Hedgehog

Symbol: Hh	Flybase ID: {Flybase_ID}
Synonyms: {Name}	{GadFly}
Function: {Short_Function}	{LocusLink}
Keywords: {Keywords}	{Interactive_Fly}

{Summary}

Regulating the growth and patterning of the wing and other appendages in adult segment polarity
In the embryo hh maintains wg transcription at the boundary of each segmental unit
Involved in the development of:
- wing (Mohler 1988; Basler, 1994; Tabata, 1994)
- leg (Diaz-Benjumea et al.1994)
- eye discs (Heberlein et al.1995; Dominguez 1999)
- germ-cell migration (Deshpande et al.2001)
- optic lamina (Huang and Kunes 1996,1998)
- gonad (Forbes et al.1996;Zhang and Kalderon 2000)
- abdomen (Struhl et al.1997)
- gut (Pankratz and Hoch 1995)
- tracheal system (Glazer and Shilo 2001).

Ci signaling complex
- Stimulation of cells by Hh releases the complexes (Cos-2, Fu, Su (fu), Ci) from microtubules and induces the phosphorylation of both the Fu (Therond, 1996) and Cos-2 (Robbins, 1997) components of the complexes.
Costal-2
- Hh induces the phosphorylation of Cos2 (Robbins, 1997)
Engrailed
- Hh induces the expression of en at late stages of wing disc development (Blair, 1992; Sanchez-Herrero, 1996; Strigini, 1997; Alves, 1998)
- In embryos homozygous for loss-of-function alleles of hh, expression of en is lost from ectodermal cells after gastrulation (DiNardo, 1988), indicating that the activity of hh, like that of wg, is also required for the maintenance of en transcription. Although ubiquitous expression of wg results in the ectopic activation of en (Noordermeer, 1992), the distribution of En protein is unaltered in heat-shocked HS-hh embryos (Ingham, 1993: Fig 2C). Thus, although necessary for the maintenance of en, expression of hh is not sufficient for its activation.
Fused
- Hh induces the phosphorylation of Fu (Therond, 1996)
- in a fu mutant there is no expression of wg in the ventral ectodermal cells of each parasegment in the embryo, though transcript is still found in other cells where wg expression is independent of hh function. The phenotype stays the same even if Hh is overexpressed with HS-hh. (Ingham, 1993)
- transcription of hh persists for longer in embryos lacking wild-type fu activity (Fig 2a), suggesting that fu may act 'downstream' of hh to regulate wg transcription (Ingham, 1993)
Wingless
- in a fu mutant there is no expression of wg in the ventral ectodermal cells of each parasegment in the embryo, though transcript is still found in other cells where wg expression is independent of hh function. The phenotype stays the same even if Hh is overexpressed with HS-hh. (Ingham, 1993)
- In en-Gal4/UAS-wg; hh^- embryos Winglessis spreads posterior to the engrailed domain as if a barrier had been lifted or Wingless movement enhanced (Figure 5B). Wingless protein distribution is symmetrical, and this is reflected in the cuticle pattern: in contrast to en-Gal4/UAS-wg embryos, en-Gal4/UAS-wg; hh^- embryos lack rows 2–4 and, instead, have an extra expanse of naked cuticle (Figure 5C). At the positions where rows 5 and 6 normally form, lies a thin stripe of small denticles. Naked cuticle is specified equally in the anterior and posterior directions, as shown by marking the winglessexpressing cells with GFP (Figure 5C). Thus, in the absence of hedgehog, wingless action is symmetric. (Sanson, 1999)
  - This effect is due to Hedgehog signaling since the cuticle phenotype of en-Gal4/UAS-wg; ci^- embryos is identical to that of en-Gal4/UAS-wg; hh^- embryos (Figure 5G). This requirement is dose sensitive, since in hedgehog or cubitus interruptus heterozygotes, Wingless produced in the engrailed domain generates occasional breaches of naked cuticle in the denticle belts (Figures 5H and 5K ). (Sanson, 1999)
Changes in transcriptional activity that are eleicited by Hh signaling require Ci (Forbes, 1993)
Hh signaling reduces phosphorylation of Ci155, which appears to decrease processing in cultured cell line (Chen, 1999)
In the ventral ectoderm, Hh signals in both directions leading to the expression of the patched (ptc) gene in all neighboring cells (Nakano et al., 1989; Hooper and Scott, 1989; Ingham,, 1991).
In the ectoderm two observations suggest that anterior and posterior cells might have different abilities to respond to Hh: (1) only the anterior neighbouring cells express wg in a Hh-dependent manner; (2) Hh patterns only those cells posterior to its expression domain in the dorsal ectoderm (Heemskerk, 1994)
In smo clones (no Hh signaling) dpp-lacZ, Ptc and anterior En expression is inhibited, suggesting that they are direct targets for regulation by Hh signaling (Strigini, 1997)
Hh patterns the vein 3-4 region (Strigini, 1997)
A Ptc-related protein, Dispatched (Disp), is specifically required for the controlled release of Hh-Np (Burke, 1999).
- In the absence of Disp function, Hh-expressing cells accumulate high levels of Hh but fail to secrete it; as a consequence, Hh target genes are not activated in responding cells. This requirement is completely overridden when Hh-Nu is expressed in disp mutant cells (Burke, 1999), indicating that it is only needed for the secretion of the lipid-modified form of Hh-N
- Disp is required in the embryonic ectoderm as well as in the posterior compartment of the imaginal disc (Burke, 1999), implying that the release of Hh- Np is essential for both the long-range and short-range modes of Hh signaling
Reduction of the hh dosage (hh^ts2) enhanced the partial fusion of L3 and L4 observed in kn¹/col¹ wings (Vervoort, 1999)
The anterior displacement, partial loss of L3 and increased width of the L3–L4 intervein that are observed upon overexpression of hh in its own domain (UAS-hh/engrailed (en)-Gal4 driver,(Figure 1e) were suppressed by reducing col dosage (Figure 1f).(Vervoort, 1999)
The expression of col in the L3–L4 intervein was completely lost in hhts2 discs raised to 30°C (null condition); it was also lost, or severely reduced, at 25°C (Figure 4b) (Vervoort, 1999), in contrast to dpp, the expression of which is lost at 30°C but is normal at 25°C (Strigini, 1997). This indicates that col activation requires Hh activity, and that it requires Hh levels superior to those required for activation of dpp.

Appears as if hh is sequestered by Ptc

Regulation of Hh transcription:
- Initial transcription of hh in posterior cells (in embryos) is not dependent on en (Lee, 1992; Mohler, 1992; Tabata, 1992; Tashiro, 1993)
- In imaginal wing discs en activity is both necessary and sufficient to drive hh expression in a cell autonomous manner (Burke, 1999)
- Mutant clones in the posterior compartment lacking both en and invected have a loss of hh expression (Sanicola, 1995)
Hh regulates other proteins:
- Hh binding causes removal of Ptc from surface (Denef, 2000)
- Hh causes phosphorylation, stabilization, and accumulation of Smo at cell surface [comparable effects when Ptc is removed by RNAi] (Denef, 2000)

{Structure}

Hh-Np is tethered to the membrane but is released through the fxn of Disp (Burke, 1999)
In the embryonic ectoderm Hh has been shown to accumulate preferentially basolaterally (Taylor et al. 1993; Tabata, 1994).

Hh protein processing:
- Hh family proteins are synthesized as ~45-kD precursor proteins that undergo an intramolecular cleavage (Lee, 1994; Bumcrot et al. 1995) that is catalyzed by the C-terminal portion of the precursor (Lee, 1994; Porter et al. 1995).This reaction yields a 25-kD C-terminal fragment that has no other known function and an ~19- kD N-terminal fragment (referred to as Hh-N) that is sufficient for all known Hh signaling activity.
- This autocleavage of Hh proceeds via a thioester intermediate that undergoes a nucleophilic attack by cholesterol, resulting in the covalent coupling of cholesterol to the C terminus of Hh-N to yield the processed form of the signaling moiety, denoted Hh-Np ("p" standing for processed) (Porter et al.1996b).
  - cells expressing an unmodified form of Hh-N (generated by a C-terminally truncated form of the coding region, which circumvents the autoproteolysis and hence the cholesterol coupling step) secrete large quantities of this unmodified form, Hh-Nu, into the medium (Bumcrot et al. 1995; Porter et al. 1995). In line with these findings, expression of the Hh-Nu in the embryonic ectoderm was found to have effects consistent with an increased range of Hh activity (Porter et al. 1996a). Little Hh-Np is found in medium conditioned by cells expressing the full-length protein (Pepinsky et al. 1998; Zeng et al. 2001).
  - One interpretation of these results is that Hh-Nu can move further than Hh-Np due to the absence of the cholesterol modification. Intriguingly, Ptc contains a sterol-sensing domain (SSD, reviewed by Osborne and Rosenfeld, 1998), which has been shown in proteins such as HMG CoA reductase (Gil et al., 1985) and SREBP cleavage-activating protein (SCAP, Hua et al., 1996) to be able to monitor sterol levels in membranes. One possibility is that Ptc interacts directly with the cholesterol moiety of Hh-Np via its SSD, thus sequestering Hh and restricting its motility (Beachy et al., 1997)
  - Such cholesterol-mediated membrane anchoring could thus explain the restricted range of Hh in the Drosophila embryo and in certain contexts in vertebrate embryos (such as tooth and hair development), where Hh appears to act at short range, but it seems at odds with the long-range signaling activities of the protein in the limbs and neural tube.
- Shh (Pepinsky et al. 1998), as well as Drosophila Hh (Chamoun et al. 2001), is palmitoylated on its most N-terminal cysteine.
  - The sightless/skinny hedgehog (sig/ski) gene, encodes a polytopic transmembrane protein with similarity to mammalian acyl transferases that catalyze O-linked acyl transfers, most likely occuring through a thioester intermediate(Chamoun et al. 2001).
    - Hh-N is rendered inactive in sig/ski mutants (Chamoun et al. 2001; Lee and Treisman 2001), however an un-acylable form of Shh retains some activity when expressed in transgenic Drosophila imaginal discs (Chamoun et al. 2001; Lee and Treisman 2001).
    - In contrast, studies of Shh modification in tissue culture cells suggest that palmitoylation is in some way dependent on cholesterol addition, as only a small fraction of a form of Shh-N that lacks cholesterol, generated by a mutant form of the cDNA, is palmitoylated (Pepinsky et al. 1998).
    - In line with this, the unmodified form of Shh can elicit equivalent responses in some in vitro assays when administered at significantly higher concentrations (20–30•) than mature native protein. In other contexts, however, notably the ventralization of neural plate explants, both acylated and unmodified forms of the protein appear to have equivalent or very similar levels of activity (Pepinsky et al. 1998; Kohtz et al. 2001). Replacement of the N-terminal Cys by a hydrophobic residue is itself sufficient to increase signaling activity, indicating that it is the hydrophobicity per se, rather than the specific nature of the palmitoyl moiety, that potentiates activity (Taylor et al. 2001). In Drosophila embryos, Hh accumulates in characteristic membrane-associated patches (Taylor et al. 1993; Tabata, 1994) that most likely correspond to lipid rafts (Rietveld, 1999), that is, membrane microdomains that function as platforms for intracellular sorting and signal transduction. The lipid modifications of Hh may play a role in targeting them to rafts; testing this proposition will require a comparison of the subcellular localization and trafficking of the modified and unmodified forms of the protein.[Ingham, 2001]
Hh-Np can freely traverse cells lacking the Hh-binding activity of Ptc, before being bound and endocytosed by ptc in genetically wild-type cells (Chen, 1996).
- This sequestering activity of Ptc helps explain why the ptc gene itself is a target of Hh activity: by up-regulating ptc transcription, Hh effectively promotes its own sequestration, a negative feedback mechanism that restrains the spread of Hh protein from its source (Chen, 1996).
- Hh-Nu appears to be immune to ptc sequestration impling that ptc sequestration depends critically on the cholesterol moiety in Hh-Np (Chen, 1996).
- This might suggest that cholesterol mediates interaction between Hh-Np and Ptc, perhaps via the latter’s SSD, several findings argue against this:
  - Not least of these are the facts that Hh-Nu efficiently activates the pathway by abrogating Ptc activity and that lipid modification has no significant effect on the in vitro binding affinity of Hh for Ptc. Moreover, Hh-Nu appears to be endocytosed with Ptc in responding cells (Burke, 1999), all of which raises questions about the basis of Hh sequestration. Remarkably, whereas recent studies in chick embryos suggest that Ptc1 has a similar role in sequestering Shh in the vertebrate neural tube (Briscoe et al. 2001), in vivo analysis of an unmodified form of Shh-N (Shh-Nu) reveals a quite different effect of cholesterol modification on its behavior (Lewis et al. 2001). In this case, absence of the cholesterol moiety severely limits the range of the Shh-Nu protein. Therefore, although it retains activity comparable to that of the wild-type protein at short range (as assayed by its ability to promote normal hair, whisker, tooth, and lung development and to promote the specification of the most posterior digits in the hand and foot plates), it fails to spread across the developing limb bud, leading to a contraction in the expression domains of target genes and an accompanying loss of intermediate digits (Lewis et al. 2001). These findings point, instead, to a requirement for cholesterol modification for the efficient movement of Shh-N through the limb mesenchyme, a requirement that seems at odds with the properties of the Hh-Nu form in Drosophila. It is possible that this disparity may reflect a difference in the experimental conditions under which the unmodified forms of the respective proteins have been assayed, or in the cellular milieu in which the endogenous forms normally operate (Lewis et al. 2001), but it is also notable that in vertebrates, Hh proteins are subject to an additional restraining influence, namely, that imposed by Hip1 (Chuang and McMahon 1999), an Hh-binding protein that has no counterpart in Drosophila. Like Ptc, expression of Hip1 is up-regulated in response to Hh signaling (Chuang and McMahon 1999), but unlike Ptc, there is no evidence that it acts by directly regulating Smo. Therefore, Hip1 adds a second layer of control to the Hh negative feedback mechanism, a layer that is exclusive to vertebrates. One scenario that could reconcile the immunity of Drosophila Hh-Nu to Ptc sequestration with the attenuated range of Shh-Nu in the mouse would be if Shh-Nu were to bind Hip with equal or greater affinity than its cholesterol-coupled counterpart. In this case, Shh-Nu would still be capable of antagonizing Ptc at short range, but would not be free to move beyond cells immediately adjacent to its source. But how might cholesterol coupling allow Shh-Np to override Hip sequestration? In Drosophila, movement of the cholesterol-modified form of Hh-Np depends critically on the activity of tout velou (ttv) (see Fig. 7; Bellaiche et al. 1998), a homolog of the human EXT genes that were identified through their association with the bone disorder multiple exostoses (Stickens et al. 1996). These genes encode GAG transferases (Lind et al. 1998), implying that TTV (and its vertebrate homologs) generates a proteoglycan that mediates the transfer of Hh-Np between cells (Bellaiche et al.1998; Thé et al. 1999). That the activity of TTV is required in Hh-receiving cells even in the absence of Ptc (Bellaiche et al. 1998) implies that the hypothetical proteoglycan may interact directly with Hh-Np and possibly present it to Ptc, a process similar to that proposed above for Hip. If both the proteoglycan and Hip compete for Hh-Np binding and if cholesterol coupling is obligate for interaction with the former, then it is easy to envisage how in vertebrates, the absence of cholesterol from Shh-Nu would block its movement and lead to its sequestration by Hip. In Drosophila, in contrast, where there is no Hip1 to bind unsequestered Hh-Nu, the latter might remain free simply to diffuse away from its source, abrogating Ptc activity in its wake. Investigating the properties of Shh-Nu in a Hip mutant background should therefore help to resolve this issue. [Ingham, 2001]

Mouse homologue: Sonic hedgehog, Desert hedgehog, Indian hedgehog (Echelard, 1993)
- Dhh is most closely related to Drosophila hedgehog. Ihh and Shh are more related to one another, representing
  a more recent gene duplication event.
hh genes have been identified in several other invertebrate species including the leech and sea urchin (Chang et al.1994) as well as in the cephalochordate amphioxus (Shimeld 1999).One notable exception is the nematode worm Caenorhabditis elegans ,which has no hh ortholog (Aspock et al.1999)but does possess severa lgenes encoding proteins homologous to the Hh receptor Patched (Kuwabara et al.2000) [passage taken from Ingham and McMahon, 2001]
Vertebrate Hh homologues play key roles in the morphogenesis of the neural tube, somites, axial skeleton, limbs, lung and skin (reviewed in Neumann and Cohen, 1997; Ingham, 1998; Ruiz i Altaba, 1999; McMahon, 2000)
Structural analysis of Hh-N provided initial excitement as it revealed a striking conservation with zinc hydrolases, suggesting that the Hh ligand might have an enzymatic activity (Hall et al. 1995). However, an absence of conservation of key histidines that coordinate the zinc ion in hydrolases and biochemical analyses in cell culture seem to argue against an enzymatic role for the signaling moiety (Fuse et al. 1999).

hh mutant embryos are short and show a sever 'lawn of denticle' phenotype (Nusslein-Volhard, 1984)
If Hh signal is not transmitted, levels of ptc expression and apparent levels of Ci protein are not elevated, dpp is not transcribed, and Fu protein is not phosphorylated (Tabata, 1992; Basler, 1994; Capdevila, 1994; Felsenfeld and Kennison, 1995; Sanicola et al., 1995 Therond, 1996)
Reduction of the hh dosage (hh^ts2) enhanced the partial fusion of L3 and L4 observed in kn¹/col¹ wings (Vervoort, 1999)
hh^ts2 is a temperature sensitive allele. It behaves as a null at 30°C and as a strong hypomorphic mutant at 25°C (Strigini, 1997; Ma, 1993)
in a gain of function allele called Moonrat (hh^Mrt) hh in addition to its normal expression in the posterior compartment of the wing disc, is ectopically expressed along the DV boundary within the anterior compartment (Felsenfeld and Kennison, 1995)

uba1>hh clones in the A compartment express dpp-lacZ but not hh-lacZ and do not have smooth borders like Tuba1>en clones do (Zecca, 1995)
Overexpression or misexpression of hh (UAS-hh/en-Gal4 or UAS-hh/apterous (ap)-Gal4) led to an expanded expression of col anteriorly (Figure 4c), or ectopic expression dorsally (Figure 4d), respectively. (Vervoort, 1999)
In embryos overexpressing hh with a HS-hh construct wg is ectopically expressed anterior to each normal wg domain, but more anterior cells don't express wg. ptc, however, is expressed in all cells except engrailed expressing cells. (Ingham, 1993)
The distribution of En protein is unaltered in heat-shocked HS-hh embryos (Fig 2C). (Ingham, 1993)
In embryos overexpressing hh with a HS-hh construct the ventral denticles characteristic of the posterior part of each belt are eliminated and replaced by those typical of the second and third row of wild-type belts (Fig 3a, d) (Ingham, 1993). The denticle belts are also reduced in the lateral extension so that they become less trapezoidal and more rectangular. This phenotype is very reminiscent of that of ptc mutants.

{Reagents}