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).
The glypican family of heparan sulfate proteoglycans has been implicated in formation of morphogen gradients. The role of the glypican Dally-like protein (Dlp) in shaping the Wingless gradient was studied in the Drosophila wing disc. Surprisingly, Dlp has opposite effects at high and low levels of Wingless. Dlp promotes low-level Wingless activity but reduces high-level Wingless activity. Evidence is presented that the Wg antagonist Notum acts to induce cleavage of the Dlp glypican at the level of its GPI anchor, which leads to shedding of Dlp. Thus, spatially regulated modification of Dlp by Notum employs the ligand binding activity of Dlp to promote or inhibit signaling in a context-dependent manner. Notum-induced shedding of Dlp could convert Dlp from a membrane-tethered coreceptor to a secreted antagonist (Kreuger, 2004).
Notum encodes a secreted member of the alpha-hydrolase superfamily, which includes serine proteases, lipases, and other enzymes in which a Ser-Asp-His catalytic triad comprises the active site. Mutation of Ser237 to Ala was shown to remove Notum activity in vivo and in vitro (Giraldez, 2002). On the basis of its similarity to pectin acetylesterase enzymes from plants and plant pathogens and on the basis of its ability to compete with the N-deacetylase-N-sulfotransferase enzyme for use of Dlp as a substrate, it has been proposed that Notum might function as a GAG deacetylase. If this were the case, it would be expected that Notum would be unable to modify a form of Dlp lacking GAG side chains (Kreuger, 2004).
Dally and Dlp each contain five putative GAG addition sites and have been shown to carry predominantly heparan sulfate (HS, as shown for vertebrate glypicans). GAG biosynthesis is initiated by addition of a xylose residue to the hydroxyl group of a serine in a serine-glycine (SG) motif in the stem region of the glypican (Lindahl, 1998). Mutant forms of Dally and Dlp proteins were prepared in which all the putative GAG-addition sites were mutated. For Dally, the serine residues in the five SG motifs were mutated to alanine. For Dlp, four SG motifs were deleted by removing a block of 25 residues, and the fifth was mutated to AG (Kreuger, 2004).
The glycosylation states of epitope-tagged Dally and Dlp and the GAG addition site mutants were compared by anion exchange chromatography. Glypicans isolated from mammalian cells in culture are highly negatively charged and are typically retained on a strong anion exchange matrix up to 1 M NaCl. When expressed in Schneider S2 cells, a considerable fraction of Dally bound to Q-Sepharose in 0.3 M salt and some was able to bind in up to 1 M salt. Dally migrated as a broad band in SDS-PAGE. The more negatively charged forms that were bound to Q-Sepharose at higher salt exhibited a higher apparent molecular weight. This may reflect the presence of more and longer HS side chains as well as differences in charge due to sulfation. Dally behaves like a conventional glypican (Tsuda, 1999). In contrast, most of the Dlp protein bound to Q-Sepharose in 0.15 M salt, but none bound at higher salt concentrations. Dlp migrated as a more tightly resolved band in SDS-PAGE. These observations suggest that Dally and Dlp differ in the extent or quality of GAG modification. Removal of the GAG addition sites considerably reduced retention of both mutant proteins on Q-Sepharose. The proportion of the mutant form of Dally that bound was reduced at all salt levels and none was bound above 0.3 M salt. Very little of the mutant form of Dlp was able to bind to Q-Sepharose, even in physiological salt (Kreuger, 2004).
To further evaluate their GAG content, lysates of S2 cells transfected to express wild-type or mutant Dally or Dlp were digested with heparitinase, and the remaining glycan stub was detected with 3G10 antibody. Dally-expressing cells showed elevated GAG labeling in a broad band that comigrated with the major form of endogenous glypican in untransfected cells. Although the level of expression of the mutant form of Dally was comparable, GAG labeling was not detectable above background. The level of GAG modification on overexpressed Dlp was lower than that seen on the endogenous glypicans. This indicates that Dlp has a lower GAG content than Dally, consistent with its poor retention on Q sepharose. The mutant form of Dlp showed much reduced GAG labeling, barely distinguishable from background levels (Kreuger, 2004).
Dally resembles a conventional glypican, being extensively modified by GAG side chains. Dlp appears to have less heterogeneous and less extensive GAG modification than Dally. The mutant form of Dlp has low levels of residual GAG and would be expected to be a poor substrate for Notum, if Notum acts directly on the GAG side chains. Coexpression of Notum and HA-tagged Dlp in S2 cells caused a large shift in the electrophoretic mobility of Dlp in SDS-PAGE to a faster migrating species, and the Notum S237A mutant had no effect. S2 cells contain endogenous Notum, so there is always a background level of Dlp processing. Interestingly, Notum caused a shift in the mobility of the low-GAG form Dlp comparable in magnitude to the shift in the wild-type protein, though the processing efficiency was lower. This would be difficult to explain if the shift were due to alteration in the amount or in the negative charge of the remaining GAG due to deacetylation. Further, no increase in deacetylase activity was detected in lysates of S2 cells overexpressing Notum, nor was there any detectable decrease in the degree of HS acetylation in larvae overexpressing Notum. These observations suggest that Notum does not act as a GAG-modifying enzyme (Kreuger, 2004).
The mobility shift of Dlp caused by Notum is ~15 kDa. Proteolytic cleavage at the N or C termini of Dlp could cause the apparent size reduction, although other modifications such as removal or attachment of lipids, sugars, or phosphate groups also could cause anomalous migration in SDS-PAGE. To test the possibility of proteolytic processing, forms of Dlp were prepared with epitope tags close to the two ends. For Dlp-HA-C, an HA epitope tag was inserted at serine 732, just before the putative cleavage site for addition of the GPI anchor (S733). To produce GFP-Dlp-HA-C, the HA tag was placed at the corresponding position in GFP-Dlp, which contains a GFP moiety inserted in place of residue G68, close to the N terminus of the protein following removal of the signal peptide at residue 41. S2 cells were transfected with Dlp-HA, Dlp-HA-C, or GFP-Dlp-HA-C together with normal or mutant forms of Notum. Notum induced comparable mobility shifts in all three forms of Dlp. The C-terminally HA-tagged forms of Dlp were processed more efficiently than Dlp-HA, so that more of the faster migrating from was observed without addition of wild-type Notum (Notum is expressed in S2 cells). These observations are difficult to reconcile with Notum acting as a protease (Kreuger, 2004).
The possibility was considered that the shift in Dlp mobility could be due to cleavage of the GPI anchor. Lysates were prepared from S2 cells transfected with Dlp-HA with or without Notum, and half of each was treated with phospholipase-C (PI-PLC) to cleave the GPI anchor of Dlp. Interestingly, PI-PLC cleavage caused a mobility shift in Dlp comparable to that caused by Notum. Notum had no additional effect on PI-PLC-treated Dlp. Although the magnitude of this shift in apparent molecular weight is larger than would be expected from the mass of the GPI anchor alone, anomalous migration of proteins in SDS-PAGE has been observed following removal of GPI anchors (Kreuger, 2004).
To further evaluate the possibility that the mobility shift caused by Notum might be due to cleavage of the GPI anchor, a Triton X-114 phase separation analysis was performed. Following phase separation, integral membrane proteins and GPI-anchored proteins are recovered mainly in the detergent phase, while other proteins are mainly in the aqueous phase. In cells cotransfected to express Dlp and Notum, processing of Dlp was efficient and the faster migrating form of Dlp was mainly recovered in the aqueous phase. In cells transfected to express Dlp, the slower migrating form was mainly recovered in the detergent phase, consistent with it having a GPI anchor. The faster migrating form partitioned between aqueous and detergent phases. These observations are consistent with the suggestion that the faster migrating form of Dlp produced by Notum lacks a GPI anchor (Kreuger, 2004).
It was next asked if Notum could act on forms of Dlp that lack a GPI anchor. The predicted GPI anchor site of Dlp consists of the sequence SDA (at S733) followed by a characteristic hydrophobic motif. Mutation of any of the three residues of the GPI anchor site to proline has been shown to reduce GPI addition in other glypicans. The SDA motif was mutated to PDA in GFP-Dlp-S988P (residue numbers are for GFP-Dlp). A truncated version of GFP-Dlp was also prepared in which a stop codon was inserted after S987 to mimic the effects of cleavage of the protein chain at this position without addition of GPI. Coexpression with Notum had little or no effect on these forms of GFP-Dlp. GFP-Dlp-S988P migrated predominantly at an intermediate position, faster than the GPI-anchored form but slower than the Notum-processed form. This presumably reflects the full-length protein that has not been cleaved for GPI addition (this form appears as a minor band in most preparations). The major band of GFP-Dlp-S988P is unaffected by Notum, though a small amount of the protein does appear to have had GPI added (perhaps at S987) and was subject to Notum activity. The truncated GFP-Dlp-S987Z protein did not produce any of the slower migrating GPI anchored form and was not affected by Notum. GFP-Dlp-S987Z migrated slightly faster than the Notum-modified form of GFP-Dlp. In Triton X-114 phase separation analysis, GFP-Dlp-S987Z partitioned into the aqueous phase, also consistent with the lack of a GPI anchor. The form of Dlp cleaved by PI-PLC comigrated more closely with the Notum-processed form, consistent with the possibility that Notum cleavage was in the GPI moiety (Kreuger, 2004).
Next a version of Dlp was prepared that was not GPI anchored, in order to ask if it would be a substrate for Notum. GFP-Dlp was fused at residue S975 to residue R23 of the transmembrane protein CD2 and expressed in S2 cells. This protein contains the entire ectodomain (CRD and stem) of Dlp, but lacks the GPI addition signals (the fusion is at the equivalent position in Dlp to the S987Z construct; numbering differs because the HA tag was removed). GFP-Dlp-CD2 was not cleaved by PI-PLC, verifying that it does not contain a GPI anchor. GFP-Dlp-CD2 was also not a substrate for Notum. If Notum acted as a protease to cleave in the Dlp protein, it would be expected that GFP-Dlp-CD2 fusion would be a substrate for Notum. Likewise, if Notum acted on the GAG side chains or on another posttranslational modification, it would be expected that GFP-Dlp-CD2 would be affected. Taken together, these observations indicate that Notum can induce modification of Dlp in a manner that resembles cleavage of the GPI anchor. Given that the alpha-hydrolases includes lipases, it is possible that Notum might act directly to cleave within the GPI anchor. Alternatively, Notum could induce the activity of an endogenous phospholipase (Kreuger, 2004).
Can cleavage at the GPI anchor cause Dlp to be shed from the cell surface? The faster and slower migrating forms of Dlp-HA were efficiently recovered from the cell lysates. In addition, the faster migrating cleaved form of Dlp-HA was recovered from the culture medium of cells overexpressing Notum. The slower migrating GPI-anchored form of Dlp-HA was not recovered from the medium. Very little Dlp-HA was recovered from medium conditioned by cells transfected with Dlp alone or with the Notum mutant. This indicates that Notum can release Dlp from the cell surface, causing it to be shed into the medium. Notum does not induce detectable shedding of HA-tagged Dally (Kreuger, 2004).
Dlp-HA or Dally-HA were expressed under en-Gal4 control in the wing disc to compare the effects of the endogenous Notum protein on Dlp and Dally. Notum is expressed at the dorsoventral boundary under control of Wg. En-Gal4 produced a uniform level of Dally-HA across the wing pouch. In contrast, Dlp-HA protein levels were lower at the dorsoventral boundary. Notum caused loss of Dlp, but had little effect on Dally. This presumably reflects shedding of Dlp protein from cells in the disc in the region where Wg levels are highest (Kreuger, 2004).
The finding that Notum can cleave and release Dlp from cells raised the possibility that Dlp might be released together with bound Wg. This could explain how Dlp acts to reduce Wg activity where Wg and Notum levels are high. Therefore the possibility was tested of synergistic action of Notum and Dlp on Wg function. Overexpressed Dlp binds and sequesters Wg at the cell surface, leading to reduced Wg activity. Coexpression of Notum with Dlp reduced the ability of Dlp to bind and retain Wg (Giraldez, 2002). To examine the effects of Notum on Dlp activity, transgenes expressing Notum or Dlp were selected at levels that were on the threshold for altering wing morphology. When expressed under en-Gal4 control, each transgene caused loss of some of the posterior wing margin bristles, indicating mild reduction of Wg activity. Expression of the two transgenes together caused scalloping of the wing, a typical defect caused by more severe reduction of Wg activity. This observation suggests that expression of Notum enhanced the ability of Dlp to reduce Wg activity. In view of the finding that Notum can cause Dlp to be released from the cell surface, it is suggested that coexpression of Notum causes Dlp to be released together with bound Wg (Notum reduced the level of Wg bound in the disc; Giraldez, 2002). Release of Wg bound to Dlp could reduce the level of Wg available for signaling and cause the observed wing scalloping phenotype. Notum causes release of Dlp from cells at the DV boundary, where Wg levels are highest (Kreuger, 2004).
Therefore, Notum can induce cleavage of Dlp in a manner that resembles PI-PLC cleavage of the GPI anchor. Notum and PI-PLC produce comparable shifts in the electrophoretic mobility of Dlp. Although the magnitude of this shift is larger than would be expected from the mass of the GPI anchor, other studies have shown that the effects of removing GPI anchors are not predictable. Examples of increased or decreased mobility have been reported, and the magnitude of the shifts can be large. Notum-induced processing also renders Dlp soluble in the aqueous phase following detergent phase separation of soluble and membrane-associated proteins, consistent with removal of the GPI anchor. Also, a Dlp-CD2 fusion protein that is not GPI anchored, and is therefore not a substrate for PI-PLC, is insensitive to Notum. This would not be expected if Notum acted on the GAG side chains or if Notum was a protease cutting within the Dlp core protein. These observations are consistent with two modes of Notum action. Notum might act directly to cleave the GPI anchor or it might modify Dlp in some way that makes Dlp a substrate for an endogenous phospholipase. The exact nature of the Notum-induced cleavage remains to be determined; however, one intriguing possibility is that Notum might cleave within the glycan linker of the GPI anchor (Kreuger, 2004).
Can the effects of Notum on Dlp provide an explanation for the apparently opposing activities of Dlp at high and low levels of Wg? How could Notum-induced cleavage of Dlp lead to reduced Wg activity, whereas removal of Dlp by RNAi increases Wg activity? If Wg remains bound to Dlp when Dlp is cleaved and shed from the cell, bound Wg would also be shed and so become unavailable for signaling. Shedding of Dlp as a consequence of Notum-induced cleavage could reduce peak levels of available Wg. Notum is expressed at the source of Wg and so would be expected to shed Dlp and reduce Wg activity where Wg levels are highest. In this way, removal of Dlp protein as a consequence of Notum-induced cleavage could have a different effect than failure to express Dlp. In the absence of Dlp, Wg would not bind Dlp and be shed with it, so that more Wg might be available to interact with Dally and/or the Wg receptor complex. This could increase the effective concentration of Wg locally near the site of Wg production (Kreuger, 2004).
This model is consistent with the observed synergy between low-level expression of Dlp and Notum. Overexpression of Dlp is thought to increase the number of Wg binding sites and shift the equilibrium toward more Wg bound to Dlp. At high levels of Dlp, this can reduce the amount of Wg available for signaling and produce a Wg loss-of-function phenotype. A level of Dlp overexpression was chosen that produces a mild defect due to reduced availability of Wg. Coexpression of Notum would lead to shedding of the Dlp bound Wg and thus remove this fraction of Wg from the pool on the cell surface so that it would no longer be able to contribute to the pool of Wg in equilibrium with the receptor. This would be expected to further reduce Wg activity and increase the severity of the defect, as observed (Kreuger, 2004).
Localized cleavage of Dlp induced by regulated expression of Notum may provide a unifying explanation for the opposing effects of the Dlp glypican in different regions of the tissue. A mathematical model of Wg gradient formation has invoked a need for an elevated level of Wg turnover in cells close to the source of Wg. The mechanism described here would be sufficient to provide the reduction of Wg activity in cells closest to the source of the secreted ligand that is needed for formation of a robust morphogen gradient (Kreuger, 2004).
Wingless is a morphogen required for the patterning of many Drosophila tissues. Several lines of evidence implicate heparan sulfate-modified proteoglycans (HSPGs) such as Dally-like protein (Dlp) in the control of Wg distribution and signaling. dlp is required to limit Wg levels in the matrix, contrary to the expectation from overexpression studies. dlp mutants show ectopic activation of Wg signaling at the presumptive wing margin and a local increase in extracellular Wg levels. dlp somatic cell clones disrupt the gradient of extracellular Wg, producing ectopic activation of high threshold Wg targets but reducing the expression of lower threshold Wg targets where Wg is limiting. Notum encodes a secreted protein that also limits Wg distribution, and genetic interaction studies show that dlp and Notum cooperate to restrict Wg signaling. These findings suggest that modification of an HSPG by a secreted hydrolase can control morphogen levels in the matrix (Kirkpatrick, 2004).
By a number of cellular and molecular markers, compromising dlp function produced ectopic activation of high-threshold Wg target genes in the wing imaginal disc. dlp adult escapers have ectopic mechanosensory bristles and dlp third instar larvae show expanded expression of Ac, a marker for sensory organ precursor cells. Wg signaling was abnormally elevated along the DV boundary of dlp mutant discs by a number of other measures, including expansion of the zone of nonproliferating cells (ZNC) and ectopic expression of Cyclin A. These findings are consistent with the expansion of sensory organ precursor cells produced by RNAi inhibition of dlp function. These phenotypes show that Dlp serves to limit activation of high-threshold target genes near the DV boundary (Kirkpatrick, 2004).
The expansion of Wg signaling found in dlp mutants is accompanied by locally elevated levels of extracellular Wg. The increase in Wg in the matrix of dlp mutant wing discs is not associated with increases in Wg production or altered expression of D-fz2, a known regulator of Wg levels. These findings argue against the model that dlp controls Wg levels indirectly via control of Wg expression or other regulatory genes. It is well established that Wg binds heparan sulfate, and therefore it is likely that Dlp controls Wg levels through a direct interaction (Kirkpatrick, 2004).
The expansion of Wg-directed patterning in the wing disc of dlp mutants was a surprising finding on a number of counts: (1) mutations in genes encoding heparan sulfate biosynthetic enzymes compromise rather than increase the levels of Wg in the matrix; (2) ectopic expression of Dlp results in higher levels of Wg in the matrix. These findings suggest that loss of Dlp would destabilize Wg on cell surfaces and in the matrix, thereby reducing Wg signaling. However, exactly the opposite result was obtained in dlp mutants: loss of Dlp increases extracellular Wg and signaling. These findings emphasize that while mutations compromising synthesis of all HSPGs might destabilize Wg in the matrix, loss of any one proteoglycan can have very different effects (Kirkpatrick, 2004).
The phenotypes of dlp mutants suggest that Dlp serves to limit Wg distribution and/or alter Wg stability, thus affecting the Wg gradient in the wing disc. Genetic mosaic analysis of dlp clearly demonstrates that the ectopic activation of Wg signaling, as measured by Arm, Cyclin A, and Ac expression, occurs non-cell autonomously, providing support for this model. Recent mathematical modeling of morphogen gradients demonstrates that binding of morphogens to receptors or coreceptors on cell surfaces or in the matrix can have profound effects on diffusion and subsequently limit morphogen activity. Consistent with such modeling, it has been demonstrated experimentally that a nonheparin binding isoform of the secreted growth factor VEGF adopts a broader, shallower distribution than heparin binding forms and the differential localization of VEGF-A isoforms in the matrix controls the vascular branching pattern. It is also well established that heparan sulfate proteoglycans mediate endocytosis of extracellular ligands, and the findings suggest the possibility that Dlp might mediate endocytic control of Wg levels in the matrix. dlp mutants display locally elevated levels of extracellular Wg without an increase in production, and therefore Wg turnover must be affected in some way (Kirkpatrick, 2004).
In contrast to the higher levels of extracellular Wg found near the DV boundary in dlp mutant wing discs, there is no apparent change in the low level of Wg at the edges of the gradient. However, genetic mosaic analysis of dlp in wing development shows that loss of dlp can modestly reduce the activation of a low-threshold Wg target gene in those regions where Wg levels are low. Since this effect on Wg signaling is not a consequence of reduced levels of Wg in the matrix, it must result from an alteration of cell responsiveness or the bioactivity of the Wg that is present. The findings are consistent with studies showing that RNAi of dlp in the wing disc also inhibits transcriptional activation of Wg target genes in regions where Wg is low. It is interesting to note that dlp expression, like that of the Wg receptor D-fz2, is highest outside the presumptive wing margin. Perhaps the high level of dlp expression in this region serves, in part, to enhance cellular responses to Wg. The ability of Dlp to enhance Wg signaling as well as limit Wg distribution is not unprecedented: the other glypican in Drosophila, Dally, enhances Dpp signaling in regions of the wing disc where Dpp levels are modest in addition to regulating the distribution of Dpp protein (Kirkpatrick, 2004).
Mutations in Notum produce ectopic activation of Wg signaling and alter the Wg gradient, similar to what was observed in dlp mutants. Notum encodes a protein with homology to plant hydrolases and has been proposed to alter the structure of Dlp, thus affecting its affinity for Wg. Coexpression of Notum with Dlp decreases Dlp-mediated stabilization of Wg protein in the wing pouch. dlp and Notum mutants fail to complement: this is strong genetic evidence that Dlp and Notum cooperate to control Wg-mediated patterning. Biochemical studies show that Notum can cleave Dlp from the cell surface. The finding that heterozygosity for dlp ameliorates patterning abnormalities produced by ectopic Notum suggests that Dlp is a principal substrate for Notum activity. The results suggest that Notum-mediated release of Dlp from the cell surface is required for Dlp to limit Wg signaling (Kirkpatrick, 2004).
Notum and dlp are expressed in complementary patterns in the wing disc, with Notum levels highest near the DV boundary and dlp levels highest elsewhere. Thus, a gradient of Dlp cleavage may be established across the disc, with some Dlp remaining on cell surfaces in regions distant from the DV boundary, enhancing cellular responses to Wg. Cleavage of Dlp would remove this coreceptor activity, but shed Dlp-Wg complexes also could limit Wg signaling by promoting Wg clearance or could serve as dominant-negative inhibitors of signaling. Coexpression of Notum and Dlp in the wing disc produced more intracellular vesicles compared to expression of Dlp alone: this is consistent with a role for shed Dlp in promoting endocytosis and clearance of Wg from the matrix (Giraldez, 2002). Such a role is also consistent with the non-cell-autonomous effects of dlp somatic cell clones on extracellular Wg distribution. Loss of dlp in clones might locally reduce Wg turnover as well as reducing cell surface binding sites for Wg, permitting increased diffusion of Wg and accumulation on wild-type cells at clone boundaries. Spatial regulation of Dlp activity by Notum can explain how Dlp primarily limits Wg signaling near the DV boundary and enhances signaling away from the boundary, but other Wg-dependent factors may also influence the ability of Dlp and Notum to downregulate Wg levels. Notably, recent mathematical modeling suggests that elevated degradation of Wg close to its source is necessary to enhance the robustness of the morphogen gradient. Together, Dlp and Notum may provide this locally increased turnover and hence stabilize the Wg gradient. Enzymatic modification of a proteoglycan to influence its cell surface localization may enable it to play both positive and negative roles in signaling and provides another potential mechanism for regulating morphogen distribution in tissues (Kirkpatrick, 2004).
In humans, mutations in glypican-3 (GPC3) were identified as the genetic basis of a human overgrowth and tumor susceptibility syndrome, Simpson Golabi Behmel Dysmorphia (SGB) (Pilia, 1996). SGB patients display both prenatal and postnatal overgrowth, a number of morphological abnormalities including renal dysplasia and skeletal defects, as well as a high incidence of tumors (Neri, 1998). Subsequently, loss of GPC3 expression has been found associated with a number of cancers, including breast, mesothelioma, and ovarian neoplasias (Lin, 1999; Murthy, 2000; Xiang, 2001). Given the established role of proteoglycans as molecules facilitating growth factor signaling at the cell surface, the effects of loss of GPC3 on growth and tumor development are a bit puzzling. The current findings provide a molecular mechanism for understanding the capacity of glypicans to serve as tumor suppressors. Dlp restricts the cellular domain of Wg signaling during wing development, and loss of dlp results in ectopic growth factor signaling. Clearly, ectopic signaling produced by loss of GPC3 could readily contribute to tumor development and growth in humans. Recently, analysis of a gene-trap mutant in mouse Ext1 has provided further evidence for the capacity of heparan sulfate proteoglycans to limit the range of morphogen activity during chondrocyte differentiation (Koziel, 2004). Mice partially defective for heparan sulfate biosynthesis as a consequence of hypomorphic mutations in Ext1 show ectopic Indian Hedgehog signaling and altered Hedgehog distribution at the growth plate (Kirkpatrick, 2004).
The respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient has been examined. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, it was found that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. These studies reveal that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, this analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient (Baeg, 2004).
Sfl encodes a homolog of the Golgi enzyme HS N-deacetylase/N-sulfotransferase that is required for the modification of HS. It was of interest to determine whether the retention of Wg at the cell surface involves HSPGs in receiving cells, since it has been proposed that HSPGs are unlikely to be required in Wg-receiving cells. In the wing blade, Wg, originating from the D/V border, is detected in an irregular pattern of puncta in receiving cells; these puncta correspond to the internalized Wg protein. The intensity and number of puncta decreases from the source of Wg. Furthermore, using an extracellular labeling method, a gradient of Wg protein that appears broader, shallower, and with less puncta is observed. Both in sfl mutant wing discs and in large sfl mutant clones, a striking decrease in the number of Wg puncta was observed. This decrease is not due to a change in wg transcription because it is not affected in sfl mutant cells. Further, lack of Sfl activity does not appear to disrupt the overall amount of Wg produced by wg-expressing cells but is associated with a dramatic decrease in extracellular Wg. These results suggest that HSPGs are required for sequestering extracellular Wg in receiving cells (Baeg, 2004).
To gain further insights into the role of HSPGs in receiving cells to shape the Wg gradient, an examination has been made of the distribution of Wg in patches of WT cells located within a large sfl mutant territory. In such cases, bright spots of Wg were be detected within the patch of WT cells, indicating that sfl-expressing cells are able to sequester extracellular Wg, unlike neighboring cells that lack sfl. This result is consistent through an analysis of more than 10 clones. Further, clones of cells that overexpress Notum-GT (Golgi-tethered), which acts cell autonomously in receiving cells, were generated. Notum, which encodes a member of the α/β-hydrolase superfamily, antagonizes Wg signaling and it has been proposed that Notum acts by altering the ability of the cell surface glypican molecules Dally and Dlp to stabilize extracellular Wg (Giraldez, 2002). Consistent with the conclusion that HSPGs are required in receiving cells to capture extracellular Wg, a decrease in the formation of Wg puncta was observed in cells overexpressing Notum-GT. These results are consistent with at least two nonexclusive models: (1) HSPGs could be required for Wg stability and/or trapping of Wg at the cell surface such that it does not diffuse away; (2) HSPGs could be involved in promoting Wg movement throughout tissues. The role of HSPGs in sequestering and/or stabilizing the ligand is supported by previous observations that overexpression of either Dlp or Dally results in the accumulation of extracellular Wg (Baeg, 2004).
Because HSPGs have been implicated in endocytosis of ligands such as FGF, possibly HSPGs also play a role in Wg internalization. A role for HSPG in Wg endocytosis would be consistent with the absence of puncta in sfl clones, and also the observed accumulation of extracellular Wg following overexpression of Dlp-HA (Giraldez, 2002). However, much of Wg proteins appeared in intracellular vesicles, instead of outlining the cell surface, in discs overexpressing both Dlp-HA and Notum. If HSPGs were directly involved in Wg internalization, fewer intracellular vesicles would be detected in discs overexpressing Dlp-HA and Notum since Notum acts to decease the affinity of Dlp for Wg. Furthermore, if the primary function of HSPGs were to internalize Wg, then extracellular Wg accumulation in cells lacking HSPGs activity would be expected, which is not the case. However, the data does not rule out the possibility that HSPGs play a direct role in Wg endocytosis, and, thus, further analysis will be required to clarify this issue. Taken together, these results suggest that the primary role of HSPGs is to trap and/or stabilize extracellular Wg in receiving cells where it is then able to interact with its signaling receptor as well as other factors that are responsible for its internalization, and thus contributes to shaping the Wg gradient (Baeg, 2004).
Previous ectopic expression studies have shown that Dlp can trap extracellular Wg and prevent activation of the Wg signaling pathway. Because Dlp appears to be a major HSPG required to regulate Wg signaling, its endogenous distribution was examined in the wing imaginal disc using a polyclonal antibody against Dlp and a staining method that primarily detects extracellular proteins. The specificity of the Dlp antibody was confirmed by misexpressing dlp using the ap-Gal4 driver. In the third instar wing imaginal disc, Dlp was detected throughout the disc; however, a significant decrease in the level of Dlp was detectable at the D/V border. This domain of low Dlp expression correlates with the region where high level of Wg signaling is required to induce the expression of short-range target genes. It is noted that since dlp mRNA expression is uniform throughout the disc, the down-regulation of Dlp at the D/V border must occur post-translationally. Interestingly, an optical cross section of the disc has revealed that endogenous Dlp localizes mostly on the basolateral surface of the cell where extracellular Wg is detected. The subcellular localization of Dlp protein was also examined using a GFP-dlp expressed under the control of a Gal4 driver. Consistent with the Dlp antibody result, it was found that GFP-Dlp localizes predominantly to the basolateral membrane. Altogether, these observations suggest that Dlp can bind to extracellular Wg and that Dlp levels need to be reduced for high-level Wg activity in cells near the D/V boundary (Baeg, 2004).
In a genome-wide RNAi screen in S2R+ cells to identify genes that either up- or down-regulate Wg signaling, Dlp was identified as both a negative and positive regulator of Wg signaling under stimulated and nonstimulated conditions, respectively. The cell-based assay devised consists of the activation of a Tcf/Arm-dependent Wg-reporter gene upon induction of S2R+ cells by expressing a wg cDNA by transient transfection. The activity of the Wg pathway and the effect of the addition of various dsRNAs on the pathway were assayed by monitoring Luciferase-reporter activity using a luminescence-based plate reader. Using this assay, the addition of dsRNAs of positive transducers of Wg signaling, such as arm, decrease the Top12X-HS-Luciferase reporter activity, while dsRNAs to negative Wg regulators, such as Daxin, increase its activity. Interestingly, under condition of Wg induction, dlp was found to act as a negative regulator of Wg signaling, since dlp dsRNA led to a twofold increase in luciferase activity. This increase is significant since it is comparable to that of daxin dsRNA. In the absence of Wg induction, dlp was found to positively regulate Wg signaling, since dlp dsRNA leads to a fivefold decrease in luciferase activity, a decrease that is similar to that observed by the addition of arm dsRNA. These results suggest Dlp acts as a positive regulator of the Wg pathway when Wg level is low and negatively influences signaling when Wg is abundant. These results are consistent with in vivo results that demonstrate that Dlp has both a positive and negative role in Wg signaling (Kirkpatrick, 2004). The observations in S2R+ are consistent with the hypothesis that low Dlp levels at the D/V boundary supports high-level Wg signaling, while further away from the D/V boundary where the Wg concentration is lower, Dlp positively influences Wg signaling. Overall, the result from the S2R+ RNAi experiments indicates that (1) Dlp is not an essential component of the Wg signal transduction pathway; and (2) Dlp can either have a negative or positive impact on Wg signaling depending on the level of Wg available. The negative effect of Dlp is consistent with previous in vivo studies that have shown ectopic Dlp expression can trap extracellular Wg and prevent activation of the Wg signaling pathway. Therefore, a reduction of Dlp levels at the D/V border would be expected to contribute to high-level Wg signaling. The positive effect of Dlp in Wg signaling needs to be understood in the context of the previous findings that loss of HSPG activity results in wg loss-of-function phenotypes, as shown by a decrease in dll expression in clones mutant for enzymes involved in GAG biosynthesis. One attractive model is that Dlp would act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with the signal transducing Fz receptors in cells located at a distance from the D/V boundary where low level of Wg is available. Finally, these observations are consistent with a recent study (Kirkpatrick, 2004) that showed that (1) ectopic activation of Wg signaling at the wing margin occurs in dlp mutant tissues, and (2) a cell autonomous reduction in Wg signaling in dlp clones located distal to the Wg-producing cells (Baeg, 2004).
The distribution of Dlp protein is reminiscent of the down-regulation of Dfz2 transcription near the D/V border. Wg-mediated repression of DFz2 expression has been shown to affect the shape of the Wg gradient, resulting in a gradual decrease in Wg concentration. Because these results indicate that HSPGs affect Wg distribution, the functions of the two seven transmembrane Wg receptors, Fz and DFz2, were examined to evaluate how the signal transducing receptors cooperate with HSPGs in shaping the Wg gradient. To determine the role of Fz and DFz2 in Wg movement, the distribution of Wg in fz DFz2 double-mutant clones was examined. In these clones, an expansion of wg expression was observed, which is consistent with the previously described Wg “self-refinement” process, by which Wg signaling represses wg expression in cells adjacent to wg-expressing cells. Unexpectedly, within these clones, Wg puncta are still present, indicating that Fz/DFz2 receptor activities are not required for Wg spreading (Baeg, 2004).
To determine whether Wg is present in endosomes in the absence of Fz/DFz2 activities, wing discs were labeled with the endosomal marker Texas-red dextran. More than 50% of Wg puncta co-localize with red dextran, indicating that Wg is internalized in the absence of Fz/DFz2 activities. These observations are consistent with results in the embryo, and altogether suggest that internalization of Wg can be accomplished by proteins other than Fz/DFz2. Interestingly, this observation contrasts with the role of HSPGs in Wg distribution, since wing discs lacking GAGs show alteration in Wg puncta in receiving cells. The extracellular distribution of Wg was examined in fz DFz2 mutant clones. Interestingly, accumulation of extracellular Wg was detected throughout these clones, thus revealing that Wg can bind to the cell surface and that Fz/DFz2 receptors are required somehow for Wg degradation. To exclude the possibility that accumulation of extracellular Wg results from increased wg expression or secretion in fz DFz2 clones that cross D/V boundary, small clones that do not include the D/V boundary were generated. Accumulation of extracellular Wg was detected in these clones, which is reminiscent of the finding that overexpression of a dominant-negative form of DFz2 (ΔDFz2-GPI) driven by en-Gal4 in embryonic tissue prevents Wg decay within the en domain. It has been proposed that endocytosis of a Wg/receptor complex is responsible for down-regulating Wg levels. Further, because Wg is still organized in a graded manner in these clones, as shown by the distribution of the Wg puncta, it indicates that Wg movement can occur in the absence of Fz/DFz2 (Baeg, 2004).
There is a third member of the Frizzled family encoded by DFz3 that could influence the distribution of Wg in tissues. DFz3 expression is similar to that of wg, and a constitutively activated form of Arm up-regulates its expression in the wing disc, suggesting that DFz3 is transcriptionally regulated by Wg signaling. Based on these observations, little or no DFz3 protein would be expected to be present in cells that lack Fz/DFz2 activity, suggesting that internalization of Wg in Fz/DFz2 mutant cells is unlikely to be mediated by DFz3. Another candidate that could affect Wg distribution is Arrow, which is a Drosophila homolog of a low-density lipoprotein (LDL)-receptor-related protein (LRP) and has been shown to be essential in cells receiving the Wg signal. However, because a soluble form of the Arrow fails to bind Wg and Fz receptors in vitro, and because Arrow functions after DFz2 engages Wg, it is unlikely that Wg internalization in Fz/DFz2 mutant cells is mediated by Arrow. Finally, as is case for FGF endocytosis, HSPGs themselves possibly play a role in Wg internalization (Baeg, 2004).
In summary, there is a Fz/DFz2 receptor-independent mechanism that organizes Wg distribution, and Fz/DFz2 proteins play a role in Wg gradient formation by decreasing the level of extracellular Wg. Regulation of extracellular Wg levels by Fz/DFz2 may occur through receptor-mediated endocytosis, or by some other mechanisms. If Wg degradation occurs by receptor-mediated endocytosis, it indicates that there may exist more than one way to generate Wg puncta since these are still present in the absence of Fz/DFz2 receptor activity. These findings also emphasize that the amount of Fz/DFz2 receptors at the cell surface must be precisely regulated to achieve the proper spreading of Wg, an observation that is underscored by the transcriptional down-regulation of DFz2 expression near the source of Wg (Baeg, 2004).
To further examine the role of Fz/DFz2 receptors in Wg gradient formation, DFz2 was overexpressed at the D/V boundary using the C96-Gal4 driver, and the effect on Wg distribution and wing patterning was examined. Analysis was focused on DFz2 since DFz2 has been shown to bind Wg with high affinity and to stabilize it. Further, DFz2 expression is down-regulated by Wg signaling, and this regulation has been shown to play a critical role in the overall shape of the Wg gradient. Interestingly, ectopic expression of DFz2 results in wing notching and ectopic bristles at the wing margin of adult wing. Previous studies have shown that wing nick phenotypes result from an inhibition in Wg signaling activity while the presence of ectopic bristles on the wing blade corresponds to an increase in Wg signaling. Thus, based on the wing phenotypes, it appears that overexpression of DFz2 paradoxically both increases and decreases Wg signaling (Baeg, 2004).
Overexpression of the DFz2 could interfere with Wg signaling and its distribution in a number of ways. For example, an increase in DFz2 could increase the efficiency of Wg signaling, if the amount of receptor is limiting. Further, since wg expression in the wing disc is restricted to the D/V margin, and Wg diffuses from it, trapping of Wg near these cells most likely will have an effect on Wg short- and long-range activity since the shape of the Wg gradient will be disrupted. To distinguish between these possibilities, Wg distribution was examined in discs with clones of cells that overexpress DFz2 at the D/V boundary. These clones were associated with two effects on Wg distribution. (1) The level of Wg was increased in the clones of cells where DFz2 was overexpressed, indicating that an increase in the level of DFz2 in receiving cells leads to an increase in trapping extracellular Wg. This observation is consistent with the occurrence of extra bristles on the wing blade since they reflect high levels of Wg signaling activity. (2) A dramatic reduction in Wg puncta was detected in WT cells located adjacent to the cells overexpressing DFz2, suggesting that Wg movement from the D/V margin into the wing blade is impaired as a result of the excess trapping of Wg by cells that overexpress DFz2. To demonstrate that Wg accumulation correlates with an increase in Wg signaling and that the absence of Wg puncta correlate with an absence of Wg signaling, the effect of DFz2 overexpression on the expression of senseless was examined. sen expression is expanded in cells overexpressing DFz2, yet sen is not expressed in WT cells near a clone of cells overexpressing DFz2. This is consistent with the observation that more Wg can be detected in cells overexpressing DFz2 and that less Wg puncta are present in WT cells near a clone of cells overexpressing DFz2 (Baeg, 2004).
It has been proposed that Fz proteins contribute to Wg turnover. Thus, it is intriguing to note that overexpression of DFz2 leads to an accumulation of extracellular Wg. This may reflect saturation of the endocytotic pathway when DFz2 is overexpressed or an inability of the regulatory pathways that normally control Dfz2 endocytosis in the wing disc to appropriately respond under this overexpressed condition. Another possibility is HSPGs themselves might play an important role. Wg endocytosis and the stoichiometry of Fz to HSPGs is essential to promote proper Wg internalization. Detailed biochemical and cell biological studies are now required to clarify the role(s) of these receptors in Wg movement (Baeg, 2004).
Finally, whether overexpression of DFz2 at the D/V boundary could affect long-range activity of Wg was examined, using wing disc overexpressing DFz2 driven by C96-Gal4 driver. Interestingly, Dll expression is dramatically shortened in wing disc overexpressing DFz2 at the D/V boundary when compared to that of WT disc. It is concluded that DFz2 has multiple roles in Wg signaling: First, it transduces Wg signaling and its level is limiting in amount; and second, it affects Wg short- and long-range activity by modulating the availability of extracellular ligand (Baeg, 2004).
In this study, the respective roles of HSPGs and Fz/DFz2 receptors in Wg distribution and gradient formation were examined. Interestingly, it was found that loss of Dlp activity significantly increases the level of Wg activity in S2R+ cells upon Wg induction, indicating that Dlp acts as a negative regulator in Wg signaling and that it is not required for transducing the Wg signal. Interestingly, the in vivo results show that Dlp protein levels are low near the D/V boundary. Thus, low levels of Dlp near the source of Wg production may allow for activation of high threshold Wg target gene. It is of interest to note that Notum is highly expressed along the D/V boundary (Giralez, 2002), which would be predicted to further diminish HSPGs activity. In addition, it was found that Dlp positively influences Wg signaling in S2R+ cells when Wg is not induced, suggesting that it is required for Wg signaling in cells where Wg level is low. A possible explanation for this result is that Dlp may act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with signal transducing Fz receptors. In the wing imaginal disc, given that Dlp is required for Wg signaling in cells where Wg levels are low, HSPG activity is possibly required for Wg signaling by somehow facilitating Wg movement. The binding of extracellular Wg to the low-affinity HSPG receptors in receiving cells may result in the association of Wg to cell membranes. Ligand movement could then occur by a mechanism that directly involves HPSGs where subsequent cycles of Wg dissociation/reassociation with HSPGs might promote the movement or require other yet to be identified extracellular molecules. To distinguish these possibilities, the role of HSPGs in Wg movement will require further detailed analysis. Regardless, these results clearly indicate that the primary role of HSPGs is to sequester and/or stabilize extracellular Wg in receiving cells. The imposition of the HSPG-mediated Wg accumulation and the Fz-dependent degradation mechanism would thus contribute to the Wg morphogen gradient. It is important to note that the expression levels of some of the critical components of each systems (i.e., dally, DFz2) are also regulated by the Wg pathway itself, indicating that the slope of the Wg gradient is established by the delicate balance between these two systems (Baeg, 2004).
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 ‘all hair’ 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).
Studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. This study shows that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, the role of Dlp was analyzed in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, these results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila (Yan, 2007).
There are three main important findings in this work. First, Dlp was identified as an essential molecule required for tracheal development. Dlp is required for Btl-mediated tracheal branching during embryogenesis while both Dlp and Dally are involved in the formation of air sac tracheoblasts in the wing disc. Second, the data show that other HSPGs cannot replace Dlp for Btl signaling during embryogenesis and that both Dlp and Dally are not essential for Htl-mediated mesodermal cell migration. These data demonstrate that different FGFs may require different HSPGs to execute their effective signaling activities during development. Third and most importantly, strong evidence is provided that Dlp controls Btl signaling only in FGF-receiving cells in both embryonic and larval tracheal systems. This mechanism of HSPG activity in FGF signaling is very different from its roles in regulating the signaling activities of morphogens including Wnt, Hh and Dpp. Together, these new findings further define novel mechanisms and the specificities of HSPGs in FGF signaling during development (Yan, 2007).
Extensive biochemical and cell culture studies suggest that HSPGs are the part of the FGF/FGFR signaling complex. However, the mechanisms of HSPGs in FGF signaling during development are less known. Embryos mutant for two HSPG biosynthesis enzymes, sgl and sfl, exhibit defects in both Btl- and Htl-mediated FGF signaling. An important issue remaining to be solved is which HSPG core proteins are involved in these signaling events. The data in this work provide strong evidence that Dlp is the key molecule required for Btl signaling during embryonic tracheal development, while both Dlp and Dally are involved in the Btl mediated air sac tracheoblasts formation in the wing disc. The results provide several novel insights into the specificity of individual HSPG in FGF signaling. First, Dlp is involved in Btl signaling, but not in Htl signaling. These findings indicate that different FGF/FGFR complexes may require different HSPGs for their signaling activities. Second, Dlp is highly active and specific for Btl signaling; overexpression of the other three Drosophila HSPGs fail to rescue tracheal defects in dlp embryos. The specific activity of Dlp in Btl signaling could be due to the Dlp protein core or the HS GAG chains attached to the Dlp core protein. In this regard, it is especially surprising that Dally, which has 22% identity with Dlp and also bears a GPI anchor, cannot rescue tracheal phenotypes associated with dlp embryos. As Dlp is involved in several other signaling pathways such as Hh, it is unlikely that Dlp core protein interacts with the ligands directly. In this regard, it is worthwhile to note that ectopic expression of Dally also fails to rescue Hh signaling in dlp embryos. It is proposed that Dlp may have unique HS GAG chains that might provide high and specific activity for ligands such as Bnl and Hh (Yan, 2007).
The biosynthesis of HS GAG chains is determined by the HSPG protein core in which the GAG attachment sites and other protein parts such as the N-terminal cystenine-rich domain control both quantity and quality of the attached GAG chains. Detailed structure and functional studies of Dlp will further help to define specific requirements of the core protein or GAG attachment sites in FGF signaling. Furthermore, the unique GAG chains may be modified by specific enzymes. In this regard, it is particularly important to note that 6-O sulfation of HS is critical for Btl signaling, as Drosophila heparan sulfate 6-O-sulfotransferase is specifically expressed in embryonic tracheal system and is required for Btl signaling during embryogenesis. Recent study has shown that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts. In this regard, it is suggested that Dlp may be the optimal substrate for sulfation enzymes during embryogenesis. Therefore, the activity of Dlp in FGF signaling during embryogenesis cannot be replaced by other HSPGs including Dally, Syndecan and Perlecan (Yan, 2007).
Although Dlp is essential for Btl signaling during embryogenesis, both Dally and Dlp are involved in Btl signaling in air sac tracheoblast cells. Similarly, previous studies have shown that both Dally and Dlp are involved in regulating Wg, Hh and Dpp distribution in the wing disc. The different functions of the same HSPG in embryos and discs may reflect temporal and developmental stage dependent regulation of HSPG functions (Yan, 2007).
While it is well established that HSPGs can regulate FGF signaling by facilitating FGF/FGFR interaction, it is unknown whether HSPGs can also control FGF distribution, thereby modulating FGF signaling. This is a particularly important issue as in many developmental contexts FGF ligand is produced in one type of cell and acts on other cells to initiate its biological activity. One important finding of this work is that HSPGs control tracheal morphogenesis by regulating FGF signaling only in FGF-receiving cells, but not by regulating the secretion or distribution of FGF ligand in its producing cells and surrounding cells. Several important results support these conclusion: (1) dlp mutant embryos can suppress the phenotype of overexpressing Bnl in the tracheal cells. (2) Ectopic expression of Dlp in tracheal cells, rather than FGF expression cells, can effectively restore tracheal defects associated with dlp embryos. (3) Embryos rescued by prd-Gal4/UAS-dlp in dlp backbround is very similar to btl mutant embryos rescued by prd-Gal4/UAS-btl-GFP. (4) HSPGs are required for FGF signaling in its receiving cells in the air sac, but are dispensable in the columnar epithelial layer which includes FGF producing cells and other surrounding cells. Detailed analyses thus demonstrate the specific and distinct requirement of HSPGs in FGF signaling during tracheal development. Moreover, embryonic and larval data together suggest this is likely a general mechanism for HSPG function in FGF signaling in Drosophila (Yan, 2007).
Two major models are proposed for the role of HSPGs in FGF signaling. In one model, low affinity HS/GAG chains on the cell surface limit the diffusion of FGF ligand, thereby increasing its local concentration and the probability that it will interact with high-affinity FGFRs. In the second model, HSPGs facilitate the dimerization or oligomerization of FGF ligands thereby inducing receptor clustering and signal transduction. The experimental data cannot exclude either of these mechanisms. However, the results are in favour of the second case, since it is shown that HSPGs are not required in FGF concentration gradient in FGF producing cells, but are essential in FGF-receiving cells. Finally, a recent study showed that dynamin-mediated vesicle internalization is a crucial step to regulate FGF signaling in Drosophila tracheal system. Mutants in awd (abnormal wing disc) or shi (shibire), which encodes for a nucleoside diphosphate kinase and Drosophila dynamin, respectively, have increased levels of Btl in tracheal cell surface, increased FGF signaling activity and ectopic tracheal branching. In this regard, HSPGs may control FGF signaling by stabilizing the FGF/FGFR complex from degradation or internalization in FGF receiving cells. Further experiments using HSPG and awd/shi double mutant are needed to test this possibility (Yan, 2007).
Over the past several years, extensive studies in Drosophila and other model systems have established the essential roles of HSPGs in developmental signaling pathways including Wg, Hh and Dpp. In Drosophila embryo and wing imaginal disc, HSPGs are involved in the transport of morphogens including Wg, Hh and Dpp by a restricted diffusion mechanism. Narrow stripes of clones mutant for HSPGs can impede the movement of morphogens to further cells. However, in all of these cases, the first mutant cells adjacent to the morphogen source can still transduce signals arguing that HSPGs are not essential for morphogen signaling activity, but rather control the distributions or local concentrations of morphogens. The novel results from this work point out a major difference for a role of HSPGs in FGF signaling from their roles in morphogen signaling, as removal of HSPGs (dally-dlp or sfl) from FGF receiving cells can effectively block FGF signaling. Although the graded FGF activity may play an essential role in tracheal morphogenesis, the data from this work argue that the main function of HSPGs in FGF signaling is not to regulate the distribution of FGF ligand. Consistent with the different roles of HSPGs in FGF and morphogen signaling, it was found that Dlp acts cell-autonomously in FGF signaling while it functions non-autonomously in Hh signaling in embryos. These results suggest that Bnl transportation may be different from morphogen movement in the epithelial cells of the wing pouch. Indeed, morphogen molecules diffuse through the same layer of cells, columnar epithelial cells, while FGF is transported between different layers of tissues, from columnar epithelia to tracheoblasts. Moreover, leading air sac cells are always in close proximity with underlying columnar epithelia. They also extend multiple filopodia toward ligand gradient and presumably actively pursue the FGF ligands while wing disc morphogens including Wg, Hh and Dpp need to transport many cell diameters from their sources to reach their receiving cells. Studies in vertebrate also suggest that a graded distribution of FGF8 protein can be generated by the decay of fgf8 mRNA and this RNA gradient is translated into a protein gradient. In this case, no active transport mechanism is required to form a FGF gradient. In mammalian limb and lung development different FGFs are often expressed in different layers of cells, such as epithelium and mesenchyme, and signal through each other. It is interesting to determine whether HSPGs function similarly in these systems as in Drosophila (Yan, 2007).
Decapentaplegic, a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. This study characterized activity, stability, and gradient formation of a truncated form of Dpp (DppΔN), which lacks a short domain at the N-terminus essential for its interaction with Dally. DppΔN shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, a model is proposed in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex (Akiyama, 2008)
The Dpp pathway is regulated by multiple cell surface and extracellular factors. In the developing wing, Dally is one of the key molecules that modulate Dpp signaling. It affects the shape of the Dpp ligand gradient (protein distribution) as well as its activity gradient (spatial patterns of signaling activity). Dally and Dpp expressed in S2 tissue culture cells are coimmunoprecipitated, suggesting that Dally forms a complex with Dpp. It was also observed that Dally colocalizes with Dpp and Tkv in cells. In addition, Dally enhances Dpp signaling in a cell autonomous fashion. These findings suggest that Dally acts as a Dpp co-receptor at least in some developmental contexts. Interestingly, however, in embryos and in imaginal disc cells close to Dpp-expressing cells, Dpp can mediate signaling without Dally, indicating that HS is not absolutely required for all BMP-dependent processes in vivo (Akiyama, 2008)
DppΔN, which does not bind to heparin, fails to interact with Dally. The easiest interpretation of this result is that wild-type Dpp interacts with Dally via its HS chains. However, a recent study using Surface Plasmon Resonance showed that binding of BMP4 to Dally is not fully inhibited by excess HS. Also, a mutant form of Dally, which does not undergo HS modification, is able to significantly rescue dally mutant phenotypes. These findings suggest that the BMP-glypican interaction is not entirely dependent on the HS chains. One possible explanation for the failure of DppΔN to bind to Dally is that Dpp normally binds to Dally through both the HS chains and its protein core, and DppΔN has reduced affinities for both sites (Akiyama, 2008)
Although DppΔN lacks the ability to interact with Dally, it shows normal in vitro protein stability and signaling activity. Therefore, this truncated form of Dpp provided a powerful system to gain insight into the functions of Dally in distribution and signaling of the Dpp morphogen: this molecule was used to elucidate the consequences of lacking the ability to bind HSPGs. In the wing disc, DppΔN cannot form a normal gradient: only a low level of DppΔN was detected in the Dpp-receiving cells. Notably, this pattern of DppΔN resembles the Dpp ligand and activity gradients observed in dally mutant wing discs. A series of in vitro and in vivo Dpp stability assays suggested that DppΔN forms a shallow gradient because it is remarkably unstable in the matrix and that the stability of Dpp depends on its interaction with Dally (Akiyama, 2008)
Genetic experiments revealed that Tkv and Dally have opposite effects on Dpp gradient formation during wing development. Dally and Tkv share some common properties as components of the Dpp signaling complex: they both autonomously enhance Dpp signaling and limit migration of Dpp by binding to Dpp protein. Nevertheless, tkv and dally mutually suppress one another’s pMad gradient phenotypes. Consistent with the genetic interactions observed in dally and tkv mutants, the pMad phenotype produced by overexpression of tkv was significantly restored by coexpression of dally. These observations indicate that dally antagonizes tkv in Dpp signaling. Since it has been proposed that Tkv promotes Dpp degradation by receptor-mediated endocytosis, dally may stabilize Dpp by inhibiting this process (Akiyama, 2008)
Altogether, these studies suggest that Dally serves as a co-receptor for Dpp and regulates its signaling as well as gradient formation by disrupting the degradation of the Dpp-receptor complex. In this model, the Dpp signaling complex with Dally co-receptor would remain longer on the cell surface or in the early endosomes to mediate signaling for a prolonged period of time. In contrast, in the absence of Dally, the complex would be relatively quickly degraded. This possible role of Dally can account for the shrinkage of the Dpp gradient in dally mutant wing discs. However, the possibility cannot be excluded that DppΔN is lost from the cell surface by lack of retention and further diffuses away (Akiyama, 2008)
A previous kinetic analysis of FGF degradation in cultured mammalian vascular smooth muscle cells also showed that HSPG co-receptors can enhance FGF signaling by stabilizing FGF. In these cells, the intracellular processing of FGF-2 occurred in stages: low molecular weight (LMW) intermediate fragments accumulated at the first step. Blocking HS synthesis by treatment of cells with sodium chlorate substantially reduced the half-life of these LMW intermediates, indicating that HSPGs inhibit a certain step of the intracellular degradation of FGF-2. HSPGs have also been implicated in the endocytosis and degradation of Wg. Wg protein is endocytosed from both apical and basal surfaces of the wing disc and degraded by cells to downregulate the levels of Wg protein in the extracellular space. It has been proposed that Dally-like (Dlp), the second Drosophila glypican, regulates the Wg gradient by stimulating the translocation of Wg protein from both the apical and basal membranes to the lateral side, a less active region of endocytosis, thereby inhibiting degradation of Wg protein (Akiyama, 2008)
Interestingly, DppΔN behaved differently in vivo from the previously reported mutant Xenopus BMP4 lacking the heparin-binding site. Although the action range of BMP4 is restricted to the ventral side during Xenopus embryogenesis, the truncated BMP4 migrated further in the embryo. In addition, heparitinase treatment of embryos also resulted in long-range diffusion of BMP4. These findings led to the conclusion that HSPGs trap BMP4 in the extracellular matrix to restrict its distribution in the Xenopus embryo. This activity of HSPGs seems to be opposite to that of Dally in the Dpp receiving cells of the Drosophila wing, where the major role of Dally is to stabilize Dpp protein. In general, ligands that fail to be retained on the cell surface can have any of the following fates: they may (1) migrate further and act as a ligand somewhere else, (2) be degraded by extracellular proteases, or (3) be internalized by endocytosis and degraded intracellularly. Theoretically, a mixture of all these phenomena can happen at the same time in a given tissue. However, which of these predominates may depend on cellular and extracellular environmental conditions such as concentrations of proteases in the matrix and the rate of endocytosis. Therefore, in the absence of HS, whether a major fraction of ligands is degraded or migrates further can be tissue-dependent, and the effects of HSPGs on BMP gradients will vary depending on the developmental context: a mutant BMP4 molecule moves further in a frog embryo, but DppΔN is degraded in Drosophila wing (Akiyama, 2008)
Although the results presented in this study support a role for HSPGs in Dpp stability, they do not rule out the possible involvement of HSPGs in migration of Dpp protein from cell to cell. The gradient of DppΔN is significantly narrower than that of wild-type Dpp, raising the argument that HS-binding plays a role also in normal Dpp migration in a tissue. Further studies will be required to determine whether or not HSPGs affect morphogen movement. This study also provides new insight into functional aspects of Dpp processing. Mature forms of Dpp generated by differential cleavages are likely to show different affinities for proteoglycans in the matrix. Therefore, they may have different half-lives and/or spatial distribution patterns in vivo. The biological significance of the occurrence of differently processed forms remains to be elucidated (Akiyama, 2008)
Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. This study demonstrates that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. Wg is secreted apically in the disc epithelium and apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action (Gallet, 2008).
Previous studies failed to observe Dlp at the apical surface of the wing disc epithelium despite the fact that it has been extensively shown in vertebrates that GPI-linked proteins are mainly targeted to the apical surface of epithelial cells. By performing extracellular labeling and kinetic experiments, this study demonstrated that Dlp is targeted to the apical surface before being endocytosed and readdressed to the basolateral compartment. Blocking endocytosis allowed demonstration that apical surface accumulation takes place at the expense of its basolateral location, showing that Dlp is first targeted to the apical domain of the epithelium before being sent to the basolateral compartment. The GPI anchor of Dlp is essential for its internalization, as Dlp tethering by a transmembrane domain (e.g., GFP-Dlp-CD2) abolishes its capacity to be internalized. It is also concluded that Dlp apical targeting followed by its rapid internalization is essential for full Hh signal transduction and for shaping the Wg gradient because when Dlp internalization is blocked, both processes are impaired (Gallet, 2008).
Interestingly, a rescue was observed of the first row of dlp mutant cells by the wild-type surrounding cells. Two mechanisms could explain this: (1) GPI-linked proteins are inserted in the outer leaflet of the plasma membrane and are exposed to the extracellular space. Dlp could interact with extracellular proteins located in nearby cells that could aid in flipping Dlp from the outer leaflet of the plasma membrane of one cell to the next, and/or (2) Dlp might be carried by argosomes, which are large extracellular particles resembling low-density lipoproteins containing lipophorins, esterified cholesterol, and triglycerides surrounded by a phospholipid monolayer. These argosomes not only bear GPI-linked proteins such as HSPGs but also morphogens such as Wg or Hh and are able to travel over several rows of cells (Gallet, 2008).
Whereas numerous data have clearly demonstrated the role of the HSPG in Hh signaling through regulation of Hh stabilization, movement, and reception, the function of Dlp in Hh signaling remained unclear. This study clearly demonstrates that Dlp is exclusively necessary in Hh-receiving cells for full-strength Hh signaling. Strong colocalization of Ptc, Hh, and Dlp is observed in endocytic vesicles in Hh-receiving cells. Ptc is probably responsible for the Hh/Dlp internalization, because it was not possible to detect Dlp-Hh-containing endocytic vesicles in the absence of Ptc. Overexpressing Dlp increased the number of Ptc-Hh-internalized vesicles, whereas absence or tethering Dlp at the cell surface (e.g., GFP-Dlp-CD2) lowered the number of Ptc/Hh vesicles. Therefore, it can be imagined that the presence of Dlp increases Ptc-Hh internalization to elicit a high level of pathway activation. Dlp could also stabilize Ptc/Hh within the intracellular compartment, allowing a stronger and/or a longer signaling. The role of Dlp is clearly different from other Hh coreceptors. Indeed, already identified Hh coreceptors such as Ihog and Brother of Ihog (Boi) (Cdo and Boc, respectively, in vertebrates) stabilize Hh at the cell surface, whereas Dlp overexpression does not increase Hh binding on receiving cells in vivo or in vitro (Gallet, 2008).
It has been demonstrated that Hh is apically secreted by wing disc epithelial cells; however, the compartment by which target cells receive Hh signal (e.g., apical, lateral, or basal) remains controversial. Nevertheless, the data suggest that endocytosis from the apical surface is necessary to sustain full-strength Hh signaling because blocking endocytosis inhibits Dlp internalization from the apical surface and decreases Hh signaling activation. Moreover, a colocalization between Hh and extracellular Dlp is observed at the apical side of Hh-receiving cells but not at the basolateral part of the cell. It has also showed previously that in ptc mutant embryos, Hh accumulates at the apical side of receiving cells. Nevertheless, although blocking endocytosis strongly stabilized Dlp at the apical cell surface, it also stabilized Ptc at both poles of the epithelial cells, raising the possibility that part of the signal transduction occurs basally. Unfortunately, it was not possible to see any Hh accumulation at either pole of the receiving cells when endocytosis was blocked. Hence, a model is favored in which Hh can trigger its signal both basally and apically, where the apical signaling is amplified by Dlp and is necessary for a full-strength Hh signal (Gallet, 2008).
Blocking endocytosis impairs Hh signaling both in embryos and in wing discs. However, it has been previously published that inhibiting endocytosis in imaginal discs using a thermosensitive allele of shi (shits) does not impaired Hh signaling: Ci and collier (an Hh target gene) are unaffected but Ptc is stabilized. How can the differences with these data be explained? It is important to note that the shi null allele is cell lethal; therefore, either a dominant-negative or a shits allele, which may not fully inhibit endocytosis, must be used. Accordingly, Ptc stabilization is observed in only 30% of discs, but each time such a stabilization is observed, a decrease of dpp expression was observed. Increasing the level of ShiDN expression increased the penetrance of the phenotype but triggered lethality, making analysis difficult (Gallet, 2008).
Wg is mainly secreted via the apical pole of producing cells. Strikingly, in those cells, Wg is strongly localized in endocytic vesicles that are abundant apically but also in multivesicular endosomes. Dlp overexpression decreases the level of Wg at the apical surface of cells while increasing Wg stability along their lateral compartment. Therefore, it is proposed that Wg is secreted apically and is then endocytosed with the help of Dlp. Once internalized, Dlp targets Wg by transcytosis to the lateral compartment, where it is stabilized and can spread farther away to activate long-range target genes (Gallet, 2008).
Intriguingly, Dlp seems to play antagonistic roles in Wg signaling. Although it inhibits Wg activity near the Wg source, it is also necessary for Wg pathway activation far from the Wg source. This functional duality is directly related to its pattern of expression. Indeed, Dlp is expressed at low levels along the Wg source (e.g., the D/V axis) owing to repression by the Wg pathway itself, whereas it is expressed at higher levels far from the Wg source. The results could explain how Dlp functions antagonistically in Wg signaling. The two Wg receptors DFrizzled2 (Dfz2) and Arrow are involved in both the signal transduction and the internalization of Wg, mainly through the apical surface to shape its gradient by targeting Wg to the lysosome. Therefore, it is proposed that low Dlp-dependent transcytosis of Wg in producing cells and neighboring ones allows a high level of Wg at the apical surface and hence a strong activation of the pathway. On the contrary, higher Dlp-dependent transcytosis of Wg in more distant cells reduces both Wg pathway activation and degradation, promoting Wg movement along the basolateral compartment. Accordingly, it is observed that, in absence of dlp, extracellular Wg is absent from the lateral compartment of distant cells (Gallet, 2008).
This model supposes that Dlp is able to internalize Wg independently of the receptors DFz2 and Arrow. Interestingly, several groups found some internalized Wg in the absence of the Wg receptors Dfz2 and Arrow. Moreover, Dlp overexpression stabilizes Wg at the cell surface at the expense of short-range signaling activity, in accord with the fact that Wg must be endocytosed by Dfz2/Arrow to promote strong signaling. The results further support the view that Wg may form two different complexes: on the one hand a Dlp-Wg complex involved in Wg transcytosis and stabilization, and on the other hand a DFz2-Arrow-Wg signaling complex that shapes the Wg morphogen gradient and signals. Interestingly, when GFP-Dlp-CD2 is overexpressed, although Wg is stabilized at the apical surface over a very long range, where it should activate its pathway, a much stronger inhibition of Wg signaling is observed and an absence of internalized Wg. Therefore, GFP-Dlp-CD2 may titrate Wg from its receptors and prevent internalization, giving rise to both the stabilization of Wg and the inhibition of the pathway. Under physiological conditions, Dlp targeting of Wg to the lateral compartment supports its stabilization and spreading at the expense of its internalization/degradation from the apical surface by its receptors (Gallet, 2008).
Cell fate determination during developmental patterning is often controlled by concentration gradients of morphogens. In the epithelial field, morphogens like the Hedgehog (Hh) peptides diffuse both apically and basolaterally; however, whether both pools of Hh are sensed at the cellular level is unclear. This study shows that interfering with the amount of apical Hh causes a dramatic change in the long-range activation of low-threshold Hh target genes, without similar effect on short-range, high-threshold targets. Genetic evidence is provided that the glypican Dally upregulates apical Hh levels, and that the release of Dally by the hydrolase Notum promotes apical Hh long-range activity. The data suggest that several pools of Hh are perceived in epithelial tissues. Thus, it is proposed that the overall gradient of Hh is a composite of pools secreted by different routes (apical and basolateral), and that a cellular summation of these components is required for appropriate developmental patterning (Ayers, 2010).
Morphogens form long-range concentration gradients to signal positional information to cells within a complex tissue. Within an epithelium, the exact apicobasal cellular position of long-range gradient formation is not well understood. The morphogen Hh appears to be released both apically and basolaterally; however, which pool of Hh represents the functional long-range Hh was previously unknown. These studies have provided firm evidence that the wing imaginal disc uses its apical space to form a long-range gradient of Hh responsible for target gene activation. These data also suggest that short-range activity of Hh may be regulated by Hh released into the basolateral space. This implies that morphogen-receiving cells must integrate the apicobasal value of the extracellular Hh gradient present at different planes (Ayers, 2010).
In addition, it was found that morphogenic long-range gradients are shaped by extracellular matrix proteins. In the case of Hh, the glypican Dally positively regulates apical Hh levels in the producing cells, and Dally release promotes long-range spreading in this plane. Finallywe show direct genetic evidence is shown that the enzyme Notum regulates Dally release, thereby controlling the formation of a functional long-range gradient of Hh. A model is therefore proposed in which Dally controls the apical accumulation of Hh in the Hh-producing cells, and cleavage of Dally by Notum helps to shape the long-range gradient (Ayers, 2010).
It is believed, based on various observations in other organisms, that apical release and formation of a functional long-range Hh gradient may be a conserved mechanism. First, studies in Caenorhabditis elegans and Drosophila embryos have hinted at a role of apical secretion for Hh. Indeed, active apical secretion of exosomes containing Hh-like peptides in C. elegans epithelial cells has been demonstrated. In accordance, this study shows that the protein Dispatched (Disp), which is essential for Hh release in invertebrates and vertebrates, regulates apical Hh release in Drosophila embryos. Moreover, the formation of C. elegans cuticle requires the apical secretion of exosomes by Che14, the homolog of Disp. Shh aggregates in the apical lumen of the chick neural tube have been described, whereas an endogenously tagged Shh:GFP has been used that enabled visualization of the Hh gradient in mouse embryos. At embryonic day 8.5 (E8.5), when Shh is produced solely in the notochord, Shh:GFP (in Nodal Vesicular Particles [NVPs]) was found mostly apically in the neural tube (although the notochord has direct contact with the basal membrane of the neural tube). This apically concentrated Shh:GFP forms a gradient that drops exponentially along the dorsal-ventral axis (i.e., farther from the source). Similar observations have been seen when using antibodies against Shh. Thus, although apical gradients have been described in several organisms, these gradients need to be functionally challenged, something that was developed in this study (Ayers, 2010).
A tradeoff has often been observed between the long-range and the short-range Hh pathway targets; for example, increased range of dpp is often coupled with a decreased En range. Several different explanations for this could be suggested. First, increasing the spreading of Hh apically (for example, in overexpression of Sec:Dally) may result in a lower level of apical Hh close to the source, perhaps below the level required for En expression, while increasing the range of the low level needed for dpp expression farther from the Hh source. However, other data presented in this study do not fit this model. Indeed, a secreted form of Dally (Sec:Dally) did not reduce Hh apical levels at or close to the source, but seemed to just increase and elongate the levels of spreading Hh, especially far in the A compartment, whereas basolateral levels of Hh were reduced. Also, when levels of lumenal Hh were reduced (by blocking Hh on the PPM with Ptc1130x), only a reduction in expression of long-range targets (dpp) was observed, not a change in En or Ptc expression. Furthermore, along with short-range signaling, the basolateral Hh gradient was unchanged in this genotype. Thus, two different secretion/signaling mechanisms of Hh are thought to occur from the Hh-secreting cells to the receiving cells. This tradeoff is often observed between increased dpp expression and decreased En expression when trafficking of Hh in its secreting cells is perturbed (e.g., in ShiDN expression), suggesting that apical and basolateral pools of Hh in the producing cells are not independent. Furthermore, because this tradeoff is also observed when Dally in the P compartment is modified, it seems that Dally partially controls the balance between apical and basolateral Hh gradient formation. The data also indicate that basolateral Hh activity is formed independently of Notum, as short-range signaling is not affected in mutants of this protein. Having said this, it is not ruled out that transcription of short-range targets, such as En, may be a response to reception of both apical and basolateral Hh pools (Ayers, 2010).
Although often decreased, En expression was never lost completely; it is often unaffected in the first row of cells. Therefore, it is proposed that Hh may be secreted to the basolateral membrane, and here may signal to the adjacent receiving cells by both cell-cell contact (resulting in high En expression in the first row of receiving cells) and very limited diffusion (to activate En in up to three cells away from the A-P boundary) (Ayers, 2010).
This study suggests that the Hh that forms a basolateral gradient may be loaded in a different form to that released into the apical lumen. This is because at the lateral position, the Hh trapped by expression of Ptc1130X in the disc proper illustrated a very different staining (highly membranous and nonpunctuate) than that found apically (which was highly punctuate). Also, the presence of basolateral Hh was found in only up to 2-3 cells away from the source, indicating a very limited ability to disperse; this could also be due to a different extracellular environment compared with the apical lumen, where Hh dispersal is higher. Although it may be basolaterally targeted, poorly diffusing Hh that is responsible for En expression in the three rows of cells, this is extremely difficult to prove. Indeed, little is known about polarized trafficking in wing imaginal discs. Although several methods have been described with which it was possible to modify apical Hh levels, no way has been found to specifically enrich or block basolateral Hh in the P compartment without affecting the apical Hh pool. Therefore, it has not been possible to directly test whether it is this gradient of Hh responsible for the pathway activation in the first row of cells in the A compartment (Ayers, 2010).
Evidence has been found for the involvement of the glypican Dally and the hydrolase Notum in the long-range apical spreading of Hh. Reduction of either of these proteins in Hh-producing cells reduces the range within which dpp is expressed, whereas short-range target En is untouched. Furthermore, Dally has been found to positively regulate apical Hh levels, and Dally release aids in long-range spreading of Hh, whereas Notum appears to be essential just for the latter. But what could the exact role of GPI-tethered Dally be in the Hh-secreting cells? It has been shown that the Heparan Sulfate of Glycosaminoglycan chains can bind and sequester both Hh and Lipophorins, which are thought to carry Hh. Therefore, GAG chain binding of Hh at the apical surface may work as a platform by which to stabilize and associate Hh with components necessary for its release and long-range spreading (Ayers, 2010).
In addition to regulating apical Hh accumulation, secretion of Dally appears to play a role in the long-range spreading and activity of Hh. It has been suggested that the secreted form of Dally increased dpp expression due to its mediation of Hh binding to the receptor complex. It cannot be ruled out that secreted Dally could have a role in stabilizing the Hh-receptor complex interaction and signaling, but it is believed that Dally shed from the P compartment is mainly involved in the formation of a long-range Hh gradient, due to changes seen in the apical Hh gradient profile. In addition, if secreted Dally was necessary for Hh-receptor interaction and signaling, then the absence of Dally in the P compartment should affect all Hh target genes. On the contrary, it was found that dally mutant clones in the P cells only affect the expression of the long-range target dpp. If promotion of ligand-receptor interaction was the only role of Dally released from the P compartment, then one would expect expression of secreted Dally to induce a higher level of signaling in the A compartment close to the Hh source, as Hh would be more highly sequestered to its receptors. On the contrary, a decrease is seen in En expression. Therefore, it is proposed that secreted Dally augments movement and extension of the Hh gradient, as opposed to solely increasing signaling (Ayers, 2010).
Lastly, analysis of Notum, a protein described as a GPI anchor cleaver (Traister, 2008), indicates that Notum in the P compartment promotes Hh long movement through its regulation of Dally. Therefore, it is proposed that after apical accumulation and clustering of Hh by GPI-anchored Dally, Notum cleaves and releases Dally, preparing Hh for its long apical voyage (Ayers, 2010).
Hedgehog (Hh) acts as a morphogen in various developmental contexts to specify distinct cell fates in a concentration-dependent manner. Hh signaling is regulated by two conserved cell-surface proteins: Ig/fibronectin superfamily member Interference hedgehog (Ihog) and Dally-like (Dlp), a glypican that comprises a core protein and heparan sulfate glycosaminoglycan (GAG) chains. Dlp core protein can interact with Hh and is essential for its function in Hh signaling. In wing discs, overexpression of Dlp increases short-range Hh signaling while reducing long-range signaling. By contrast, Ihog has biphasic activity in Hh signaling in cultured cells: low levels of Ihog increase Hh signaling, whereas high levels decrease it. In wing discs, overexpression of Ihog represses high-threshold targets, while extending the range of low-threshold targets, thus showing opposite effects to Dlp. It was further shown that Ihog and its family member Boi are required to maintain Hh on the cell surface. Finally, Ihog and Dlp have complementary expression patterns in discs. These data have led to a proposal that Dlp acts as a signaling co-receptor. However, Ihog might not act as a classic co-receptor; rather, it may act as an exchange factor by retaining Hh on the cell surface, but also compete with the receptor for Hh binding (Yan, 2010).
Previous studies have shown that Dlp is specifically required for Hh signaling in cell-based assays and in embryos. However, the molecular basis of this specificity was unknown. This study shows that overexpression of Hh can restore naked cuticle in sugarless (sgl) and sulfateless (sfl) embryos, but not in dlp embryos. It was further demonstrated that the specificity of Dlp in Hh signaling results from its core protein. The Dlp core protein can restore Hh signaling autonomously in dlp embryos and in dlp-RNAi cells. In addition, the Dlp core protein interacts with Hh and promotes Hh signaling in the disc. Overexpression of Dlp increases Hh signaling strength, but reduces signaling range as well as Hh gradient range. These data suggest that the Dlp core protein could act as a classic co-receptor in Hh signaling by facilitating Hh-Ptc interaction (Yan, 2010).
Recent studies have shown that the vertebrate glypican-3 core protein directly promotes Wnt signaling in cancer cells, but inhibits sonic hedghog signaling during development. That the same glypican has opposite effects on Wnt and Hh is interesting because Dlp can inhibit high-threshold Wg signaling when overexpressed in discs. In addition to the essential role of the core protein, the attached GAG chains are important for the non-cell-autonomous functions of Dlp. The current results demonstrated that wild-type Dlp can rescue non-cell-autonomously in dlp embryos, whereas the core protein mainly acts in its expression domains. Interestingly, the CD2 forms of Dlp also lose the non-autonomous activity in dlp embryos. Several studies suggest that the GPI anchor of Dlp can be cleaved by the hydrolase Notum and that the GAG chains of Dlp can recruit lipoprotein particles. Thus, it will be interesting to determine the mechanism of Dlp non-autonomous activity (Yan, 2010).
This study suggests that the GPI anchor of Dlp is not essential for its activity in Hh signaling. Most importantly, two CD2 forms of Dlp, Dlp(-HS)-CD2 and GFP-Dlp-CD2, can effectively rescue Hh signaling in dlp embryos. In addition, CD2 forms of Dlp can also signal in cultured cells and discs. It is important to note that although the GPI, but not the CD2, form of Dlp is colocalized with Ptc in intracellular vesicles, both forms have similar activities in promoting Hh signaling in the disc. These data argue that colocalization of Dlp with Ptc in endocytic vesicles is not essential for Dlp function in Hh signaling. Consistent with this view, several studies have shown that endocytosis is not essential for Hh signaling in Drosophila wing discs. The current conclusion differs from a recent publication arguing that the GPI anchor of Dlp is required for its function in Hh signaling (Gallet, 2008). In that study, it was shown that overexpression of GFP-Dlp-CD2 can reduce Hh signaling in the wing discs. However, this study did not observe any dominant-negative effect of GFP-Dlp-CD2, which can rescue dlp mutant embryos and enhance Hh signaling strength in cells and discs. A possible explanation for the discrepancy is that expression of Dlp enhances signaling strength but also reduces signaling range. ap-Gal4 was always used, allowing use of the ventral disc as an internal control. The observed dominant-negative effect might reflect the reduced signaling range rather than signaling strength (Yan, 2010).
Previous studies in both Drosophila and vertebrates have demonstrated positive roles of the Ihog family proteins in Hh signaling. Ihog and Ptc synergize in mediating Hh binding to cells. These observations suggest that Ihog functions as a co-receptor for Hh. However, the new data argue that Ihog does not simply act as a classic co-receptor that only increases the binding of ligand to the signaling receptor. By altering Ihog levels in vivo and in vitro, it was shown that Ihog has biphasic activity in Hh signaling, with too much or too little Ihog leading to reduced signaling. Overexpression of Ihog leads to the accumulation of Hh, and knockdown of Ihog results in reduced Hh levels, suggesting that one major activity of Ihog is to retain Hh on the cell surface. Moreover, knockdown of both Ihog and Boi dramatically reduces Hh levels and signaling. This reduction of Hh signaling activity is likely to be due to an absence of Hh on the cell surface of the double-mutant tissue. However, a high level of Ihog causes a large amount of Hh to accumulate on the cell surface, but also reduces signaling strength. One explanation for this result is that Ihog can compete with Ptc for Hh binding. Thus, a low level of Ihog is required to maintain Hh on the cell surface, whereas a high level of Ihog can sequester Hh from its receptor. In other words, depending on the context, Ihog can either provide Hh for the receptor by retaining Hh on the cell surface, or compete with the receptor for Hh binding. This activity of Ihog is very similar to the recently proposed 'exchange factor' model, which allows the exchange of Hh between Ptc and Ihog. A recent study has demonstrated that Ihog also interacts with Ptc and that Ihog, Ptc and Hh form a triple complex (Zheng, 2010). The close association between the Ihog and Ptc receptors may thus allow them to exchange Hh ligand. It will be important to determine whether the triple complex has a greatly reduced ability to signal, a prediction from the mathematical exchange factor model (Yan, 2010).
Although this is the first demonstration of a biphasic co-factor in Hh signaling, similar biphasic co-factors have been reported. Drosophila Cv-2 enhances BMP signaling at low concentrations, but inhibits signaling at high concentrations. Syndecan-1 shows a similar concentration-dependent activation or inhibition of BMP signaling in Xenopus. Interestingly, Dlp has biphasic activity in Wg signaling, depending on its protein levels. All these co-factors are likely to act by a similar mechanism, suggesting that the biphasic co-factor is a recurring motif in different morphogen systems (Yan, 2010).
This study has shown that Dlp and Ihog play distinct roles in Hh signaling. Expression of Dlp enhances Hh signaling strength, but reduces signaling range. By contrast, expression of Ihog reduces Hh signaling strength, but extends signaling range. In addition, the Dlp level is elevated in the high Hh signaling area, whereas the Ihog level is reduced in that region. It is important to consider from a system point of view what these two co-factors provide for the Hh morphogen. For morphogens to work, they should be able to generate sharp boundaries between target genes with different thresholds. They also need to diffuse over a certain range without being lost in the extracellular space. The positive feedback of Dlp expression in the high Hh signaling areas helps to sharpen the boundaries between high- and low-threshold target genes. The negative-feedback regulation of Ihog might ensure that a strong Hh signal is attained in the areas close to the Hh source and also allow the Hh gradient to diffuse to those areas distant from the source (Yan, 2010).
Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. This paper reports the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and a localized region on the Dlp surface important for mediating its function in Hh signaling was identified. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex (Kim, 2011).
Glypicans modulate the activity of multiple growth factors active
during development, and defects in glypican function lead to
widespread and diverse developmental malformations. Much of the activity of glypicans can be attributed to interactions between their heparan sulfate attachments and heparan-binding growth factors, but recent work has demonstrated important functional roles for the protein cores of glypicans, notably in
Hh and Wnt signaling. In particular, the protein cores
seem likely to mediate functions that are specific to particular
glypicans. The crystal structure of the N-terminal
globular region of a glypican, DlpδNCF, adopts an
elongated, all α-helical fold with no evident homology to previously
determined structures. The high level of sequence conservation
among the N-terminal protein cores of glypicans (greater
than 40% sequence identity exists between Drosophila and
human glypicans in this region) indicates that the DlpδNCF structure provides a sound basis for the design and interpretation of
experiments with other glypicans. The absence of any
apparent active site-like cavities or sources of conformational
flexibility in the DlpδNCF structure suggests that the protein
regions of glypicans exert their effects by serving as binding
proteins, consistent with proposed roles as coreceptors or in targeting
ligands to specific subcellular compartments (Kim, 2011).
One reason Dlp has been proposed as an Hh coreceptor is the
ability of Dlp lacking heparan sulfate to coimmunoprecipitate
with Hh. The inability to detect a high-affinity interaction
between DlpδNCF and HhN using purified proteins suggests that
Dlp may interact with Hh as part of a larger complex and additional
factors are needed to promote Dlp/Hh interactions.
One candidate for such a factor is the Hh coreceptor Ihog, which
is an essential component of the Hh receptor complex, but
this study shows that purified DlpδNCF does not form a high-affinity
complex with either an active fragment of Ihog (IhogFn12) or an
HhN:IhogFn12 complex. This result does not rule out Ihog as
important for mediating Hh:Dlp interactions but suggests that
an additional factor or factors may be needed. An obvious candidate
for such a factor is Patched, a key cell-surface component
of the Hh signaling pathway, but assessing its role in Hh-containing
complexes awaits purification of suitable amounts of this
12-pass integral membrane protein. An additional element complicating
interpretation of results of studies of receptor-ligand
interactions in solution arises from the absence of membrane
tethering and ligand multivalency. Restricting components to a
membrane surface orients them and greatly enhances their local
concentration, and a multivalent ligand greatly increases the
avidity of binding. Interactions between a monovalent
ligand and cell-surface components may thus not be strong
enough to be observed with soluble components in solution (Kim, 2011).
The inability to observe an interaction between ShhN and the
N-terminal domain of glypican-3 is puzzling, however, given that
the protein region of glypican-3 has been reported to bind to
ShhN with nanomolar affinity. Several possibilities may
explain this discrepancy: (1) ShhN may interact with the glypican-
3 stalk region, which was present in the earlier study but
not in the current experiment; (2) attachment of histidine-tagged ShhN
to Ni-NTA agarose may have blocked a glypican interaction site
in these studies; or (3) an unidentified cofactor that promotes
high-affinity interaction was present in the earlier studies but absent
in the earlier studies. Calcium ions, for example, are required to
promote high-affinity interactions between ShhN and CDO, the mammalian homolog of Ihog (Kim, 2011).
The ability to identify a localized region on the C lobe of the
Dlp surface important for proper Dlp function in Hh signaling is
further consistent with Dlp participating in Hh signaling primarily
as a binding protein, although the nature and number of binding
partners remains to be determined. Curiously, the identified surface
is composed largely of hydrophilic residues, which is unusual
for protein-protein interfaces. This surface also occurs on
the opposite surface of Dlp relative to the disordered Dlp-specific
insertion that follows the furin-like processing site, suggesting
that interactions mediated by this region are likely independent
of furin-like processing and this insertion. Glypican mutations
previously associated with functional impairments, for example
those in glypican-3 that cause Simpson-Gohlabi-Behmel syndrome and those in glypican-6 that cause omodysplasia, appear to result in severe truncations or complete loss of expression of the affected glypican. Whether the C-lobe region
identified on Dlp is generally involved in glypican function
or is specific to Dlp or positive regulators of Hh signaling is
an interesting question for future investigation. The results presented
in this study establish a molecular foundation to guide design and
interpretation of studies investigating the molecular bases of glypican function (Kim, 2011).
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