wingless
Wingless Regulation during Head Formation The mechanisms of
action of cephalic gap genes remain poorly understood. orthodenticle (otd), which
establishes a specific region of the anterior head, has been proposed to act in a combinatorial fashion with the
cephalic gap genes empty spiracles (ems) and buttonhead (btd) to assign segmental identities in this region. To test this
model, a heat-inducible transgene was used to generate pulses of ubiquitous otd expression during embryonic
development. Ectopic otd expression causes significant defects in head formation, including the duplication of sensory
structures derived from otd-dependent segments. However, these defects do not appear to result from the
transformation of head segment identities predicted by the combinatorial model. To determine if the combinatorial model is correct, focus was placed on the dorsomedial papilla (dmp), antennal sense organ (anso), and dorsolateral papilla (dlp). The epidermal portions of these structures serve as markers of the ocular, antennal, and intercalary segments, respectively. According to the combinatorial model, ubiquitous otd expression should cause a transformation of the intercalary segment to a second antennal segment, without affecting the identity of the more anterior ocular segment. This would be indicated by duplication of the anso, loss of the dlp, and no change in the dmp. In a significant fraction of the cuticles that developed after an early pulse of otd expression, the anso is indeed duplicated. Significantly, however, the dlp is generallly not lost in these embryos, indicating that at least part of the intercalary segment is still present. These results do not indicate a transformtion of segmental identity, but rather the specific duplication of otd-dependent sensory structures (Gallitano-Mendel, 1998).
It is likely that misexpression of otd causes intrasegmental transformations rather than intersegmental transformations. The results correlate with specific
regulatory effects of otd on the expression of the segment polarity genes engrailed (en) and wingless (wg).
In wild-type embryos, en is expressed in each of the head segments. In the anterior head, en expression first appears during germ band extension in the antennal primordium, as a stripe 1-2 cells in width. Expression subsequently appears in the ocular segment (a small spot), the intercalary segment (a small stripe), and eventually in the clypeolabral region. Early induction of otd causes a broadening of the en antennal stripe to a width of as many as eight cells. In wild-type embryos, wg expression first apppears in the forgut primordium at the blastoderm stage. Subsequently, a broad anterior cephalic stripe forms. Following germ-band extension, wg is activated in a discrete stripe or spot in each head segment, anterior and adjacent to the engrailed counterpart. Ubiquitous otd expression causes the reduction or loss of wg in the antennal, intercalary, gnathal, and trunk segments. More anterior wg expression in the forgut, clypeolabral region, and ocular segment is not reduced (Gallitano-Mendel, 1998).
Mutant embryonic cuticles were examined for en or wg: en mutant embryos lack ansos; wg mutant embryos exhibit severe disruptions in head formation. However, unlike in en embryos, the anso is not missing but instead is frequently duplicated. These results indicate that en is required for anso formation. They also suggest that wg plays an inhibitory role in the specification of this sensory strucure. In double en;wg mutant embryos, the anso is absent, indicating that, although the absence of wg permits the formation of multiple ansos, this requires en activity. Although ubiquitous otd represses wg expression in the antennal segment and all segments posterior to it, otd induction has the opposite effect in the ocular segment, positively regulating wg expression. It is concluded that cephalic gap genes define head morphology through the direct modulation of segment polarity gene expression (Gallitano-Mendel, 1998).
Wingless expression in the notum Two types of sensory organs, large bristles (macrochaetes) and
small bristles (microchaetes), develop in fixed numbers at
constant positions on the dorsal part of the mesothorax (also called the
notum) of Drosophila. The accurate
positioning of the macrochaetes is established within the epithelial sheets of the
notum region of the wing imaginal discs during the third
larval to early pupal stage. For convenience, this region of the wing disc will be referred to as the 'thoracic disc' to
distinguish it from the wing pouch region. Initially, in the
thoracic disc, group of cells (termed proneural clusters) are formed and
characterized by the expression of the proneural genes achaete
(ac) and scute (sc). These proneural clusters form around the positions where
macrochaetes will form. Next, one or a few sensory mother cells (SMCs) are
singled out from the proneural cluster, and each SMC
subsequently undergoes two rounds of cell division to form
four progeny cells that differentiate into the components of a
sensory bristle. Thus, precise positioning of the macrochaete on the
notum depends on the complex expression pattern of the ac
and sc genes in the thoracic disc. ac and sc expression patterns are controlled
through the action of enhancer-like cis-regulatory elements. These
elements are presumed to respond to a prepattern established
by local specific combinations of factors. The identity of these pattern producing factors is largely unknown (Tomoyasu, 1998).
Two large bristles, an anterior-dorsocentral bristle (aDC) and a
posterior-dorsocentral bristle (pDC) are formed along the
anterior/posterior (A/P) axis on the notum. It has been shown
that wg activity is necessary for the formation of both aDC
and pDC. Wingless is expressed in an anterior-dorsal (medial) to posterior-ventral (lateral) stripe in the thoracic discs.
However, the SMCs are not induced all along the wg
expression domain, but induced only adjacent to the dorsal
posterior side, behind the wg expression domain in the anterior
compartment of the thoracic disc. This suggests that Wg signaling
alone is insufficient to induce SMCs in aDCs and pDCs, and
that another factor(s), which resides on the dorsal posterior
side of the thoracic disc, is also required for inducing these
SMCs. One candidate factor is Dpp. In the thoracic disc, Dpp
is induced in a stripe of cells located posterior to the
dorsocentral SMCs. This expression pattern and the
property of Dpp as a morphogen suggests that Dpp signaling
may also participate in prepattern formation of the
macrochaetes on the notum (Tomoyasu, 1998).
The role of Dpp signaling in
dorsocentral bristle formation has been examined by either ectopically activating or conditionally reducing Dpp signaling. Ubiquitous activation of
Dpp signaling in the notum region of the wing imaginal disc induces additional dorsocentral proneural clusters all along the
dorsal side of the wg expression domain, and alters wg expression. Conditional loss-of-function of Dpp signaling during
disc development results in the inhibition of dorsocentral proneural cluster formation and expansion of the wg expression
domain. These results suggest that Dpp signaling has two indispensable roles in dorsocentral bristle formation: induction of
the dorsocentral proneural cluster in cooperation with Wg signaling and restriction of the wg expression domain in the notum
region of the wing imaginal disc (Tomoyasu, 1998).
There is a substantial distance between dorsocentral SMCs
and the dpp expression domain in wild-type discs.
One explanation for the existence of this gap is that the
highest level of Dpp signaling inhibits the formation of
proneural clusters. A down shift of the Dpp activity slope would
release the area in which proneural induction is inhibited by
the highest levels of Dpp signaling.
The mechanism by which the highest levels of
Dpp signaling inhibits proneural induction is unclear and
should be studied at the molecular level.
It is worth noting that the effective range of wg from its
source for proneural cluster induction seem to be different from
that of dpp. The dorsocentral proneural cluster is formed within
approximately five cell diameters from the wg expression
domain, whereas it can be formed more than ten cell diameters
from the dpp source. This difference must contribute
to the oval shape of the proneural cluster, which is longest
along the A/P axis. wg expression is not uniform in the notal
stripe: it is lower at the A/P compartment border. It
is possible that the difference in wg expression levels along the
A/P axis also affects the precise positioning of the dorsocentral
proneural cluster (Tomoyasu, 1998).
In developing Drosophila notum, wingless expression is regulated by positive and negative Decapentaplegic signaling so that only notal cells receiving optimal levels of Decapentaplegic signal express wingless. This Decapentaplegic-dependent regulation of notal wingless expression includes multiple mechanisms involving pannier and u-shaped. In the medial notum, Pannier and U-shaped form a complex. The expression of pannier and u-shaped is positively regulated by Decapentaplegic signals emanating from the dorsal-most region. The Pannier/U-shaped complex serves as a repressor and a transcriptional activator, respectively, for wingless and u-shaped expression. In the more lateral region, wingless expression is up-regulated by U-shaped-unbound Pannier. wingless expression is also weakly regulated by its own signaling (Sato, 2000).
To further clarify the notion that notal wg expression
occurs only in cells receiving optimal levels of Dpp signal, wg expression was examined in Mothers against dpp (Mad) and thickveins (tkv) mutant clones generated at two different stages. Mad encodes a
transactivator acting downstream of Dpp signals, while tkv encodes a type I receptor for Dpp. Early and late clones were generated at late first
instar and late second instar, respectively; resultant clones
were observed in late third instar. A dorsal shift of wg expression was detected in both early and late clones homozygous for Mad1-2, a hypomorphic mutant allele of Mad. In contrast, in the case
of tkva12 (a strong hypomorphic mutant allele of tkv), a dorsal shift of wg expression was observed only in late clones. Little or no wg expression could be detected in any of the early clones. These results suggest that Dpp-signaling activity in cells within early tkva12 clones is much lower than that in Mad1-2 and late tkva12 clones, and hence, medial-notal cells in Mad1-2 and late tkva12 clones but not early tkva12 clones possess residual levels of Dpp-signaling activity, sufficient to induce wg expression. Consistent with this, twin spot analysis in the wing pouch where Dpp signals are autonomously required for cell proliferation has showen that Mad1-2 mutant clones are recovered much more frequently than tkva12 clones. The absence of wg misexpression in late medial tkva12 clones situated along the anterior notal edge is possibly due to Bar-dependent repression of wg expression in the future anterior notum (Sato, 2000).
achaete-scute (ac-sc) expression in the notum is affected in several allelic combinations of pnr, whose function is prevented by ush gene product (Ush) as a result of direct binding to Pnr. Since pnr appears involved in notal wg expression, and pnr and ush are expressed in the
future medial notum in a graded fashion with peaks within the dpp expressing dorsal-most region, the late third instar notum was examined by staining for wg protein and pnr or USH RNA. ush and wg expression areas were found to abut on each other except for the future scutellum,
while almost all wg expressing cells were situated in a
ventral-most region of the pnr expression domain. Although somewhat ambiguous, a similar relationship among wg, pnr and ush expression areas was detected in small discs at an early third instar stage, the earliest stage
of notal wg expression. It may thus follow that
wg expression occurs in lateral-notal cells expressing pnr
but not ush throughout third instar larval notal development.
wg-LacZ expression occurs in a region much
broader than the authentic wg expression domain; wg-LacZ
expression was always observed to be expanded medially or
dorsally, suggesting that the authentic wg expression
domain shifts ventrally as a disc grows (Sato, 2000).
To determine the role of pnr in wg expression, examination was made of wg expression on various pnr mutant backgrounds. Strong wg misexpression occurs in medial pnr-null-mutant (pnrVX6) clones. wg-LacZ or Wg signals are detected in the entire medial notum transheterozygous for pnrVX6 and pnrVI, from which most, if not all, pnr activity is absent. In contrast, a significant reduction of wg expression occurs in pnr-null-mutant (pnrVX6) clones generated within the authentic wg
expression domain. These findings indicate that
pnr is involved in both negative and positive regulation of
notal wg expression; Pnr serves as a positive regulator of wg
expression in the future lateral notum including the authentic wg expression domain, while it is a negative factor of wg
expression in the medial notum. Consistent with this,
ubiquitous or clonal expression of wild-type pnr induces
wg misexpression in the notum ventral to the authentic wg
expression domain, while no or little wg misexpression
occurs in the future medial notum (Sato, 2000).
As in the case of ac-sc expression, ush appears to serve as a negative factor for notal wg expression, since (1) wg is misexpressed in ush-null-mutant (ush1) clones in the future medial notum and (2) virtually all endogenous wg expression is abolished when
wild-type ush is overexpressed throughout the notal
region. In contrast to medial ush1clones, no appreciable change in wg expression is detected in ush1clones
generated within the authentic wg expression domain. That the authentic wg expression domain is demarcated by medial ush expression may indicate that medial ush expression is involved in the establishment of
the dorsal boundary of the authentic wg expression domain.
Based on the fact that Pnr mutants such as PnrD1
and PnrD4, lacking ability to bind to Ush, are still capable of
activating ac-sc in the presence of ush activity, wild-type Pnr has been proposed to be inactivated by ush
through direct interactions of Ush with Pnr. However, the results presented here show that this may not be the case in notal wg expression and ac-sc expression for the DC macrochaetae formation. If Ush serves only as the inhibitor
of Pnr as predicted, a wild-type copy of pnr added in trans to
PnrD1 would not decrease the area of wg expression, since
wild-type Pnr is considered to either activate wg expression
or neutralize the negative function of Ush or both. The results presented here are apparently at variance with this consideration. Both wg and ac-sc misexpression found in the future medial notum of Pnr14/PnrVI
discs are abolished in PnrD1/+ discs with no loss of wg and ac-sc expression in the authentic wg expression domain. This
negative effect of wild-type pnr is reversed by halving the
copy number of endogenous ush. It is concluded
that, in the medial notum, Pnr forms a complex with Ush
and the resultant Pnr/Ush complex represses wg and ac-sc
expression directly or indirectly to establish the dorsal
boundaries of the authentic wg expression domain and the
ac-sc expression area for the DC macrochaetae formation (Sato, 2000).
Notal wg expression is regulated not only by dpp signaling but also by Pnr and Ush. Thus, pnr and ush expression may be under the
control of Dpp signaling or conversely, Dpp signaling is
regulated by pnr and ush. The second possibility, however,
seems to be unlikely, since neither pnr nor ush mutant
clones exhibit any appreciable change in brinker (brk)-LacZ expression. brk is a general Dpp target gene whose expression is negatively regulated by Dpp signaling. Loss of Dpp signaling causes cell-autonomous
brk misexpression in the wing pouch and notum of wing
imaginal discs. To determine the feasibility of the first possibility, pnr
and ush expression was examined in tkva12, Mad1-2 or tkvQ253D(tkvQD) clones; tkvQD is a constitutively active form of tkv. pnr and ush are misexpressed in lateral UAS-tkvQD clones generated in late second instar, an observation indicating that pnr and ush expression is under the control of Dpp
signaling. Unlike wg expression, pnr and
ush expression are abolished not only in early tkva12
clones but also in late tkva12 and early
Mad1-2 clones, both expressing wg, suggesting that pnr and ush expression requires higher levels of Dpp-signaling activity than those required for wg expression. Loss of ush expression in tkva12 and Mad1-2 clones might be a secondary effect due to the loss of pnr expression, since the maintenance of ush expression requires both pnr and ush activities. pnr and ush expression may be independently initiated by Dpp signaling, since pnr expression normally occurs in ush mutant clones and no ush misexpression is induced by ubiquitous pnr expression. It is concluded that the graded expression of pnr and ush is determined by Dpp signaling and hence, Pnr and Ush act downstream of Dpp (Sato, 2000).
In the larval notal region, dpp expression is not continuous but is broken by the authentic wg expression domain, thus suggesting that notal development could be regulated by Dpp signals emanating separately from dorsal
and ventral sources up to the wg expression domain. As
anticipated, the expression of dad (dad-LacZ), a downstream component of Dpp signaling whose expression is positively regulated by Dpp signaling, is detected not only in medial but also in lateral notum. However, double-staining of dad-LacZ and either PNR or USH RNA expression shows that unlike dad-LacZ, pnr and ush are not induced in the postero-lateral notum in spite of the presence of active Dpp signals. In addition, ectopic wg expression induced by tkvQD is restricted to the antero-lateral notum. It may thus follow that an unidentified factor represses the expression of a fraction of Dpp target genes, which include pnr, ush and wg but not dad, in the postero-lateral notum (Sato, 2000).
Wg signaling represses wg transcription for refinement of
its own expression domain in the wing margin. Thus, an examination has been made of notal wg expression on Wg-signaling mutant backgrounds. In contrast to
wing-margin, wg expression in the notum is activated
by its own signaling though much less effectively. armadillo (arm) and disheveled (dsh) encode Wg signal transducers and wgts is a temperature-sensitive Wg secretion mutant. Weak partial wg misexpression is noted in about 50% of lateral clones (19 of 40 clones)
expressing Deltaarm, which constitutively activates Wg signaling. Ectopic wg expression was also detected in a cell non-autonomous fashion when wg misexpressing clones were induced in the lateral notum. In contrast, wg transcription is considerably reduced in dsh null mutant clones. When wgts mutant discs are incubated at a non-permissive
temperature, for 48 h, an appreciable reduction of wg expression is detected in the authentic wg domain without any significant change in pnr and ush expression. Taken together, these results indicate that Wg signaling weakly activates wg transcription in the future lateral notum. The failure of induction of wg misexpression in Deltaarm and wg clones in future medial notum may indicate that wg expression due to auto-activation is repressed by Pnr/Ush complexes in the medial notum. One unexpected finding is that, in the hinge region, strong wg misexpression occurred only in cells surrounding wg expressing cells, suggesting possibly a new type of Wg-dependent wg expression (Sato, 2000).
The entire notal ush expression area is included in the
notal pnr expression domain and hence, notal ush expression might be positively regulated by pnr. This possibility using a pnr hypomorphic mutant and a significant reduction of notal ush expression was in pnr hypomorphic mutant flies. Thus, it is concluded that Pnr is involved in the up-regulation of notal ush expression. In the case of wg expression, Ush free of Pnr serves as an activator, while a Pnr/Ush complex serves as a repressor. To determine which forms of Pnr are involved in ush expression, examination was made of USH RNA expression in the notum expressing pnr ubiquitously and the notum transheterozygous for pnrD1 and pnrVl. Neither wild-type Pnr free of Ush nor PnrD1, incapable of binding to Ush but capable of activating wg expression, could induce ush expression. It may thus follow that a Pnr/Ush complex (but not Pnr free of Ush) is required for ush expression as a positive transcriptional regulator (Sato, 2000).
A summary is presented of wg regulation in the notum. In both future medial and lateral notal regions, dpp is expressed and Dpp signaling is active. However, ventral Dpp signals are neutralized by an unknown mechanism as far as
pnr, ush and wg expression is concerned. Notal wg expression, except for that in the scutellum, is regulated through four different pathways, three under the control of Dpp signals emanating from the dorsal-most region. pnr and ush expression is up-regulated by Dpp signaling, but ush expression is much narrower than that of pnr, possibly because of the requirement of higher Dpp-signaling activity for ush expression than that for pnr expression. In the future medial notum, Pnr and Ush form a
complex repressing wg expression, while Ush-unbound Pnr activates lateral wg expression. The authentic wg domain and the medial notum abut one another. Unlike wg expression, ush expression in the future medial notum is positively regulated by the Pnr/Ush complex. This regulation appears required for the maintenance of medial ush expression. Dpp signaling is also capable of activating notal wg expression through an unidentified factor X. This route includes neither Pnr nor Ush. In addition, wg expression is weakly up-regulated by its own signaling in the lateral notum (Sato, 2000).
The morphogen gradient of Wingless provides positional information to cells in Drosophila imaginal discs.
Elucidating the mechanism that precisely restricts the expression domain of wingless is important in understanding the two-dimensional
patterning by secreted proteins in imaginal discs. In the pouch region of the wing disc, wingless is induced at the dorsal/ventral compartment
boundary by Notch signaling in a compartment-dependent manner. In the notum region of the wing disc, wingless is also expressed across the
dorsal/ventral axis, however, regulation of notal wingless expression is not fully understood. Notal wingless expression is
established through the function of Pannier, U-shaped and Wingless signaling itself. Initial wingless induction is regulated by two transcription factors, Pannier and U-shaped. At a later stage, wingless expression expands ventrally from the pannier expression domain by a
Wingless signaling-dependent mechanism. Interestingly, expression of pannier and u-shaped is regulated by Decapentaplegic signaling
that provides the positional information along the anterior/posterior axis, in a concentration-dependent manner. This suggests that the
Decapentaplegic morphogen gradient is utilized not only for anterior/posterior patterning but also contributes to dorsal/ventral patterning
through the induction of pannier, u-shaped and wingless during Drosophila notum development (Tomoyasu, 2000).
A hierarchy of the activity of these genes during notum development is presented. dpp is initially induced at
the dorsal region of the A/P compartment boundary by Hh
signaling. Dpp signaling induces two target genes,
pnr and ush. Analyses of pnr expression in put-ts and tkva12
cells suggest that different thresholds are set for the induction of these genes: low levels for pnr and high levels for ush. wg is induced by Pnr where ush is not expressed. Simultaneously, the Pnr-Ush complex represses
wg expression at the dorsal-most region of the presumptive
notum. In the later stage, the wg expression
domain expands ventrally from the pnr expressing region
and wg starts to be expressed in the non-pnr-expressing
cells. During this process, Wg signaling plays a crucial
role and this separation does not occur in the Wg signaling
mutants. The Pnr-Ush complex acts as a repressor for the induction of wg and of DC enhancer-lacZ expression (DC enhancer is an enhancer of the achaete-scute
proneural gene complex that activates gene expression in
the dorsocentral area). It is interesting that Ush does not
simply inhibit Pnr function but switches the activator function of Pnr to a repressor function. Based on the result that the extra doses of Pnr cannot revert the repressor activity of
Pnr-Ush, it has been proposed that the activator function of Pnr and the repressor function of the Pnr-Ush complex do not simply compete with each other on the
notal wg enhancer element. However, it also seems to be
possible that Pnr and the Pnr-Ush complex compete for the
binding site at the notal wg enhancer, but the ability of Pnr-Ush complex to bind this site may be greater than that of
Pnr. It is also worth noting that FOG-1, a mammalian homolog of Ush, represses the transactivation of alpha-globin and
EKLF promoter by GATA-1, but enhances the transactivation of NF-E2 p45 promoter by GATA-1 in a culture cell
system. Dorsocentral (DC) bristles are ectopically formed but
postvertical bristles on the head are missing in a loss-of-function allelic combination for ush or in pnrD1 heterozygous flies. These
observations suggest that the Pnr-Ush complex acts as a
repressor for the DC enhancer, but acts as an activator for
the enhancer of postvertical bristles. Only a cis-regulatory
element of the DC enhancer has been analyzed at the
nucleotide level. Additional studies of the molecular
analyses of the cis-regulatory elements of both wg and DC
or other enhancers of the achaete-scute complex seem to be
necessary in order to reveal the functions of Pnr and Ush (Tomoyasu, 2000).
Generally, at least two different coordinate axes are
necessary for positional specification in a two-dimensional
field. Morphogen gradients of Dpp and Wg provide this
axial information during Drosophila imaginal disc development. In both wing and leg discs, Dpp is induced at the A/P compartment boundary by Hh
signaling. In the leg disc, wg is also induced by Hh signaling.
Mutual repression between Dpp and Wg signaling separates
each expression territory, localizing dpp in the dorsal and
wg in the ventral regions abutting the A/P border (a compartment-independent manner). In contrast, wg is induced by Notch signaling only
at the D/V compartment boundary in the wing pouch (a
compartment-dependent manner). Then, secreted Dpp and Wg
proteins provide positional information along the A/P and
D/V axes, respectively, to establish Cartesian-like coordinates in the pouch field. Relative positions of dpp and wg
expression domains in the notum are more similar to those in
the wing pouch (in both cases, the expression domains are
orthogonal). However, a D/V compartment boundary does
not exist in the notum. The results described here reveal that
another compartment-independent mechanism acts to
pattern the presumptive notum. Namely, the D/V axis,
provided by Pnr, Ush, and Wg, is initially established by
the Dpp gradient, which mainly contributes the positional
information along the A/P axis. One of the key issues of this
patterning model is that Dpp signaling seems to act preferentially along the A/P axis of the notum. This is because two
target genes, pnr and ush, are induced farther from the Dpp
source along the A/P axis than along the D/V axis. One
possible explanation for this phenomenon is that the diffusion of Dpp protein may be positively regulated along the A/P axis. However, such asymmetric induction is not observed
on the dad induction; dad is one of the
Dpp signaling targets in the wing disc. This suggests that diffusion of Dpp protein is not
directionally regulated in the notum region. An alternative
explanation would be that an effective range of Dpp
morphogen gradient is established in a relatively short
range. Cells that respond to Dpp would proliferate or
migrate preferentially along the A/P axis. pnr mRNA is
detected mainly in the posterior-dorsal region of the presumptive notum. GFP expression of UAS-gfp pnrmd237 is seen along the entire dorsal side of the presumptive notum. This difference between the staining pattern of
pnr mRNA and GFP expression of UAS-gfp pnrmd237
in the
late third larval stage seems to be caused by a long half-life
of gal4 and/or gfp products, suggesting that cells that once
have expressed pnr mRNA proliferate preferentially along
the A/P axis. However, it seems to be difficult to explain the
determination of pnr and ush expression domains only by
the Dpp morphogen gradient. The existence of Tkv*-insensitive cells for inducing pnr and ush indicates that some
regional subdivision may occur independently of Dpp
signaling. Discontinuous expression of dpp in the A/P
border of the notum also suggests the existence of a Dpp-independent subdivision. D/V subdivision of the presumptive notum seems to be achieved by several parallel mechanisms, including Dpp signaling (Tomoyasu, 2000).
Because Drosophila is a holometabolous insect, it should
destroy larval tissues and replace them with a different
population of cells to form the adult structure during the
pupal stage. Thus, formation of epidermal structure should
occur reiteratively during embryogenesis and metamorphosis. Patterning of larval epidermal structure takes place
during embryogenesis; however, patterning of adult structure is mainly performed in larval stage imaginal discs. The Dpp morphogen gradient has been shown here to
regulate pnr and ush expression to pattern the presumptive
notum, which forms the dorsal structure of the adult, in the
wing imaginal disc. pnr
and ush are necessary for the formation of amnioserosa, the dorsal
structure of the embryo, and both pnr and ush expressions are also positively regulated by Dpp in a concentration-dependent manner during embryogenesis. Furthermore, dorsal closure during embryogenesis and thorax closure in metamorphosis is also analogous,
because both processes are regulated by the same signaling
cascade, JNK signaling. These similarities between embryogenesis
and metamorphosis presumably reflect the evolutionary
history of the development in holometabolous insects (Tomoyasu, 2000).
pannier encodes a GATA transcription factor that is involved in various biological processes, including heart development, dorsal closure during embryogenesis as well as neurogenesis and regulation of wingless (wg) expression during imaginal development. This study demonstrates that pnr encodes two highly related isoforms that share functional domains but are differentially expressed during development. Moreover, two genomic regions of the pnr locus are described that drive expression of a reporter in transgenic flies in patterns that recapitulate essential features of the expression of the isoforms, suggesting that these regions encompass crucial regulatory elements. These elements contain, in particular, sequences mediating regulation of expression by Decapentaplegic (Dpp) signaling, during both embryogenesis and imaginal development. Analysis of pnr alleles reveals that the isoforms differentially regulate expression of both wg and proneural achaete/scute (as/sc) targets during imaginal development. Pnr function has been demonstrated to be necessary both for activation of wg and, together with U-shaped (Ush), for its repression in the dorsal-most region of the presumptive notum. Expression of the isoforms define distinct longitudinal domains and, in this regard, it is shown that the dual function of pnr during regulation of wg is achieved by one isoform repressing expression of the morphogen in the dorsal-most region of the disc while the other laterally promotes activation of the notal wg expression. This study provides novel insights into pnr function during Drosophila development and extends knowledge of the roles of prepattern factors during thorax patterning (Fromental-Ramain, 2008).
Focus was placed on reporter expression in the wing disc where pnr is necessary for the development of thoracic macrochaetae. A DNA fragment, 15.7 kilobases (kb) in length and including the 5′ untranslated sequences of exon 1 (construct A15.7), directs expression of lacZ in the dorsal-most domain of the disc. The 15.7 kb DNA fragment was dissected by 5′-end deletion, it was observed that the genomic sequences contain two distinct regions responsible for reporter expression. The 3.2 kb DNA fragment adjacent to pnr (construct E3.2) drives expression of the reporter along the A/P border of the notal region of the disc where Dpp is expressed, and also in a central cluster of cells. Expression remains similar in lines carrying the reporter under the control of the 9.3 kb fragment (construct C9.3), suggesting that the supplementary 6.1 kb DNA fragment does not contain essential regulatory sequences. When the DNA fragment inserted upstream of the reporter is the 12 kb fragment (construct B12), lacZ expression is reinforced in comparison of expression seen with lines carrying construct C9.3. Expression of the reporter fully covers dorsal domain of the disc when the promoter sequences include the distal DNA fragment (construct A15.7). Thus, a second domain responsible for expression in the disc appears to be located in the distal region of construct A15.7 (Fromental-Ramain, 2008).
It is concluded that reporter expression depends on activity of two domains, a proximal one located in the 3.2 kb fragment adjacent to pnr (construct E3.2) and a distal one corresponding to the 5′-end of construct A15.7. These observations are reinforced by the fact that both the distal fragment (construct H6.4) and the proximal fragment (construct J1.8) inserted in front of an heterologous hsp43 (heat shock protein43) minimal promoter direct reporter expression in the wing disc. In contrast, the intervening fragment (construct I6.1) does not promote expression when placed in front of this heterologous promoter (Fromental-Ramain, 2008).
Interestingly, the location of the two domains suggests that they may correspond to alternate promoters of the pnr isoforms. Indeed, sequence analysis of the pnr locus and characterization of the mRNAs expressed during development led to the prediction that pnr may encode two isoforms. Isoform-α (pnr-α) encodes the Pnr protein as it has been identified, whereas the putative isoform-β (pnr-β) encodes a truncated version of the Pnr-α protein, lacking the 52 N-terminal amino acids. However, Pnr-α and Pnr-β share functional domains and the N-terminus of Pnr-α does not contain any obvious functional signature. In vitro experiments revealed that both Pnr-α and -β associate with Ush and equivalently activate a reporter driven by promoter sequences including GATA sites in a cultured cell line (Fromental-Ramain, 2008).
Several reports have implicated Pnr as a key transcriptional regulator during expression of both ac/sc and wg in the presumptive notum. The current study extends previous work and importantly demonstrates that Pnr function is achieved by two structurally related isoforms with distinct expression domains. Moreover, the isoforms display distinct transcriptional activities, including antagonism during regulation of wg expression (Fromental-Ramain, 2008).
In the presumptive notum of Drosophila, wg expression is regulated by different mechanisms, acting downstream of Dpp. It was shown that expression of pnr and ush are activated by Dpp signaling. pnr RNA is expressed in the dorsal-most domain of the disc, including the authentic domain of wg expression, whereas ush RNA appears restricted to the future medial notum, abutting on the authentic domain of wg expression. ush appears to serve as a negative factor for wg expression since wg was misexpressed in mutant cells lacking ush activity and induced in the dorsal-most territories of the disc. Thus, it was proposed that Ush-unbound Pnr activates wg in its authentic domain of expression, while Pnr-Ush complexes repress wg in the dorsal-most domain of the disc. Moreover, mosaic clones lacking pnr function and induced in the dorsal-most area of the disc, exhibit mis-expressed wg expression, suggesting that wg can be activated by a Pnr-independent mechanism (Fromental-Ramain, 2008).
This study demonstrates that pnr encodes two isoforms which are differentially expressed during development and are likely regulated by Dpp signaling in embryos and/or wing discs. Isoform-β is expressed in the dorsal-most area of the presumptive notum in a pattern similar to that of both the UASGFP reporter driven by pnrGal4 and the reporter present in lines carrying construct G5.6. Isoform-β expression, visualized using the reporter present in lines carrying construct G5.6, delimits the authentic domain of wg expression revealed by in situ hybridization. This observation slightly differs from a previous report where overlapping expression is described of both UASGFP driven by pnrGal4 and Wg (Garcia-Garcia, 1999). However, overexpression of UASGFP driven by pnrGal4 probably led to an overestimate of the overlap of pnr and wg expression. In the current study, pnr-β expression is visualized with a reporter driven by regulatory sequences from the pnr locus and is compared with wg mRNA expression. The distinct experimental approaches used to detect pnr and wg expression may explain these differences. Nevertheless, this study demonstrates that pnr-β expression is identical to that of the UASGFP reporter driven by pnrGal4. Moreover, pnr-α expression laterally extends with respect to the domain of pnr-β expression and functional analysis of pnr alleles revealed that pnr-α mediates wg activation (Fromental-Ramain, 2008).
The pnr-α isoform corresponds to pnr as it was previously identified. It is weakly expressed in the dorsal territory of the wing disc, with stronger accumulation along the A/P border of the notal region of the disc and in a central cluster of cells. These features of pnr-α expression are reproduced in lines carrying construct C9.3. Doubly-stained wing discs for wg RNA and reporter expression driven by construct C9.3 revealed that the lateral domain of reporter expression coincides with the authentic domain of wg expression, suggesting that isoform-α may activate wg during imaginal development. In addition, the expression domains of reporters driven by construct C9.3 or G5.6 are very similar to those of pnr-α and pnr-β. Together, they define an expression domain similar to that of the pnr RNA as described by in situ hybridization with a cDNA probe detecting both isoform (Fromental-Ramain, 2008).
Previous analysis have shown that both pnr mutant flies (pnrGal4/pnrVX6) and the homozygous pnrGal4 flies exhibit expanded wg expression towards dorsal-most territories of the disc. This correlates with severe reduction of pnr-β accumulation, while pnr-α accumulation is dramatically increased. This suggests that Pnr-α activates wg, while Pnr-β, possibly together with Ush, represses expression in the dorsal-most domain. Finally, analysis of the pnrV1 allele also supports the hypothesis of antagonistic roles of pnr isoforms during wg expression. Homozygous pnrV1 flies exhibit increased pnr-α and wild-type pnr-β, associated with expansion of wg expression towards the dorsal-most territories of the wing disc. As the pnrV1 allele likely encodes functional proteins, it is concluded that Pnr-α is a crucial factor during wg activation and that Pnr-β antagonizes Pnr-α function (Fromental-Ramain, 2008).
Further evidence for antagonistic activities of the pnr isoforms during thorax patterning is provided by comparison of wg expression between homozygous (pnrV1) and (pnrGal4/pnrVX6) flies. Indeed, pnr-β expression is not affected in pnrV1, although it is severely impaired in the (pnrGal4/pnrVX6) combination, while wg expression fully covers the dorsal-most domain of the disc only in the case of the (pnrGal4/pnrVX6) combination. It is concluded that Pnr-α activates wg whereas Pnr-β, possibly together with Ush, mediates repression in the dorsal-most domain of the disc. Moreover, pnr isoforms display antagonistic activities and the wg expression is regulated by the molecular ratio between activating pnr-α and repressing pnr-β (Fromental-Ramain, 2008).
These conclusions are further supported by experiments where isoforms are ubiquitously overexpressed using the c765 line. Expression of wg is ectopically induced in the lateral domains of the presumptive notum by overexpressed pnr-α (Garcia-Garcia, 1999), whereas this study shows that it is repressed in the medial domain by overexpressed pnr-β. wg expression in the medial domain does not totally depend on pnr, since mutant cells lacking pnr activity exhibit reduced wg expression. The complete repression of wg in its authentic domain after overexpression of isoform-β does not depend on repression of isoform-α by isoform-β, but rather on a competition for the notal wg enhancer between isoform-β and a factor(s) responsible for pnr-independent expression of wg (Fromental-Ramain, 2008).
Identification of the isoforms led to revisiting the role of pnr during regulation of ac/sc and ush targets in the wing disc. Both overexpressed pnr-α and overexpressed pnr-β lead to activation of proneural expression and development of ectopic sensory bristles suggesting that the isoforms may act as subunits of the multiprotein proneural complex as it has been previously identified. However, the current analysis of the pnrV1 and pnrGal4 alleles do not argue in favor of such a model during regulation of ac/sc expression. Both the reduced pnr-β expression associated with homozygous pnrGal4 animals and the increased pnr-α expression observed in homozygous pnrV1 animals exhibit a loss of DC bristles and impaired proneural expression at the DC site of the wing/thorax discs. As the domains of isoform expression stay the same in mutants animals, this suggest that the mutant phenotypes result from antagonistic activities of the Pnr proteins. This hypothesis is reinforced by the observation that proneural expression is reduced in both (pnrGal4/+) and (pnrV1/+) animals and is totally abolished in homozygous mutant animals. Thus, proneural expression at the DC site of the imaginal disc relies on the stoichiometry between Pnr-α/Pnr-β. Additional evidence is provided by molecular analysis of the vertebrate complex, homologous to the proneural complex encompassing Pnr, Chip and the heterodimer (Ac/Sc)-Da. Indeed, the vertebrate hematopoietic-specific complex contains only one GATA molecule, that does not support the notion that both the Pnr-α and Pnr-β isoforms simultaneously belong to the Drosophila complex necessary for ac/sc activation during Pnr-driven proneural development (Fromental-Ramain, 2008).
Previous analysis have shown that ush expression is also regulated by Pnr. ush expression is abolished in the dorsal-most domain of (pnrVX6/pnrV1) disc. Since the (pnrVX6/pnrV1) combination was predicted to correspond to a loss of pnr function, it was postulated that Pnr mediates activation of notal ush expression. It has also been reported that ush expression is lost in (pnrD1/pnrV1) disc, except at the A/P border of the notal region. Since pnrD1 encodes a mutant protein carrying a single amino acid exchange in the DNA binding domain that disrupts interaction with the negative regulator Ush, it was hypothesized that the (Pnr-Ush) complex serves as a transcriptional activator of ush expression. However, the current analysis revealed a strong induction of pnr-α expression at the A/P border of the disc while pnr-β expression is not modified. Hence, expression of the (PnrD1-α) protein is induced at the A/P border in (pnrD1/pnrV1) discs and it is suggested that Pnr-α-Ush is involved in the repression of ush expression. Moreover, it is also suggested that the ush expression depends on the stoichiometry between Pnr-α and Pnr-β since ush expression is abolished in the dorsal-most domain of the pnrD1/pnrV1 discs outside the A/P border. The pnrV1/pnrD1 combination is consequently characterized by ectopic sensory bristles and increased proneural expression in the DC area (Fromental-Ramain, 2008).
Pnr is involved in regulation of both the ac/sc and ush targets during neural development and the stoichiometry of the isoforms is a crucial determinant during regulation of gene expression. These characteristics may explain the paradoxical observations that increased pnr-α expression in homozygous pnrV1 displays reduced ac/sc expression and loss of DC bristles whereas overexpressed pnr-α in (pnrGal4/UAS pnr α) leads to activated ac/sc expression and additional macrochaetae. The DC enhancer would require lower Pnr-α concentration for repression than the notal ush enhancer, probably reflecting different affinities of the binding sites for the Pnr-α-Ush effector. At low concentration, the Pnr-α-Ush heterodimer antagonizes Pnr-β activity, leading to reduced ac/sc expression at the DC site and loss of DC bristles. Overexpressed pnr-α mediates repression of ush, leading to reduced concentration of the Pnr-α-Ush heterodimer and consequently, ac/sc expression at the DC site results from activating Pnr-β. Hence, overexpressed pnr-α displays ectopic sensory organs. In contrast, overexpressed pnr-β would repress pnr-α involved together with Ush in repression of ac/sc and would also directly activate proneural expression to promote development of ectopic sensory organs. Both overexpressed pnr-α or pnr-β activates proneural expression, leading to ectopic sensory organs but they act by distinct mechanisms. During activation of proneural expression, overexpressed pnr-β appears to directly stimulate ac/sc through binding to their regulatory sequences whereas overexpressed pnr-α indirectly acts in repressing ush expression (Fromental-Ramain, 2008).
The present data highlight the merit of revisiting pnr function during development since pnr isoforms are expressed in domains that define a novel subdivision of the wing disc. The biological significance of the subdivision is of critical importance since the isoforms exhibit antagonistic activities during regulation of targets genes. A challenging issue will be to understand how the Pnr isoforms molecularly interact with the regulatory sequences of the target genes ac/sc, ush and wg. Sequence analysis revealed that the DC enhancer contains several Pnr binding sites and some of them are involved in regulation of ac/sc expression during neural development (Garcia-Garcia, 1999). These binding sites may correspond to targets for Pnr-β and (Pnr-α)-Ush complexes. Mutagenesis of the Pnr binding sites would be required to understand how the isoforms interact with the regulatory element to antagonistically regulate proneural expression, to clarify the role of Ush during regulation of Pnr target genes, and to resolve the question on how upon dimerization Ush can convert Pnr from an activator to a repressor (Fromental-Ramain, 2008).
The roles played by signaling pathways and neural activity during the development of circuits have been studied in several different contexts. However, the mechanisms involved in maintaining neuronal integrity once circuits are established are less well understood, despite their potential relevance to neurodegeneration. This study demonstrates that maintenance of adult Drosophila olfactory sensory neurons requires cell-autonomous neuronal activity. When activity is silenced, development occurs normally, but neurons degenerate in adulthood. These detrimental effects can be compensated by downregulating Glycogen synthase kinase-3β (Gsk-3β). Conversely, ectopic expression of activated Gsk-3β or downregulation of Wnt effectors also affect neuron stability, demonstrating a role for Wnt signaling in neuroprotection. This is supported by the observation that activated adult neurons are capable of increased Wingless release, and its targeted expression can protect neurons against degeneration. The role of Wnt signaling in this process is non-transcriptional, and may act on cellular mechanisms that regulate axonal or synaptic stability. Together, evidence is provided that Gsk-3β is a key sensor involved in neural circuit integrity, maintaining axon stability through neural activity and the Wnt pathway (Chiang, 2009).
During development, electrical activity and cell signaling pathways have
been shown to collaborate to eliminate neurons or synaptic connections. In the mature vertebrate nervous system, many neurons are eliminated, although the majority survive through the life of the animal. Little is known about the mechanisms underlying the maintenance of mature neurons in a circuit, or, indeed, whether there is any active requirement at all for maintenance. Given the fundamental role of electrical activity in neuronal function, it was asked whether activity is the sensor for the integrity of the nerve cell. In order to test this rigorously, a preparation was required from which both spontaneous and evoked activity could be removed and the consequences on neuronal maintenance
assessed. The OSNs are unique in that they allow the manipulation of
spontaneous and odor-induced activity in the mature animal. Experiments in
this system implicate a role for activity in neuronal maintenance. Further, this study showed that Gsk-3β, a molecule that is a sensor for nutrients and Wnt
signals, acts 'downstream' of neuronal activity in this process. The
results thus integrate a key property of neurons, electrical activity, with
Gsk-3β signaling. Although Gsk-3β can receive inputs from a variety of
pathways, the expression of Wg in the adult antennal lobe and the observation
that degeneration could be rescued by expression of Dsh and Wg, suggested that
this is a major signaling pathway acting on neuronal survival in the mature
animal. It was also established that Wnt signaling acts through a
non-transcriptional pathway involving Gsk-3β. The action of Wnt signaling
during the formation and plasticity of neuronal circuits is mediated through
the 'divergent canonical pathway', as demonstrated by several studies in both
vertebrates and Drosophila (Chiang, 2009).
Spontaneous activity is essential for both the development and maintenance of OSN projections in the mouse olfactory bulb. In Drosophila, the formation of the peripheral
olfactory map is independent of ORs or activity, and is possibly hardwired.
However, as in mammals, OSN maintenance requires neural activity. The availability of Or83b-null mutants and the conditional
TARGET system was exploited to demonstrate that OSN terminals within the antennal lobe
glomeruli develop normally in the absence of activity, but show local
degeneration in older animals in which the most distal ends exhibit signs of
beading, blebbing and, eventually, fragmentation, which are hallmarks of axon
degeneration (Chiang, 2009).
In vertebrates, electrically silent visual system neurons and OSNs retract
when placed in an environment of active neurons because of synaptic
competition for target sites. When all neurons in a given field are silenced
to the same extent, elimination does not occur, suggesting that differences in
activity, rather than the absolute activity state, determine the stability of
connections. Contrary to expectation from these findings, neurons innervating
a single glomerulus retracted their contacts and showed degeneration when
silenced. Further, clones that drive the light chain of tetanus toxin (TNT-G) in small subsets of OSNs also
degenerate, indicating that activity influences neuron survival by exerting
autonomous effects at the level of individual cells (Chiang, 2009).
Enhanced neuronal activity has been shown to trigger the activation of a
wide range of genes including transcription factors, cell adhesion molecules,
membrane excitability proteins, translational regulators such as dFmr (Fmr1)
and signaling molecules such as Wnt. The observation that Wg is expressed in the adult
brain led to a test of the role of Wg/Wnt signaling in stability. It was found that
downregulation of Wg pathway members compromises OSN stability, resulting in
phenotypes similar that resulting from a lack of neuronal activity. Several
studies argue for a link between neuronal activity and Wnt/Wg signaling during
formation of LTP and in activity-dependent dendritic morphogenesis. At the
Drosophila neuromuscular junction, activity-dependent Wg secretion
results in structural outgrowth mediated by Gsk-3β in the motoneurons and
nuclear localization of the cleaved C terminus of DFz2 in the postsynaptic
muscle cells (Ataman, 2008; Chiang, 2009 and references therein).
Evidence is provided for a requirement of Wg/Wnt signaling for
stabilization of adult OSNs, although a transcriptional output of the pathway
is not required. Non-transcriptional roles for Wnt signaling have been
demonstrated previously, well-studied examples being in Wnt7a-induced growth
cone and axon remodeling of the mossy fibers and in
Drosophila neuromuscular junction synaptogenesis and plasticity
(Ataman, 2008). The output of signaling in these systems is mediated by Gsk-3β, which regulates microtubule cytoskeletons. Gsk-3β phosphorylates the Microtubule-associated protein 1B (mammalian homolog of Futsch) and Tau,
thereby influencing microtubule stability. Neuronal activity regulates Gsk-3β enzymatic activity through a series of phosphorylation and dephosphorylation events, whereby activity-regulated PP1 phosphatase and PI3K-Akt kinase regulate phosphorylation of Gsk-3β serine 9 (Chiang, 2009).
Activation of Gsk-3β in rat hippocampus inhibits LTP with associated
synaptic impairments reminiscent of Alzheimer's disease. A genetic
link between late-onset Alzheimer's disease and the Wnt pathway co-receptor
LRP6 has been demonstrated in human subjects and
there is a possibility that Alzheimer's disease-associated synaptic
impairments are due to aberrant GSK-3β kinase activity. The
phenotypes observed in OSNs with activated Gsk-3β or a chronic
blockage of activity are tantalizingly similar to those described during
neurodegeneration in Alzheimer's disease models(Chiang, 2009).
A model is proposed whereby neuronal activity acts together with Gsk-3β
to maintain circuit stability in the adult olfactory system. Activity appears to
lead to Wg release, which acts in an autocrine manner to impinge upon
Gsk-3β activity. It is also possible that autonomous activity could
signal to Gsk-3β through other pathways, one of them being the energy
status of the neuron. In adult OSNs, Wnt signaling appears to be
non-transcriptional and Gsk-3β possibly acts cell-autonomously to
regulate the dynamics of the microtubule cytoskeleton. The results
support the emerging view of a role for the Wnt pathway in neuroprotection,
and the approach provides a system in which to examine the structural and
molecular mechanisms that operate during altered physiological states in a
genetically tractable organism (Chiang, 2009).
Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. This study used chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, wingless (wg), encoding a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), was identified as a potential Repo target gene. Repo regulates wg expression in vivo, and local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ (Kerr, 2014).
The diversity of genes directly regulated by Repo-a critical transcriptional regulator of glial cell development in Drosophila-has not been thoroughly explored. ChIP studies from Drosophila S2 cells identified several potential Repo targets that have been shown to govern fundamental aspects of glial development or function. For example, known targets were identified that actively promote glial cell fate specification (e.g., pointed, distalless;, blood-brain barrier formation (e.g., gliotactin, loco, coracle, Nrv1, engulfment activity (e.g., dCed-6), neurotransmitter metabolism (e.g., EAAT1, Gs2), ionic homeostasis (e.g., fray), and neuron-glia signaling during nervous system morphogenesis (e.g., Pvr). For at least two potential targets, gs2 and Cp1, this study demonstrated a key requirement for Repo in their transcriptional activation during development (Kerr, 2014).
Given the broad roles of this collection of genes in glial cell biology, this work supports the hypothesis that Repo transcriptionally
regulates a diverse class of genes that modulate many aspects of glial cell development. For instance, Pointed, which is now a predicted Repo target, is a key glial factor that activates glial fate at very early developmental stages. Likewise, Repo appears to regulate Gliotactin, Coracle, and Nrv1, which are molecules essential for formation of the pleated septate junction-based blood-brain barrier at mid to late embryogenesis in Drosophila. At the same time, EAAT1 and GS2 are activated late in the embryonic glial program, with expression being retained even in fully mature glia, and these transporters are critical for synaptic neurotransmitter recycling. Since EAAT1 and GS2 are both activated by Repo, and primarily expressed in CNS glia, these data argue that Repo is directly upstream of multiple key glial factors required for glutamate clearance from CNS synapses (Kerr, 2014).
Mammalian excitatory glutamatergic synapse formation is modulated by multiple soluble glia-derived factors including TSPs, Hevin/Sparc, and glypicans 4 and 6. These factors, along with other secreted glial factors that remain to be identified, are essential for initial synapse formation and (with the exception of TSPs) can promote postsynaptic differentiation through membrane insertion and clustering of AMPA receptors. This study identified Wg as a novel glia-derived factor essential for postsynaptic structure and function in vivo at the Drosophila glutamatergic NMJ. Combined with previous findings that NMJ glia can also release a TGF-β family member to regulate presynaptic growth in a retrograde manner (Fuentes-Medel, 2012), these studies provide compelling evidence that Drosophila glia function as a major integrator of synaptic signals during developmen (Kerr, 2014).
Previous work has demonstrated that Wg/Wnt signaling potently modulates the coordinated assembly of both presynaptic and postsynaptic structures at the Drosophila NMJ (Speese, 2007). Loss of Wg, or its receptor DFz2, leads to a dramatic decrease in synaptic boutons and disrupted clustering of postsynaptic glutamate receptors (Packard, 2002). Although previous studies supported evidence implicating motor neurons in Wg release, the presence of alternative cellular sources remained an open and important question. The surprising discovery of Wg as a candidate Repo target gene by ChIP led to an exploration of the possibility that NMJ glia could act as an additional in vivo source of NMJ Wg. Consistent with this idea, this study found that peripheral glia expressed Wg, SPGs were able to deliver Wg::GFP to the NMJ, and knockdown of SPG Wg significantly reduced NMJ Wg levels and led to a partial phenocopy of wg mutant phenotypes (Kerr, 2014).
Interestingly, it was found that loss of glia-derived Wg could account for some, but not all, wg loss-of-function phenotypes. For example, whereas depletion of glia-derived Wg disrupted clustering of postsynaptic glutamate receptors, it had no effect on the formation of synaptic boutons. In contrast, depletion of neuronal Wg led to defects in both glutamate receptor clustering as well as bouton formation. Although only neuronal Wg regulated bouton growth, these data argue that both glial and neuronal Wg are capable of modulating the assembly of glutamate receptor complexes. Thus, this study has identified two in vivo sources of Wg at the NMJ: the presynaptic neuron and local glial cells (Kerr, 2014).
Regarding the modulation of neurotransmission, both glial and neuronal Wg was found to have important roles, which, as in the case of the development of synaptic structure, were only partially overlapping. Loss of glial or neuronal Wg resulted in postsynaptic defects in neurotransmission, including increased mEJP amplitude (a postsynaptic property), decreased nerve-evoked EJPs, and decreased quantal content. Consistent with Repo regulating glial Wg expression, these phenotypes were mimicked by loss of repo function. The most notable difference in functional requirements for glial versus neuronal Wg is in mEJP frequency (a presynaptic function): depletion of glial Wg resulted in a dramatic increase in mEJP frequency, whereas manipulating neuronal Wg had no effect. Thus both glial and neuronal Wg are critical regulators of synaptic physiology in vivo, likely modulating NMJ neurotransmission in a combinatorial fashion, although glial Wg has the unique ability to modulate presynaptic function (Kerr, 2014).
The increase in mEJP amplitude is consistent with findings that GluR cluster size was increased upon loss of glia- or neuron-derived Wg, and that in general this was accompanied by minor changes in GluRIIA signal intensity. A potential explanation is that neuron- and glia-derived Wg regulate the levels of GluRIIA subunits. Previously, it was demonstrated that downregulation of the postsynaptic Frizzled Nuclear Import (FNI) pathway also increased GluRs at the NMJ (Speese, 2012). This suggests that glia- and neuron-derived Wg may act in concert via the FNI pathway to stabilize the synapse by regulating GluR expression (Kerr, 2014).
An important property of the larval NMJ is the ability to maintain constant synaptic function throughout development via structural and functional modifications. The combined functions of glial and neuronal Wg likely contribute to this mechanism, as together
they positively regulate synaptic growth and function as well as organize postsynaptic machinery. However, a previous study suggested that the transcription factor Gooseberry (Gsb), in its role as positive regulator of synaptic homeostasis in neurons,
may be antagonized by Wg function (Marie, 2010). Mutations in gsb block the increase in neurotransmitter release observed when postsynaptic GluRs are downregulated. Furthermore, Marie (2010) showed that the gsb mutant defect can be rescued by a heterozygous wg mutant allele. However, the specific role of Gsb in this process is unclear, as rapid synaptic homeostasis was normal in the mutant, and defects appeared restricted to a long-term decrease in GluR function. It will be important to define the specific role of Gsb in synaptic homeostasis and to manipulate Wg function in alternative ways before a clear relationship between Wg and Gsb can be established (Kerr, 2014).
How could neuronal versus glial Wg differ in regulating NMJ development and physiology? One possibility is that the level
or site of Wg delivery by each cell type is different. For example, since SPGs invade the NMJ only intermittently (Fuentes-Medel, 2009), it is possible that they release most of their Wg outside of the NMJ, whereas the presynaptic neuron, which is embedded in the muscle cell, delivers it more efficiently and directly to the postsynaptic muscle cell. Alternatively, the Wg morphogen released by glia versus that released by neurons could be qualitatively different through alternative post-translational modifications such as glycosylation. Either mechanism would allow for glia to modulate specific aspects of NMJ physiology independently from neuronal Wg, perhaps in an activity-dependent manner (Kerr, 2014).
Although glia-derived Wg does not modulate NMJ growth, Drosophila glia can indeed regulate synaptic growth at the NMJ in vivo. It has been demonstrated previously that Drosophila glia release the TGF-β ligand Maverick to modulate TGF-β/BMP retrograde signaling at the NMJ and thereby the addition of new synaptic boutons (Fuentes-Medel, 2012). The discovery that glia-derived Wg can exert significant control over the physiological properties of NMJ synapses expands the mechanisms by which Drosophila glia can control NMJ synapse development and function. In the future it will be important to understand how glial Wg and TGF-β signaling integrate to promote normal NMJ growth, physiology, and plasticity (Kerr, 2014).
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continued:
Biological Overview
| Evolutionary Homologs
| Targets of Activity
| Protein Interactions
| mRNA Transport
| Developmental Biology
| Effects of Mutation
| References
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