EvoprintHD of hedgehog

BIOLOGICAL OVERVIEW

hedgehog is a segment polarity gene in Drosophila; its biochemical and functional homolog in vertebrates is sonic hedgehog. Segment polarity genes and their influences constitute an important aspect of Drosophila development. What is meant by segment polarity, and why is this significant?

Once segmentation is established (through the expression of pair rule genes), the anterior portion of each parasegment (the term for embryonic segments) takes on a different fate from the posterior portion. Segment polarity refers to this seeming polarization within each segment, resulting in differing cell fates. The requisite polarity in segments, ultimately responsible for proper development of Drosophila wings and legs, is established through the action of segment polarity genes. An analogy can be made to human segment polarity. Call the thumb the anterior aspect of the human hand; the posterior aspect would be the fifth or "little" finger. Without the action of segment polarity genes, humans might well be "all thumbs," or worse, no thumbs.

The earliest action of hedgehog establishes the polarity of the 14 parasegments in the trunk (thorax and abdomen) of the fly, segments fated to develop into head, thorax and abdomen. Particularly interesting to developmental biologists is segmental specialization in the wing. The part of the embryo destined to become the adult wing is composed of the posterior part of parasegment 2, and the anterior part of parasegment 3. hedgehog, induced by Engrailed, is secreted by posterior cells (derived from the anterior part of parasegment 3) and diffuses a short distance. It acts on the adjacent (more anterior) segment to overcome repression by Patched and this results in the induction of decapentaplegic. DPP then defines the compartment border between the anterior and posterior halves of the wing (Zecca, 1995). For more of the confusing nomenclature surrounding any discussion of segments, and an attempt to clarify it, see engrailed.

The HH signaling pathway is traversed by means of phosphorylation. This is a process by which an enzyme attaches phosphate residues to other signaling molecules. Cyclic AMP-dependent protein kinase A (PKA) is one such enzyme. PKA signals downstream to repress wingless and dpp. HH overcomes the repressive conspiracy of Patched and PKA and allows the transcription of wg and dpp. The two signaling molecules WG and DPP proceed to define the the border between anterior and posterior compartments in segmentation.

Cells producing Wingless and DPP do not always overlap, as they do in the eye and in the segmentation process. In the leg, in the antennal portion of the eye-antennal disc and in the wing, cells producing Wingless and DPP are separate. In wing for example, DPP synthesis takes place in the dorsal aspects of the disc, while Wingless synthesis is in the anterior aspect (Diaz-Benjumea, 1994b).

In summary, hedgehog is a necessary element in the establishment of polarity during segmentation of the fly, and during the development of appendages. It is made by anterior compartments in the embryo (that become the posterior compartments of the developing adult). HH acts through Smoothened and Patched (probable components of the HH receptor). cAMP-dependent protein kinase A and Fused are the targets of SMO and PTC respectively. Signals from PKA and Fused integrate downstream of Fused to overcoming repression of target genes (wg and dpp) in adjacent compartments.

Very little information is available about gene expression during the larval period, a developmental interval critical to the formation of the adult. To what extent does gene expression during this period resemble that in the embryonic stages, and how does gene expression during the larval period contribute to segment polarity in the adult? In fact, all the genes expressed during embryonic segment polarity also play a similar role in the formation of the adult. Most of the larval cuticle of Drosophila is secreted by large, polyploid cells that derive directly, without cell division, from cells in the epidermis of the embryo. By contrast, cells destined to form the cuticle of the adult abdomen are present as clusters of small, non-dividing diploid cells (the anterior dorsal, posterior dorsal and ventral histoblast nests) located at stereotyped postions in the larval epidermis. These cells, just as do their embryonic counterparts, express engrailed, hedgehog, wingless, patched, cubitus interruptus and sloppy paired in a stereotyped manner dependent on their positions within each segment. Each segment is subdivided into an anterior (A) and posterior (P) compartment, distinguished by activity of the selector gene engrailed (en) in P but not A compartment cells. The ventral epidermis of each abdominal segment forms a flexible cuticle, the pleura, with a small plate of sclerotised cuticle, the sternite, centered on the ventral midline. The pleura is covered with a uniform lawn of hairs, all pointed posteriorly, whereas the sternite contains a stereotyped pattern of bristles. Posterior compartments are to a large degree devoid of hairs and bristles, while the sternite cuticle of the A compartment consists of an anterior-to posterior progression of six types of cuticle distinguished by ornamentation and pigmentation. Just anterior to the posterior compartment, A6 is unpigmented, with hairs and none of the larger ornaments called bristles. A5 is darkly pigmented with hairs and bristles of large size. A4 and A3 are darkly and lightly pigmented respectively with moderately sized hairs and bristles. A2 is lightly pigmented with hairs, and A1, adjacent to the next more anteriorly located "posterior" compartment is unpigmented without hairs (Struhl, 1997a).

Hedgehog (Hh), a protein secreted by engrailed expressing P compartment cells, spreads into each A compartment across the anterior and the posterior boundaries to form opposing concentration gradients that organize cell pattern and polarity. Anteriorly and posteriorly situated cells within the A compartment respond in distinct ways to Hh: they express different combinations of genes and form different cell types. patched is expressed at both boundaries. patched is expressed in a graded fashion within each stripe, just anterior to each P compartment. ci peaks at high level in those cells abutting Hh- secreting cells of the P compartment and declines progressively in cells further away. wingless is also expressed in this domain and sloppy paired is expressed in the same region as wingless. decapentaplegic is expressed only in the ventral pleura in those A compartment cells neighboring P compartment cells within the same segment. dpp is not expressed immediately behind posterior compartments. Essentially, this leaves the patched expressing stripe immediately posterior to each P compartment and the adjacent more posterior cuticle, to be decorated with bristles, like a "no-mans land," as far as secretion of signaling proteins is concerned. Anterior compartment cells form polarized structures that, in the more anterior part of the compartment, point down the Hh gradient and, in the posterior part of the compartment, point up the gradient - therefore all structures point posteriorly. It has been shown that ectopic Hh can induce cells in the middle of each A compartment to activate en. Where this happens, A compartment cells are transformed into an ectopic P compartment and reorganize pattern and polarity both within and around the transformed tissue. Hh could pattern the A compartment by a simple gradient mechanism; the concentration of Hh would be read as a scalar to determine the type of cuticle secreted. Although this role is supported by the experiments in which Hh is ectopically expressed, there are some instances in which the slope of the presumed concentration gradient of Hh does not correspond with the orientation of hairs and bristles. Interestingly, only a subset of A compartment cells respond to ectopic Hh by activating engrailed and subsequently adopting a P identity. These cells occur in the middle of the A compartment, and therefore are not normally exposed to Hh secreted by the normal P compartment. It is clear that the A compartment is finely structured in terms of cell identity and fate (Struhl, 1997a).

A second paper (Struhl, 1997b) deals directly with the instances in which cell polarity does not correspond to the presumed concentration gradient of Hh and considers whether Hh acts directly or by a signal relay mechanism. Mutations in Protein Kinase A (PKA) or smoothened (smo) were used to activate or to block Hh signal transduction in clones of A compartment cells. For cell type, a scalar property (differing with position along the AP axis), both manipulations cause strictly autonomous transformations: the cells affected are exactly those and only those that are mutant. Hence, it is infererd that Hh acts directly on A compartment cells to specify the various types of cuticular structures that they differentiate (Struhl, 1997b).

By contrast, these same manipulations cause non-autonomous effects on cell polarity, a vectorial property. For example, PKA mutant clones in the A3 and A4 region alter polarity of hairs and bristles both within the clone and outside it. In general, wild-type cells positioned laterally and posteriorly to the mutant clone form hairs and bristles that point centripetally towards the clone; thus, behind the clone, cells form hairs and bristles that point anteriorly. The region of wild-type tissue showing this reversed polarity can be up to 4 cell diameters wide. Of great interest is the effect of PKA and smo mutant clones in the anterior portion of A compartment, the "no-mans land" described above. PKA and smo mutant clones in the anterior region of the A comparment alter cell type much as they do in the posterior portion, but some clones of smo cells in the A1 region form hairs that have reversed polarity and these hairs point anteriorly. Consequently, it is surmised that Hh influences cell polarity indirectly, possibly by inducing other signaling factors (Struhl, 1997b).

Evidence is presented that Hh does not polarize abdominal cells by utilizing either Decapentaplegic or Wingless, the two morphogens through which Hh acts during limb development. If Hh were to work through Wg to influence polarity, removal of wg from clones of cells that are activated in the Hh pathway should eliminate that influence. Neither the change in cell type nor the alterations in cell polarity cause by the loss of PKA activity appear to be due to the ectopic expression of wg. Likewise, eliminating dpp from PKA mutant clones fails to alter the polarity phenotype (Struhl, 1997b).

How might Hh polarize cells via a signal-relay mechanism? One clue is that within and surrounding some PKA mutant clones the hairs and bristles point inwards, towards the center. A simple model is that the loss of PKA activity in these cells mimics reception of Hh and hence induces them to secrete a diffusible polarizing factor, 'X'. Because mutant cells in the center of the clone would be surrounded by X-secreting cells, they might be exposed to higher levels of X than mutant cells at the periphery. If cells were oriented by the direction of maximal change (the vector) in the concentration of X, cells both inside and outside of the clone would point towards the center of the clone. Such a propagation model does not demand that X be diffusible, because polarity could be organized by local cell-cell interactions, which spread as in a game of dominoes (Struhl, 1997b).

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein while A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. Clones of cells were produced with altered activities for the engrailed, patched and smoothened genes. The results confirm (1) that the state of engrailed, whether 'off' or 'on', determines whether a cell is A or P type and (2) that Hedgehog signaling, coming from the adjacent P compartments across both A/P and P/A boundaries, organizes the patterning of all the A cells. Four new aspects of compartments and the expression of engrailed in the abdomen have been uncovered. (1) engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. (2) The polarity changes induced by the clones were examined, and a working hypothesis was generated, as follows: polarity is organized, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. (3) It has been shown that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from one another. (4) When engrailed is removed from the P cells (of segment A5, for example) the P cells transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6) (Lawrence, 1999a).

The cells of the dorsal epidermis of the adult abdomen in Drosophila exhibit two properties: (1) a scalar property, shown by the identity of the cuticle they secrete, and (2) a vectorial property, indicated by the orientation of hairs and bristles. The scalar properties are represented by the presence of subdomains within both the A and P compartments. ptc-;en- cells at the front and the back of the A compartment give different transformations, confirming that there are two domains in A. These domains correspond largely to the territories of a1, a2 (no bristles) and a3, a4, a5 cuticle (with bristles). Removal of the Notch (N) gene from these two regions gives different outcomes: N- clones in a2 cuticle make epidermal cells, while those in a3 do not. It follows that the cells composing a2 (non-neurogenic) and a3 (neurogenic) are fundamentally distinct. The P compartment is also subdivided. Thus, the loss of en from posterior P cells converts them from making p1 cuticle to either a1 or a2, depending on whether they can receive the Hh signal. The removal of en from anterior P cells causes them to make either a5 or a3 cuticle, again depending on whether they can receive Hh (Lawrence, 1999a).

Why should there be such a subdivision of the compartments? Perhaps it is connected with making a distinction between A/P and the P/A borders, for if both were simply an interface between A and P cells, they would differ only in their orientation. It is not known what agent discriminates between the two domains in either compartment; perhaps one regulatory gene would be sufficient for both: its expression could flank the segment boundary, redefining nearby regions of the A and P compartments. The domains are not maintained by cell lineage. Analogous domains are found in the legs, where A compartment cells respond to Hh by expressing high levels of either Decapentaplegic or Wingless, depending on whether they are located dorsally or ventrally in the appendage. This dorsoventral bias in response is established early in development, and then maintained, not by lineage, but by feedback between Wg- and Dpp-secreting cells (Lawrence, 1999a).

The vector property of the epidermis is represented by the orientation of adult hairs. A model has been proposed where Hh crosses over from P to A and elicits production of a `diffusible Factor X' that grades away anteriorly from the A/P border, and has a long range; the cells are oriented by the vector of this gradient. For simplicity, this discussion will be restricted to the posterior domain of the A compartments. The A/P boundaries cannot be unique sources of X, for polarity changes also occur when cells from one level of A confront those from another (e.g. when a5 and a3 cells meet at the edge of ptc-;en- clones). This suggests that away from the compartment boundaries, cells also produce X, the quantity depending on the amount of Hh received. It is therefore imagined that a gradient of X would be formed both by the graded production of X (high near the A/P boundary, low further away) and also by its further spread into territory (a3) where Hh is low or absent. Note that this model fits with most of the results for it makes the A/P boundaries the organisers: whenever ectopic A/P boundaries are generated by the clones, their orientation correlates with the polarity of territory nearby; this is most clearly seen at the back of en-expressing clones. The line where polarity switches from normal to reversed does not occur at a fixed position in the segment but rather appears to be related to the position of nearby A/P borders (Lawrence, 1999a).

en- clones in the P compartment make A cuticle. In the anterior part of P these clones have normal polarity. In the posterior part of P the whole clone displays reversed polarity, as do some cells outside the clone. In order to understand this (at least, in part), consider the behaviour of ptc- clones in the A compartment: they behave differently depending on their distance from the A/P border, the presumed source of X. At the back of the A compartment they are near that border and have little or no effect on polarity, but when closer to the front of A, they repolarize several rows of cells in the surround. This is explained as follows: near the source of X, where the ambient level is high, limited production of X might not much affect the concentration landscape. But far from the source, where the local concentration of X would be low, any effects would appear greater. Likewise, if there were a polarizing factor similar to X in the P compartment, then clones of en minus cells that produce complete or partial borders might become ectopic sources of this factor: they would produce altered polarities only in an environment where the level of the factor were low. This argument suggests that a polarising factor 'Y' for the P compartment might emanate from the A/P border and spread backward. Thus the evidence is consistent with the idea that polarizing signals spread in both directions from the A/P boundaries. The P/A (segment) boundaries might act to stop these factors trespassing into the next segment, just as they appear to block the movement of Wingless protein (Lawrence, 1999a).

During Drosophila eye development, Hedgehog (Hh) protein secreted by maturing photoreceptors directs a wave of differentiation that sweeps anteriorly across the retinal primordium. The crest of this wave is marked by the morphogenetic furrow, a visible indentation that demarcates the boundary between developing photoreceptors located posteriorly and undifferentiated cells located anteriorly. Evidence is presented that Hh controls progression of the furrow by inducing the expression of two downstream signals. The first signal, Decapentaplegic (Dpp), acts at long range on undifferentiated cells anterior to the furrow, causing them to enter a 'pre-proneural' state marked by upregulated expression of the transcription factor Hairy. Acquisition of the pre-proneural state appears essential for all prospective retinal cells to enter the proneural pathway and differentiate as photoreceptors. The second signal, presently unknown, acts at short range and is transduced via activation of the Serine-Threonine kinase Raf. Activation of Raf is both necessary and sufficient to cause pre-proneural cells to become proneural, a transition marked by downregulation of Hairy and upregulation of the proneural activator, Atonal (Ato), which initiates differentiation of the R8 photoreceptor. The R8 photoreceptor then organizes the recruitment of the remaining photoreceptors (R1-R7) through additional rounds of Raf activation in neighboring pre-proneural cells. Dpp signaling is not essential for establishing either the pre-proneural or proneural states, or for progression of the furrow. Instead, Dpp signaling appears to increase the rate of furrow progression by accelerating the transition to the pre-proneural state. In the abnormal situation in which Dpp signaling is blocked, Hh signaling can induce undifferentiated cells to become pre-proneural but does so less efficiently than Dpp, resulting in a retarded rate of furrow progression and the formation of a rudimentary eye (Greenwood, 1999).

Hh, secreted by maturing photoreceptor cells, is normally responsible for inducing cells within and ahead of the morphogenetic furrow to initiate photoreceptor differentiation. Nevertheless, cells that lack Smoothened (Smo) function, and hence the ability to transduce Hh, can form normal ommatidia. These findings suggest that Hh can induce photoreceptor differentiation in Smo-deficient cells through the induction of other signaling molecules in neighboring wild-type tissue. As a first step toward identifying such secondary signals and analyzing their roles, the consequences of creating clones of cells homozygous for smo³, an amorphic mutation, have been examined on two early markers of retinal development, the expression of Ato and Hairy, which are expressed in adjacent dorso-ventral stripes within and anterior to the morphogenetic furrow. Ato expression has two prominent phases in the developing eye. In the first phase, Ato is expressed uniformly in a narrow dorso-ventral swath of cells that demarcates the anterior edge of the furrow. This uniform swath then breaks up into small clusters of Ato expressing cells and resolves into the second phase, a spaced pattern of single Ato expressing cells (the future R8 photoreceptor cells). The first phase of Ato expression is severely reduced or absent in clones of smo³ cells, similar to large clones that lack Hh. However, the second phase of expression still occurs, even though it is displaced posteriorly, indicating that it is delayed. This displacement is more severe in the middle of the clone than along the dorsal and ventral borders, producing a crescent shaped distortion of the line of spaced single cells that express Ato. It is concluded that cells within smo mutant clones can be induced to express Ato even though they cannot receive Hh, provided that they are located near to wild-type cells across the clone border. Equivalent effects have been observed for Hairy expression. Hairy is normally expressed at peak levels in a dorso-ventral stripe positioned immediately anterior to the Ato stripe, but is abruptly downregulated in more posteriorly situated cells. Clones of smo³ cells have only a modest effect on Hairy expression anterior to the furrow, causing a slight, but consistent, posterior displacement of the anterior edge of the stripe. However, they are associated with a pronounced failure to repress Hairy expression in some, but not all, posteriorly situated smo³ cells. As in the case of Ato expression, the exceptional mutant cells that retain the normal downregulation of Hairy are those positioned close to the lateral and posterior borders of the clones. Just within the lateral border, a line of cells is typically observed, one or two cell diameter lengths wide, where Hairy expression is repressed. Along the posterior border, the zone of mutant cells in which Hairy expression is repressed is usually wider (Greenwood, 1999).

These results are interpreted to indicate that (1) Hh normally induces cells to express a secondary signal (or signals) that can activate Ato expression and repress Hairy expression; (2) this signal acts non-autonomously, allowing it to move from wild-type cells where it is induced by Hh to nearby smo³ cells where it regulates Ato and Hairy expression; and (3) the range of this signal is short, restricting its action to only one or two cells across the lateral borders of smo³ mutant clones. A somewhat greater range of action is apparent along the posterior borders of such clones, perhaps because the adjacent wild-type cells were induced by Hh to send this signal at an earlier time than those along the lateral (more anterior) borders of the clone, allowing the signal more time to accumulate to higher levels and to move deeper into mutant tissue (Greenwood, 1999).

To examine how the posterior displacements in Ato and Hairy regulation in smo³ clones influence subsequent ommatidial development, the expression of the protein Elav, a marker of photoreceptor differentiation was examined. Clones of smo³ cells are capable of differentiating as photoreceptors, in agreement with previous findings. However, there is a significant delay. In wild-type tissue, Elav expression initiates immediately posterior to the morphogenetic furrow with the specification of the R8 cell and continues as other photoreceptors are recruited into the ommatidial cluster. In clones of smo³ cells, there is a clear posterior displacement in the onset of photoreceptor differentiation in mutant cells: photoreceptor differentiation is first seen at the posterior, and occasionally lateral, edges of the clone, correlating with the effects of neighboring wild-type tissue on Hairy and Ato expression and indicating a general delay in photoreceptor differentiation. However, as seen in more posteriorly situated clones, most or all of the smo³ tissue eventually differentiates as normally patterned ommatidia. Thus, Hh signal transduction is not autonomously required for presumptive eye cells to express Ato, downregulate Hairy, or differentiate as photoreceptors. This is in contrast to the general requirement for Hh signaling revealed by experiments in which Hh signaling is blocked throughout the entire disc using temperature-sensitive hh mutations. In the latter case, loss of Hh signaling causes a rapid and complete block in photoreceptor differentiation and furrow progression. Hh signaling appears to induce at least two secondary signals that are essential for the normal recruitment of undifferentiated cells to form the R8 photoreceptors. One of them appears to be the short-range signal that can induce Ato expression and repress Hairy in clones of smo minus cells. The second, Decapentaplegic (Dpp), appears to act at longer range to prime cells to receive this short range signal (Greenwood, 1999).

One candidate for a secondary signal, which acts downstream of Hh in the developing retina, is the TGF-beta homolog Dpp. Dpp is induced by Hh just anterior to the morphogenetic furrow. Moreover, experiments in other discs have established that Dpp can act at long range from its source to mediate the organizing activity of Hh on more anteriorly situated tissue. However, previous studies have shown that Dpp signaling is not essential for either photoreceptor differentiation or propagation of the furrow once photoreceptor differentiation initiates at the posterior edge of the eye primordium. These findings challenge the notion that Dpp mediates the organizing activity of Hh in front of the furrow. To test whether Dpp has such an organizing role, two kinds of experiments were performed. In the first, Dpp or activated Thickveins (Tkv), a type I TGFbeta receptor required for all known Dpp activities, was ectopically expressed anterior to the furrow. In the second, Dpp expression or Tkv activity was blocked. The results of these experiments indicate that Dpp signaling is both necessary and sufficient to upregulate Hairy expression anterior to the furrow and to maintain the normal rate of furrow progression, but that it is neither necessary nor sufficient to activate Ato expression and initiate photoreceptor differentiation in more posterior cells (Greenwood, 1999).

The dpp^blk mutation is associated with a deletion of cis-acting regulatory sequences that are essential for Hh-dependent transcription of dpp in the eye. As a result, Dpp signaling in the eye disc is abolished or severely reduced anterior to the furrow, and the resulting eye is greatly reduced in size in both the dorsal-ventral and antero-posterior axis. Hairy expression in wild-type and dpp^blk disks were compared, using the upregulation of Cubitus interruptis (Ci), a protein that is stabilized in response to Hh signaling, as a marker of the position at which the furrow should normally form. In wild-type eye discs, Ci accumulates to peak levels in a dorso-ventral stripe of cells just posterior to the stripe of peak Hairy expression, consistent with the finding that Hairy expression is repressed in response to Hh signaling within the furrow, but is activated by Dpp signaling anterior to the furrow. In contrast, the stripe of maximal Hairy expression is displaced posteriorly in dpp^blk discs relative to the stripe of maximal Ci expression. Moreover, the furrow appears to have moved only a small distance from the posterior edge of the presumptive eye primordium, even in eye discs from mature third instar larvae, consistent with the 'small eye' phenotype observed in the adult. These results indicate that Dpp signaling is normally required to activate high level Hairy expression in a stripe positioned just anterior to the furrow. They also indicate that Dpp signaling is necessary to sustain the normal rate of furrow progression. Finally, they suggest that Dpp signaling influences the response of cells to peak levels of Hh signal transduction: Hairy expression is downregulated in these cells in wild-type discs, but not in dpp^blk discs (Greenwood, 1999).

This analysis indicates that Hh orchestrates Hairy and Ato expression as well as furrow progression in a manner that depends on Dpp signaling ahead of the furrow. Yet Dpp signaling is not sufficient to activate Ato or to initiate photoreceptor differentiation. Conversely, cells devoid of Smo, and hence unable to receive Hh, can still be induced by neighboring wild-type cells to express Ato and make photoreceptors. These findings argue for an additional signal involved in mediating the organizing activity of Hh on photoreceptor differentiation and furrow progression. The signal transduction pathway downstream of receptor tyrosine kinases involving Ras and Raf has been shown to have multiple roles during the formation of the Drosophila eye, most notably in mediating signals by the Sevenless and Epidermal Growth Factor receptors. Further, misexpression of activated forms of Ras, or the EGF receptor, anterior to the furrow, can initiate photoreceptor differentiation. It was therefore asked whether this signaling pathway might also be utilized in the induction of Ato. To constitutively activate the Raf transduction pathway in the eye disc, a myristylated and truncated form of human Raf, which is called Raf*, was expressed in clones of cells using the Flp-out technique. Under conditions that cause expression of constitutively active Raf* in most cells, the anterior stripe of peak Ato expression is observed to expand several cell diameter lengths forward, to fill the region just anterior to the position of the furrow where Hairy is normally expressed at peak level. However, the anterior limit of peak Ato expression does not extend past that of Hairy expression. Examination of Hairy under the same conditions reveals that the normally high levels of expression just anterior to the furrow are diminished. This reduction in Hairy expression, alone, is not likely to account for the expansion of Ato expression, because Ato expression is not altered in eye discs lacking hairy activity. However the combined loss of both Hairy and the related repressor protein Emc causes ectopic Ato expression anterior to the furrow. In the presence of indiscriminate Raf* activity, Emc expression, like that of Hairy, appears to be diminished anterior to the furrow. Hence, the expansion of Ato expression can be attributed to the concomitant reduction in both Hairy and Emc expression (Greenwood, 1999).

Constitutive expression of Raf* also induces ectopic photoreceptor differentiation anterior to the furrow, as assayed by the expression of the neuronal antigen 22C10. Similar to the expansion of Ato induced by Raf*, ectopic photoreceptor differentiation is restricted to a dorso-ventral stripe of cells just anterior to the normal position of the furrow. Not all the cells within this column differentiate as photoreceptors. Rather, there is a saltatory pattern of differentiation that can be attributed to the process of lateral inhibition, which normally restricts the number of proneural cells that differentiate as photoreceptors. Similar results are obtained under conditions that create small, rare clones of cells expressing Raf*. In this case, small, isolated clusters of photoreceptors, located anterior to the endogenous furrow and surrounded by what appears to be an ectopic furrow (marked in this experiment by the expression of a dpp-lacZ reporter gene) are observed. As is observed for neural differentiation induced by widespread expression of Raf*, small clones of Raf* expressing cells initiate ectopic photoreceptor development only when they are close to the furrow. These findings indicate that during normal development, Dpp signaling anterior to the furrow creates a pool of cells; these cells are primed to enter the proneural pathway, but blocked from doing so by the expression of high levels of Hairy and Emc. These cells are referred to as being 'pre-proneural'. Release from this block requires an additional signal, which is induced at short range by Hh signaling and transduced by activation of the Raf pathway. Raf, but not the Drosophila EGF receptor, is required for Ato expression and photoreceptor differentiation. The results seen with activated Raf suggest that endogenous Raf may normally regulate Ato expression. Therefore, Ato expression was examined in clones of cells that lack Raf function. Clones of raf minus cells marked by the expression of a nuclear-localized form of beta-galactosidase were generated using the Flp-out technique. Raf minus cells do not express Ato. Moreover, the absence of Ato expression is cell-autonomous, indicating that Raf is normally required to transduce a signal that is essential for Ato expression (Greenwood, 1999).

Raf normally mediates signals from receptor tyrosine kinases. Two candidate receptor tyrosine kinases, which could activate Raf ahead of the furrow, are the Drosophila EGF receptor (Egfr) and one of the Drosophila FGF receptor homologs, Heartless (Htl). Egfr is expressed at high levels ahead of the furrow, and is required for photoreceptor differentiation. Similarly, two ligands for Egfr, Spitz and Vein, are active within the furrow and regulate photoreceptor differentiation. However, Ato is expressed in Egfr minus clones, indicating that Egfr is not essential for the Raf-dependent activation of Ato expression. Htl is also expressed in the morphogenetic furrow. Similar to Egfr, however, removal of Htl has no effect on Ato expression. It is possible that both Egfr and Htl can receive the Ato inducing signal, accounting for the ability of cells lacking one or the other function to initiate Ato expression. Alternatively, Raf may transduce a signal that is received by another receptor (Greenwood, 1999).

It is envisaged that pre-proneural cells are metastable, having a latent proneural capacity that is actively held in check by proneural repressors such as Hairy and Emc. How does activation of Raf precipitate the transition to the proneural state? Because the simultaneous loss of both Hairy and Emc activities causes an expansion of Ato expression similar to that resulting from the expression of activated Raf, it has been suggested that Raf activation may normally induce transition to the proneural state by blocking the expression or activity of these repressors. Consistent with this possibility, Hairy contains potential phosphorylation sites for MAPK, a kinase downstream of Raf in the signaling pathway. Daughterless expression is also upregulated in the furrow and is necessary to maintain Ato expression. Moreover, Daughterless, like Hairy, contains phosphorylation sites for MAPK, raising the possibility that Raf activity may directly potentiate proneural activators at the same time that it downregulates the activities of their repressors. Similar events may also occur in mammalian neural differentiation, as NGF-induced differentiation of the mammalian neuronal cell line PC12 is mediated by the phosphorylation of HES-1, a Hairy related protein (Greenwood, 1999).

GENE STRUCTURE

cDNA clone length - 2.2 kb

Bases in 5' UTR - 385

Exons - three

Bases in 3' UTR - 570

PROTEIN STRUCTURE

Amino Acids - 471

Structural Domains

The HH protein has a novel sequence with a hydrophobic N-terminal region (Lee, 1992 and Tashiro, 1993).

The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. A 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing has been identified and crystallized. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins. Inteins are central portions of self-splicing proteins that are excised posttranslationally. The amino- and carboxy-terminal flanking regions, termed exteins, are subsequently ligated to form a mature protein. Inteins typically contain an endonuclease activity in addition to self-splicing properties and have been inserted in a wide variety of archaeal, bacterial, and chloroplast proteins as well as yeast vacuolar ATPases (Hall, 1997).

date revised:28 Dec 96 

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