dispatched

EVOLUTIONARY HOMOLOGS

Sensory organs are often composed of neuronal sensory endings accommodated in a lumen formed by ensheathing epithelia or glia. Lumen formation in the C. elegans amphid sensory organ requires the gene daf-6. daf-6 encodes a Patched-related protein that localizes to the luminal surfaces of the amphid channel and other C. elegans tubes. While daf-6 mutants display only amphid lumen defects, animals defective for both daf-6 and the Dispatched gene che-14 exhibit defects in all tubular structures that express daf-6. Furthermore, DAF-6 protein is mislocalized, and lumen morphogenesis is abnormal, in mutants with defective sensory neuron endings. It is proposed that amphid lumen morphogenesis is coordinated by neuron-derived cues and a DAF-6/CHE-14 system that regulates vesicle dynamics during tubulogenesis (Perens, 2005).

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA results in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor Patched and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (Carstea, 1997).

An integrated human-mouse positional candidate approach was used to identify the gene responsible for the phenotypes observed in a mouse model of Niemann-Pick type C (NP-C) disease. The predicted murine NPC1 protein has sequence homology to the putative transmembrane domains of the Hedgehog signaling molecule Patched, to the cholesterol-sensing regions of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and SREBP cleavage-activating protein (SCAP), and to the NPC1 orthologs identified in human, the nematode Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae. The mouse model may provide an important resource for studying the role of NPC1 in cholesterol homeostasis and neurodegeneration and for assessing the efficacy of new drugs for NP-C disease (Loftus, 1997).

Niemann-Pick type C disease is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to the lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. It is concluded that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol (Neufeld, 1999).

The secreted protein Sonic hedgehog exerts many of its patterning effects through a combination of short- and long-range signaling. Three distinct mechanisms, which are not necessarily mutually exclusive, have been proposed to account for the long-range effects of Shh: simple diffusion of Shh, a relay mechanism in which Shh activates secondary signals, and direct delivery of Shh through cytoplasmic extensions, termed cytonemes. Although there is much data (using soluble recombinant Shh [ShhN]) to support the simple diffusion model of long-range Shh signaling, there has been little evidence to date for a native form of Shh that is freely diffusible and not membrane-associated. Evidence is provided for a freely diffusible form of Shh (s-ShhNp) that is cholesterol modified, multimeric and biologically potent. The availability of s-ShhNp is regulated by two functional antagonists of the Shh pathway: Patched (Ptc) and Hedgehog-interacting protein (Hip). A gradient of s-ShhNp across the anterior-posterior axis of the chick limb is shown, demonstrating the physiological relevance of s-ShhNp (Zeng, 2001).

To address how s-ShhNp, a protein with two lipophilic modifications, is soluble in an aqueous environment it is proposed that Shh might bind additional proteins to bury its hydrophobic moieties. Conditioned media was isolated from cells transfected with full-length Shh or ShhN and subjected to analysis by gel filtration chromatography. These conditioned media were isolated under serum-free conditions in which they are still biologically active to verify that s-ShhNp is not just binding to proteins present in serum. s-ShhNp migrates at about six times its native molecular weight. In contrast, ShhN migrates through the column close to its predicted molecular weight. To determine whether Shh can multimerize with itself to form the large Shh complex, s-ShhNp was immunoprecipitated from the conditioned media of cells co-transfected with Shh and a Flag-tagged Shh construct. Antibodies to the Flag epitope are able to co-immunoprecipitate untagged Shh, suggesting that Shh multimerizes with itself (Zeng, 2001).

It is speculated that mammalian homologs of the Drosophila protein Dispatched (Disp) might be involved in either the packaging of s-ShhNp or the targeting of ShhNp to lipid rafts, given that amorphic disp mutants are insensitive to HhNp. Additionally, Tout velu (TTV), an enzyme involved in heparin sulphate biosynthesis, has been implicated as a necessary component in Hh-receiving cells. Although the function that mammalian TTV homologs have in Shh target cells has not been analyzed, biochemical data is presented consistent with the proposed function of TTV in Drosophila. That is, ttv mutations are insensitive to HhNp but responsive to HhN, suggesting that Hh's lipid modifications are necessary for TTV to exert its normal biological effects. There is a central difference between ShhN and ShhNp: ShhNp can form large, stable multimers whereas ShhN cannot. Therefore, TTV homologs might regulate the biosynthesis of a molecule that is necessary to recognize s-ShhNp but is not necessary for signaling by ShhN (Zeng, 2001).

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, recessive ENU-induced mutations were identified in six genes that prevent normal specification of ventral cell types in the spinal cord. The genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components, were identified and cloned. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. These data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh (Caspary, 2002).

In contrast to Drosophila disp, which is expressed ubiquitously in embryos and imaginal discs, Disp1 shows a restricted expression pattern. At e8.5–9.5 Disp1 is expressed in tissues that require Hh signaling, including the notochord, ventral neural tube, somites, branchial arches, and limb buds. The pattern of expression of Disp1 is restricted by e10.5 to high levels in head mesenchyme, neural tube, somites, and limb ectoderm. While Disp1 expression generally correlates with tissues known to possess active Hedgehog signaling, it is interesting to note that the Disp1 expression becomes localized to the anterior half of the forelimb buds, away from the posterior source of Shh (Caspary, 2002).

Disp1 is a 1521 aa protein with 10–13 transmembrane domains, including a sterol sensing domain (SSD). Topology programs predict that C829 lies in a large extracellular loop just C-terminal to the SSD. This extracellular loop in Disp1 contains six cysteines, including C829, that are conserved in all dispatched family members. The cysteine-to-phenylalanine change in the icb mutation (the mutation in Disp1) is likely to disrupt a disulfide bond and cause destabilization of either the loop or the protein. Because this mutation should disrupt Disp1 function and because icb blocks Shh signaling at a point between Shh and expression of Ptch1, the step likely to be affected by Disp1, it is concluded that the missense mutation blocks most or all function of Disp1 and is responsible for the icb phenotype. Thus, although there are two Ptch genes and two Disp genes in the mouse, it appears that, in both cases, only one of the homologous genes plays a central role in Hedgehog signaling (Caspary, 2002).

Drosophila disp is required in Hh-producing cells to allow release of active Hh, and clones of homozygous disp mutant cells in imaginal discs appear to accumulate high levels of Hh protein. The distribution of Shh in embryos at e9.5 was examined by confocal microscopy. In wild-type embryos, Shh protein made in the notochord spreads to the ventral neural tube and then activates Shh expression in the floor plate. High levels of Shh protein are present in the notochord cells of Disp1^icb embryos; however, there is no detectable Shh protein in the ventral neural tube. Thus, the Shh made in the Disp1^icb notochord fails to induce Shh expression in the ventral neural tube. Given that Disp1^icb acts upstream of Patched, the results suggest that mouse Disp1, like Drosophila disp, is required for the spread of Shh ligand from the cells where it is synthesized. Further, the resemblance of the Shh Ihh, Smo, and Disp1^icb phenotypes suggests that Disp1 is required for the spread of both Shh and Ihh ligands (Caspary, 2002).

The Shh expression pattern revealed an interesting difference between the Disp1^icb and the Smo^bnb mutant phenotypes: the notochord in Disp1^icb is intact and expresses high levels of Shh protein, whereas the notochord in Smo^bnb is small and expresses lower levels of Shh. The notochord begins to degenerate at e9.0 in Shh mutants, similar to what is observed in Smo^bnb embryos. Thus, Shh signaling is required, directly or indirectly, for maintenance of Shh expression in the notochord. The robust notochord of e9.0 Disp1^icb mutants suggests that Disp1 is not required for Shh activity within the notochord. Similarly, Drosophila Hh produced in posterior compartment wing cells can activate the signaling pathway locally, but not at a distance, in the absence of disp. Together, the results suggest that Disp1 is not required for juxtacrine signaling by Shh and is specifically required for release of Shh to an extracellular compartment from which Shh can move to more distant cells. Long-range signaling by Shh is important for patterning the mouse neural tube, somites, and limb bud. In contrast to the case in Drosophila, where disp is expressed ubiquitously, localized expression of Disp1 in the mouse embryo could play a decisive role in determining where Shh can act at a distance (Caspary, 2002).

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development, which involves both short- and long-range signaling from localized Hh sources. One unusual aspect of Hh signaling is the autoproteolytic processing of Hh followed by lipid modification. As a consequence, the N-terminal fragment of Hh becomes membrane anchored on the cell surface of Hh-producing cells. A key issue in Hh signaling is to understand the molecular mechanisms by which lipid-modified Hh protein is transported from its sites of synthesis and subsequently moves through the morphogenetic field. The dispatched gene, which encodes a putative multipass membrane protein, was initially identified in Drosophila and is required in Hh-producing cells, where it facilitates the transport of cholesterol-modified Hh. The mouse dispatched (Disp) gene has now been identified. The complete Disp cDNA (4721bp) encodes a predicted protein of 1521 amino acids with a relative molecular mass of 170,047. Both Disp and a second gene, Disp-related, encode proteins with twelve predicted membrane-spanning domains as well as stretches of sequences similar to a conserved domain known as the sterol sensing domain (SSD). Proteins containing the SSD include several classes of proteins that are involved in different aspects of cholesterol homeostasis or cholesterol-linked signaling. Notably, Ptch, the Hh receptor, also contains an SSD. Disp-null mice phenocopy mice deficient in the smoothened gene, an essential component for Hh reception, suggesting that Disp is essential for Hh signaling. This conclusion is further supported by a detailed molecular analysis of Disp knockout mice, which exhibit defects characteristic of loss of Hh signaling. Evidence is provided that Disp is not required for Hh protein synthesis or processing, but rather for the movement of Hh protein from its sites of synthesis in mice. Taken together, these results reveal a conserved mechanism of Hh protein movement in Hh-producing cells that is essential for proper Hh signaling (Kawakami, 2002).

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. One of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. A biochemical assay has been developed that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins (Ma, 2002).

Perhaps the most striking and unexpected aspect of these results is the extreme nature of the pattern disruptions in mDispA^-/- embryos. The mDispA^-/- mutant phenotype is more severe than that of mutations in any single gene encoding a Hh protein. In contrast, the Drosophila disp mutant phenotype is less severe than the hh phenotype. This discrepancy appears in part due to disruption of signaling by multiple Hh proteins, because the mDispA^-/- phenotype resembles that of Smo^-/- and that of the Shh^-/-; Ihh^-/- double mutant. However, the phenotype also appears to owe its severity, at least in part, to a distinct balance in the relative importance of long-range and short-range signaling in Drosophila and in the mouse. In Drosophila disp mutants, short-range Hh signaling is intact and contributes to maintenance of target gene expression and to patterning. In mDispA^-/- embryos, some signal response is retained in cells that express Shh (Ptch-lacZ is expressed in the notochord), but this response appears not to contribute to morphological pattern. The null function phenotype for the mDispA gene thus permits dissection of the relative importance of long- and short-range Hh signaling in mouse embryos and reveals a near absolute dependence on long-range signaling in patterning of the mouse embryo (Ma, 2002).

Polarized trafficking of proteins is critical for normal expression of the epithelial phenotype, but its genetic control is not understood. The putative zinc finger transcription factor lin-26 is essential for normal epithelial differentiation in the nematode Caenorhabditis elegans. To identify potential effectors of lin-26, mutations that result in lin-26-like phenotypes have been characterized. The phenotypic and molecular analysis of one such mutant line, che-14, is reported. Mutations in che-14 result in several partially penetrant phenotypes affecting the function of most epithelial or epithelial-like cells of the ectoderm, including the hypodermis, excretory canal, vulva, rectum and several support cells. The defects are generally linked to the accumulation of vesicles or amorphous material near the apical surface, suggesting that secretion is defective. The CHE-14 protein shows similarity to proteins containing sterol-sensing domains, including Dispatched, Patched and NPC1. A fusion protein between full-length CHE-14 and the green fluorescent protein becomes localized to the apical surface of epithelial cells that require che-14 function. Deletions that removed the predicted transmembrane domains or extracellular loops of CHE-14 abolish apical localization and function of the protein. It is proposed that CHE-14 is involved in a novel secretory pathway dedicated to the exocytosis of lipid-modified proteins at the apical surface of certain epithelial cells. These data raise the possibility that the primordial function of proteins containing a sterol-sensing domain is to control vesicle trafficking: CHE-14 and Dispatched in exocytosis, Patched and NPC1 in endocytosis (Michaux, 2000).

Genetic analyses in Drosophila have demonstrated that a transmembrane protein Dispatched (Disp) is required for the release of lipid-modified Hedgehog (Hh) protein from Hh secreting cells. Analysis of Disp1 null mutant embryos has demonstrated that Disp1 plays a key role in hedgehog signaling in the early mouse embryo. A hypomorphic allele in Disp1(Disp1Delta²), was used to extend the knowledge of Disp1 function in Hh-mediated patterning of the mammalian embryo. Through genetic combinations with null alleles of patched 1 (Ptch1), sonic hedgehog (Shh) and Indian hedgehog (Ihh), it has been demonstrated that Disp1 genetically interacts with Hh signaling components. Since Disp1 activity is decreased a progressive increase in the severity of hedgehog-dependent phenotypes is seen, that is further enhanced by reducing hedgehog ligand levels. Analysis of neural tube patterning demonstrates a progressive loss of ventral cell identities that most likely reflects decreased Shh signaling, since Disp1 levels are attenuated. Conversely, increasing available Shh ligand by decreasing Ptch1 dosage leads to the restoration of ventral cell types in Disp1Delta2/Delta2 mutants. Together, these studies suggest that Disp1 actively regulates the levels of hedgehog ligand that are available to the hedgehog target field. Further, they provide additional support for the dose-dependent action of Shh signaling in patterning the embryo. Finally, in-vitro studies on Disp1 null mutant fibroblasts indicate that Disp1 is not essential for membrane targeting or release of lipid-modified Shh ligand (Tian, 2004).

Disp1 function is essential for Shh and Ihh signaling in the mouse, and Disp1 gene dose regulates the level of Shh signaling activity in vivo. To determine whether Disp1 activity is required in Shh-producing cells for paracrine signaling in Shh target fields, a Shh^GFP-Cre (here shortened to Shh^Cre) knock-in allele and a Disp1 conditional allele were used to knock down Disp1 activity specifically within Shh-producing cells. The resulting facial and neural tube phenotypes support the conclusion that the primary and probably exclusive role for Disp1 is within hedgehog protein-producing cells. Furthermore, using an allele that produces N-Shh (a noncholesterol modified form of the Shh protein), it has been demonstrated that N-Shh is sufficient to rescue most of the early embryonic lethal defects in a Disp1-null mutant background. Thus, Disp1 activity is only required for paracrine hedgehog protein signaling by the cholesterol modified form of Shh (N-Shhp), the normal product generated by auto-processing of a Shh precursor protein. In both respects, Disp function is conserved from Drosophila to mice (Tian, 2005).

Disp1 function is essential for Shh and Ihh signaling in the mouse, and Disp1 gene dose regulates the level of Shh signaling activity in vivo. To determine whether Disp1 activity is required in Shh-producing cells for paracrine signaling in Shh target fields, a Shh^GFP-Cre (here shortened to Shh^Cre) knock-in allele and a Disp1 conditional allele were used to knock down Disp1 activity specifically within Shh-producing cells. The resulting facial and neural tube phenotypes support the conclusion that the primary and probably exclusive role for Disp1 is within hedgehog protein-producing cells. Furthermore, using an allele that produces N-Shh (a noncholesterol modified form of the Shh protein), it has been demonstrated that N-Shh is sufficient to rescue most of the early embryonic lethal defects in a Disp1-null mutant background. Thus, Disp1 activity is only required for paracrine hedgehog protein signaling by the cholesterol modified form of Shh (N-Shhp), the normal product generated by auto-processing of a Shh precursor protein. In both respects, Disp function is conserved from Drosophila to mice (Tian, 2005).

Evidence for a role of vertebrate Disp1 in long-range Shh signaling

Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures with Shh-expressing cells revealed a significant reduction in the range of the contact-dependent Shh response in Disp1^-/- neuralized EBs. These observations support a dual role for Disp1, not only in the secretion of Shh from the source cells, but also in the subsequent transport of Shh through tissue (Etheridge, 2010).

These results suggest that Ptch1 and Disp1 act in concert to mediate the transport of Shh through tissues. The similarities between Ptch1 and Disp1, such as their ability to trimerize and their putative proton channel, indicates that their function might be conserved with that of the resistance-nodulation-cell division (RND) family of proton-driven transporters in bacteria. In general, the role of Disp1 is in the secretion of Shh, whereas Ptch1 is involved in the uptake of Shh, and the function of both is necessary for long-range Shh signaling. These observations are consistent with a model in which reiterated secretion (by Disp1) and uptake (by Ptch1) are involved in the long-range transport of Shh. The non-directionality of this process, combined with the incomplete secretion of all internalized Shh, would sufficiently distribute Shh in a gradient away from the source (Etheridge, 2010).

Based on these results the following model is proposed. Disp1 is active in MVBs and mediates the loading of Shh onto exosome/lipoprotein-like particles, which are then secreted. These particles are specifically recognized by Ptch1 at the surface of adjacent cells, which traffics them into early/late endosomes, where the particles are disassembled. Shh can either be degraded, trafficked to the apical surface or trafficked into MVBs, where it would be loaded onto exosomes again for re-secretion. This model accounts for the putative function of Disp1 as a proton-driven transporter and explains the high molecular weight complex that Shh is found in outside of cells, the pH-dependent action of Disp1 and Ptch1 in the intracellular trafficking of Shh and the role of Disp1 in the re-secretion of Shh (Etheridge, 2010).