The Dpp pathway is regulated by multiple cell-surface and extracellular factors (O’Connor et al., 2006
). 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) ( Fujise et al., 2003
; Belenkaya et al., 2004
). Dally and Dpp expressed in S2 tissue culture cells are coimmunoprecipitated, suggesting that Dally forms a complex with Dpp (; (Kirkpatrick et al., 2006
)). We also observed that Dally colocalizes with Dpp and Tkv in cells (Akiyama and Takeo, unpublished result). In addition, we previously demonstrated that Dally enhances Dpp signaling in a cell autonomous fashion (Fujise et al., 2003
). These findings suggest that Dally acts as a Dpp co-receptor at least in some developmental contexts (Kramer and Yost, 2003
). 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.
We demonstrated that DppΔN
, which does not bind to heparin, failed 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 (Kirkpatrick et al., 2006
). 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.
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: we were able to use this molecule 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 (, ; (Fujise et al., 2003
)). 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.
Our 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 (Lecuit and Cohen, 1998
; Tanimoto et al., 2000
; Fujise et al., 2003
). Nevertheless, tkv
mutually suppress one another’s pMad gradient phenotypes. Consistent with the genetic interactions observed in dally
mutants, the pMad phenotype produced by overexpression of tkv
was significantly restored by coexpression of dally
. These observations indicate that dally
in Dpp signaling. Since it has been proposed that Tkv promotes Dpp degradation by receptor-mediated endocytosis (Entchev et al., 2000
; Teleman and Cohen, 2000
may stabilize Dpp by inhibiting this process.
Altogether, our 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 (Fujise et al., 2003
). However, we cannot exclude the possibility that DppΔN
is lost from the cell surface by lack of retention and further diffuses away.
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 (Sperinde and Nugent, 2000
). 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 (Marois et al., 2006
). Wg protein is endocytosed from both apical and basal surfaces of the wing disc and degraded by cells to down-regulate the levels of Wg protein in the extracellular space. Marois et al. (2006)
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.
behaved differently in vivo from the previously reported mutant Xenopus
BMP4 lacking the heparin-binding site (Ohkawara et al., 2002
). 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
Although the results presented here 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 ( and ), 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. Our 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.