Wnt and Hedgehog: Secretion of Lipid-Modified Morphogens

Abstract

Morphogens are signaling molecules produced by a localized source, specifying cell fate in a graded manner. The source secretes morphogens into the extracellular milieu to activate various target genes in an autocrine or paracrine manner. Here we describe various secreted forms of two canonical morphogens, the lipid-anchored Hedgehog (Hh) and Wnts, indicating the involvement of multiple carriers in the transport of these morphogens. These different extracellular secreted forms are likely to have distinct functions. Here we evaluate newly identified mechanisms that morphogens use to traverse the required distance to activate discrete paracrine signaling.

Keywords: Hh; Wg; Wnt; cancer; exosomes; lipoprotein; tissue patterning; vesicles.

Figure I. Cartoon Representing Different Molecular Mechanisms of Exovesicle Biogenesis.

Figure 1. Hedgehog (Hh) and Wnt Signaling in the Drosophila Wing Disc and Vertebrate Neural Tube.

(A) Cartoon representing Hh signaling in wing discs. Hh is produced in the posterior domain (green; P) and generates a concentration gradient (arrows) along the anterior domain (white; A). Engrailed (En) and Patched (Ptc) are short-range targets of Hh, expressed by up to three or four cells abutting the AP boundary. Collier (Col) requires intermediate Hh levels, whereas Decapentaplegic (Dpp) and Iroquois (Iro) are long-range targets, expressed ∼10–15 cells away along the AP boundary. Stabilization of cubitus interruptus (Ci¹⁵⁵) requires the lowest levels of Hh. Higher levels of this protein are seen near the AP boundary, while cells away from the AP boundary see lower levels of stabilized Ci. (B) Cartoon representing Wingless (Wg) signaling in wing discs. Wg expression is seen at the dorsoventral (DV) boundary in a narrow strip of cells and at the boundary of the wing pouch (blue). Wg forms an extracellular gradient on either side of the DV boundary (arrows) and activates targets in a concentration-dependent manner. Senseless (Sens) requires high Wg levels and Distalless (Dll) intermediate levels with graded expression, while Vestigial (Vg) is a lowest-threshold target. (C) Sonic hedgehog (Shh) (green) and Wnt (blue) during vertebrate neural tube pattering. These emanate from two sources: dorsally (BMPs/Wnts from the epidermis) and ventrally (Shh from the notochord). The roof plate (RP) and floor plate (FP) form the secondary signaling centers, which also secrete BMP/Wnt and Shh, respectively. They form reciprocal gradients to specify specific neural progenitors. Wnt/BMPs specify the dorsal sensory neurons (dI1–dI5) while Shh specifies the ventral motor neurons; highest to lowest: v3, MN, v2, v1, and v0.

Figure 2. Molecular Players Involved in Exovesicular Release of Hedgehog (Hh) and Wingless (Wg).

Hh (in green) and Wg (in yellow) are secreted from the Golgi apparatus and anchored on the cell membrane via their lipid modifications. Exosomal release requires endocytosis of Hh (via Shibire, Dispatched) and Wg (maybe mediated by Evi; precise role unclear) with sorting in Rab5-containing early endosomes (EEs). From EEs, Hh and Wg are sorted onto intraluminal vesicles (ILVs) by inward budding of multivesicular body (MVB)-limiting membrane. The endosomal sorting complex required for transport (ESCRT) machinery is required for release of Sonic hedgehog (Shh) (Tsg101, Shrub, and Vps4) and Wg (Hrs, Tsg101, and Vps28) on exosomes. Fusion of the MVB with the cell surface results in the release of ILVs in the form of exosomes: Rab27 for Hh, while for Wg, Rab11, syntaxin-1A, and Ykt-6 are thought to be involved in MVB fusion. Endocytosed Hh fails to populate tubular lysosomes, which are known for their role in the degradation of endocytic cargo; in the case of Wg, this possibility has not been probed (marked ??). Besides exosomes, Hh and Wg can also be secreted on microvesicles by plasma membrane budding. In the case of Hh, cell-surface accumulation of Hh on the expression of a dominant-negative form of Vps4 has led to the proposition of microvesicle-mediated Hh secretion; however, for Wg exovesicles this mode of secretion has not yet been proposed (marked ??). Molecular players involved in exovesicular secretion and identified in vitro and in vivo/in vivo, are highlighted in blue text; molecules identified in specific cell types or in cell-based assays only are in red text.

Figure 3. Transport of Morphogens Across Tissue: Multiplicity of Carriers.

(A) Cartoon representing the various extracellular forms of Hedgehog (Hh) and their polarized transport. Hh secretion and transport are seen on both the apical and the basolateral side of the Drosophila wing imaginal disc. On the apical side, Hh is secreted on exovesicles in the lumen, which is necessary for its long-range transport. Additionally, a carrier involving Hh and Dally (cleaved by Notum) is also secreted in the apical lumen and is necessary for long-range signaling by Hh. Basally, Hh-containing exovesicles are seen predominantly on cytonemes, which are filopodial structures. The hemolymph is in contact with the wing imaginal disc on the basal side. Hemolymph contains circulating lipoprotein particles. Hh interacts with lipoproteins, which in turn act as carriers for long-range transport. The identity of a short-range carrier involved in Hh signaling remains elusive and we speculate that the soluble forms of Hh could be crucial for transporting Hh at short distances to activate high-threshold targets like Patched (Ptc). (B) Cartoon representing the various extracellular forms of Wingless (Wg). Wg is also ferried by multiple carriers to facilitate its long-range spread. Secreted complexes of Wg with Flotillin and Swim are necessary for its long-range transport. Wg is also transported basolaterally on lipoprotein particles, which help it to spread over long distances. Exovesicles carrying Wg are necessary for its short- as well as long-range transport, thus differentiating them from exovesicles, which are uniquely employed only for long-range Hh spread.