-6 motors together with other components of the Centralspindlin complex can function as general brakes of microtubule-microtubule sliding during both cell division and postmitotic neurite formation (Del Castillo, 2014).

The fact that microtubule sliding is inhibited by Pav-KLP in mature, but not young, neurons suggests that Pav-KLP itself is temporally regulated. One potential mechanism that could affect the ability of Pav-KLP (MKLP-1) to regulate microtubule sliding is Pav-KLP phosphorylation. Phosphorylation of Ser710 in MKLP-1 (Ser743 in Drosophila Pav-KLP) has been shown to promote its binding to protein 14-3-3, preventing MKLP-1 from clustering on microtubules. Future studies using phosphomimetic variants of Pav-KLP may help to test this mechanism (Del Castillo, 2014).

Regulation of axon regeneration by the RNA repair and splicing pathway

Mechanisms governing a neuron's regenerative ability are important but not well understood. This study has identified Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. This study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma (Song 2015).

Failure of damaged axons to regenerate is the primary cause of permanent disabilities after CNS injury and the irreversible neurologic dysfunction of neurodegenerative diseases. The ability of a neuron to regenerate its axon after trauma is governed by the interaction between its intrinsic growth capacity and the local environment. Notwithstanding the discoveries of extracellular factors and intrinsic pathways that reduce the regenerative capacity of axons, effective therapies have not yet emerged because removing the known inhibitory cues only partially restores regeneration, thus indicating the presence of additional inhibitory machineries that remain to be discovered (Song 2015).

Studies using model organisms such as Caenorhabditis elegans have begun to identify new genes important for axon regeneration, illustrating the power of the genetic approach. To identify more factors that control axon regeneration, a Drosophila sensory neuron injury model that exhibits class-specific axon regeneration was established and it was demonstrated that the class IV dendritic arborization (da) neuron is capable of regenerating its axon in the periphery but exhibits limited regrowth inside the CNS, resembling its mammalian counterpart at the phenotypic and molecular levels (Song, 2012). Using this model, a candidate-based genetic screen was performed focusing on axotomy-regulated genes from several organisms and Drosophila Rtca (CG4061), a cellular RNA-processing enzyme with unknown biological function, was identified as an inhibitor of CNS axon regeneration. Furthermore, it was found that Drosophila Archease, a RNA ligase cofactor, functions downstream of Rtca as a pro-regeneration factor. Rtca and Archease are components of the RNA repair and splicing pathway, and they regulate the unconventional mRNA splicing of Xbp1, a stress sensor. Thus, Xbp1 acts as a substrate, readout and downstream effector for the regulation of axon regeneration by the RNA repair and splicing pathway (Song 2015).

To assess axon regeneration, a previously described protocol (Song, 2012). Briefly, with a two-photon laser, the axons of class IV da neurons (labeled with pickpocket (ppk)-CD4tdGFP) was severed in the ventral nerve cord (VNC) of second-instar larvae 48 h after egg laying (AEL), the degeneration of the remaining axons was confirmed after 1 d (72 h AEL) and their regeneration was assessed after 2 more days (120 h AEL). Using this model, the effect was measured of RtcaNP5057, an insertional loss of function (LOF) allele with a P-element inserted in the 5'-UTR, disrupting mRNA splicing and reducing transcript expression. Compared to wild types, which showed limited regrowth, new axons regrew extensively from the retracted axon stems and extended into the commissure region, forming elaborate branches and reconnected commissure segments in RtcaNP5057 larvae. Similar phenotypes were seen in transheterozygotes of RtcaNP5057 over a deficiency line, Df(1)BSC718, that lacks the Rtca locus and in a Rtca deletion allele, RtcaΔ, generated from imprecise excision of RtcaNP5057. Even stronger phenotypes were seen in RtcaΔmat, in which both the zygotic and maternal transcripts were removed. RtcaNP5057 is homozygous viable and fertile, so these larvae were derived from homozygous mutant mothers. The mothers of RtcaNP5057/Df(1)BSC718 transheterozygotes and RtcaΔ mutants were heterozygous for the wild-type allele and may provide maternal wild-type Rtca transcripts. The fact that RtcaΔmat mutants, in which both the zygotic and maternal transcripts were removed, showed a stronger phenotype than RtcaΔ zygotic mutants confirmed the maternal effect. Thus, the phenotype of RtcaNP5057 mutants compared to RtcaNP5057/Df(1)BSC718 transheterozygotes and RtcaΔ mutants is likely stronger because no wild-type maternal transcripts were provided to RtcaNP5057 mutants. The function of Drosophila Rtca is cell autonomous, as its RNA interference knockdown in class IV da neurons (ppk-Gal4>RtcaRNAi) but not in glial cells (repo-Gal4>RtcaRNAi) recapitulated the enhancement of regeneration. The regeneration phenotype was further quantified by assessing the following metrics, as described previously (Song, 2012): regeneration percentage, terminal branching and commissure regrowth. The enhancement of regeneration is unlikely to be due to developmental defects of axon outgrowth because, first, the overall axon patterning of class IV da neurons in the uncut VNC is grossly normal and second, reducing Rtca function in RtcaΔ mutants or transheterozygotes of RtcaNP5057 over Df(1)BSC718 or via Rtca RNAi in class IV da neurons did not result in obvious defects of axon terminal patterning in the VNC (Song 2015).

Tests were performed to see whether reducing Drosophila Rtca function would trigger a regenerative response in neurons normally incapable of regeneration by severing their axons in Rtca mutants. Indeed, Rtca removal in class III da neurons (labeled with 19-12-Gal4>CD4tdGFP, repo-Gal80), which unlike class IV da neurons did not regrow their axons that were severed in the periphery, elicited substantial regeneration in RtcaΔmat mutants and after RNAi knockdown of Rtca specifically in class III da neurons, leading to significant increases in the regeneration percentage, regeneration index and regeneration length (Song 2015).

Conversely, overexpression of Rtca in class IV da neurons (ppk-Gal4>Rtca) mildly reduced their regenerative potential in the peripheral nervous system (PNS). In wild-type class IV da neurons, which regenerated about 74% of the time, new axons extended beyond the lesion site and followed the axonal track. In contract, Rtca overexpression caused the incidence of regeneration to be reduced to 48% and the length of the new axons to be significantly shortened as well. These data indicate that Drosophila Rtca is an inhibitor of axon regeneration: not only does its removal cell-autonomously enhance axon regeneration in the CNS and enable regeneratively incompetent neurons such as class III da neurons to regrow their axons in the PNS, its overexpression in regeneratively competent neurons impedes axon regeneration in the periphery (Song 2015).

The inhibitory function of Drosophila Rtca is, furthermore, not limited to sensory neurons. Rtca overexpression in motor neurons also suppressed motor axon regeneration after nerve crush, as demonstrated by the reduced elaboration of growth cones (Song 2015).

The expression pattern of Drosophila Rtca was examined via two approaches. First, the P-element inserted in Rtca 5'-UTR (RtcaNP5057) contains Gal4 in the same orientation as Rtca and thus can allow inference of Rtca expression via a UAS reporter. It was found that the Rtca-Gal4>CD4tdGFP reporter colocalized with the class IV da neuron marker ppk-CD4tdTomato, confirming its presence in class IV da neurons. Although Rtca-Gal4 expression was observed in other tissues in the PNS and VNC, the analyses indicate that Drosophila Rtca functions cell autonomously in neurons to inhibit axon regeneration (Song 2015).

A polyclonal antibody was generated against Drosophila Rtca. The protein was present in wild-type but not in RtcaΔmat null class IV and class III da neurons, and was enriched in the nucleus. Drosophila Rtca was also present in other types of multidendritic neurons (Song 2015).

To begin to understand the mechanisms underlying Drosophila Rtca's role in regeneration, attempts were made to determine how it genetically interacts with the known axon regeneration regulators Pten (phosphatase and tensin homolog) and the cytoskeletal regulator Rac1 GTPase. Deletion of Pten, a negative regulator of the mammalian target of rapamycin (mTOR) pathway, has been shown to increase CNS axon regeneration in both mammals and flies. Overexpression of Rtca in a Pten hypomorphic mutant background (PtenMGH6; ppk-Gal4>Rtca) or overexpression of Pten in Rtca null mutants (RtcaΔ; ppk-Gal4>Pten) largely abolished the enhancement of axon regeneration as seen in the VNC in PtenMGH6 or RtcaΔ mutants. This suggests that Rtca and Pten are likely to function in parallel pathways. Notably, double mutation of Rtca and Pten (RtcaNP5057; PtenMGH6) did not further improve regeneration, as compared to Rtca mutation alone, indicating the presence of additional brakes on regeneration. The regeneration phenotype in Rtca mutants appeared to be comparable to if not stronger than that in PtenMGH6 mutants or that seen with Akt overexpression (ppk-Gal4>Akt) (Song 2015).

Because Rac is required for regenerative axon outgrowth in C. elegans, this study overexpressed Rac1 in class IV da neurons (ppk-Gal4>Rac1). An increase was found in the number of axons initiating the regenerative response in the VNC but not in terminal branching or commissure regrowth; that is, there was a partial improvement in regeneration. Conversely, overexpressing a dominant negative (DN) form of Rac1 abolished the enhancement of CNS axon regeneration seen in Rtca null mutants (RtcaΔmat; ppk-Gal4>Rac1DN), whereas Rac1DN overexpression alone in class IV da neurons did not result in obvious axon regeneration defects in the PNS. It thus seems likely that Rac1 functions downstream of Rtca in a pathway that converges on regulation of the cytoskeleton (Song 2015).

Rtca is a RNA processing enzyme that possesses RNA-3'-phosphate cyclase activity and catalyzes the ATP-dependent conversion of a 3' phosphate to a 2',3'-cyclic phosphodiester at the end of RNA molecules (Genschik, 1997). The RNA 2',3'-cyclic phosphate ends are important in RNA metabolism—for example, as intermediates during RNA repair by ligases (Popow, 2011; Remus, 2013). Rtcb (RNA 2',3'-cyclic phosphate and 5'-OH ligase) represents a new type of RNA ligase that joins 2',3'-cyclic phosphate and 5'-OH RNA ends to yield a 3'-5' phosphodiester splice junction. Specifically, Rtcb is known to possess cyclic phosphodiesterase activity, which hydrolyzes the 2',3'-cyclic phosphate to a 3'-phosphate, as well as ligase activity, which then joins the RNA 3'-phosphate to a 5'-OH RNA end. In addition, the specificity and efficacy of Rtcb's ligase activity can be enhanced by Archease (Desai, 2014; Popow, 2014), which is a small acidic protein conserved among Eukarya, Bacteria and Archaea. In Escherichia coli, RtcA and RtcB are encoded in a single operon, suggesting that they might cooperate to provide a healing and sealing function in an RNA repair pathway (Tanaka, 2011). In one scenario, healing would refer to the restoration of ligatable 2',3'-cyclic phosphate ends in the event of the inciting RNA damage directly generating RNA 3'-phosphates, or of the 2',3'-cyclic phosphate products of RNA transesterification being further processed to a 3'-phosphate by a 2',3'-cyclicphosphodiesterase. However, this model cannot readily be reconciled with the subsequent finding that RtcB readily joins 3'-phosphate to 5'-OH ends or 2',3'-cyclic phosphate to 5'-OH ends (Song 2015).

Therefore, the exact relationship between RtcA and RtcB remains undetermined. Notably, the RtcBA operon in E. coli is regulated by the σ54 coactivator RtcR, suggesting that the RNA repair functions are induced in response to cellular stress. Although the biological function of Rtca remains unknown, the enzyme is speculated to act in some aspect of cellular RNA processing . Taking into account these findings and the observation that loss of Rtca function enhances axon regeneration, it is hypothesized that the Rtca-Archease-dependent RNA repair and splicing pathway regulates axon regeneration. Specifically, it is speculated that axon injury triggers a type of cellular stress leading to RNA damage and splicing, producing RNA 3'-phosphates that need to be processed and rejoined by the Rtcb ligase, which is catalyzed by Archease. Because Rtca converts RNA 3'-phosphate to 2',3'-cyclic phosphate, it can slow the ligation process and impede regeneration. Consequently, silencing Rtca promotes axon regeneration. Following this reasoning, the role of Archease in axon regeneration was investigated (Song 2015).

To determine the role of the Drosophila Archease in axon regeneration, regeneration of class IV da neuron axons in the periphery was examined. To maximize the phenotype, the PNS axon injury protocol was modified as described previously (Song, 2012): axotomy was induced at 72 h AEL, degeneration was confirmed at 96 h AEL and regeneration was assayed at 120 h AEL. The Archease (CG6353) LOF mutant allele ArcheasePBc01013, which is an insertional allele with a P-element inserted into the 5'-UTR disrupting its mRNA splicing and eliminating Archease transcripts. Unlike in wild-type neurons, which exhibited substantial regrowth of their severed axons, axon regeneration was significantly impaired in ArcheasePBc01013 neurons, as revealed by a significant drop of the regeneration percentage, regeneration index and regeneration length. This phenotype was confirmed in transheterozygotes of ArcheasePBc01013 over either of the two deficiency lines, Df(3R)ED6076 or Df(3R)BSC678, that lack the Archease locus. The ArcheasePBc01013 mutation is larval lethal. ArcheasePBc01013 mutants and ArcheasePBc01013/DfED6076 and ArcheasePBc01013/DfBSC678 transheterozygotes showed similar phenotypes, suggesting that ArcheasePBc01013 is likely an amorphic allele. The function of Archease is required cell-autonomously, as class IV da neuron-specific knockdown of Archease (ppk-Gal4>ArcheaseRNAi) but not glial cell knockdown (repo-Gal4>ArcheaseRNAi) was sufficient to phenocopy the regeneration failure. Moreover, loss of function of both Rtca and Archease (RtcaNP5057; ArcheasePBc01013) completely abolished the axon regeneration-promoting effect in the VNC seen in RtcaNP5057 mutants, producing many retracted or stalled axon stems. This epistasis analysis indicates that Archease is a pro-regeneration factor downstream of Rtca and that they act in opposing ways to regulate axon regeneration (Song 2015).

What might be the RNA substrates processed by this Drosophila Rtca-Archease-dependent RNA repair and splicing pathway for the regulation of axon regeneration? This study investigated X-box binding protein 1 (Xbp1) as a candidate substrate for three reasons. First, cellular stress such as endoplasmic reticulum (ER) stress triggers an adaptive intracellular signaling cascade known as the unfolded protein response (UPR). One main branch of the UPR is the activation of Ire1, which cleaves Xbp1 pre-mRNA in the cytoplasm, converting the unspliced Xbp1μ, a putative transcriptional repressor, into the unconventionally spliced Xbp1s by eliminating an intron (26 nucleotides in mammals, 23 in flies) that changes the open reading frame of the third exon, resulting in a new protein that acts as a transcriptional activator (Yoshida, 2001). Xbp1s directly activates ER stress target genes to facilitate refolding and also degradation of misfolded proteins (Ron, 2007). Second, the RNA ligase Rtcb and its cofactor Archease are involved in the unconventional splicing induced by the UPR, and Archease is required for the splicing of the Xbp1 mRNA (Jurkin, 2014). Third, loss of xbp1 function in C. elegans results in severely reduced axon regeneration (Nix, 2014). To determine the function of Xbp1 in axon regeneration in the PNS and CNS, a mutant allele, Xbp1k13803, was used that has a P-element inserted into its 5'-UTR, thus reducing transcripts (Ryoo, 2007). Axon regeneration in the periphery was mildly reduced in these mutants. This defect was stronger in transheterozygotes of Xbp1k13803 over a deficiency line, Df(2R)BSC484, that lacks the Xbp1 locus, suggesting that Xbp1k13803 is likely a hypomorphic allele. Class IV da neuron-specific (ppk-Gal4>Xbp1RNAi) but not glia-specific (repo-Gal4>Xbp1RNAi) RNAi of Xbp1 reproduced the impairment of regeneration, indicating it functions cell-autonomously. Moreover, double mutation of Rtca and Xbp1 (RtcaNP5057; Xbp1k13803) dampened the enhancement of CNS axon regeneration seen in RtcaNP5057 mutants, indicating that Xbp1 is indeed a pro-regeneration factor downstream of Drosophila Rtca. Consistent with these lines of evidence, overexpression of the spliced form Xbp1s in class IV da neurons significantly enhanced axon regeneration in the VNC, and it also promoted axon regeneration in the periphery when overexpressed in class III da neurons. The observations that Rtca; Xbp1 double mutation did not completely eliminate the enhanced regeneration phenotype in Rtca mutants and that Xbp1s overexpression led to milder enhancement of regeneration as compared to Rtca LOF, suggest that additional substrates contribute to regeneration regulation (Song 2015).

To directly assess the nonconventional splicing of Xbp1 mRNA in vivo, a heat-shock model was used. Fly larvae of various genotypes underwent a 40°C heat shock and the abundance of Xbp1 splice variants was assessed using semiquantitative RT-PCR. The Xbp1s/Xbp1μ ratio was then quantified. Heat-shock induced the expression of the spliced form, Xbp1s. In contrast to the enhanced expression of Xbp1s in Rtca mutants, Xbp1s levels were greatly reduced in Archease LOF mutants. Double mutants of Rtca and Archease resembled Archease mutants in the reduction of Xbp1 splicing. Taken together, these data indicate that Drosophila Rtca and Archease in the RNA repair and splicing pathway negatively and positively regulate the stress-induced Xbp1 mRNA splicing, respectively, so that Xbp1 acts as a readout and effector for the regulation of axon regeneration (Song 2015).

Having established the role of Rtca in axon regeneration in Drosophila, the study went on to determine whether its function is evolutionarily conserved in mammals. The expression pattern of the mammalian ortholog of Drosophila Rtca was examined in vitro. Antibodies raised against human RTCA recognized rat Rtca in the cell bodies and processes of cultured hippocampal neurons. Moreover, the expression of Rtca transcripts in the dorsal root ganglion (DRG) in vivo increased progressively throughout development, reaching the highest level in adults. Using quantitative RT-PCR, it was found that Rtca transcript levels in the DRG were significantly reduced following lesion of the sciatic nerve peripherally, but not lesion of the central axon branch of DRG neurons with a spinal cord hemisection. Since the peripheral processes of DRG neurons are capable of regeneration, whereas their central axons that project into the spinal cord fail to regrow after injury, the selective suppression of Rtca following peripheral injury supports the hypothesis that the persisting expression of Rtca is inhibitory to axon regeneration in the CNS. Furthermore, in agreement with the overexpression phenotype in flies, overexpression of Rtca in cultured hippocampal neurons reduced axon complexity and markedly reduced proximal axonal branching without affecting total axon length, indicating an inhibitory function of Rtca (Song 2015).

It was next asked whether knocking out Rtca during development enhances axon regeneration of adult mouse retinal ganglion cells (RGCs) in vivo. For this purpose, a mutant allele was generated with a lacZ cassette inserted after the third exon to disrupt splicing and reduce transcription (to ~19%), thereby generating RtcalacZ_loxP (RtcaIns/Ins) mutant mice. Rtca protein level was also reduced to ~18% in the mutants, suggesting this is a hypomorphic allele. Homozygous RtcaIns/Ins mice were born, although at less than the Mendelian ratio. By adulthood, there were no obvious differences in RGC number or RGC axon morphology among mutant (RtcaIns/Ins), heterozygous (RtcaIns/+) and wild-type (Rtca+/+) animals. Since the lacZ cassette is inserted into the Rtca locus, it can be used as a reporter for examining Rtca expression. β-Galactosidase staining was observed in the RGC layer of RtcaIns/+ mice but not in Rtca+/+ littermates . Moreover, β-galactosidase immunostaining in RtcaIns/+ mice showed distinct expression of the lacZ reporter in NeuN+ neurons in the retina, which was absent in Rtca+/+ littermates, indicating that Rtca is indeed expressed in RGCs. To assess RGC axon regeneration, optic nerve crush was perfored in RtcaIns/Ins, RtcaIns/+ and Rtca+/+ littermate mice at two developmental time points, postnatal day (P) 35 and 2-3 months old, and the extent of axon regeneration was measured in the optic nerve after 2 weeks or 3 weeks, respectively. RtcaIns/+ and Rtca+/+ mice injured at P35 did not exhibit substantial axon regrowth beyond the crush site, whereas RtcaIns/Ins mutant mice showed a substantial increase in the number of regenerating axons at various distances from the injury site, with some regenerating axons extending over 1.5 mm beyond the crush site. Mice operated on at 2-3 months old showed a milder regeneration enhancement phenotype. In these animals, curving, turning and looping of axons were observed, indicative of new axon growth, and the furthest distance that axons traveled beyond the injury site was about 3.5 times longer in the mutants. The crush site was further marked by the presence of ED1 staining, which labels infiltrating macrophages. Whereas axons rarely penetrated beyond the ED1+ region in sibling controls, a large number of axons were seen hundreds of microns beyond the ED1+ region in RtcaIns/Ins mutants. Reducing Rtca function did not affect RGC survival after injury, confirming that this increase in regenerating axons was not secondary to an increase in RGC numbers. The finding that reducing Rtca expression increased the regenerative potential of adult RGCs thus provides evidence for a potentially conserved role of Rtca as an anti-regeneration factor (Song 2015).

These findings reveal an important role of the RNA repair and splicing pathway in regulating the intrinsic axon regeneration potential in response to PNS and CNS injury in Drosophila. Rtca and Archease integrate the injury signals triggered by axotomy and lead to the activation of downstream effectors such as the stress response cascade involving Xbp1 splicing, affecting the ability of a neuron to regenerate. Axon injury has been suggested as a cellular stress, and the mTOR pathway, a potential determinant of neuronal regeneration competence, could be inactivated under stress conditions such as hypoxia or DNA damage (Lu, 2014). Notably, Xbp1 splicing has been observed in RGCs after optic nerve injury and forced activation of Xbp1 promotes RGC survival (Hu, 2014). This work implicates proper splicing of Xbp1 as also important for axon regeneration in Drosophila (Song 2015).

Moreover, recent work in C. elegans also suggests the involvement of stress response pathways, such as heat-shock, hypoxia and UPR, in axon regeneration (Nix, 2014). However, how the injury signal is relayed to the stress response is unclear. This work identifies a missing link and implicates the Rtca-Archease-dependent RNA metabolism machinery as a regeneration regulator. A priori, axonal injury could either signal directly to the stress pathways, which then recruit Rtca-Archease, or alternatively, Rtca and Archease may represent injury response elements upstream of the stress pathways. The results showed that the Xbp1-dependent UPR pathway acts downstream of Rtca-Archease in controlling axon regeneration, and the remaining question is whether and how it impinges on other stress pathways, such as hypoxia or DNA damage. It will be important in future studies to identify other substrates, in addition to Xbp1, that are modified by Rtca-Archease, and to search for response genes downstream of Xbp1. This study raises the prospect of manipulating Rtca, Archease and Xbp1 as potential therapeutic interventions for treating nervous system injury (Song 2015).

As a first step to determining whether the Rtca pathway may have an evolutionarily conserved function in axon regeneration, this study has examined CNS axon regeneration after optic nerve crush in a hypomorphic mouse mutant allele of Rtca and evidence was obtained suggesting that this is indeed the case. The enhancement of RGC axon regeneration phenotype in the Rtca mutant is modest as compared to that seen in Pten, Klf4 or Socs3 knockouts. This may be due to the residual Rtca function in this hypomorphic allele or to developmental compensation. Future experiments using mammalian injury models to examine the Rtca null allele and to assess other components of the RNA repair and splicing pathway are therefore warranted to further define its potential role in axon regeneration (Song 2015).

Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development

Axonal growth and targeting are fundamental to the organization of the nervous system, and require active engagement of the cytoskeleton. Polymerization and stabilization of axonal microtubules is central to axonal growth and maturation of neuronal connectivity. Studies have suggested that members of the Tubulin Polymerization Promoting Protein (P25alpha/TPPP) family are involved in cellular process extension. However, no in vivo knockout data exists regarding its role in axonal growth during development. This study reports the characterization of Ringmaker (Ringer; CG45057), the only Drosophila homolog of long p25alpha proteins. Immunohistochemical analyses indicate that Ringer expression is dynamically regulated in the embryonic CNS. ringer null mutants show cell misplacement, and errors in axonal extension and targeting. Ultrastructural examination of ringer mutants revealed defective microtubule morphology and organization. Primary neuronal cultures of ringer mutants exhibit defective axonal extension, and Ringer expression in cells induced microtubule stabilization and bundling into rings. In vitro assays showed that Ringer directly affects tubulin, and promotes microtubule bundling and polymerization. Together these studies uncover an essential function of Ringer in axonal extension and targeting through proper microtubule organization (Mino, 2016).

DISCO Interacting Protein 2 regulates axonal bifurcation and guidance of Drosophila mushroom body neurons

Axonal branching is one of the key processes within the enormous complexity of the nervous system to enable a single neuron to send information to multiple targets. However, the molecular mechanisms that control branch formation are poorly understood. In particular, previous studies have rarely addressed the mechanisms underlying axonal bifurcation, in which axons form new branches via splitting of the growth cone. This study demonstrates that DISCO Interacting Protein 2 (DIP2) is required for precise axonal bifurcation in Drosophila mushroom body (MB) neurons by suppressing ectopic bifurcation and regulating the guidance of sister axons. DIP2 localizes to the plasma membrane. Domain function analysis revealed that the AMP-synthetase domains of DIP2 are essential for its function, which may involve exerting a catalytic activity that modifies fatty acids. Genetic analysis and subsequent biochemical analysis suggested that DIP2 is involved in the fatty acid metabolization of acyl-CoA. Taken together, these results reveal a function of DIP2 in the developing nervous system and provide a potential functional relationship between fatty acid metabolism and axon morphogenesis (Nitta, 2016).

The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation

Developmental axon pruning is essential for wiring the mature nervous system, but its regulation remains poorly understood. This study shows that the endosomal-lysosomal pathway regulates developmental pruning of Drosophila mushroom body γ neurons. The UV radiation resistance-associated gene (Uvrag) functions together with all core components of the phosphatidylinositol 3-kinase class III (PI3K-cIII; see Phosphotidylinositol 3 kinase 59F) complex to promote pruning via the endocytic pathway. By studying several PI3P binding proteins, this study found that Hrs, a subunit of the ESCRT-0 complex, required for multivesicular body (MVB) maturation, is essential for normal pruning progression. Thus, the existence of an inhibitory signal that needs to be downregulated is hypothesized. Finally, the data suggest that the Hedgehog receptor, Patched, is the source of this inhibitory signal likely functioning in a Smo-independent manner. Taken together, this in vivo study demonstrates that the PI3K-cIII complex is essential for downregulating Patched via the endosomal-lysosomal pathway to execute axon pruning (Issman-Zecharya, 2014).

Neuronal remodeling is an essential step of nervous system development in both vertebrates and invertebrates. One mechanism used to remodel neuronal circuits is by the elimination of long stretches of axons in a process known as axon pruning. With a few exceptions, the current dogma is that axon pruning of long stretches of axons occurs via local axon degeneration while axon pruning of short stretches occurs via retraction. While in some cases remodeling is directly affected by experience or neural activity, in cases of stereotypical pruning the identity of the axon that is destined to be pruned does not depend on experience or neural activity. Because of mechanistic similarities to Wallerian degeneration and dying back neurodegenerative diseases, understanding the molecular mechanisms of axon pruning should result in a broader insight into axon fragmentation and elimination during development and in disease (Issman-Zecharya, 2014).

The neuronal remodeling of the Drosophila mushroom body (MB) during development is a unique model system to study the molecular aspects of axon pruning. The stereotypic temporal and spatial occurrence of MB axon pruning combined with mosaic analyses provide a platform to perform genetic screens and molecular dissections of these processes in unprecedented resolution. The MB is comprised of three types of neurons that are sequentially born from four identical neuroblasts per hemisphere. Out of the three MB neuronal types, only the γ neurons undergo axon pruning, indicating that the process is cell-type specific. During the larval stage, γ neurons project a bifurcated axon to the dorsal and medial lobes. At the onset of metamorphosis, the dendrites of the γ neurons as well as specific parts of the axons are eliminated by localized fragmentation in a process that peaks at about 18 hr after puparium formation. Subsequently, γ neurons undergo developmental axon regrowth, which is distinct from initial axon outgrowth, to occupy the adult specific lobe (Issman-Zecharya, 2014).

Axon pruning of MB γ neurons depends on the cell-autonomous expression of the nuclear steroid hormone receptor, ecdysone receptor B1 (EcR-B1). The expression of EcR-B1 is regulated by at least three distinct pathways: the cohesin complex, the TGF-β pathway, and a network of nuclear receptors comprised of ftz-f1 and Hr39. While expression of EcR-B1 is required for pruning, it is not sufficient to drive ectopic pruning either in γ neurons or in other MB neurons that do not undergo remodeling. This raises two possible nonmutually exclusive scenarios: (1) additional molecules are required to initiate pruning and (2) an inhibitory signal needs to be attenuated in the MB for pruning to occur. Additionally, the ubiquitin pathway is also cell-autonomously required in γ neurons for pruning, but the target that must be ubiquitinated remains unknown. Thus, while understanding of the cellular sequence of events culminating in the elimination of specific axonal branches is quite detailed, understanding of the molecular mechanisms remains incomplete (Issman-Zecharya, 2014).

In a forward genetic screen, this study identified a cell-autonomous role for the UV radiation resistance-associated gene (UVRAG) in MB γ neuron pruning. UVRAG was originally identified based on its ability to confer UV resistance to nucleotide excision repair deficient cells. It was later shown to function as a tumor suppressor gene deleted in various types of cancers including colon and gastric carcinomas. UVRAG interacts with Atg6 (also known as Beclin1), another tumor suppressor gene, and together they promote autophagy in vitro. Their tumor suppression capabilities were first attributed to their autophagy-promoting function. However, a mutant form of UVRAG isolated from colon carcinomas promoted autophagy normally in cell culture. Both UVRAG and Atg6 are subunits in the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex, involved in autophagy and endocytosis. Recent studies have found that UVRAG mediates endocytosis in an Atg6-dependent manner suggesting that as part of the PI3K-cIII complex, both proteins regulate various aspects of vesicle trafficking. Two studies have recently identified new and seemingly unrelated functions for UVRAG in regulating DNA repair in response to UV-induced damage and ER to Golgi trafficking. Finally, an in vivo study has shown that UVRAG affects organ rotation in Drosophila by regulating Notch endocytosis in what seemed to be an Atg6-independent manner. A unifying understanding of the various aspects of UVRAG physiological function in vivo is still lacking. Likewise, although the PI3K-cIII complex has been extensively studied and implicated in autophagy, cytokinesis and endocytosis, its physiological roles during the normal course of development are not known (Issman-Zecharya, 2014).

This study reports that UVRAG and the PI3K-cIII complex mediate the endosome-lysosome degradation of Ptc to promote axon pruning. Furthermore, the results suggest that Ptc represses pruning via a Smo- and Hh-independent manner. This study provides evidence for the existence of a pruning inhibitory pathway originating at the membrane of MB neurons (Issman-Zecharya, 2014).

This study shows that the endosomal-lysosomal pathway is cell-autonomously required for developmental axon pruning of mushroom body (MB) γ neurons. Genetic loss-of-function experiments indicate that UVRAG, a tumor suppressor gene previously linked to both endocytosis and autophagy, promotes pruning as part of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex and that UVRAG is required in MB neurons for the formation of phosphatidylinositol 3-phosphate (PI3P). The ESCRT-0 complex, which is recruited to the PI3 moiety on endosomal membranes, is required for pruning, indicating that endosome to multivesicular body maturation is critical for the normal progression of axon pruning and suggesting that it involves receptor downregulation. Genetic loss-of-function and gain-of-function experiments suggest that downregulation of the Hedgehog receptor Patched (Ptc) by the endocytic machinery is instrumental in promoting pruning. Finally, the results suggest that Ptc inhibits pruning in a smo-independent and likely also hh-independent manner (Issman-Zecharya, 2014).

A recent study suggested that UVRAG is required for Notch endocytosis during organ rotation in Drosophila in an Atg6-independent manner. While the current study shows that Atg6 is required for pruning, these seemingly contradicting results can be easily explained by specific allele differences. The Atg600096 allele, used in the previous study, is a P element insertion about 100 bp upstream of the Atg6 gene that does not necessarily create a null allele. Indeed, this study could also not see any effect of this allele on axon pruning. This study used an Atg61 null allele created by homologous recombination resulting in a strong effect on pruning. Furthermore, the data clearly show that the entire PI3K-cIII complex is required for axon pruning (Issman-Zecharya, 2014).

The PI3K-cIII complex has been implicated in a wide variety of membrane trafficking processes ranging from autophagy to endocytosis to cytokinesis. How the PI3K-cIII is regulated to participate in these different processes and its physiological roles in vivo are not well understood. While its role in promoting autophagy is supported by several studies, deleting the catalytic unit, Vps34, in sensory neurons does not affect autophagy, but rather endocytosis. Whether this is a common feature of PI3K-cIII function in neurons remains to be further elucidated. One attractive hypothesis is that the PI3K-cIII function is determined by its complex composition. Indeed, it appears that in vitro, UVRAG and Atg14 are mutually exclusive subunits defining two distinct populations of the PI3K-cIII complex (Funderburk, 2010; Itakura, 2009). The current study is consistent with these findings, suggesting that UVRAG may define an endocytosis-specific PI3K-cIII complex at least in neurons. The full spectrum of the various PI3K-cIII complexes physiological roles in vivo remains to be further studied (Issman-Zecharya, 2014).

The PI3K-cIII complex phosphorylates PI to form PI3P on endosomal membranes. Indeed, this study found that UVRAG is essential for efficient PI3P formation and that PI3P is abundant throughout development. It is thus hypothesized that a PI3P binding protein mediates the effect of UVRAG and the PI3K-cIII complex on axon pruning. This study has identified Hrs, a subunit of the ESCRT-0 complex and a PI3P binding protein, as required for axon pruning. The role of ESCRT-0 in MVB maturation led to a hypothesis that the endolysosomal pathway is required to downregulate a signal that originates at the plasma membrane. While signaling can still occur in the early endosome, it is terminated at the MVB (Issman-Zecharya, 2014).

What is the identity of this transmembrane protein? Using genetic loss-of-function and gain-of-function experiments, it is suggested that Patched (Ptc) is at least one of the transmembrane proteins that is responsible for mediating the PI3K-cIII pruning defect. Strikingly, mutating ptc on the background of a Atg6 mutant significantly suppressed its pruning defect. Furthermore, overexpression of Ptc in WT brains resulted in a weak to mild pruning defect, depending on the Gal4 driver. Finally, overexpressing Ptc on the background of an endosomal defect significantly exacerbated the pruning defect. Together, these data suggest that Ptc mediates an inhibitory signal that needs to be attenuated for the normal progression of pruning. Interestingly, Ptc inactivation by endocytosis followed by lysosomal degradation was proposed before as a mechanism to activate the Hh pathway. What is the nature of this signal? Ptc is known to be the Hedgehog (Hh) receptor. Binding of Hh to Ptc relieves the Ptc-induced suppression of another transmembrane protein, Smoothened (Smo). Once derepressed, Smo initiates the intracellular Hh signal that culminates in the expression of specific nuclear transcription factors. Therefore this study tested the role of Smo and Hh in developmental axon pruning and, to surprisingly, demonstrated that both molecules seem to be irrelevant for pruning. Overexpressing Ptc mutant transgenes within MB neurons to identify the domains that are important for pruning inhibitions confirmed that Smo inhibition was not required to inhibit pruning. In contrast, the results suggest that the ligand binding domain is important. Because the results suggest that Hh is not required for pruning inhibition, it will be interesting to investigate in the future what other ligands might bind to Ptc. In this regard it is interesting to mention that a recent study has shown that Ptc is a lipoprotein receptor. The precise mechanism of Ptc action in MB neurons remains to be further elucidated in future studies (Issman-Zecharya, 2014).

This study has uncovered a role for the endocytic machinery in downregulating an inhibitory signal that is dependent on Ptc during MB axon pruning. A recently published study has shown that the Rab5/ESCRT endocytic pathways are required to downregulate neuroglian (Nrg) to promote dendrite pruning of sensory neurons in Drosophila. Both studies highlight that a combination of both promoting and inhibitory signals during developmental pruning is likely important to provide fail-safe mechanisms to regulate the process in a temporal, spatial, and cell-type specific resolution (Issman-Zecharya, 2014).

Drosophila Dunc-115 mediates axon projection through actin binding

A central step in organizing the central nervous system development is the growth cone of an axon navigating through guidance cues to reach its specific target. While a great deal of this process has been understood especially in identifying the extracellular guidance cues and their membrane receptors, much less is known about how guidance signals are further relayed to the actin filaments that are central to the mobility of the growth cone. Previous results have shown that Drosophila gene dunc-115 regulates axon projection in the eye and the central nervous system. Furthermore, Dunc-115 has a villin-headpiece (VHD) domain, implying the possibility of binding to actin. To further characterize Dunc-115's functions, this study has identified the isoform Dunc-115L as a possible downstream target in relaying guidance cues further down to the cytoskeleton. Specifically, it was shown that Dunc-115 regulates neural connections in both the eye and the central nervous system in Drosophila and that Dunc-115 contains an actin-binding domain potentially capable of binding to actin filaments. This report shows that Dunc-115 binds to actin via its VHD domain directly, suggesting a possible mechanism for how Dunc-115 relays guidance signals (Roblodowski, 2017).

MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2

Injury-induced (Wallerian) axonal degeneration is regulated via the opposing actions of pro-degenerative factors such as SARM1 and a MAPK signal and pro-survival factors, the most important of which is the NAD+ biosynthetic enzyme NMNAT2 that inhibits activation of the SARM1 pathway. This study investigated the mechanism by which MAPK signaling facilitates axonal degeneration. MAPK signaling was shown to promote the turnover of the axonal survival factor NMNAT2 in cultured mammalian neurons as well as the Drosophila ortholog dNMNAT in motoneurons. The increased levels of NMNAT2 are required for the axonal protection caused by loss of MAPK signaling. Regulation of NMNAT2 by MAPK signaling does not require SARM1, and so cannot be downstream of SARM1. Hence, pro-degenerative MAPK signaling functions upstream of SARM1 by limiting the levels of the essential axonal survival factor NMNAT2 to promote injury-dependent SARM1 activation. These findings are consistent with a linear molecular pathway for the axonal degeneration program (Walker, 2017).

Islet coordinately regulates motor axon guidance and dendrite targeting through the Frazzled/DCC receptor

Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. This study shows that the LIM homeodomain factor Islet (Isl) controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra)/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring (Santiago, 2017).

The RP3 motor neurons innervate the NetrinB-expressing muscles 6 and 7 and are enriched for fra mRNA during the late stages of embryonic development, and it was reported previously that, in the absence of fra or Netrin, there are significant defects in the innervation of muscles 6 and 7. This phenotype is also detected in the absence of hb9/exex or isl/tailup, two transcription factors expressed in RP3 as well as in other ventrally projecting motor neurons, suggesting that hb9 or isl may regulate fra. Interestingly, Hb9, Isl, and the LIM homeodomain factor Lim3 were all recently shown to bind directly to the fra locus in vivo, as determined by a genome-wide DNA adenine methyltransferase identification (DAM-ID) analysis performed in Drosophila embryos. However, DAM-ID results do not provide information about the functional significance of the detected binding events or about the cell types in which they occur. To determine whether Hb9, Isl, or Lim3 regulate the expression of fra in embryonic motor neurons, situ hybridization experiments were performed and fra mRNA expression was analyzed with single-cell resolution in embryos mutant for these factors. Only isl is required for fra expression in the RP3 motor neurons at stage 15, when RP axons have reached the ventral muscle field but their final targets have not been selected. 80% of RP3 neurons in abdominal segments A2-A7 in isl/+ embryos are positive for fra mRNA versus 38% in isl mutant embryos. A significant difference was also observed in fra mRNA levels in RP3 neurons between isl mutants and heterozygotes when quantifying pixel intensity from the fra in situ, whereas no difference was detected in the signal of the isl-H-tau-myc transgene. No change was detected in the number or position of RP3 neurons in isl mutants, consistent with previous data demonstrating that Isl is not required for the generation or survival of Drosophila motor neurons. Importantly, no requirement was found for either hb9 or lim3 in regulating fra mRNA expression in any RP motor neurons, demonstrating that isl's effect on fra is specific and could not have been predicted simply from similarities in loss of function phenotypes or from transcription factor binding data (Santiago, 2017).

Hb9 has been shown to be required for robo2expression in RP3. Interestingly, just as hb9 is not required for fra expression in RP neurons, isl is not required for robo2expression. A previous study reported that isl; hb9 double mutants have a stronger intersegmental nerve b (ISNb) phenotype than either single mutant, but muscle 6/7 innervation defects were not quantified. This study scored motor axon guidance defects in isl; hb9 double mutants and found that the double mutants display significantly more muscle 6/7 innervation defects than either single mutant. Similarly, embryos mutant for both robo2and fra have a stronger motor axon phenotype than either robo2or fra single mutants. Note that, because robo2, fra double mutants have severe defects in midline crossing, motor axon phenotypes should be interpreted with caution . These results show that Hb9 and Isl act in parallel to regulate distinct downstream programs in RP3 neurons, demonstrating how combinations of transcription factors result in specific cell surface receptor profiles and axon trajectories (Santiago, 2017).

To determine whether isl and fra act in the same genetic pathway during RP3 guidance, embryos mutant for both genes were examined. In isl-null mutants, 20% of hemisegments lack muscle 6/7 innervation, whereas fra-null mutants have a significantly stronger phenotype (34% of hemisegments). isl, fra double mutants do not have more muscle 6/7 innervation defects than fra single mutants, consistent with isl and fra acting in the same pathway. If fra acts downstream of Isl during motor axon targeting, then it was reasoned that restoring Fra expression in isl mutant neurons might rescue muscle 6/7 innervation. Indeed, it was found that pan-neural overexpression of Fra in isl mutants partially but significantly rescues these defects. The difference between genotypes was most noticeable when hemisegments were counted in which a growth cone stalls at the 6/7 cleft as well as those in which it fails to reach it. In isl mutants, a growth cone stalls at or fails to reach the 6/7 cleft in 27% of hemisegments compared with 15% of hemisegments in sibling mutants overexpressing Fra. The data was also analyzed by comparing the number of embryos with 6/7 innervation defects. In isl mutants, 0% of embryos have no 6/7 innervation defects in A2-A6, 44% have one defect, and 56% have two or more defects. In contrast, in isl mutants overexpressing Fra, 29% of embryos have no innervation defects, 29% have one defect, and 41% have two or more defects. The incomplete rescue could be due to differences in the timing or levels of GAL4/UAS-mediated expression of Fra compared with its endogenous regulation or could indicate that Isl regulates additional downstream effectors important in this process. Nevertheless, these data strongly suggest that Fra is an essential downstream effector of Isl during the guidance of the RP3 axon to its target muscles (Santiago, 2017).

To further investigate the relationship between isl and fra, whether ectopic expression of isl is sufficient to induce fra expression was tested. These experiments used the apterous (ap) neurons. The axons from this subset of interneurons form a single fascicle on either side of the midline that are labeled by ap-Gal4. The ap neurons express low levels of fra, do not express isl, and do not cross the midline. Fra overexpression causes ectopic midline crossing of ap axons. Overexpression of Isl with ap-Gal4 produced high levels of midline crossing, phenocopying the effect of Fra overexpression. In stage 17 control embryos, ap axons cross the midline in 12% of segments, whereas in embryos overexpressing UAS-Isl with ap-Gal4, ap axons cross the midline in 60% of segments. This phenotype is dose-dependent because embryos with two copies of an UAS-Isl insertion display significantly more ectopic midline crossing than embryos with one insert (Santiago, 2017).

To determine whether Isl overexpression results in fra induction, the expression of fra mRNA in situ was examined in ap neurons. In stage 15 wild-type embryos, a low percentage of ap neurons express fra (25% of ventral ap clusters were fra+). In contrast, in embryos overexpressing isl from two UAS-Isl inserts, 37% of the ventral ap clusters were fra+. To test whether the ectopic crossing phenotype depends on fra function, Isl was overexpressed in embryos homozygous for a null allele of fra. Strikingly, removing fra completely suppresses the crossing phenotype, indicating that fra is required for Isl to produce its gain-of-function effect. Although it cannot be ruled out that Isl affects the expression of other genes in the Fra pathway to cause midline crossing, these results demonstrate that ectopically expressing Isl causes an increase in fra expression and a fra-dependent axon guidance phenotype and suggest that the functional relationship between isl and fra may be used in multiple contexts (Santiago, 2017).

Fra mutants have defects in RP axon midline crossing, as shown by retrograde labeling of single motor neurons. In addition, Netrin-Fra signaling controls the medio-lateral position of dendrites in several groups of motor neurons. Therefore, it was asked whether isl regulates midline crossing or RP3 dendrite development through fra. A genetic strategy was used to label single motor neurons by mosaic expression of a membrane-tethered GFP transgene under the control of lim3b-GAL4, which labels RP motor neurons, sensory neurons, and several other motor and interneuron populations. RP3 neurons were identified by the stereotyped position of the RP3 cell body and by the targeting of its axon to muscles 6 and 7. Because of the axon targeting defects observed in isl and fra mutants, cell body position was used to identify RP3 neurons in mutants. By this approach, significant midline crossing defects were detected in RP3 axons in fra mutants. Surprisingly, however, no defects were observed in RP axon midline crossing in isl mutants (Santiago, 2017).

Isl and fra expression both initiate earlier than stage 13, the time at which RP axons cross the midline. Therefore, whether isl is required for fra expression was examined during the early stages of commissural axon guidance. Interestingly, isl was not required for fra expression at stage 13 in any of the ventrally projecting RPs. In contrast, in stage 15 isl mutant embryos from the same collection, a decrease in was observed fra expression in RP1 and RP3. The temporal pattern of fra expression in RP motor neurons is dynamic, so that a larger proportion of RP1 and RP3 neurons express fra mRNA during late embryogenesis than during the stages of midline crossing. A requirement was detected for isl in regulating fra in RP1 and RP3 as early as stage 14, when the RP motor axons have exited the CNS. Taken together, these results suggest that isl is not essential for early fra expression but required for fra expression during the late stages of motor neuron differentiation. The stages at which a requirement was detected for isl in regulating fra correspond to when RP3 axons are exploring their ventral muscle targets, consistent with a model in which Isl instructs the final stages of RP3 axon targeting through Fra (Santiago, 2017).

Another essential feature of Drosophila larval motor neurons that is established late in embryogenesis is the morphogenesis and targeting of their dendrites in the ventral nerve cord. Motor neuron dendrites begin to form as extensions off the primary neurite at stage 15, a stage when a requirement is detected for isl in regulating fra. By early stage 17 (15 hr after egg laying, AEL), RP3 has assumed its stereotyped morphology, consisting of a small ipsilateral projection extending from the soma and a large dendritic arbor forming off the contralateral primary neurite (Santiago, 2017).

The FLP-out genetic labeling strategy was used to visualize individual late-stage RP motor neurons and analyze their dendrites. Focus was placed on the large contralateral arbor of the RP motor neurons that spans one side of the nerve cord in wild-type embryos and forms branches that extend into several medio-lateral zones. Analyses using isl-tau-myc and lim3a-tau-myc transgenes confirmed that the RP cell bodies retain their stereotyped positions in isl mutants and that the relative dorsal-ventral positions of RPs 1/4, 3, and 5 are preserved, allowing identification of distinct classes of RP motor neurons (Santiago, 2017).

Most RP3 neurons in late-stage isl/+ embryos neurons form contralateral dendritic arbors that send projections into the zone between the medial FasII+ axon pathways and the intermediate FasII+ pathways, hereafter referred to as the 'intermediate zone,' consistent with previously published images of RP3 neurons from wild-type embryos. Interestingly, the dendritic morphology of RP3 was distinct from that of a related neuron, RP5, that also expresses Isl and Lim3b-Gal4 and that can be unambiguously identified in both wild-type and mutant embryos because its cell body is found in a more ventral position than the other RP neurons. In wild-type embryos, the RP5 axon targets muscles 12 and 13 (VL1 and VL2) as well as other ventral muscles. Most RP5 neurons in isl/+ embryos exclusively target their dendrites to the lateral zone of the neuropile. Furthermore, the difference observed in the dendritic targeting of RP3 and RP5 neurons correlates with a difference in fra expression. Although fra expression in RP3 and RP5 neurons in control embryos is comparable when RP axons are crossing the midline, by stage 15, significantly fewer RP5 than RP3 neurons express fra. Interestingly, isl is not required for the low levels of fra expression in late-stage RP5 neurons, in contrast to its role in promoting high levels of fra in late-stage RP3 neurons (Santiago, 2017).

Finally, endogenous Netrin expression was monitored in late-stage nerve cords using a Myc-tagged NetB knockin allele, and significant enrichment of Netrin protein was detected in the area between the intermediate and medial FasII+ axon bundles. This area corresponds to the zone where contralateral dendritic projections from RP3 neurons were detected, suggesting that high levels of Fra in RP3 may instruct the formation of dendritic arbors in this region in response to Netrin (Santiago, 2017).

RP motor neuron dendrites were examined in isl mutant embryos to determine whether Isl regulates dendritic position or morphogenesis through Fra or other effectors. No significant difference in the morphology or medio-lateral position of RP5 dendrites was observed between heterozygous and mutant embryos. In striking contrast, many RP3 neurons in isl mutants fail to extend contralateral dendrites into the intermediate zone. Instead, the dendrites of these RP3 neurons remain fasciculated with the intermediate FasII+ axon pathways and do not send medial extensions toward the midline. To more quantitatively measure medio-lateral position and to address the possibility that defects in targeting are secondary to defects in outgrowth, RP3 neurons were traced using Imaris software and total contralateral dendrite lengths and the total number of dendrite tips were measured. Total length of contralateral dendrites was also measured in the intermediate zone of the neuropile, defined as the area between the medial FasII+ and the intermediate FasII+ axon pathways. Although RP3 neurons displayed increased variability in the size of their dendritic arbors in isl mutants, there was no significant difference in the total length or tip number of RP3 dendrites between isl mutants and heterozygotes, suggesting that targeting defects in isl mutants are not likely due to reduced outgrowth. However, the ratio of RP3 dendrites in the intermediate zone over total RP3 dendrite length was significantly reduced in isl mutants, confirming that isl mutant RP3 dendrites are shifted laterally relative to controls (Santiago, 2017).

The dendrites of RP3 neurons were examined in fra/+ and fra mutant embryos. As with isl mutants, cell body position was used to identify RP3 neurons, and neurons with ambiguous positions were excluded. In fra mutant RP3 neurons whose axons fail to cross the midline, a single dendritic arbor forms off the ipsilateral primary neurite, and this arbor was traced. A significant lateral shift was observed in the position of RP3 dendrites in fra mutants both by scoring for the presence of dendrites in the intermediate zone and by quantitative analysis of the dendrites of traced neurons. The lateral shift in fra mutants was more pronounced than in isl mutants, consistent with the observation that some RP3 neurons retain fra expression in the absence of isl. Of note, the lateral shift phenotype did not correlate with whether the RP3 axon had crossed the midline because it was detected at similar frequencies in both contralateral and ipsilateral arbors. Curiously, several RP3 contralateral dendritic arbors appeared reduced in size in fra mutants, whereas this phenotype was not seen in control embryos. However, as in isl mutants, there was no significant change in the total dendrite length or tip number in fra mutants compared with their sibling controls, although there was increased variability in the sizes of dendritic arbors in the mutants. These findings are consistent with previous reports that Netrin-Fra signaling does not play a major role in regulating the outgrowth of motor neuron dendrites in the nerve cord (Santiago, 2017).

A single-cell labeling method allows precise description of the axon targeting defects in isl and fra mutants and determine whether they correlate with defects in dendrite position. Axon and dendrite targeting occur at approximately the same developmental stage, and there is no evidence that one process depends on the other. Importantly, previous studies using retrograde labeling of motor neurons in mutant embryos were not able to address this question because they relied upon motor axons reaching the correct muscles to be visualized (Santiago, 2017).

To determine whether defects in dendrite position correlate with defects in axon targeting, both phenotypes were scored in single labeled RP3 neurons in embryos with muscles fully preserved following dissection. All of the RP3 axons that could be scored in isl heterozygous embryos innervated the muscle 6/7 cleft . In contrast, 18 of 26 isl mutant RP3 axons innervated muscles 6/7, and eight stalled at the 6/7 cleft or earlier along RP3's trajectory or bypassed the choice point. In fra mutant embryos, 10 of 22 RP3 neurons failed to innervate the muscle 6/7 cleft and stalled at or bypassed the choice point. This phenotype is stronger than the frequency at which a complete loss of muscle 6/7 innervation in isl or fra mutants was detected by scoring with anti-FasII. To determine whether this enhancement was due to the heat shock (H.S.) step that is required for genetic labeling, defects were scored using anti-FasII in embryos heat-shocked for either 5 min or 1 hr; it was found that the 1-hr H.S. mildly enhances muscle innervation defects in isl mutants (to 30.4%) whereas a 5-min H.S. does not (to 24.7%, data not shown). Importantly, the two H.S. protocols did not result in any difference in the frequency of dendrite targeting defects observed in isl mutants because 7 of 17 RP3 dendrites in isl mutants are shifted laterally in embryos treated with 1-hr H.S, and 9 of 16 dendrites are shifted after 5-min H.S (Santiago, 2017).

Surprisingly, no correlation was detected between axon and dendrite defects in isl mutants. Although 5 of 26 RP3 neurons displayed defects in both axons and dendrites in isl mutants, 12 of 26 neurons showed defects in one process but not the other). A similar analysis in fra mutants revealed that 8 of 22 RP3 neurons displayed defects in both muscle 6/7 innervation and dendrite position, whereas 8 of 22 displayed normal targeting in one process but not the other. These data suggest that axon and dendrite targeting can occur independently within an individual RP3 neuron and that the central targeting defects observed in isl mutants are not likely to be secondary to defects in muscle innervation (Santiago, 2017).

It was next asked whether isl and fra regulate dendrite development in other classes of motor neurons. RP1 and RP4 also express isl, fra, and lim3b-Gal4. A requirement was detected for isl in regulating fra expression in RP1, but not in RP4, at stage 15. Interestingly, most RP1 neurons, like RP3 neurons, retain high levels of fra at this stage, whereas few RP4 neurons express fra in late-stage control embryos. Previous descriptions of RP1 and RP4 neurons indicate that they form contralateral dendritic arbors of distinct morphologies; RP1's dendritic arbor is taller and found more medially. However, because the axons of RP1 and RP4 target adjacent muscles external to muscles 6 and 13, and their cell bodies are found close to the midline at a similar dorsal-ventral position, they could not nr unambiguously distinguished in single-cell labeling experiments. Nevertheless, when RP1 and RP4 neurons were scored together, a significant lateral shift was observed in the position of RP1 and RP4 dendrites in isl mutants compared with heterozygous siblings: 3 of 22 RP1 and RP4 dendritic arbors were excluded from the intermediate zone in isl heterozygous embryos (14%) compared with 16 of 22 in mutant embryos. A similar phenotype was detected in RP1 and RP4 dendrites in fra mutants. Specifically, 6 of 24 RP1 and RP4 dendritic arbors were excluded from the intermediate zone in heterozygotes (25%) compared with 17/19 in fra mutants. Although additional work will be necessary to determine whether the defects in dendrite position that were detect in RP1 and RP4 neurons in isl mutants correlate with changes in fra expression, these data demonstrate that Isl is required for high levels of fra expression in at least two classes of motor neurons (RP1 and RP3), both of which require isl and fra for dendritic targeting (Santiago, 2017).

To directly test whether isl regulates RP3 dendrite position through its effect on fra expression, a UAS-HA-Fra transgene was overexpressed using lim3b-GAL4 in isl mutants and the hsFLP technique was used to sparsely label RP motor neurons. Strikingly, in isl mutants overexpressing Fra, 0 of 21 RP3 contralateral dendritic arbors were excluded from the intermediate zone compared with 8 of 22 (36%) in sibling mutants lacking the UAS-Fra transgene. To quantitatively measure dendrite position, traces of RP3 dendrites were obtained. A robust rescue of the lateral shift phenotype was detected in isl mutants, as measured by the length of dendrites in the intermediate zone over the total dendrite length. Indeed, the ratio of dendrites in the intermediate zone in rescued mutants was higher than in heterozygous controls, perhaps reflecting a gain-of-function effect caused by artificially high levels of Fra from transgenic overexpression. Importantly, Fra overexpression did not have any effect on total dendritic arbor lengths or tip numbers, strongly arguing that the rescue that was observe is not caused by an increase in the total size of the arbors. Although it cannot be ruled out that Isl regulates dendrite position in part through additional effectors, the observation that cell-type-specific overexpression of Fra in isl mutants rescues dendrite targeting provides compelling support for the model that fra acts downstream of isl to control RP3 dendrite morphogenesis. Together with the demonstration that isl directs RP3 motor axon targeting through the regulation of fra, it is concluded that isl coordinately regulates the targeting of axons in the periphery and of dendrites in the CNS through a common downstream effector (Santiago, 2017).

In the vertebrate spinal cord, the position of motor neuron cell bodies correlates with the targeting of their axons in the periphery (Catela, 2015). This myotopic map may be established through the action of transcription factors that coordinately control cell migration and axon guidance. In particular, Lhx1 and Isl1 are expressed in limb-innervating lateral motor column (LMC) motor neurons and regulate the trajectory of their axons as well as the medio-lateral settling position of their cell bodies . Lhx1 and Isl1 regulate axon guidance through EphA4 and EphB receptors, respectively, and a recent study suggests that Lhx1 regulates cell body position through a distinct effector, the Reelin signaling protein Dab-1 (Santiago, 2017).

In Drosophila, unlike in vertebrates, the position of motor neuron cell bodies does not necessarily correlate with the targeting of their axons in the periphery because neurons that innervate adjacent muscles can be found far apart within a segment. Instead, recent studies have shown that both the larval and the adult Drosophila nervous systems use a myotopic map in which the position of motor neuron dendrites, rather than their cell bodies, correlates with the position of their target muscles (Brierley, 2009, Mauss, 2009). This may be a conserved feature of motor systems across phyla because the dendritic patterning of at least four motor neuron pools in the spinal cord correlates with muscle target identity in the mouse (Santiago, 2017).

Slit-Robo, Netrin-Fra, and Sema-Plexin signaling have been shown to control motor neuron dendrite targeting in Drosophila, and rescue experiments suggest that these guidance receptors act cell-autonomously in this process (Brierley, 2009, Mauss, 2009, Syed, 2016). In addition, the initial targeting of motor neuron dendrites in the embryo is largely unaffected by manipulations that affect the position or the activity of pre-synaptic axons or the presence of muscles, suggesting that this process is likely under the control of cell-autonomous factors, although these remain unidentified (Santiago, 2017).

This study has addressed several key questions about how motor neuron dendrite targeting is specified in Drosophila. First, it was shown that fra expression in two classes of motor neurons (RP3 and RP5) correlates with the medio-lateral position of their dendrites. Previous studies suggested that different classes of motor neurons express different levels of guidance receptors to direct the position of their dendrites, but this has not been demonstrated. Second, Isl, which was previously shown to regulate axon targeting in a subset-specific way, was also shown to regulate dendrite targeting. Third, it was found that Isl regulates both processes through fra. Surprisingly, no correlation was found between axon and dendrite phenotypes in isl mutants. The absence of a correlation suggests that the dendrite positioning defects are not secondary to defects in target selection, consistent with a previous study in which the general patterning of motor neuron dendrites was not disrupted in muscle-less embryos. However, additional experiments that disrupt axon targeting and monitor the medio-lateral position of dendrites will be necessary to confirm that the two occur independently (Santiago, 2017).

Future work will also be necessary to identify additional transcription factors that specify motor neuron dendrite development. A role has been identified for Hb9 in regulating robo2and robo3 expression, but it is not known whether these receptors regulate motor neuron dendrite development. No change was detected in robo1 mRNA levels in RP3 neurons in either hb9 or isl mutants. Robo signaling could be regulated post-transcriptionally. Comm is required for midline crossing of motor neuron dendrites and may endogenously regulate their medio-lateral position. The temporal pattern of comm expression does not support a role in dendrite targeting, however, because comm is not expressed in RP motor neurons at late stages of embryogenesis (Santiago, 2017).

The functional consequences of dendrite targeting defects remain to be explored. It is likely that shifting the position of motor neuron dendrites alters their connectivity, but testing this hypothesis will require identifying the pre-synaptic neurons that impinge on the RPs during locomotive behavior. Forcing a shift in the position of dendrites of dorsally projecting motor neurons does not abolish their connectivity with known pre-synaptic partners but does change the number of contacts established. In mice, the ETS factor Pea3/Etv4 is required for the dendritic patterning of a subset of motor neurons, and electrophysiological recordings reveal changes in connectivity in Pea3 mutant spinal cords. It will be of high interest to investigate whether analogous defects are detected in isl or fra mutant embryos (Santiago, 2017).

Drosophila Isl was initially described as a subset-specific regulator of axon guidance. More recently, Wolfram (2012) demonstrated that Isl also acts instructively to establish the electrophysiological properties of RP motor neurons through repression of the potassium ion channel Shaker. The curreng data show that, in addition to regulating the axonal trajectory and the electrophysiological properties of the RP3 neuron, Isl also establishes its dendritic position. Terminal selectors have been defined as transcription factors that coordinately regulate gene programs conferring multiple aspects of a neuron's identity, including its neurotransmitter phenotype, ion channel profile, and connectivity. Unlike the early-acting factors that function transiently to specify cell fate, terminal selectors are expressed throughout the life of an animal and are required for the maintenance of neural identity. Although there are several examples of transcription factors that act this way, it remains unclear how widespread a phenomenon it is. Does Isl fit the criteria for a terminal selector? Isl is not required for all aspects of RP3 identity because RP neurons retain expression of other motor neuron transcription factors in isl mutants, and their axons exit the nerve cord. Future work will be necessary to determine whether Isl is required throughout larval life for the maintenance of RP3's physiological and morphological features and to what extent Isl coordinately establishes multiple features of RP neuron identity (Santiago, 2017).

Co-expressed transcription factors could act synergistically to regulate specific downstream programs, in parallel through completely distinct effectors, or by some combination of the two mechanisms. Indeed, examples of all of these scenarios have been described. Both in vitro and in vivo studies demonstrate that, in vertebrate spinal motor neurons, Isl1 forms a complex with Lhx3 and that the Isl1-Lhx3 complex binds to and regulates different genes than Lhx3 alone or than a complex composed of Isl1 and Phox2b, a factor expressed in hindbrain motor neurons. In a subset of spinal commissural neurons, Lhx2 and Lhx9 act in parallel to promote midline crossing through upregulation of Rig-1/Robo3. In Drosophila dorsally projecting motor neurons, Eve, Zfh1, and Grain act in parallel to promote the expression of unc5, beat1a, and fas2, although Eve also regulates additional targets important for axon guidance that are not shared by Zfh1 or Grain (Santiago, 2017).

This study shows that Isl and Hb9 act in parallel through at least two distinct effectors and proposes that they regulate their targets by different mechanisms. Hb9 likely indirectly promotes robo2 expression by repressing one or multiple intermediate targets because its conserved Engrailed homology repressor domain is required for its function in motor axon guidance and for robo2 regulation. In vertebrate motor neurons, Isl1 forms a complex with Lhx3 to directly activate several of its known targets. A recent genome-wide DAM-ID analysis found that Isl binds to multiple regions within and near the fra locus in Drosophila embryos, suggesting that it may directly activate fra. The finding that lim3 is not required for fra expression in RP motor neurons, together with evidence that Isl can alter the electrical properties of muscle cells independently of Lim3, suggest that Drosophila Isl does not need to form a complex with Lim3 for all of its functions. Future research will be necessary to detect Isl binding events in embryonic motor neurons, although these experiments are challenging when binding occurs transiently or in a small number of cells. Interestingly, overexpression of Isl using ap-Gal4 or hb9-Gal4 induces fra only in certain subsets of these neurons, consistent with a model in which Isl binds to the fra locus in a cell-type-specific manner. The generation of many large-scale datasets for transcription factor binding sites presents the field with the task of reconciling these data with clearly defined genetic relationships during specific biological processes. This study and others have initiated this effort, but it will be important to investigate the functional significance of other putative transcription factor-effector relationships to achieve a better understanding of how transcriptional regulators control cell fate (Santiago, 2017).

Axon death pathways converge on Axundead to promote functional and structural axon disassembly

Axon degeneration is a hallmark of neurodegenerative disease and neural injury. Axotomy activates an intrinsic pro-degenerative axon death signaling cascade involving loss of the NAD+ biosynthetic enzyme Nmnat/Nmnat2 in axons, activation of dSarm/Sarm1, and subsequent Sarm-dependent depletion of NAD+. This study has identified Axundead (Axed) as a mediator of axon death. axed mutants suppress axon death in several types of axons for the lifespan of the fly and block the pro-degenerative effects of activated dSarm in vivo. Neurodegeneration induced by loss of the sole fly Nmnat ortholog is also fully blocked by axed, but not dsarm, mutants. Thus, pro-degenerative pathways activated by dSarm signaling or Nmnat elimination ultimately converge on Axed. Remarkably, severed axons morphologically preserved by axon death pathway mutations remain integrated in circuits and able to elicit complex behaviors after stimulation, indicating that blockade of axon death signaling results in long-term functional preservation of axons (Neukomm, 2017).

The large and small SPEN family proteins stimulate axon outgrowth during neurosecretory cell remodeling in Drosophila

Split ends (SPEN) is the founding member of a well conserved family of nuclear proteins with critical functions in transcriptional regulation and the post-transcriptional processing and nuclear export of transcripts. In animals, the SPEN proteins fall into two size classes that perform either complementary or antagonistic functions in different cellular contexts. This study shows that the two Drosophila representatives of this family, SPEN and Spenito (NITO), regulate metamorphic remodeling of the CCAP/bursicon neurosecretory cells. CCAP/bursicon cell-targeted overexpression of SPEN had no effect on the larval morphology or the pruning back of the CCAP/bursicon cell axons at the onset of metamorphosis. During the subsequent outgrowth phase of metamorphic remodeling, overexpression of either SPEN or NITO strongly inhibited axon extension, axon branching, peripheral neuropeptide accumulation, and soma growth. Cell-targeted loss-of-function alleles for both spen and nito caused similar reductions in axon outgrowth, indicating that the absolute levels of SPEN and NITO activity are critical to support the developmental plasticity of these neurons. Although nito RNAi did not affect SPEN protein levels, the phenotypes produced by SPEN overexpression were suppressed by nito RNAi. It is proposed that SPEN and NITO function additively or synergistically in the CCAP/bursicon neurons to regulate multiple aspects of neurite outgrowth during metamorphic remodeling (Gu, 2017).

The SPEN family has been evolutionarily conserved, with representatives from protists and plants to animals. The family includes mouse MINT (Msx2-interacting nuclear target protein) and human SHARP (SMRT/HDAC1 associated repressor protein). SPEN, SHARP, and MINT are unusually large proteins of ~3575 to 5500 amino acids in length. In addition to SPEN, the Drosophila genome contains one other SPEN-like gene, spenito (nito), that encodes a much smaller, 793 amino acid protein . Although SPEN and NITO share conserved N-terminal RRMs and the SPOC domain, their overall sequence similarity is only 28%, suggesting that SPEN and NITO may function similarly in some processes, but differently in others. For example, SPEN and NITO display functional antagonism during eye development (Jemc, 2006) but act synergistically in regulating Wingless signaling in wing imaginal discs and cultured Kc cells (Chang, 2008). In addition, NITO was identified as a splicing factor involved in Sxl regulation and sex determination in Drosophila, while SPEN had no such effect (Yan, 2015). Several studies in vertebrates also suggest that the relationship between these two proteins is context-dependent (Chang, 2008). Similar to SPEN, NITO is broadly expressed in Drosophila tissues (Chang, 2008). Therefore, this study investigated the function of NITO and its possible interactions with SPEN in the context of remodeling of the CCAP/bursicon neurons (Gu, 2017).

Several previous studies have examined the role of SPEN during neuronal differentiation. In embryos, SPEN contributes to neuronal cell fate specification and regulates the growth, pathfinding, and fasciculation of PNS and CNS axons. SPEN also regulates proliferation and differentiation of Drosophila photoreceptor neurons. In addition to its role in neuronal differentiation, SPEN has also been shown to modify late-onset, progressive neurodegeneration in a model for spinocerebellar ataxia in the mature Drosophila retina. This study reports that SPEN regulates developmental plasticity in mature neurons (Gu, 2017).

SPEN overexpression specifically inhibited neurite outgrowth during metamorphic remodeling of the CCAP/bursicon cells, with little or no effect on the larval morphology of these neurons. The reasons for the stage-dependence of SPEN activity in these cells are unknown, but one intriguing possibility is that the stage-dependence results from a direct or indirect link to the ecdysone titer during metamorphosis. This is suggested by work on the human SPEN ortholog, SHARP. SHARP expression is steroid-inducible. In addition, the RRM domains of SHARP interact directly with the steroid receptor RNA cofactor SRA, which acts as a scaffold to bring together nuclear receptors, corepressors, and coactivators. SHARP inhibits the transcriptional activity of SRA-stimulated estrogen and glucocorticoid receptors. No Drosophila ortholog of SRA has yet been identified. Nevertheless, in future studies, it will be of interest to examine interactions between SPEN and signaling by the ecdysone receptor (EcR) during neuronal remodeling. (Gu, 2017).

The overexpression of spen and nito produced axon growth and branching phenotypesthat were qualitatively similar (although not identical) to the loss-of-function effects of both genes. Such similarities in GOF and LOF phenotypes are often observed in systems where the stoichiometric ratio of gene products to the concentration of other cellular components is important. Thus, the current observations indicate that SPEN may need to be maintained at a specific level, or within an expression window, to properly regulate axonal outgrowth. Recent studies observed strong genetic interactions between spen and multiple factors controlling Myosin II activity. Notably, many cellular movements, including growth cone migration and branching, depend upon dynamic cytoskeletal rearrangements that can be disrupted by either increasing or decreasing the stability or function of cytoskeletal components. Thus, the interaction between SPEN and Myosin II provides one possible explanation for the similar responses of the CCAP/bursicon neurons to reduced versus enhanced levels of SPEN activity. Interestingly, SPEN also interacts genetically with crinkled (Myosin VIIA) in controlling wing vein development and wing bristle positioning, suggesting a more general function of SPEN in cytoskeletal rearrangements. If SPEN and NITO regulate common cellular processes, then this explanation may also apply to NITO (Gu, 2017).

The current results showed that NITO and SPEN act additively or synergistically to regulate CCAP/bursicon cell remodeling. This finding is in agreement with other studies showing that SPEN and NITO are both positive regulators of Wg signaling in developing wing discs and Drosophila Kc cells, and of programmed cell death in the eye disc induced by the pro-apoptotic factors Head involution defective and Reaper. In contrast, SPEN and NITO are antagonistic in regulating photoreceptor number, rhabdomere morphology, and regular ommatidial spacing in the adult eye. Furthermore, SPEN and NITO may play completely different functions in some situations, as NITO regulates Sxl level and its alternative splicing, while SPEN has no such effects (Yan, 2015). Thus, the interaction between SPEN and NITO depends on the cellular context. In mammals, the large and small SPEN-like proteins display similar context-dependence in the regulation of Notch signaling. The large SPEN family proteins MINT and SHARP both suppress Notch signaling by competing with the N intracellular domain (NICD) for binding to the core transcription factor RBP-J. The NITO ortholog, RBM15, also complexes with RBP-J, but it either stimulates or represses expression of a reporter in different cell lines. Therefore, in Drosophila and in vertebrates, large and small SPEN family proteins act redundantly or synergistically in some cellular contexts and antagonistically in others, or they perform completely different roles (Gu, 2017).

Recent insights into the molecular interactions with SPEN family proteins provide some clues to mechanisms underlying these context-dependent differences in function. In different systems, the large SPEN family proteins function as either transcriptional corepressors or coactivators. For example, human SHARP and RBM15 were identified as crucial factors required for the long non-coding RNA Xist-mediated silencing of X-chromosomes by directly interacting with the Xist, recruiting nuclear corepressor, SMRT, activating histone deacetylase3 (HDAC3), and deacetylating histones to exclude Pol II and repress transcription. Other studies have revealed functions of the SPEN proteins in transcription activation. MINT enhances transcriptional activation at the osteocalcin promoter and associates with an actively phosphorylated and processive form of RNA polymerase II. SHARP enhances beta-catenin/T cell factor (TCF)-mediated transcription. In differentiating Drosophila hemocytes, SPEN binds to many target gene promoters in association with a known activating histone modification pattern (Gu, 2017).

The small SPEN proteins have not been shown to function as transcriptional coactivators or corepressors and instead play important roles in alternative splicing, selection of alternative polyadenylation sites, and nuclear export of mRNAs. However, both the large and small SPEN proteins may function together in protein complexes that first associate with nascent transcripts. These messenger ribonucleoprotein (mRNP) complexes are dynamic structures that participate in transcription, pre-mRNA splicing, and nuclear export, and they change in protein composition to permit nuclear export once pre-mRNAs are completely spliced. Importantly, several studies have detected associations among the large and small SPEN proteins in these structures . Given the strong evolutionary conservation of the large and small SPEN proteins, similar interactions are likely to explain some of the overlapping functions of SPEN and NITO during neuronal remodeling in Drosophila (Gu, 2017).

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