shifted: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - shifted

Synonyms -

Cytological map position - 6C13--D1

Function - receptor binding

Keywords - Hedgehog pathway, wing

Symbol - shf

FlyBase ID: FBgn0003390

Genetic map position - 1-17.9

Classification - WIF family, EGF repeats

Cellular location - secreted



NCBI links: Precomputed BLAST | Gene | UniGene | HomoloGene
BIOLOGICAL OVERVIEW

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development and human diseases. Lipid modified Hedgehog is able to migrate to and program cells far away from its site of production despite being associated with membranes. To investigate the Hh spreading mechanism, Shifted (Shf) was characterized as a component in the Drosophila Hh pathway. Shf, discovered by Calvin B. Bridges in 1913 (see Thomas Hunt Morgan and His Legacy by Edward B. Lewis), is the ortholog of the human Wnt inhibitory factor (WIF), a secreted antagonist of the Wingless pathway. In contrast, Shf is required for Hh stability and for lipid-modified Hh diffusion. Shf colocalizes with Hh in the extracellular matrix and interacts with the heparan sulfate proteoglycans (HSPG), leading to the suggestion that Shf could provide HSPG specificity for Hh. Shifted acts over a long distance and is required for the normal accumulation of Hh protein and its movement in the wing. Human WIF inhibits Wg signaling in Drosophila without affecting the Hh pathway, indicating that different WIF family members might have divergent functions in each pathway (Gorfinkiel, 2005; Glise, 2005).

Understanding the Hedgehog (Hh) pathway is a central issue in developmental biology. Besides its role in morphogenesis and patterning, this pathway also has implications in human diseases. The Drosophila imaginal disc is a relative simple model that has served to address some of the questions regarding Hh production, secretion, signal reception, and transduction along with its role in morphogenesis. The Drosophila wing disc is a two-sided sac made of a columnar epithelial layer, containing presumptive thorax and wing blade domains and an overlying squamous peripodial membrane. The columnar epithelial cells constitute a single-layered sac of polarized cells with their apical surfaces oriented toward the disc lumen. In this epithelium, two populations of cells with different adhesion affinities divide the field into posterior (P) and anterior (A) compartments (García-Bellido, 1973). In the P compartment cells, Hh protein is produced under the control of the engrailed (en) gene and signals to the cells of the A compartment. Hh is synthesized as a precursor 45 kDa protein that undergoes autoproteolytic cleavage. Concomitant with this reaction, a cholesterol molecule is covalently linked to its COOH terminal end. This processed protein (Hh-Np) is further modified by acylation, and a palmitic acid molecule is added to its N terminus. The acyl transferase enzyme encoded by the gene sightless (also named skinny hedgehog/central-missing/rasp) catalyzes the addition of palmitic acid. In mutants for this enzyme, there is no production of an active form of Hh and, consequently, there is no signaling in the A compartment cells (Gorfinkiel, 2005).

A key issue that remains unclear in Hh signaling is the molecular mechanisms by which a Hh protein highly modified by lipids is able to diffuse long distances despite its association with membranes. Hitherto, proteins of the extracellular matrix such as HSPG have been implicated in regulating the signaling activity of secreted morphogen molecules. Thus, it has been described that the Drosophila EXT family of proteins encoded by the genes tout velu (ttv), brother of tout velu (botv), and sister of tout velu (sotv), which are essential for the synthesis of the HSPG, are not only required for the diffusion of lipid-modified Hh but also for Wingless (Wg) and Decapentaplegic (Dpp) diffusion and signaling. These EXT proteins are glycosyl transferases that catalyze the formation of heparan sulfate glycosylamino glycan chains, which are attached to a core protein. Recently, the glypican proteins Dally and Dally-like (Dlp), which code for the HSPG protein core, were found to be required for Hh diffusion and Dlp was also shown to be needed for reception of the Hh signal in Drosophila cultured cells and embryos. These data indicate that HSPGs are important for the formation of morphogen gradients and signal reception, but it is not known how the specificity of HSPGs for the different ligands is accomplished (Gorfinkiel, 2005).

The secreted protein encoded by the shf locus is required for normal levels of Hh in the extracellular matrix of the Hh-producing cells and for diffusion of Hh in both A and P compartment cells. Shf is a secreted factor ortholog of the vertebrate Wnt inhibitory factor (WIF). Despite WIF being involved in Wnt signaling in vertebrates, Shf/Dwif does not seem to be involved in the Drosophila Wg pathway (Gorfinkiel, 2005).

shf encodes a secreted protein containing a WIF module and 5 EGF-like repeats. Homologs to WIF-1 have been found in human, mouse, Xenopus, and Zebrafish (Hsieh, 1999). Human WIF-1 (HWIF-1) antagonizes the activity of Wnt in inducing the secondary axis in Xenopus embryos. Moreover, Wg and Xwnt8 bind to the WIF domain in coimmunoprecipitation experiments (Hsieh, 1999). However, it should be noted that so far there have been no reports of a WIF loss-of-function phenotype in vertebrates (Gorfinkiel, 2005).

Despite the above indications that WIF is involved in Wnt signaling in vertebrates, thus there is no evidence for a role of shf in the Wg pathway in Drosophila. Df(1)2.5 imaginal discs rescued by overexpressing C3G show a wild-type Wg expression pattern and Dll, one of the Wg targets in the wing, is expressed normally. Neither was it possible to detect any effects on the Wg pathway when Shf was overexpressed. These observations are rather striking considering the generally conserved function of homologous proteins of Drosophila and vertebrates. To test if the human WIF was able to block Wg signaling in Drosophila, transgenic flies were generated containing human WIF-1 and it was ectopically expressed in wing imaginal discs. Overexpression of HWIF-1 is unable to rescue the shf2 phenotype in the wing. However, ectopic expression of HWIF-1 in the wing disc using the MD638-GAL4 or hh-GAL4 lines, gives rise to a Wg lack-of-function phenotype, as observed by the nicks at the wing margin. Moreover, hh-GAL4/UAS-HWIF-1 wing discs show that the distribution of Wg is altered in the posterior compartment cells, as shown by the accumulation of Wg mainly outlining the cell surface, and by a reduced number of punctate vesicle structures of internalized Wg. A similar alteration in Wg distribution has also been observed when Dlp is ectopically expressed in the wing disc, and it has been suggested that Wg is sequestered by Dlp and is less available for binding to its receptor. Thus, the observed pattern of Wg accumulation in hh-GAL4/UAS-HWIF-1 wing discs would be in line with a role for HWIF-1 in sequestering Wg, as has been previously described (Hsieh, 1999). It is concluded that HWIF-1 behaves as an antagonist of Wg signaling both in Drosophila and vertebrates and that Shf and HWIF-1 are homologs in structure although their function is not conserved (Gorfinkiel, 2005).

In Drosophila, there are three other genes that code for WIF-containing proteins. These are derailed, derailed-2, and doughnut (Oates, 1998; Savant-Bhonsale, 1999). These proteins do not have the EGF-like repeats and instead they have a domain related to tyrosine kinase (RYK) domain. It has been proposed that the WIF domain has a conserved function in both the WIF-1 and RYK proteins because derailed is the receptor for Wnt5. The results do not support this idea of a conserved function for the WIF module (Gorfinkiel, 2005).

The other characteristic domain in Shf is the EGF domain, which seems to be essential for Shf function because shf2 and shf919 alleles have a substitution in a conserved cysteine residue of the EGF-repeats. EGF repeats are found in the extracellular domain of membrane-bound proteins or in secreted proteins such as those involved in cell-cell adhesion. It is possible that Shf interacts with the HSPG proteins of the extracellular matrix through its EGF domain (Gorfinkiel, 2005).

It is tempting to speculate that the WIF domain could have a redundant function in blocking Wg or stabilizing Wg at the cell surface, since the wg- phenotype is neither obtained by a lack- nor gain-of-function of Shf. Besides, Shf could bind to Hh (and maybe also to HSPG) through the EGF domains to control Hh diffusion. Furthermore, overexpression of the WIF domain alone does not rescue the shf phenotype, suggesting that the EGF module is the part of Shf that might interact with Hh. In view of the complexity of HSPG function in morphogen diffusion and stabilization, it could be that a combination of different extracellular matrix factors confers HSPG its specificity toward different morphogens. Shf would be expected to be one of the molecules that provides this specificity (Gorfinkiel, 2005).

The hedgehog Pathway Gene shifted Functions together with the hmgcr-Dependent Isoprenoid Biosynthetic Pathway to Orchestrate Germ Cell Migration

The Drosophila embryonic gonad is assembled from two distinct cell types, the Primordial Germ Cells (PGCs) and the Somatic Gonadal Precursor cells (SGPs). The PGCs form at the posterior of blastoderm stage embryos and are subsequently carried inside the embryo during gastrulation. This study has investigated the role of the hedgehog (hh) pathway gene shifted (shf) in directing PGC migration. shf encodes a secreted protein that facilitates the long distance transmission of Hh through the proteoglycan matrix after it is released from basolateral membranes of Hh expressing cells in the wing imaginal disc. shf is expressed in the gonadal mesoderm, and loss- and gain-of-function experiments demonstrate that it is required for PGC migration. Previous studies have established that the hmgcr-dependent isoprenoid biosynthetic pathway plays a pivotal role in generating the PGC attractant both by the SGPs and by other tissues when hmgcr is ectopically expressed. Production of this PGC attractant depends upon shf as well as a second hh pathway gene gγ1. Further linking the PGC attractant to Hh, evidence is presented indicating that ectopic expression of hmgcr in the nervous system (via the elav Gal4) promotes the release/transmission of the Hh ligand from these cells into and through the underlying mesodermal cell layer, where Hh can contact migrating PGCs. Finally, potentiation of Hh by hmgcr appears to depend upon cholesterol modification (Deshpande, 2013).

The synthesis of mevalonic acid by the enzyme Hmgcr is the rate-controlling step in the biosynthesis of isoprenoids and steroids. In mammals, one end-product of the mevalonate pathway, cholesterol, is used to modify the C-terminus of the processed Hh ligand, and this modification plays an important role in controlling the activity of this signaling molecule. Flies lack the enzymes needed for de novo cholesterol biosynthesis and depend instead upon exogenous cholesterol for this modification of the Hh ligand. Nevertheless, the mevalonate biosynthetic pathway is still used to potentiate the release/transmission of the Hh ligand, in this case through (at least in part) the geranylation of the G protein Gγ1 (Deshpande, 2009). Hmgcr as well as the downstream components in the isoprenoid biosynthetic pathway also play a pivotal role in generating the attractant that guides PGC migration both from its native source, the SGPs, and from a variety of different embryonic tissues when ectopically expressed. However, how hmgcr or the other isoprenoid pathway enzymes function in generating the PGC attractant either in the SGPs or at ectopic sites has remained unresolved and contentious. To address this problem this study has focused on the connection between the mevalonate→isoprenoid biosynthetic pathway and two proteins that have been implicated in the long distance basolateral transmission of the Hh-Np ligand, the G protein Gγ1 and the extracellular hh signaling factor Shf (Deshpande, 2013).

Previous studies have established that a rate limiting step in generating the PGC attractant either by the SGPs or by other tissues and cell types is the biosynthesis of geranylgeranyl-pyrophosphate by geranylgeranyl diphosphate synthetase (qm). The control point in the geranylgeranyl-pyrophosphate biosynthetic pathway is the production of mevalonic acid by the enzyme Hmgcr. While hmgcr seems to play a rather similar role in the release/transmission of Hh-Np from hh sending cells, in this case through the geranylation of Gγ1, an important and controversial question is whether the functioning of the hmgcr-->qm biosynthetic pathway in hh signaling has any connection to the generation of the PGC attractant. This question was addressed by determining if the PGC migration defects induced by hmgcr expression in the nervous systems depend upon Gγ1 and Shf. It was found that mutations in both gγ1 and shf dominantly suppress the migration defects induced by ectopic hmgcr. In contrast, reducing the dose of the hmgcr gene dominantly enhances the migration defects induced by hmgcr expression in the nervous system. This later finding is expected since reducing hmgcr activity in the SGPs should further compromise the ability of the attractant generated by the SGPs to compete with the attractant generated in the nervous system. The former findings show that the production/activity of the attractant generated in the nervous system by ectopic hmgcr depends on both gγ1 and shf. By themselves, these results do not exclude the possibility that gγ1 and shf only collaborate with hmgcr when it is ectopically expressed in the nervous system while they are not actually needed for the hmgcr-dependent production of the attractant by the SGPs. However, this scenario seems unlikely. For one, there are PGC migration defects in gγ1 and shf mutant embryos. For another, the Gγ1 protein must be geranylated to function in PGC migration (Deshpande, 2009). Finally, like hmgcr, ectopic expression of gγ1 and shf in the mesoderm and ectoderm perturbs PGC migration (Deshpande, 2013).

Even though Gγ1 and Shf are known to function in the release and transmission of the Hh ligand, it could be argued that these two proteins could also mediate the release/transmission of others molecules, including the 'actual' PGC attractant. Indeed, Gγ1 is likely involved in secretion of other molecules, while the fact that Shf homologs in mammals function in Wnt but not Hh signaling raises the possibility that Shf could promote signaling by an as yet unknown ligand (though not Wg). However, there is evidence that like Gγ1 and Shf, Hh itself depends upon hmgcr and the isoprenoid biosynthetic pathway not only in hh signaling but also in generating an ectopic PGC attractant in the nervous system. This comes from the differences in the effects of ectopically expressed Hh-Np (internally autoproteolytic cleavage product coupled with cholesterol addition) and Hh-N (lacking the cholesterol modification) that would be predicted based on the mechanisms proposed for their transmission. First, the apically transmitted Hh-N ligand would be expected to have a smaller effect on PGC migration when ectopically expressed in the nervous system than Hh-Np. With the caveat that expression of different UAS transgene inserts will not be precisely the same, this prediction holds. Second, the geranylation of Gγ1 in response to ectopic Hmgcr would be expected to promote the basolateral release and subsequent spreading of Hh-Np into the mesoderm. By contrast, ectopic Hmgcr should have less influence on Hh-N, which isn't readily internalized by hh sending cells and spreads mostly along the apical surface. With the same caveat, this predicted distinction is also observed. When co-expressed, Hh-Np and Hmgcr collaborate to strongly potentiate PGC migration defects, while there is a more modest collaboration between Hh-N and Hmgcr (Deshpande, 2013).

Though an imperfect mimic of Hh-Np, advantage was taken of a chimeric Hh-GFP fusion protein to analyze the effects of Hmgcr on the transmission of Hh from cells in the embryonic nervous system. Hh-GFP was found to be less effective than Hh-Np (and even Hh-N) in competing with the PGC attractant produced by the SGPs when it is ectopically expressed using the twi or elav GAL4 drivers. Since Hh-GFP appears to have near but not quite wild type activity in morphogenesis, it is surprising that it is relatively ineffective in altering PGC migration. However, a plausible reason for this discrepancy is that the demands imposed by the assays used to test Hh-GFP activity in each experimental context are quite different. The morphogenesis assay requires that Hh-GFP substitute for Hh-Np. Since animals can readily tolerate heterozygosity for hh, small deficits in the functioning of the chimeric protein might only have minimal effects on morphogenesis. In contrast, in the PGC migration assay the ectopically expressed Hh-GFP must be able to compete with the attractant(s) produced by the SGPs. If Hh-Np is the relevant endogenous PGC attractant, then even subtle deficiencies in the activity of the chimeric Hh-GFP ligand would be expected to compromise its ability to compete with the wild type protein. It would also follow that it should be possible to 'rescue' ectopic Hh-GFP by enhancing its activity. This is the case. While hh-GFP is not very active on its own, it is able to collaborate with hmgcr when co-expressed in the nervous system (Deshpande, 2013).

Previous studies have shown that expressing hmgcr in hh producing cells in the ectoderm increases the overall level of Hh protein and enhances its transmission to adjacent cells. Precisely the same sorts of effects on Hh-GFP are evident when it is 'rescued' by co-expression with hmgcr in the nervous system - Hh-GFP levels are elevated, while its transmission into and through the underlying mesodermal cell layer is appreciably enhanced. These hmgcr dependent effects, particularly on the movement of Hh-GFP from the neuroectoderm into the underlying mesoderm, would also provide a plausible explanation for why this biosynthetic enzyme plays such a pivotal role in PGC migration even though it is not directly responsible for the synthesis of the PGC attractant. In the period when PGCs are migrating through the mesoderm, the SPGs are the only cells in the embryo simultaneously expressing both hmgcr and hh. Consequently the accumulation, release and transmission of Hh-Np will be specifically potentiated in SGPs, but not in other hh expressing cells elsewhere in the mesoderm or in the ectoderm. This would provide a mechanism for ensuring that SGP derived Hh-Np out-competes Hh-Np produced elsewhere. Taken together, these findings support the idea that Hh-Np expressed in the SGPs functions as a PGC attractant. With the caveat that the activities of Hh-GFP are not identical to Hh-Np, the fact that Hh-GFP accumulates on the surface and around the PGCs further bolsters this suggestion. Moreover, in a subset of the PGCs Hh-GFP is closely associated with bulges or protrusions that could potentially be of relevance to the process of migration (Deshpande, 2013).

A number of critical questions remain. For one, it is not clear how reception of the hh signal could actually translate into directed movement. The endpoint of the signaling cascade in the canonical pathway is the transcriptional activation of target genes, including the hh receptor ptc. However, transcription is likely not involved in this instance, as ptc reporters are not activated in PGCs. Moreover, in mammals hh dependent axonal guidance and fibroblast migration are independent of transcription and involve instead the coupling of Smo activation to pathways that mediate the reorganization of the cytoskeleton. Further studies will clearly be required to establish a connection between hh signaling to the PGCs, changes in the cytoskeleton and directed movement. Another unresolved question is whether SGPs produce any other PGC attractants. Although no other plausible candidates have been identified, the current experiments do not exclude the possibility that there are other PGC attractants, even including an attractant(s) whose activity, like Hh-Np, is potentiated by the hmgcr isoprenoid biosynthetic pathway (Deshpande, 2013).


GENE STRUCTURE

cDNA clone length - 1803

Bases in 5' UTR - 260

Exons - 8

Bases in 3' UTR - 172

PROTEIN STRUCTURE

Amino Acids - 456

Structural Domains

The overall structure of Shf and the vertebrate WIF-1 protein is conserved. Both proteins contain an N-terminal signal sequence, indicating they are secreted proteins, the WIF domain, and five-epidermal growth factor (EGF)-like repeats. The sequence of the two known shf alleles shows a missense mutation (C374S in shf 2 and C363S in shf919) in the third EGF-like domain (Gorfinkiel, 2005).

The shf locus was previously mapped cytologically between positions 6D1 and 6E5 based on its inclusion in Tp(1;3)sn13a1 and exclusion from Df(1)HA32 (Craymer, 1980). Mapping the shf mutations relative to viable P element insertions in the region by using meiotic recombination allowed to positioning of shf between the insertions P{GT1}BG01406 at 6C10 and P{GT1}BG02604 at 6D1 (Glise, 2005).

Local hopping of a nearby P{Casper}cx34.6 insertion at 6D generated a line with a weak shf wing phenotype (shfP1). Inverse PCR and sequencing mapped this new insertion within the first, noncoding, exon of the CG3135 gene. A P{EY} insertion (P{EY03173}) located 20 bp 5' to the fifth exon of this same CG was obtained from the BDGP Gene Disruption Project. This line is semilethal, and adult male escapers have a typical shf wing phenotype. Excision of the P{EY03173} element either completely (13/14 lines) or largely (1/14 lines) reverted the shf phenotype. To confirm that shf is caused by disruption of CG3135, a transgene was generated expressing full-length CG3135 cDNA under the control of UAS regulatory sequences. Expressing UAS-CG3135 throughout the wing disc using MS1096-Gal4 fully rescued the shf2 wing phenotype (Glise, 2005).

Sequence from the CG3135 cDNA GH27042 shows that Shf is a protein of 456 amino acids that is an ortholog of the vertebrate WIF-1 (Hsieh, 1999). Both WIF-1 and Shf have predicted N-terminal signal sequences, followed by a single 'WIF' domain (Patthy, 2000) and five EGF-like repeats. Unlike WIF-1, Shf contains two low-complexity domains between the signal sequence and the WIF domain, and a linker sequence between the WIF and EGF domains. This linker, but not the low-complexity domains, is conserved in the predicted Shf ortholog of the mosquito Anopheles gambiae. shf is the only gene with significant similarity to WIF-1 in the annotated Drosophila genome sequence release 3 (Glise, 2005).


shifted: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 30 March 2005

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