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).

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)sn^13a1 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 (shf^P1). 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).

date revised: 30 March 2005

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