division abnormally delayed and dally-like
Is it possible to estimate the number of target genes of the homeoproteins Eve and Ftz? Eve and Ftz have been shown to bind with similar specificities to many genes, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Eve and Ftz bind at the highest levels to DNA fragments throughout the length of three probable target genes: eve, ftz and Ubx. However, Eve and Ftz also bind at only two- to ten-fold lower levels to four genes chosen in an attempt to find non targets: Adh, hsp70, rosy and actin 5C, suggesting that Eve and Ftz bind at significant levels to a majority of genes. The expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve (Liang, 1998).
At stages 10-14, 87% of cDNAs in the 8-12 hour library are likely to be directly or indirectly regulated by Eve, Ftz, Engrailed and all of the Hox proteins. These downstream genes are each expressed in unique, segmentally repeating patterns. Some are expressed at dramatically altered levels between segments. Most vary from segment to segment in the number and position of cells in which they are most prominently expressed. This is not simply because expression follows the distribution of a particular cell type. Between segments, the majority of genes are most highly expressed in differently positioned subsets of the same cell types, indicating that these patterns cannot result solely from the action of cell-type specific transcription factors. Eve, Ftz and Engrailed establish the segmentally repeating structure of the embryo. Therefore, all genes expressed in segmentally repeated patterns by stage 11 should be downstream of these three genes. This has been experimentally confirmed for eve and ftz. The expression of all 14 segmentally expressed genes tested is altered in eve and ftz mutant embryos at stage 11. These and other downstream genes can be divided into three classes: genes expressed in strong, moderate or weak segmentally repeated patterns. 33% of cDNAs fall into the strongly repeated class. For this class, staining levels vary five fold or more between cells across a transverse section of a segment along the anterior/posterior axis of the embryo. 24% of clones belong to the moderately regulated class. These genes show two- to five-fold variations in staining across the width of a segment. Finally, the weak segmentally repeated genes vary only 1.2 to 2 fold in staining between cells across a segment. Thus, most downstream genes are expressed in all cells, but each are still subject to specific and precise control by the selector homeoproteins. The more strongly regulated genes include many developmental control genes such as Enhancer of split [E(spl)] , tramtrack, division abnormally delayed (dally), and Dwnt4. A high proportion of the moderate and weakly regulated genes are involved in essential cellular functions such as splicing (e.g. RNA helicases), translation (e.g. met tRNA synthetase), general signal transduction (e.g. G-protein beta13F) and cytoskeletal structure (e.g. alpha tubulin 84B). This raises the question of whether or not modest changes in the expression of essential enzymes and structural proteins are important for morphogenesis. It is argued that they probably are. 11% of the genes picked from the 8-12 hour cDNA library do not appear to be downstream of the selector homeoproteins. Most of these genes are expressed relatively uniformly in all cells. But even these genes show some differences in expression pattern (Liang, 1998).
The expression pattern of dally, monitored by dally::lacZ enhancer-trap expression, in the developing wing along the AP axis shows a peak of expression at the A/P border cells, and dally levels are lowest in cells adjacent to this region. Furthermore, dally levels gradually increase toward the anterior and posterior distal cells. This pattern correlates with the expression patterns of several genes involved in pattern formation along the AP axis, such as tkv and master of thick veins (mtv; also known as scribbler/breakless); this suggests that dally also participates in this process. Expression of the tkv gene is controlled by two distinct pathways. (1) Hh represses tkv expression at the A/P border cells, and En regulates a high basal level of tkv in the P compartment. The activities of both hh and en genes are mediated by a putative transcription factor, Mtv. (2) tkv levels are downregulated by Dpp signaling. By this mechanism, tkv expression is maintained at low levels in the center of the disc and at higher levels toward the anterior and posterior edges. The correlation between expression patterns of dally and tkv prompted an analysis of the dally function in Dpp signaling in this tissue. Regulatory pathways controlling dally expression were analyzed and compared with those controlling tkv expression (Fujise, 2003).
Hh signaling induces dally expression at the A/P border cells. dally expression is absent in smoothened (smo) mutant clones generated in the anterior compartment, where the Hh signaling is blocked, indicating that Hh signaling is required for activation of dally at the A/P border cells. To further determine whether Hh signaling is sufficient for the induction of dally, clones were examined that ectopically express hhCD2, which encodes a membrane-tethered form of Hh, using the FLP-OUT system. In the A compartment, dally expression levels are increased in hhCD2-expressing cells and in cells immediately adjacent to them. This result shows that Hh expression is sufficient to induce dally expression in the A compartment. To determine if dally expression is controlled by en, which upregulates tkv expression, clones of en-mutant cells were induced using the FLP-FRT mosaic analysis system. Within en-mutant clones in the P compartment, dally levels are dramatically increased; this indicates that dally expression is negatively regulated by en (Fujise, 2003).
To determine whether modulation of Dpp signaling affects dally expression, the dally::lacZ expression was compared between wild-type and tkv heterozygous cells. Clones mutant for tkv were generated in a heterozygous background (tkva12/+) using the FLP-FRT system, which should, as a consequence, produce both mutant (tkva12/tkva12) and wild-type sister clones (+/+). However, tkv– cells do not survive in the wing pouch since Tkv activity is indispensable for growth and, thus, only wild-type sister clones survive. In resultant mosaic discs with wild-type and tkv-heterozygous cells, dally expression is decreased cell autonomously in wild-type (+/+) clones at the AP border and peripheral to the border. To further confirm this result, the effects were examined of tkv-hypomorphic clones on dally expression. In such clones, where tkv activity is partially compromised, the levels of dally expression are elevated. In the notum region of the wing disc, tkv-null clones can be generated in which a substantial increase of dally expression is observed. Finally, the effect of increased Dpp signaling on dally expression was tested by using the FLP-OUT method to induce clones of cells that express tkvQ253D, a constitutively active form of tkv, in the wing pouch. The level of dally::lacZ expression was found to be autonomously reduced in the tkvQ253D-expressing clones. All of these results consistently indicate that dally expression in the wing disc is negatively regulated by Dpp signaling, as has been shown for tkv. Thus, dally and tkv are regulated by the same set of molecular pathways: Hh, En and Dpp signaling (Fujise, 2003).
Although dally expression is regulated by the same set of signaling pathways that control expression of tkv the effects of Hh and En on dally are the opposite of those on tkv. In addition, dally expression is negatively controlled by Dpp signaling. Through this mechanism, relative levels of dally expression are higher at the anterior and posterior distal edges. Therefore, dally and tkv show similar patterns of expression with one exception: the level of dally expression is high in A/P border cells, where Dpp is synthesized and secreted, but by contrast, tkv expression levels are low in this region. The high levels of dally in the peripheral regions could sensitize cells to low levels of Dpp, as has been shown for tkv. These regulatory pathways appear to form negative feedback loops, which may stabilize the shape of the Dpp morphogen gradient. Thus, the regulated expression and function of Dally are crucial factors in the generation and maintenance of the Dpp morphogen gradient (Fujise, 2003).
The Drosophila gene sugarless encodes a homolog of vertebrate
UDP-glucose dehydrogenase. This enzyme is essential for the
biosynthesis of various proteoglycans. In the absence of both maternal
and zygotic activities of this gene, mutant embryos develop with
segment polarity phenotypes reminiscent of the loss of either Wingless
or Hedgehog signaling. To analyze the function of Sugarless in
cell-cell interaction processes, attention has been focused on the
requirement of Sugarless for Wingless signaling in different tissues.
sugarless mutations impair signaling by Wingless, suggesting that
proteoglycans contribute to the reception of Wingless. Overexpression
of Wingless can bypass the requirement for sugarless, suggesting that
proteoglycans modulate signaling by Wingless, possibly by limiting its
diffusion and thereby facilitating the binding of Wingless to its
receptor. Tissue-specific regulation of proteoglycans may be involved
in regulating both Wingless short- or long-range effects. For example,
dally mutations show
wing notching with loss of wing margin structures, an effect seen in
wingless and dishevelled mutants, suggesting the potential involvement
of Dally in Wg signaling. A Drosophila homolog of vertebrate Syndecans
(see Syndecan) has also been characterized. Syndecan is a transmembrane HSPG and
represents the major source of HSPGs in epithelial cells. Syndecan has
been demonstrated to function as a coreceptor for FGF signaling.
Further genetic and biochemical studies should reveal whether Dally
and/or Syndecan play a direct role in Wg signaling (Hacker, 1997).
When expressed in S2 cells, the majority (approximately 83%) of
secreted Wingless protein (WG) is bound to the cell surface and
extracellular matrix through specific, noncovalent interactions. The
tethered WG can be released by addition of exogenous heparin sulfate
and chondroitin sulfate glycosaminoglycans. WG also binds directly to
heparin agarose beads with high affinity. These data suggest that WG
can bind to the cell surface via naturally occurring sulfated
proteoglycans. Two lines of evidence indicate that extracellular
glycosaminoglycans on the receiving cells also play a functional role
in WG signaling: (1) treatment of WG-responsive cells with
glycosaminoglycan lyases reduces WG activity by 50%; (2) when
WG-responsive cells are preincubated with 1 mM chlorate, which blocks
sulfation, WG activity is inhibited to near-basal levels. Addition of
exogenous heparin to the chlorate-treated cells restores WG activity.
Based on these results, it is proposed that WG belongs to the group of
growth factor ligands whose actions are mediated by extracellular
proteoglycan molecules (Reichsman,1996).
The Drosophila UDP-glucose dehydrogenase gene is involved in Wingless
signaling. UDP-glucose dehydrogenase is an enzyme responsible for the
production of UDP-glucuronate. UDP-glucuronate, a uronic acid, is a
precursor in glycosaminoglycan (GAG) biosynthesis. GAG is a complex
carbohydrate which is linked to a distinct class of glycoproteins
called proteoglycans. A mutation in UDP-glucose dehydrogenase, called
kiwi, generates a phenotype identical to that of wingless. By rescuing
the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan
heparan sulfate, it has been shown that kiwi function in the embryo is
crucial for the production of heparan sulfate in the extracellular
matrix. Injection of heparin degrading enzyme, heparinase (and not
chondroitin, dermatan or hyaluronic acid degrading enzyme) into
wild-type embryos leads to the degradation of heparin-like
glycosaminoglycans and a 'wingless-like' cuticular phenotype.
Heparin-like GAGs are complex acidic polysaccharides that are important
soluble components of the extracellular matrix. They provide the
necessary hydration to the ECM scaffold and hence solubilize and
modulate the function of such proteins as fibroblast growth factor.
This study provides the first genetic evidence for the involvement of
heparin-like glycosaminoglycans in signal transduction (Binari, 1997).
In vitro experiments suggest that glycosaminoglycans (GAGs) and the
proteins to which they are attached (proteoglycans) are important for
modulating growth factor signaling. However, in vivo evidence to
support this view has been lacking, in part because mutations that
disrupt the production of GAG polymers and the core proteins have not
been available. The identification and characterization of Drosophila
mutants in the suppenkasper (ska) gene is described. The ska gene
encodes UDP-glucose dehydrogenase, which produces glucuronic acid, an
essential component for the synthesis of heparan and chondroitin
sulfate. ska mutants fail to put heparan side chains on proteoglycans
such as Syndecan. Surprisingly, mutant embryos produced by germ-line
clones of this general metabolic gene exhibit embryonic cuticle
phenotypes strikingly similar to those that result from
loss-of-function mutations in genes of the Wingless (Wg) signaling
pathway. Zygotic loss of ska leads to reduced growth of imaginal discs
and pattern defects similar to wg mutants. In addition, genetic
interactions of ska with wg and dishevelled mutants have been observed. These
data demonstrate the importance of proteoglycans and GAGs in Wg
signaling in vivo and suggest that Wnt-like growth factors may be
particularly sensitive to perturbations of GAG biosynthesis (Haerry,
1997).
In Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and Frizzled2 (Fz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately, suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Overexpression of Fz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz is able to substitute for Dfz2 only under conditions where the Wg ligand is present in excess. The wgPE2 mutant phenotype is also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. It is concluded that wild-type Wg signaling requires efficient interaction between ligand and the Fz2-proteoglycan receptor complex to promote the naked cuticle cell fate (Moline, 2000).
When ubiquitously expressed in a wild-type embryo, wgPE2 subtly changes the denticle pattern and shows a slight dominant-negative effect on naked cuticle formation. This contrasts with ubiquitous expression of wild-type wg, which produces uniform naked cuticle. Ubiquitous expression of wgPE2 in a wg null mutant embryo rescues denticle diversity, but does not significantly rescue naked-cuticle formation. However, coexpression of Fz2 and wgPE2 in wg null mutants produces uniform naked cuticle, as does ubiquitous expression of wild-type wg alone. Thus, the ability of wgPE2 to generate the naked-cuticle cell fate depends on overexpression of Fz2. A slight interaction was also detected with Fz under conditions of high-level coexpression, suggesting that amounts of Wg in excess of physiological concentrations permit interaction with Fz receptor (Moline, 2000).
Indeed, this observation offers an explanation for the apparent genetic redundancy of Fz and Fz2 in embryonic Wg signaling. In the absence of zygotic Fz2 receptor, Wg protein may accumulate to a level sufficient to activate Fz receptor, which then promotes normal epidermal patterning. An increased accumulation of Wg protein was detected in embryos zygotically deficient for Fz2, compared either with wild-type embryos or embryos maternally and zygotically deficient for fz. This suggests that Wg ligand is not internalized and degraded as efficiently when Fz2 is absent from the cell surface, thereby permitting interactions with Fz that are not relevant under wild-type conditions. Abnormal accumulation of Wg protein has also been observed in wgPE2 mutant embryos, which similarly show a broader and less punctate pattern of Wg antibody staining. This staining pattern is restored to a more wild-type appearance by overexpressing Fz2 in wgPE2 mutant embryos, further supporting the idea that the wgPE2 lesion compromises interaction with the Fz2 receptor (Moline, 2000).
In wgPE2 mutant embryos that are zygotically mutant for either dally or sugarless (which encodes an enzyme involved in polysaccharide synthesis), a substantial expanse of naked cuticle is produced. Both mutations are hypomorphic, semi-lethal P-element insertions that do not affect embryonic patterning in the context of wild-type Wg. Therefore, mild reductions in sugar modification suffice to restore functionality to the wgPE2 mutant ligand. Moreover, ectopic expression of dally, using a hs-dally transgene, worsens the wgPE2 mutant phenotype. These effects are specific for the wgPE2 phenotype: the hypomorphic Df(2)DE phenotype is not affected by zygotic loss of sugarless or dally and is partially suppressed, rather than enhanced, by providing ectopic dally. Thus, excess Dally improves signaling efficiency for low levels of wild-type Wg, as has been demonstrated for other hypomorphic wg phenotypes, but has the opposite effect on the partial signaling activity of wgPE2 (Moline, 2000).
Finally, overexpression of dally reverses the rescuing effect of overexpressing Fz2 in wgPE2 mutants. This suggests that ectopic Fz2 expression allows interaction with the mutant ligand because it shifts the ratio of Fz2 to Dally molecules at the cell surface, presumably increasing the number of Fz2 receptor complexes that lack Dally co-receptor and that are therefore, as free receptors, able to bind the mutant Wg ligand. As the wgPE2 genetic lesion changes an uncharged valine to a negatively charged glutamic acid, it is conceivable that introduction of a negative charge in the carboxyl terminus prevents proper binding between Wg ligand and negatively charged sulfated sugar groups (Moline, 2000).
In conclusion, it is proposed that interactions between Wg and proteoglycans are required for promoting naked-cuticle specification, but not denticle diversification, and that wgPE2 cannot promote this high-level response because of abnormal interactions with proteoglycans. It is further concluded that the Fz receptor is able to substitute for Fz2 under conditions of excess Wg ligand, but under normal circumstances, does not appear to have a major role in transducing the naked-cuticle cell fate (Moline, 2000).
A novel Drosophila Wingless (Wg) target gene, wingful
(wf: termed Notum by FlyBase), encodes a potent extracellular feedback inhibitor of Wg. In contrast to the cytoplasmic protein Naked cuticle (Nkd), the only known Wg feedback antagonist, Wf functions during larval stages, when Nkd function is dispensable. It is proposed that Wf may provide feedback control for the long-range morphogen activities of Wg (Gerlitz, 2002). A library of 2000 Gal4 enhancer trap P-element insertions
was established, each of which reports a gene expression pattern in the
wing imaginal disc. This collection was screened
with a UAS-GFP reporter for lines that show a
wg-like expression pattern. There were 11 insertions
identified that reported wg-like gene expression in the
embryonic epidermis and all imaginal discs. Four of these lines (S180, ND382,
S476, S554) contained an insertion in the wg gene itself; the other seven lines (S141, S145, S163, S330, ND337, ND339, ND634) all carried a P-element insertion at cytological position 72D, only a few base pairs upstream of gene CG13076, referred to as wingful. These enhancer trap
insertions indeed report the expression of wf, as revealed by
RNA in situ hybridization. wf is ectopically
expressed upon wg misexpression, indicating that
wf is a Wg target throughout larval development (Gerlitz, 2002).
wf codes for a presumptive protein of 671 amino acids, with an N-terminally situated signal sequence. The
wf product is readily secreted from transfected
Drosophila cells and has a noticeable propensity to
adhere to the surfaces of intact cells. The analysis of the
Wf protein sequence reveals a significant structural homology to a
subfamily of poorly characterized hydrolases related to plant pectin
acetylesterase. Together, these results
suggest that the product of the wf gene may catalyze the
hydrolytic cleavage of an extracellular substrate (Gerlitz, 2002).
To test the hypothesis that the pan-Wg-target wf encodes
an inhibitor of Wg activity, attempts were made to abolish wf function
by genetic means. From a collection of six EMS-induced lethal
mutations, located between the distal breakpoint of
Df(3L)st-f13 and the proximal breakpoint of
Df(3L)brm11, a putative
null allele of wf was identified with a stop codon at amino acid position
141, encoding a severely truncated protein. Animals homozygous for
wf141 or animals of the genotype
wf141/Df3(3L)st-f13 die during pupal stages, and
show various phenotypes. The most prominent of these are patterning
defects in the wing imaginal disc. Wg signaling plays at least two
distinct roles during wing development. Early reduction of wg
activity results in a wing-to-notum transformation, indicating a
requirement for Wg in defining the wing blade primordium, but later reductions cause the loss of wing margin and adjacent tissue, indicating its subsequent role in specifying the wing margin and organizing wing blade development. wf mutants show phenotypes opposite to both classes. Wing discs mutant for wf
are enlarged with an extended wing blade region (hence the name
wingful). Often these discs contain two wing pouches at the
expense of notal structures. Although no
apparent expansion of Distalless-lacZ (Dll-lacZ)
expression was detected along the dorsoventral axis, there was a significant
increase in the number of cells expressing neuralized, a high-threshold Wg target expressed in neural wing margin cells. Consistent with this observation, rare adult escapers mutant for wf show a dramatically increased number of
mechanosensory bristles in the wing. Wg signaling also
distinguishes between sternite and ventral pleura development in the
adult abdomen. wf adult escapers show extra sternite bristles, an effect that was also observed with ectopic expression of wg. Finally, wf adults show extra dorsocentral bristles, sensory organs on the notum whose specification has been shown to depend on wg activity. Taken together, these results show that the absence of wf function causes a gain of Wg activity in developing adult tissues. Therefore, the function of the wild-type wf product is to limit Wg signaling activity (Gerlitz, 2002).
A further prediction of the assumption that Wf functions as a Wg
feedback inhibitor is that wf overexpression should lead to
wg loss-of-function phenotypes. Three lines of
evidence are presented to show that this is, indeed, the case. (1) One of the wf enhancer trap P-element insertions was replaced with an
EP element positioning 10 UAS sites upstream of the
wf gene, rendering it transcriptionally responsive to Gal4
expression. Alterations of wf expression have unusually potent
effects, since all commonly used Gal4 drivers caused lethality in
combination with UAS-wf. The only exceptions were S168-Gal4 and scalloped-Gal4, which are expressed in the wing pouch and represent Wg targets, providing a self-regulating circuit in combination with UAS-wf. Adult animals carrying the S168-Gal4 and UAS-wf transgenes have severely reduced wings that lack all wing margin structures. (2) The expression of two target genes were analyzed in this context. S168-Gal4 expression was virtually abolished by UAS-wf expression, whereas the expression domain of wg itself is expanded. Wg is known to narrow its own domain of expression, because a reduction in Wg signal transduction causes ectopic wg transcription. (3) Finally, and perhaps most strikingly, driving expression of wf with scalloped-Gal4 results in a wing-to-notum transformation, the founding loss-of-function phenotype of the wg gene (Gerlitz, 2002).
Based on its structural features as a secreted protein with
homologies to pectin acetylesterases, Wf could exert its function by
modifying polysaccharide-based properties of cell surface proteins and
thereby impeding the intercellular movement of the Wg protein. Alternatively, Wf could counteract Wg signaling by modifying the transducing properties of Wg or one of its receptors. To distinguish between these two possibilities, tests were performed to see whether Wf also antagonizes a derivative of Wg, Nrt-Wg, that is tethered to the cell surface and
does not move through tissue. Expression of Wg or
Nrt-Wg driven by dpp-Gal4 results in a robust activation of
ectopic Dll-lacZ expression. Surprisingly, wf expression extinguishes Dll-lacZ
expression induced by tethered Wg, as well as that induced by free Wg. From this experiment it can be ruled out that the primary
function of Wf is to impede the extracellular transport of Wg.
Therefore, Wf must interfere with the signaling activities of either Wg
or its receptor components. Because no physical interaction between Wf and Wg, Frizzled-2 (Dfz2), and its LRP-like partner Arrow could be detected in tissue culture systems, the view that Wf inhibits the activity of a coreceptor component, such as
Dally or Dally-like (Dly), proteoglycans that appear to participate in
Wg reception, is favored. Wf may inhibit such receptor components via its presumptive
esterase activity, for example, by modifying Dally or
Dly glycosaminoglycan chains. A definitive proof for this mode of
action could be achieved by the genetic demonstration that in larval
dally;dly double-mutant situations, loss of
wf function has no antagonistic effect (Gerlitz, 2002).
The discovery of Wingful as an essential Wg feedback antagonist
may provide an explanation of why Nkd has no apparent role in imaginal
tissues of Drosophila . The function of Nkd may be superseded
by that of Wf, which functions in a powerful negative-feedback loop in
adult development. Conversely, when both maternal and
zygotic components of wf are removed, no obvious requirement was observed for Wf in embryonic development, possibly because the nkd
system is operative at this stage of development. Both Naked and Wf
can, however, inhibit Wg signaling throughout development if they are overexpressed, but each of them is operating more effectively at only
one of the two stages. It may not be coincidence that Nkd, as the
intracellular feedback antagonist, is used during embryonic patterning,
(where Wg functions at short range), whereas Wf, as a secreted
extracellular antagonist, primarily regulates patterning processes that
depend on long-range Wg signaling. Wf functions nonautonomously and, like Argos (a secreted feedback antagonist of the
Drosophila EGF system), may have a
different range of action compared with the primary signal, providing
an intricate means to shape the range and slope of the cellular
responses to a morphogen gradient (Gerlitz, 2002).
Animal bodies are composed of structures that vary in size and shape within and between species. Selector genes generate these differences by altering the expression of effector genes whose identities are largely unknown. Prime candidates for such effector genes are components of morphogen signaling pathways, which control growth and patterning during development. This study shows that in Drosophila the Hox selector gene Ultrabithorax (Ubx) modulates morphogen signaling in the haltere through transcriptional regulation of the glypican dally. Ubx, in combination with the posterior selector gene engrailed (en), represses dally expression in the posterior (P) compartment of the haltere. Compared with the serially homologous wing, where Ubx is not expressed, low levels of posterior dally in the haltere contribute to a reduced P compartment size and an overall smaller appendage size. One molecular consequence of dally repression in the posterior haltere is to reduce Dpp diffusion into and through the P compartment. These results suggest that Dpp mobility is biased towards cells with higher levels of Dally and that selector genes modulate organ development by regulating glypican levels (Crickmore, 2007).
Upon comparing Dpp signaling readouts in the wing and haltere, it was noticed that, in addition to a general narrowing of Dpp pathway activity, Dpp signaling was also asymmetric relative to its source (the AP organizer) in the haltere. Specifically, the P-Mad signal was stronger anterior to the AP organizer (roughly demarcated by the domain of peak P-Mad staining) than it was posterior to the organizer. To test if this asymmetry is due to asymmetric ligand distribution or differences in signal transduction, an extracellular staining protocol was used to examine the distribution of a Dpp::GFP fusion protein following its expression in AP organizer cells. In wing cells, Dpp::GFP was detected in a broad gradient on both sides of the AP organizer. In the haltere, the distribution of Dpp::GFP is limited in both directions owing to high tkv expression levels, but this restriction is stronger in the P direction. Dpp::GFP spread was abruptly halted a few cell diameters posterior to the haltere AP compartment boundary, contrasting with a tapering signal seen in the anterior direction. By contrast, the Gal4 driver used to express Dpp::GFP (ptc-Gal4) drove nearly symmetrical expression of a UAS-GFP transgene, demonstrating that the distribution of Dpp::GFP in the haltere is not due to asymmetric activity of the ptc-Gal4 driver. In both the wing and haltere, the pattern of extracellular Dpp::GFP was very similar to the P-Mad pattern, suggesting that Ubx does not affect Dpp signal transduction downstream of ligand binding, at least as detected with the anti-P-Mad antibody. Furthermore, in both the wing and haltere, a similar coincidence of extracellular Dpp::GFP and P-Mad patterns was observed when Dpp::GFP was expressed in clones. The correlation between the P-Mad and extracellular Dpp::GFP patterns in both the wing and haltere allows inference of extracelluar Dpp ligand distribution by visualizing P-Mad in the proceeding experiments (Crickmore, 2007).
In the wing, Dpp::GFP distribution and P-Mad staining were also asymmetric, owing to slightly higher levels of Tkv in the P compartment, which impedes diffusion. By contrast, because Tkv levels are similar on both sides of the AP boundary of the haltere, Tkv levels are unlikely to account for the Dpp signaling asymmetry in this appendage. This idea directly by providing uniform levels of UAS-tkv to both the haltere and wing. Under these conditions, P-Mad staining became symmetric in the wing, but remained asymmetric in the haltere. These results suggest that the more-restricted P-Mad staining in the P compartment of the wild-type haltere is due to a tkv-independent and haltere-specific anterior bias in the diffusion of Dpp (Crickmore, 2007).
In previous work, it was showed how the upregulation of the
Dpp receptor, thickveins, in the haltere causes an overall decrease
in Dpp mobility as compared with the wing, and consequently contributes to the
small size of the haltere. This study shows that the HSPG dally is
repressed in the P compartment of the haltere and that this regulation
decreases the P:A ratio and overall size of the haltere. Posterior dally repression causes Dpp diffusion to be biased away from P cells, generating an AP asymmetry in Dpp signaling. The findings reported here therefore provide another instance wherein Ubx controls the extracellular signaling environment of the developing haltere and thereby distinguishes it from the wing (Crickmore, 2007).
The movement of most or all signaling molecules through tissues is
regulated by HSPGs, including glypicans such as dally. In contrast to
receptors, HSPGs control the distribution of multiple signaling molecules.
Regulation of HSPG expression and activity by selector genes is therefore a
potentially very powerful mechanism for shaping signaling pathway activation
profiles and molding organ shapes and sizes. However, the promiscuity of HSPGs
also makes it difficult to assign the morphological consequences of their
expression patterns to the alteration of individual signaling pathways.
Indeed, it is likely that the altered dally expression pattern
in the haltere has implications for Hh, Wg and Dpp signaling,
all of which control growth and patterning. This study has focused on the
relationship between dally expression and Dpp signaling (Crickmore, 2007).
Dpp signaling is increased in dally+ clones and decreased in dally- clones. These and other findings have
suggested that Dally participates in the control of Dpp mobility. The current results
add to these earlier observations by suggesting that variations in the levels
of Dally between the cells of a tissue influence the direction and extent of
Dpp diffusion. Specifically, it is proposed that in addition to simply being
promoted by Dally, Dpp mobility is biased towards cells with higher Dally
levels. This idea derives mainly from the observation that Dally can influence
Dpp movement in a cell-non-autonomous manner. For example, when
Dally levels are increased in the haltere P compartment, there is a shift in
Dpp signaling from the A to the P compartments, as visualized by the levels of
P-Mad. Similarly, knocking down Dally levels in the P compartment of the wing
influences the extent and levels of P-Mad in the A compartment. If
discontinuities in Dally levels can non-autonomously influence Dpp signaling
across compartment borders, it follows that differences in Dally levels
between cells within a compartment can also shape the Dpp signaling landscape.
This might be important for wild-type wing development, where graded Dpp
signaling represses dally, resulting in an inverse dally
gradient that increases towards the lateral edge of the disc. It is suggested that this
inverse dally gradient helps to attract Dpp to more lateral regions
of the disc. Accordingly, in a dally-mutant wing disc, the Dpp
gradient is less broad than in a wild-type wing disc. It is
possible that other HSPGs control the mobility of signaling molecules in a
similar manner (Crickmore, 2007).
Altering dally levels in either the A or P
compartment changes relative compartment size, but only P compartment
dally levels are relevant for total organ size. Two
possible explanations are considered that link the P-specific dally repression seen in the haltere to a reduction in final organ size. Both of these scenarios
(which are not mutually exclusive) focus on the role of P cells in producing
Hh, which diffuses into A cells to instruct Dpp production and, consequently,
controls final organ size. Importantly for both models, it was found that there is
in fact less Hh detected in the P compartment of the wild-type haltere as
compared with the wing. In the
first model, the repression of dally reduces overall Hh production
simply by reducing the size of the P compartment, which is a consequence of
reduced Dpp signaling. In this scenario, fewer Hh-producing P cells result in
less total Hh production from the P compartment, and therefore less Dpp
produced in the A compartment. The logic of this potential mode of size
regulation is interesting: a selector gene (Ubx) restricts growth
factors (Wg and Dpp) from the pool of cells (the P compartment) that produces
another growth factor (Hh). In the second scenario, dally repression
may directly reduce the amount of Hh in the P compartment that can be
transported into the A compartment. In support of this idea, Hh
staining was found to be reduced in clones of cells where Dally levels are reduced
through UAS-dallyRNAi (Crickmore, 2007).
Together, dally and dlp influence the mobility of all
known morphogens in Drosophila. In addition to
the compartmental regulation of dally, it is also noted that Dlp levels
are generally lower throughout the haltere as compared with the wing. The
haltere also lacks the domain of dlp repression seen at the DV
boundary of the wing. Finally, it was also noticed that the expression of Notum-lacZ, an enhancer trap into a gene that encodes an HSPG-modifying enzyme, is
different between the wing and haltere. The
combined alteration of dally, dlp and Notum levels in the
haltere is likely to have consequences for any signaling molecule that uses
HSPGs for transport. When these observations are combined with those of
earlier work showing that the levels of both Dpp and its receptor are
regulated differently in the haltere and wing, and the observation that wg is repressed in the posterior haltere, a picture emerges in which selector
genes alter the expression of multiple components of multiple signaling
pathways to change morphogen signaling landscapes between tissues and thereby
modify organ shapes and sizes. It is hypothesized that the summation of all
signaling pathway changes may be sufficient to understand the size and shape
differences between fundamentally similar epithelia such as the wing and
haltere imaginal discs (Crickmore, 2007).
Hedgehog (Hh) proteins act through both short-range and long-range signaling to pattern tissues during invertebrate and
vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects
are not understood. A new Drosophila gene, named tout-velu and meaning ëall hairí has been identified; it is required for diffusion of Hedgehog. ttv was identified in a screen for maternal-effect mutations associated with segment polarity. ttv clones prove to have a non-cell-autonomous effect. Hh signal is shown to be unable to reach wild-type cells located anterior to ttv mutant cells. It is proposed that ttv functions in the receiving cells for the movement of Hh from sending to receiving cells.
Characterization of tout-velu shows that it encodes an integral membrane protein that belongs to the EXT gene family.
Members of this family are involved in the human multiple exostoses syndrome, which affects bone morphogenesis. Analysis of the Ttv sequence shows that there is a hydrophobic stretch at the amino terminus of the Ttv protein, indicating that Ttv might be a transmembrane protein. Ttv appears to be a type II integral protein, with the C-terminal region displayed extracellularly. These results, together with the previous characterization of the role of Indian Hedgehog in bone morphogenesis, have led to a proposal
that the multiple exostoses syndrome is associated with abnormal diffusion of Hedgehog proteins. These results show the
existence of a new conserved mechanism required for diffusion of Hedgehog. EXT-1 has been found in the extracellular reticulum, where it helps to regulate the synthesis and display of cell-surface heparin sulphate glycosaminoglycans (GAGs). Because GAGs have been implicated in receiving signaling molecules, it is plausible to speculate that Ttv is involved in the synthesis of a GAG that specifically interacts with Hh at the cell surface. Such an interaction might result in the endocytosis of Hh or, alternatively, it may facilitate the movement of Hh from one cell to the next by translocating Hh around the surface of the cell. (Bellaiche, 1998).
The Drosophila genes dally and dally-like encode glypicans, which are heparan sulphate proteoglycans anchored to the cell membrane by a glycosylphosphatidylinositol link. Genetic studies have implicated Dally and Dally-like in Wingless signalling in embryos and imaginal discs. The signalling properties of these molecules in the embryonic epidermis have been tested. RNA interference silencing of dally-like, but not dally, gives a segment polarity phenotype identical to that of null mutations in wingless or hedgehog. Using heterologous expression in embryos, the Hedgehog and Wingless signalling pathways were uncoupled; Dally-like and Dally, separately or together, were found to be unnecessary for Wingless signalling. Dally-like, however, is strictly necessary for Hedgehog signal transduction. Epistatic experiments show that Dally-like is required for the reception of the Hedgehog signal, upstream or at the level of the Patched receptor (Desbordes, 2003).
Although heparan sulphate modifications have been implicated in several signalling pathways, it remains unclear which proteins are modified
by these enzymes, and how the modifications affect a given signalling event.
Since most heparan sulphate chains at the cell surface are thought to be
carried by proteoglycans of the syndecan or glypican families, this study has examined the function of the two Drosophila homologs of glypicans,
dally and dally-like (dlp), in the embryonic epidermis. Unexpectedly, this study has found a restricted and specific role for
the fly glypicans. RNAi silencing shows that Dlp is a segment
polarity gene that is absolutely required for Hh signalling. This requirement
is specific to the Hh pathway; RNAi silencing of dlp
does not affect Wg signalling in embryos. In contrast, RNAi silencing of
dally, the other homolog of glypicans in Drosophila, does
not produce a segment polarity phenotype, suggesting that Dally is dispensable
for Wg or Hh signalling in embryos. Furthermore, RNAi silencing of both
dally and dlp does not affect Wg signalling, suggesting that
they do not function redundantly in this pathway (Desbordes, 2003).
dlp is a bona fide segment polarity gene since dlp RNAi
generates embryos that fail to maintain en and wg expression
at mid-embryogenesis, and exhibit a full segment polarity phenotype in the
cuticle at the end of embryogenesis. The late disappearance of en expression and the single stripe of rho expression in dlp embryos suggest a loss of Hh activity. This is confirmed by the fact that when hh expression is under heterologous control, ectopic wg transcription is lost in dlp RNAi embryos, whether Hh is provided autonomously (armGal4 experiments) or non-autonomously (simGal4 experiments). These experiments demonstrate unambiguously that dlp is required for Hh signalling and rule out a requirement for hh transcription (Desbordes, 2003).
Dlp is a GPI-anchored protein and is likely to be localised at the cell
surface. This leaves two plausible roles for Dlp: either it is required for
the release of active Hh from the secreting cells, or it is required for the
interpretation of the Hh signal on the receiving cells. Several possibilities have been eliminated. First, Dlp is required for the activity of
Hh-N, an engineered form of Hh which is pre-processed and unmodified by
cholesterol. This suggests that Dlp is necessary downstream of Hh processing and cholesterol modification. Downstream of these events, Hh undergoes another lipid
modification, the addition of a palmitoyl moiety. The segment polarity gene
rasp codes for an acyltransferase which is thought to be needed for
Hh palmitoylation. Thus, Dlp could be required for the function of
rasp in the signalling cells. However, whereas palmitolylation is
essential for Hh-N activity, a recent report shows that it is not strictly
required for the activity of wild-type Hh in Drosophila embryos. This
suggests that the cholesterol and palmitoylate modifications might be
partially redundant for the activity of wild-type Hh, at least in embryos.
Thus, although Dlp could still act at the level of rasp on another
function, loss of palmitoylation alone cannot account for the complete loss of
Hh signalling seen in dlp RNAi embryos. It seems therefore more
likely that dlp functions in the responding cells (Desbordes, 2003).
ptc is epistatic to dlp, indicating that Dlp
acts upstream or at the level of the Ptc receptor. One possibility is
that Dlp binds Hh and facilitates its interaction with Ptc. Increasing the
concentration of Hh in receiving cells in either armGal4/UAShh or
armGal4/UAShh-N experiments, does not abolish the requirement for Dlp. This
argues against a role of Dlp in merely increasing the concentration of Hh
ligand at the cell surface, and suggests a more specific role. Recent evidence
supports a model in which, upon Hh binding, Ptc is endocytosed and inactivated
by degradation, and this in turn indirectly activates Smoothened and the Hh
intracellular pathway. Dlp may localize Hh and Ptc in membrane microdomains
required for Ptc endocytosis and subsequent degradation (Desbordes, 2003).
Dpp functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. Evidence is shown that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thickveins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. It is proposed that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient (Fujise, 2003).
To examine the effect of dally mutations on the distribution of Dpp morphogen, Dpp-GFP was expressed in the region where it is endogenously expressed using dpp-GAL4. In wild-type discs, Dpp-GFP is detectable as intracellular punctate spots and on the surface of the receiving cells. Dpp-GFP migrates throughout the wing pouch region, forming a shallow but evident gradient. However, in dally-mutant discs, no evident gradient of Dpp distribution could be detected in the receiving cells. In general, mutant discs showed a lower level of cell surface signals, suggesting reduced stability of Dpp (Fujise, 2003).
To determine whether dally overexpression at the A/P border cells, which causes abnormal patterns of pMad, also affects Dpp ligand gradient formation, Dpp-GFP distribution was observed in discs where dally is co-expressed with Dpp-GFP using dpp-GAL4. Consistent with the pMad patterns, Dpp is restricted to the dally-overexpressing region and fails to migrate properly. This suggests that Dally binds to Dpp protein and limits its distribution. Intensity profiles of these discs show that both reduction of dally and overexpression of dally at the A/P border cells result in a shallower gradient and lower levels of Dpp in the receiving cells. Taken together, Dally regulates formation of both Dpp ligand and activity gradients. In addition, the results strongly suggest that Dally plays at least two roles in the formation of the Dpp signaling gradient: (1) it regulates the sensitivity of cells to Dpp in a cell autonomous fashion; and (2) it affects Dpp protein distribution, which is a non-autonomous effect (Fujise, 2003).
This study demonstrates that dally controls shape of both the ligand and the activity gradients of Dpp in the developing wing. How does dally contribute to the Dpp gradient formation? In vitro analyses using mammalian tissue culture cells have established that HSPGs can increase FGF signaling by stabilizing FGF/FGF receptor complexes Several lines of evidence indicate that the dosage of HSPGs is an important factor for FGF signaling. For example, sodium chlorate treatment, which inhibits the sulfation of heparan sulfate, reduces the biological response of cells to FGF; the response can be restored by an exogenous supplement of heparin. However, restoration is seen only at an optimal concentration of heparin; excess heparin competes for FGF with signaling complex, resulting in a reduction of signaling. In the Drosophila wing, ectopic expression of Dally-like, another glypican related to Dally, leads to a massive accumulation of extracellular Wg protein and compromises Wg signal transduction, suggesting that the glypicans can affect ligand stability and distribution (Fujise, 2003 and references therein).
On the basis of these studies as well as the current data, Dally would appear to have both positive and negative roles on Dpp signaling. In its positive role, Dally serves as a co-receptor for Dpp, stabilizing Dpp protein and enhancing signaling. Conversely, given that Dpp is a heparin-binding protein, Dally may bind Dpp through its heparan sulfate chains and reduce the amount of free Dpp ligands. Thus, Dally affects the Dpp gradient at two distinct steps: signal transduction (autonomous effect) and ligand distribution (non-autonomous effect). A model is proposed in which alterations in the shapes of the Dpp ligand and the activity gradients caused by dally mutations and dally overexpression are interpreted as the sum of these plus and minus effects of Dally function. In this model, Dally normally sequesters Dpp protein to some extent in A/P border cells, where dally levels are very high. Therefore, reduced levels of Dally in mutant discs may result in the release of Dpp ligand and, consequently, higher levels of signaling activity in the central region. Therefore, dally mutations may severely reduce the stability of Dpp protein as well as its signaling activity in the receiving cells. When dally is overexpressed in A/P border cells, Dpp is trapped by binding to excess Dally and fails to distribute properly (Fujise, 2003).
Although it is thought that Dally regulates the diffusion of Dpp, the results do not rule out the possibility that Dally plays a more active role in facilitating Dpp diffusion or 'carries' Dpp protein. For example, it is possible that Dally is required for the Dpp movement through the transcytosis pathway or other transport systems, such as cytonemes (Fujise, 2003).
The Drosophila transforming growth factor ß homolog Dpp acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing. Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, it was found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. A blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). These results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly (Belenkaya, 2004).
One new observation in this work is that the extracellular Dpp is broadly distributed in the wing disc. Consistent with these findings, previous biochemical analysis demonstrated that the majority of mature Dpp signaling molecules are extracellular. Importantly, the overall shape of the extracellular Dpp gradient coincides well with its activity gradient, suggesting that the extracellular Dpp gradient contributes to Dpp activity gradient in the wing disc. The observation of broadly distributed extracellular Dpp led to a re-examination of the role of Dynamin-mediated endocytosis in Dpp movement and signaling. These analyses argue that Dynamin-mediated endocytosis is not essential for Dpp movement: (1) both Dpp signaling activity and extracellular GFP-Dpp levels are not reduced in the wild-type cells behind the shits1 clones that are defective in endocytosis; (2) the extracellular GFP-Dpp is also broadly distributed in endocytosis-defective wing discs homozygous for shits1 at nonpermissive temperature. These data demonstrate that Dpp molecules are able to move across Dynamin-defective cells. Finally, it was found that extracellular Dpp accumulates on the cell surface of shits1 mutant clones, suggesting that Dpp is able to move into shits1 mutant cells and that Dynamin-mediated endocytosis is normally involved in downregulating levels of the extracellular Dpp. No accumulation of extracellular Dpp on wild-type cells was observed in front of shits1 mutant clones; this would be expected if endocytosis were required for Dpp movement (Belenkaya, 2004).
While Dynamin-mediated endocytosis does not appear to be critical for Dpp movement, Dpp signaling activity is reduced cell autonomously in shits1 mutant cells. This result argues that Dynamin-mediated endocytosis is an essential process for Dpp signaling. Studies in mammalian cell culture system have demonstrated the critical role of Dynamin-mediated internalization of activated TGF-β receptors in TGF-β signaling. SARA (Smad anchor for receptor activation), a FYVE finger protein enriched in early endosomes is involved in this process. Although the exact mechanism of endocytosis-mediated TGF-β signaling is still unclear, current data suggest a role of early endosomes as a signaling center for TGF-β. Consistent with this view, it has been shown that ectopic expression of the dominant-negative form of Rab5 (DRab5S43N) using engrailed-Gal4 leads to a reduction of Dpp signaling, while overexpression of Rab5 broadens the Dpp signaling. Rab5 localizes in early endosomes and is required for endosome fusion. Taken together, it is proposed that dynamin-mediated endocytosis is not directly involved in Dpp movement but is essential for Dpp signaling. Furthermore, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient (Belenkaya, 2004).
To investigate the role of HSPGs in Dpp morphogen gradient formation, Dpp signaling and its extracellular distribution was examined in sulfateless (sfl) and dally-dly mutant clones. dally and dly are shown to be required and partially redundant in Dpp signaling and movement in the wing disc. Two lines of evidence support the role of Dally and Dly in Dpp movement across cells: (1) Dpp signaling activity is reduced in cells behind sfl or dally-dly mutant clones; (2) extracellular Dpp levels are diminished in cells behind sfl or dally-dly mutant clones. Importantly, it was found that sfl or dally-dly mutant clones only a few cells wide can effectively block GFP-Dpp movement, suggesting that Dpp movement does not occur through 'free diffusion', by which extracellular Dpp would be expected to move across sfl or dally-dly mutant cells. Based on these observations, it is proposed that Dpp moves from cell to cell along the epithelium sheet through restricted diffusion involving Dally and Dly (Belenkaya, 2004).
If the HSPGs Dally and Dly are indeed involved in Dpp movement, observation of extracellular GFP-Dpp accumulation in front of sfl, dally-dly mutant clones would be expected. Indeed, extracellular GFP-Dpp accumulation is visible in front of sfl or dally-dly mutant clones. Consistent with this observation, Hh has been observed to accumulate abnormally in clones mutant for tout-velu (ttv) and brother of tout-velu (botv), two Drosophila EXT members involved in HS GAG chain biosynthesis. Both Wg and Dpp accumulation in front of ttv-botv clones are also observed, albeit less pronounced, compared with the case of Hh. Similarly, extracellular GFP-Dpp accumulation is relatively weak, compared with Hh accumulation. One possibility is that extracellular Dpp molecules bound by Dally and Dly in wild-type cells can still be internalized by adjacent sfl or dally-dly mutant cells through cell-cell contact, leading to a reduction of extracellular Dpp accumulation in front of sfl or dally-dly mutant cells. Consistent with this view, it was noticed that, within sfl or dally-dly mutant clones, the first row of the mutant cells immediately adjacent to wild-type cells and facing Dpp-expressing cells is still capable of transducing Dpp signaling (Belenkaya, 2004).
In addition to being required for Dpp movement, Dally and Dly are also essential for Dpp signaling in its receiving cells. Dpp signaling is reduced in sfl or dally-dly mutant cells. Reduced levels of extracellular Dpp were observed in sfl or dally-dly mutant clones. Consistent with the results in this work, clones mutant for ttv or botv as well as sister of tout-velu (sotv), members of Drosophila EXT, led to reductions in Dpp signaling and its ligand distribution when analyzed by a conventional staining protocol that revealed both extracellular and intracellular Dpp. Collectively, these data suggest that the main function of Dally and Dly in Dpp signaling is to maintain and/or concentrate the extracellular Dpp available for Dpp receptors (Belenkaya, 2004).
This study has shown that Dynamin-mediated endocytosis is not essential for Dpp movement. Dpp movement is through a cell-to-cell mechanism involving the HSPGs Dally and Dly. On the basis of these findings, it is proposed that secreted Dpp molecules in the A-P border are immediately captured by the GAG chains of Dally and Dly on the cell surface located in either the A or P compartments. The differential concentration of Dpp on the cell surface of producing cells and receiving cells drives the displacement of Dpp from one GAG chain to another toward more distant receiving cells. Alternatively, Dpp molecules bound by Dally or Dly could also move along the cell surface through a GPI linkage that is inserted in the outlet leaflet of the plasma membrane and is enriched in raft domains. In the receiving cells, Dally and Dly may present Dpp to its receptor, Tkv, that transduces Dpp signal through the Dynamin-mediated internalization process, which further downregulates extracellular Dpp levels and cell surface Tkv. Based on this model, extracellular Dpp and its receptor, Tkv, would be accumulated on the surface of Dynamin-deficient cells, and extracellular Dpp would be able to move across Dynamin-deficient cells to reach more distal cells. In sfl or dally-dly mutant clones, extracellular Dpp molecules can not be attached on the cell surface and therefore can not be transferred further to more distal cells. In this model, endocytosis is not directly involved in Dpp movement; however, through receptor-mediated internalization, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient. It remains to be determined how Dpp is transferred from one cell to another by the GAG chains of Dally and Dly and whether Dally and Dly play a role in preventing extracellular Dpp from degradation. Further studies are needed to determine whether other mechanisms are also involved in Dpp movement (Belenkaya, 2004).
During the wing development Wingless acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. This study systematically analyzes the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. Both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. Surprisingly, although Fz2 has been suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which mainly result from upregulated Dlp levels. It is further shown that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, it is demonstrated that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. It is proposed that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc (Han, 2005).
One important finding in this work is that Dally and Dlp are required for Wg gradient formation. Several recent studies have shown that extracellular Wg distribution is compromised in clones mutant for HS biosynthesis enzymes, including sfl, slalom and members of the Drosophila EXT gene. However, it is unclear which HSPG cores are involved in this process. This study shows that Wg morphogen distribution is defective in either dally or dlp mutant clones. These new findings clearly establish the requirement of Dally and Dlp in Wg morphogen gradient formation. Thus, as in the case of Hh and Dpp, the glypican members Dally and Dlp, rather than Drosophila syndecan or perlecan, are the main HSPGs involved in Wg gradient formation (Han, 2005).
Interestingly, Dally and Dlp differentially regulate the Wg extracellular gradient in distinct regions of the wing disc. Both Dally and Dlp are glypican members of HSPG family. One would expect that differences in the structure of Dally and Dlp, and their attached HS GAG chains may determine their abilities to interact with Wg, thereby leading to their specificities. This is probably one of the factors, since overexpression of Dally and Dlp has very different effects on extracellular Wg gradient. Consistent with the data in this work, previous studies have shown that Dlp is much more potent in accumulating Wg protein than Dally when overexpressed. However, the regional effects of Dally and Dlp on extracellular Wg gradient correspond well to their expression patterns. The regions with higher expression levels of Dally or Dlp have stronger extracellular Wg defects when Dally or Dlp is removed, respectively. Based on these data, it is suggested that the differential roles of Dally and Dlp in extracellular Wg distribution are at least partially determined by their restricted expression (Han, 2005).
What exact roles do Dally and Dlp play in shaping the extracellular Wg gradient? Loss-of-function results suggest that removal of Dally or Dlp leads to reduced extracellular Wg levels on the cell membrane. Furthermore, extracellular Wg levels are reduced in wild-type cells behind sfl or dally-dlp clones. These data suggest that the primary function of Dally and Dlp in Wg gradient formation is to maintain extracellular Wg proteins so that locally concentrated Wg proteins can further move to more distal cells through diffusion (Han, 2005).
Despite a positive role of Dlp in extracellular Wg distribution, surprisingly, Dlp negatively regulates Wg signaling at the DV boundary. However, ectopic Wg signaling at the DV boundary of the dlp mutant is not as great as expected. This relatively weak effect is most probably due to the low level expression of Dlp, which is downregulated by Wg signaling. The results are consistent with a previous observation that overexpression of Dlp in the wing disc leads to a blockage of Wg signaling. Dlp may compete with Fz proteins for available Wg protein at the DV boundary, thereby inhibiting Wg signaling. However, the extract mechanism of Dlp-mediated Wg inhibition needs to be further determined (Han, 2005).
Previous studies have identified Notum as a secreted inhibitor for Wg signaling. Notum is expressed at the DV boundary and has been proposed to downregulate Wg signaling by modulating Dlp activity (Giraldez, 2002). Kreuger (2004) and Selleck (Kirkpatrick, 2004) proposed that Notum negatively regulates Wg signaling by shedding of Dlp, which converts Dlp from a membrane-tethered co-receptor to a secreted antagonist. Their conclusions are mainly based on two lines of experimental data: (1) biochemical experiments clearly demonstrated that Notum can modify Dlp in a manner that resembles cleavage of the GPI anchor (Kreuger, 2004); (2) Kirkpatrick (2004) showed that transheterozygous dlp/notum flies produced ectopic mechanosensory bristles which are not seen in dlp+/- or notum+/- alone, indicating that Dlp and Notum genetically collaborate in downregulating Wg signaling (Han, 2005).
However, on the basis of the current data, it is suggested that Notum inhibits Wg signaling mainly by modifying Dally in the wing disc. (1) Genetic interaction data shown by Kirkpatrick (2004) cannot distinguish whether Dlp and Notum work in the same pathway or in two independent pathways to downregulate Wg signaling at the DV boundary (Kirkpatrick, 2004). If Dlp is indeed the main substrate for Notum, it would be expected that ectopic Wg signaling activity in dlp-notum should be similar to that in dlp mutant. However, loss-of-function analysis demonstrates that ectopic Wg signaling in dlp-notum is similar to that in notum mutant, but much stronger than that in dlp mutant. However, dally-notum clones exhibit loss of Wg signaling activity that is similar to dally mutant. (2) Dlp expression is strikingly repressed by Wg signaling and this reduction is independent of Notum. Low/absent expression of Dlp is not consistent with the view that Dlp is the main substrate for Notum. (3) It is important to mention that Notum can reduce the amount of Dally when they are co-expressed in Drosophila S2 cells (Giraldez, 2002), suggesting that Notum can modify Dally as well. Although Notum can shed Dlp, whether shed Dlp acts as a Wg inhibitor needs to be further determined. Therefore, further experiments are necessary to define the mechanism(s) of Notum-mediated Wg inhibition (Han, 2005).
One important finding of this study is that removal of the Wg receptors (Fz and Fz2) and the co-receptor Arr does not lead to a loss of extracellular Wg. Fz2 has been proposed to play a major role in Wg gradient formation in the wing disc by ectopic expression studies. Although the high capacity of Fz2 in stabilizing Wg has been demonstrated, loss-of-function results clearly show that extracellular Wg levels are not reduced in clones mutant for fz2. This is apparently not due to the overlapping function of Fz, since the extracellular Wg level is enhanced, rather than reduced, in the absence of both Fz and Fz2 functions. The results argue that Fz2 is not essential for extracellular Wg gradient formation in vivo. It is important to note that in addition to Fz and Fz2, Fz3 is also expressed in the wing disc and its expression is upregulated by Wg signaling. Although Fz3 has lower affinity than Fz2 in Wg binding and acts as an attenuator of Wg signaling, its role in Wg distribution needs to be determined (Han, 2005).
It is further demonstrated that extracellular Wg is enhanced in cells mutant for fz-fz2 or arr, suggesting that Wg receptors (Fz and Fz2) and Arr shape extracellular Wg gradient by downregulating extracellular Wg levels. The data argue that this mainly results from upregulation of Dlp. Consistent with this view, the accumulated extracellular Wg can be eliminated by loss of HSPGs in sfl-fz-fz2 or arr-botv mutant clones. Importantly, it is shown that both extracellular Wg and Dlp levels are upregulated on the cell surface of clones mutant for dsh. These data provide compelling evidence that though a feedback mechanism, Wg signaling can control the Dlp levels to regulate the extracellular Wg gradient (Han, 2005).
Another alternative possibility is that enhanced Wg levels in fz-fz2 or arr clones may be caused by impaired Wg internalization. Although a significant amount of internalized Wg vesicles has been demonstrated in fz-fz2 or arr mutant clones, this possibility cannot be ruled out, since a quantitative comparison of Wg internalization between wild-type cells and fz-fz2 or arr mutant cells is difficult. Furthermore, as mentioned above, Fz3 is expressed in the wing disc and its expression is upregulated by Wg signaling. It is possible that Fz3 may mediate the internalization of Wg in the absence of Fz and Fz2 (Han, 2005).
Evidence has been presented that Wg morphogen movement is regulated by a diffusion mechanism(s) in the wing disc. Does Wg diffuse freely in the extracellular matrix/space? In this work, it is shown that Wg fails to move across a strip of cells mutant for the HSPGs Dally and Dlp. This result suggests that Wg cannot freely diffuse in the extracellular matrix. Instead, the findings are consistent with a model in which Wg movement is mediated by the HSPGs Dally and Dlp through a restricted diffusion along the cell surface. Similar mechanisms have been proposed for Hh and Dpp. In biological systems such as imaginal discs, the restraint of Wg spreading to the surface of the epithelial cell layer is important since the folding of imaginal discs, such as the leg disc, poses a problem if the Wg gradient formation were to occur out of the plane of the epithelial cell layer through free diffusion. In agreement with this view, the model proposes that Wg gradient formation depends on Wg movement through the cell surface of the disc epithelium (Han, 2005).
Controlling the spread of morphogens is crucial for pattern formation during development. In the Drosophila wing disc, Wingless secreted at the dorsal-ventral compartment boundary forms a concentration gradient in receiving tissue, where it activates short- and long-range target genes. The glypican Dally-like promotes Wingless spreading by unknown mechanisms, while Dynamin-dependent endocytosis is thought to restrict Wingless spread. Short-term expression of dominant negative Rab proteins was used to examine the polarity of endocytic trafficking of Wingless and its receptors and to determine the relative contributions of endocytosis, degradation and recycling to the establishment of the Wingless gradient. The results show that Wingless is internalized via two spatially distinct routes: one on the apical, and one on the basal, side of the disc. Both restrict the spread of Wingless, with little contribution from subsequent degradation or recycling. As has been shown for Frizzled receptors, depleting Arrow does not prevent Wingless from entering endosomes. Both Frizzled and Arrow are internalized mainly from the apical membrane. Thus, the basal Wingless internalization route must be independent of these proteins. Dally-like is not required for Wingless spread when endocytosis is blocked, and it is proposed that Dally-like promotes the spread of Wingless by directing it to lateral membranes, where its endocytosis is less efficient. Thus, subcellular localization of Wingless along the apical-basal axis of receiving cells may be instrumental in shaping the Wingless gradient (Marois, 2006).
The data suggest that the spread of Wg is controlled by restrictive clearance. Preventing Rab5-dependent internalization increases the spread of Wg through disc tissue. While this may also elevate extracellular Wg levels by indirect mechanisms (for example by modulating extracellular Wg proteolysis or release from disc tissue), the possibility is favored that the gradient is shaped by internalization of Wg itself, for several reasons. First, Wg is actually found in Rab5- and Rab7-positive endosomes. Second, treating living discs with protease inhibitors does not cause Wg to accumulate. Finally, differential centrifugation experiments suggest that only about 6% of Wg is not membrane-associated (Marois, 2006).
Some ligands signal from endocytic compartments after internalization. Therefore, it was initially of interest to discover whether the high levels of Wg accumulating after dominant negative Rab5SN expression could increase signal transduction. While Rab5SN-expressing cells both accumulate Armadillo and reduce Senseless expression, whether internalization of Wg is required for signaling was not further investigated because of the striking and unexpected transcriptional changes caused by blocking Rab5 activity. For example, transcription of both Fz2 and Dlp increases and that of Arrow plummets within a few hours of initiating Rab5SN expression - any of these changes by themselves could alter Wg signaling. Although this phenomenon are not yet understood, one might imagine that coupling transcriptional regulation of endocytic receptors to their actual endocytosis and/or degradation would be an effective homeostatic mechanism (Marois, 2006).
Studies in tissue culture cells have shown that inhibiting Rab7-dependent lysosomal degradation can increase recycling of some proteins to the cell surface. In imaginal discs, Rab7TN expression increases the abundance of Rab11 recycling endosomes, and inhibiting recycling via Rab11SN enlarges the Rab7-positive degradative compartment. Thus, the recycling and degradative pathways may compete for some cargo in discs as they do in cultured cells. This raises the possibility that changing the balance of degradation and recycling might affect the pool of extracellular Wingless available for spreading. Indeed, Wg appears to be recycled in embryos, and inhibiting lysosomal degradation in the embryonic ectoderm extends its range. However, the data suggest that the increase in the range of Wg caused by inhibiting degradation (at least in imaginal discs) is not the result of increased recycling and extracellular spread. Although Wg protein is detected in endosomes over a broader range in imaginal disc tissue expressing Rab7TN, there is no increase in the range of extracellular Wg. Furthermore, neither extracellular nor intracellular Wg distribution is affected by inhibiting the Rab4- or Rab11-dependent recycling pathways. Inhibiting degradation probably extends the apparent range of Wg by stabilizing internalized protein, raising its levels above the threshold of detectability in more distant cells (Marois, 2006).
The idea that apical-basal polarity of epithelial cells might play a role in regulating morphogen trafficking has been suggested by the observation that wg mRNA is enriched apically in the embryonic ectoderm. Changing mRNA localization alters the distribution of Wg protein in receiving tissue, raising the intriguing possibility that Wg might be trafficked differently depending on whether it is secreted apically or basal-laterally. In support of this idea, the data show that Wingless is internalized specifically from the apical and basal (but not lateral) surfaces of the disc epithelium. Indeed, the distribution of Rab5- and Rab7-positive endosomes in general suggests that the apical and basal surfaces are more endocytically active than other regions. The apical and basal internalization mechanisms may be distinct; the known receptors for Wingless, Fz2 and Arrow are internalized mainly from the apical surface (despite their steady-state basal-lateral localization), suggesting that basal Wingless endocytosis must be independent of these proteins. One possibility is that membrane association of Wg via Palmitate is sufficient to allow its endocytosis -- perhaps by mechanisms similar to those used by gpi-linked proteins. Alternatively, Wg bound to Lipoprotein particles might be internalized via Lipoprotein receptors (Marois, 2006).
It was surprising to observe that the Wg accumulating on the basal side of disc epithelial cells after Rab5SN expression does not spread onto the lateral membrane, since no barrier to diffusion has been identified between these domains. Three possible explanations are suggested. (1) There may indeed be a 'fence' separating the lateral and basal sides of disc epithelial cells. Neurons have a fence that prevents diffusion of lipid and lipid-linked proteins between the axon and the cell body, although it does not resemble a classical intercellular junction. (2) It may be that the receptor(s) that normally internalize Wg basally are linked to cytoskeletal components, or interact with extracellular matrix (ECM), and are not free to diffuse. If these Wg receptors were of sufficiently high affinity, they might trap Wg before it could move laterally. (3) Perhaps Wg itself interacts efficiently with basal ECM components (Marois, 2006).
While it seems that endocytosis restricts the spread of Wg on the apical and basal surfaces, it is not yet clear which receptors might be responsible. A simple model would predict that removing such a receptor should produce phenotypes similar to Rab5SN expression, i.e. increased and more extensive extracellular Wg, and less Wg in endosomes. Conversely, overexpression might be expected to compress the range of Wg distribution and decrease extracellular Wg. None of the known receptors behaves in this way. Previous studies showed that at least a fraction of Wg is still internalized in the absence of both Fz1 and Fz2. Furthermore, overexpression of Fz2 causes extracellular Wg accumulation over longer distances. This study has shown that loss of Arrow actually increases the amount of Wg present in Rab5- and Rab7-positive endosomes - more consistent with a role in Wg degradation after endocytosis. The complexity of these observations may reflect different mechanisms of Wg endocytosis on the apical and basal sides of the cell - understanding both these pathways and their interplay will be necessary to understanding how the Wg gradient forms (Marois, 2006).
While internalization limits the range of Wg accumulation, the glypican Dlp extends it. It has been proposed that Dlp allows Wg to interact with disc cells, increasing local Wg concentration and restricting its diffusion to the epithelium. The data, however, show that disc cells can accumulate high levels of Wg on their surface in the absence of Dlp as long as Rab5-dependent internalization is blocked. This observation is not consistent with a model in which Dlp traps Wg on the cell surface or helps it transfer from cell to cell. Instead, it suggests that Dlp normally stabilizes Wg at the cell surface by antagonizing the effects of Rab5-dependent internalization. While Wg is normally internalized from the apical- and basal-most surfaces of disc cells, Dlp overexpression recruits Wg to the lateral cell surface. This raises the possibility that Dlp stabilizes Wg and increases its range by changing its subcellular localization to protect it from endocytosis. Polarizing the distribution of morphogens within an epithelium may have a key regulatory role in the trafficking events leading to gradient formation (Marois, 2006).
The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. The Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function (Johnson, 2006).
Converging lines of evidence suggest that membrane-associated HSPGs serve an important purpose in the assembly, function and plasticity of excitatory synapses. The ancient HSPG families of syndecans and glypicans are necessary for Drosophila to regulate distinct aspects of synaptic morphogenesis. Genetic and biochemical data indicate that Sdc and Dlp interact with LAR to control presynaptic properties. Because LAR-family RPTPs have been shown to control the formation of excitatory synapses in Drosophila, C. elegans, and mammals, these findings may represent a more general mechanism for regulating synaptic morphogenesis and function. Despite the importance of LAR-family RPTPs during cellular morphogenesis inside and outside of the nervous system, the lack of physiologically relevant extracellular binding partners has made it challenging to study this well-conserved group of receptors (Johnson, 2006).
The data show that Sdc promotes the formation of presynaptic boutons. This Sdc function appears to be mediated by LAR, as supported by parallel phenotypes, direct binding, in vivo colocalization, and three types of double mutant analysis between Sdc and LAR. Despite the fact that Sdc exhibits gain-of-function activity and endogenous expression on both sides of the synapse, neuronal and muscle-specific rescue experiments show that Sdc function is mainly presynaptic. Because LAR is required only in neurons to promote synapse growth, these findings support a model in which Sdc acts as a neuronal cell-autonomous agonist of LAR. This is somewhat surprising, given the fact that soluble forms of Sdc bind to LAR and that endogenous Sdc appears to be released from the presynaptic membrane to fill the subsynaptic reticulum. One way for Sdc to act presynaptically would be to bind to LAR even before the two proteins are presented on the neuronal surface. Because Dlp has a competitive advantage over Sdc for binding to LAR, a prebound complex of Sdc and LAR would have the ability to stimulate synapse growth before the phosphatase could be inhibited by Dlp. Such a mechanism could provide a time- and/or HSPG concentration-dependent switch from bouton addition to active zone assembly (Johnson, 2006).
Sdc could promote LAR activity in collaboration with an additional cell-type-specific membrane protein. Data from a parallel study has also identified Sdc-LAR interactions during embryonic motor axon guidance. However, in contrast to CNS pathfinding, complete loss of Sdc alone has no significant effect on motor pathfinding, suggesting that additional LAR ligands exist in the early embryo. Recent experiments with the vertebrate LAR ortholog PTP-s suggest that non-HSPG ligands may regulate its ability to promote retinal axon outgrowth. Although it remains a formal possibility, an additional ligand may not be needed to account for the NMJ growth-promoting activity of LAR because the larval synaptic phenotype in Sdc mutants is nearly as strong as the growth defect in LAR mutants (Johnson, 2006).
Active zone assembly is vital for neurotransmission at the synapse and has been proposed as a means to modulate synaptic function over time. Analysis of Dlp reveals that synaptic glypicans are required to regulate active zone morphology and function. Moreover, Dlp is limiting for active zone morphogenesis, consistent with an instructive role. Because the activities of Dlp appear opposite to those of LAR, it is proposed that the high affinity binding of Dlp to LAR induces an inhibition of receptor function. This hypothesis is supported by the double RNAi experiments showing that the LAR effect on Ena phosphorylation is epistatic to the effect of Dlp, indicating that Dlp acts upstream of LAR. Because loss of Dlp at the NMJ did not induce a significant change in the number of presynaptic boutons, the results lead to a model in which Dlp is specialized for control of active zone properties. Such a function might provide a means to independently regulate and spatially distinguish LAR inhibition from LAR activation. In any case, the presence of active zone phenotypes in dlp but not in Sdc reveals specialization among synaptic HSPGs (Johnson, 2006).
LAR regulates both NMJ growth and active zone morphogenesis. Thus, LAR appears to provide a link between two important synaptic properties that are regulated by different extracellular factors. A mechanism to couple bouton growth and active zone formation would make sense because active zones appear early in the nascent bouton. Because LAR catalytic activity is necessary for bouton addition, and yet LAR inhibition by Dlp appears necessary for proper active zone formation, LAR's role at the active zone may be primarily structural. For example, LAR may simply provide an anchorage point for synaptic components like the scaffolding protein Liprin-alpha that regulates active zone formation. Alternatively, LAR may exist in distinct yet active signaling states, one of which is dependent on PTP activity (promoting synapse growth), and one of which is dependent on recruitment of signaling molecules (controlling active zone assembly). Because loss of Dlp or LAR has opposite effects on quantal content at the NMJ, it is attractive to speculate that the Dlp-LAR pathway normally provides a means to modulate the strength of neurotransmission, either during NMJ growth or during synaptic plasticity. LAR PTPs are required for normal physiology and plasticity at mammalian hippocampal synapses (Johnson, 2006).
Sdc and Dlp are both HSPGs that bind to LAR and thus might be expected to act similarly, but the results show that their functions are different. One way to account for the specificity might be a difference in the effect of soluble versus cell-surface HSPGs on LAR. Some ligand molecules such as Ephrins function when clustered at high density (e.g., on a membrane surface) but fail to activate their receptors when presented in a soluble, monomeric form. Another possibility could be that LAR binding or signaling is differentially influenced by direct protein-protein interactions with the Sdc versus Dlp core proteins. The two HSPGs have very different core structures; Sdc is a transmembrane molecule with HS modification sites near the N terminus, whereas Dlp is a GPI-anchored protein with HS sites proximal to the membrane and a large disulphide bonded globular domain located more distally. It may also be relevant that Dlp consistently binds more effectively to LAR than Sdc, with KD measurements in solution showing an affinity approximately 2-fold higher. These results suggest a competition model in which Dlp displaces Sdc, possibly to favor the stabilization of active zones after new growth at the synapse. In this model, presynaptic growth would be initially promoted by Sdc and would then be limited or halted by Dlp binding after formation of close membrane contact between the nerve and muscle. Such a mechanism could insure a transition from growth to synapse stabilization and could participate in subsequent maintenance or plasticity of the synapse (Johnson, 2006).
Of course other molecules influence synapse size, and these might include coligands or coreceptors that may bind to Sdc, Dlp, and/or LAR. Potential candidates might include bone morphogenic protein (BMP), the type II BMP receptor Wishful thinking (Wit), or the Wnt ortholog Wingless (Wg), which have all been shown to regulate NMJ morphology in Drosophila. However, in addition to significant phenotypic differences compared to the HSPGs, overexpression studies indicate that neither BMP nor Wg are limiting for NMJ morphogenesis. In contrast, Sdc and Dlp are both limiting for different aspects of synapse development, consistent with an instructive role in this context. Consistent with this notion, Syndecan-2 is sufficient to promote dendritic spine maturation during hippocampal synaptogenesis in culture. Although vertebrate Syndecan-2 has yet to be tested at the synapse by loss of function, the colocalization and parallel biology of Synecan-2 and vertebrate LAR-family receptors strongly suggest conservation in the regulation of synaptic LAR (Johnson, 2006).
The genetic and biochemical studies described in this study have identified a partnership between HSPGs and LAR in Drosophila NMJ development that sets precedents for (1) the in vivo requirement for members of the syndecan and glypican families in synapse growth and electrophysiological function, (2) the specificity of HSPG function at the synapse, with distinct actions of Sdc and Dlp, and (3) biochemical identification of Sdc and Dlp as LAR binding partners, plus genetic evidence to place them in a pathway regulating biological function at the synapse (Johnson, 2006).
Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, this study characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. It was found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts (Kamimura, 2006; full text of article).
It was asked whether FGF signaling is impaired in mutant animals using an antibody that specifically recognizes the diphosphorylated form of MAP kinase (dpMAPK). In wild-type embryos, dpMAPK is detected in the tracheal placodes at stage 10, reflecting activation of Egfr. This dpMAPK signal was not diminished in the Hs2st; Hs6st embryos, showing that Egfr signaling is not affected by the double mutations. At stage 12, wild-type embryos show a strong dpMAPK signal in the migrating tip cells of each primary branch due to activation of FGF signaling. In contrast, the btl-dependent MAPK activation in the tip cells is disrupted in the Hs2st; Hs6st embryos. In situ RNA hybridization experiments revealed that bnl expression is not altered in the double mutant embryos, confirming that the branching defects observed in the double mutants are caused by disruption of FGF reception but not FGF expression. These results showed that HS with neither 2-O nor 6-O sulfate groups lost the ability to mediate Btl signaling (Kimimura, 2006).
The Drosophila lipoprotein particle, Lipophorin (Retinoid- and fatty acid-binding glycoprotein), bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Heparan sulfate proteoglycans are also critical for trafficking and signaling of these morphogens. Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like. Membrane-associated glypicans can recruit Lipophorin to disc tissue, and remain associated with these particles after they are released from the membrane by cleavage of their gpi anchors. The released form of Dally colocalizes with Patched, Hedgehog, and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting its distribution. These data suggest that heparan sulfate proteoglycans may influence lipid-linked morphogen signaling, at least in part, by binding to Lipophorin. They further suggest that the complement of proteins present on lipoprotein particles can regulate the activity of morphogens (Eugster, 2007).
Controlling the spread and signaling of secreted morphogens is of critical importance for pattern formation during development. Morphogens of the Wnt and Hedgehog families undergo covalent lipid modification; these lipid moieties are essential for normal trafficking and signaling. It has been shown that the cell biological mechanisms they influence are not completely understood. The lipid-linked morphogens Wingless (Wg) and Hedgehog (Hh) can be released from the plasma membrane on the Drosophila lipoprotein Lipophorin, and Lipophorin is important for their long-range but not short-range signaling activity (Panáková, 2005). How might Lipophorin association influence morphogen signaling activity? One function of Lipophorin may be to mobilize otherwise membrane-bound molecules for long-range movement. Furthermore, a variety of gpi-linked proteins are also found on these particles, raising the possibility that morphogen signaling could be subject to additional regulation by other particle-associated proteins (Eugster, 2007).
Heparan sulfate proteoglycans (HSPGs) are also essential for normal trafficking and signaling of morphogens. Sterol-modified Hh cannot accumulate in or move through tissue that does not synthesize heparan sulfate (HS). In contrast, truncated Hh that cannot be sterol modified spreads freely through tissue missing HS. The effects of HS on the Hh pathway are mediated at least in part by the glypicans Dally and Dally-like (Dlp). Whereas Dlp is required for Hh signaling in S2 cells, it appears to act redundantly with Dally in imaginal discs. Only clones of cells mutant for both dally and dlp show autonomous reduction in Hh signaling (Eugster, 2007).
While autonomous functions of Dally and Dlp are presumably exerted by membrane-linked forms of the proteins, Dlp can also be shed from the plasma membrane. Notum, an enzyme in the α/β hydrolase family, removes the gpi anchor from Dlp expressed in S2 cells, releasing the protein into the supernatant. Whether Dally can be detached from its gpi anchor by Notum or other enzymes is unresolved. Some gpi-anchored proteins are released from cell membranes by associating with lipoprotein via their lipid anchors (Panáková, 2005), suggesting yet another mechanism for glypican shedding. It is not yet clear how and to what extent shedding occurs in vivo. Furthermore, specific roles for shed glypicans would be difficult to detect genetically because mutant clones have access to released glypicans produced by wild-type tissue (Eugster, 2007).
This study examined the relationship between glypicans and Lipophorin in the Hh pathway. Lipophorin was found to bind to the HS moieties of glypicans and can be recruited to disc tissue by these proteins. Lipophorin remains associated with glypicans when they are released from the plasma membrane by gpi removal. The released form of Dally is found in endosomes containing Lipophorin, Hh, and the Hh receptor Patched (Ptc) and increases Hh signaling efficiency (Eugster, 2007).
One consequence of Lipophorin particles binding to the heparan sulfate moieties of glypicans is that membrane-associated Dally and Dlp can recruit Lipophorin particles to imaginal disc tissue. Indeed, HSPGs are autonomously required for Lipophorin's interaction with the basal side of the disc epithelium. This raises the possibility that HSPGs may recruit lipid-linked morphogens to receiving cells, at least in part, by binding to morphogen-bearing lipoprotein particles. Although Hh is present in both apical and basal pools in the wing disc, it accumulates much more strongly on the basal surface when endocytosis is blocked. This suggests that the majority of Hh protein may spread along the basal surface. ttv, botv mutant cells that cannot recruit Lipophorin basally may be compromised in their ability to interact with Hh-bearing Lipophorin particles. This idea is also consistent with the observation that Hh mutants that cannot be sterol modified (and are presumably not associated with lipoproteins) no longer depend on HSPGs to bind to receiving cells (Eugster, 2007).
These data suggest that Lipophorin must be transcytosed from the basal side of the disc to the apical lumen. Although membrane-associated Dally and Dlp are found on the basolateral membrane, removal of the gpi anchor results in apical secretion. It will be interesting to investigate whether glypican endocytosis and subsequent release might play a role in Lipophorin transcytosis. The presence of transcytosed particles in the disc lumen may explain why removal of HS from clones of cells only disrupts basal Lipophorin accumulation. Particles transcytosed by other cells would be available for apical internalization by HS-independent mechanisms (Eugster, 2007).
Dally and Dlp continue to associate with Lipophorin via HS moieties after cleavage of the gpi anchor. The fact that both glypicans remain bound to Lipophorin after shedding raises the possibility that released glypicans influence signaling from lipoprotein particles. This analysis of the role of released Dally supports this idea (Eugster, 2007).
The data indicate that released Dally is required for full-strength Hh signaling, but does not affect the range over which Hh spreads. Hh signaling promotes Ci-dependent target gene activation by three separable mechanisms that are differentially sensitive to Hh levels. Ci stabilization requires the lowest levels of Hh signaling, and thus occurs over the broadest range. Ci stabilization is insufficient for target gene activation, however. To activate transcription, full-length Ci must be released with Suppressor of Fused (Su[Fu]) from cytoplasmic complexes containing Fused and Costal-2 and translocate to the nucleus. This occurs over shorter distances than Ci stabilization. Still higher levels of Hh signaling induce other processes needed for full activity of the Ci-Su(Fu) heterodimer within the nucleus. These are less well understood, but may involve phosphorylation of Su(Fu). In dally mutant discs, Ci stabilization and nuclear translocation, which require lower levels of Hh signaling, are normal. Only Ci activation is impaired by loss of dally. Thus, released Dally appears to increase the quantitative output of the Hh signaling pathway without increasing the amount of Hh (Eugster, 2007).
Although the possibility cannot be ruled out that a small, non-particle-associated fraction of released Dally gives rise to the phenotypes that were see, the extensive cofractionation and colocalization of released Dally with Lipophorin suggests that Lipophorin may act by influencing the behavior of these particles. For example, interaction of Lipophorin with the HS moieties of released Dally might reduce the affinity of Lipophorin particles for cell-surface HS, promoting transfer of Hh-bearing particles to Ptc. Alternatively, Dally HS moieties on Hh-bearing lipoprotein particles might promote the formation of ligand-receptor complexes. IHog acts as a coreceptor with Ptc and, like released Dally, is necessary for full-strength Hh signaling (Yao, 2006). Adding soluble heparin induces IHog dimerization and increases IHog-Hh binding in vitro (McLellan, 2006). Presentation of Dally HS on the same particle with Hh may greatly increase the efficiency with which these complexes form by bringing HS, Hh, and IHog into close proximity in vivo. Furthermore, lipoprotein particles that carried multiple copies of Dally and Hh would have the potential to induce receptor crosslinking. In this way, lipoprotein particles may act as scaffolding platforms, bringing together specific ligands and increasing the diversity of combinatorial signals available for patterning during development (Eugster, 2007).
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