hedgehog

Targets of Activity (part 1/2)

Hedgehog targets in the epidermis

The secreted proteins Wingless and Hedgehog are essential to the elaboration of the denticle pattern in the epidermis of Drosophila embryos. Signaling by Wingless and Hedgehog regulates the expression of veinlet (rhomboid) and Serrate, two genes expressed in prospective denticle belts. Thus, Serrate and veinlet (rhom) partake in the last layer of the segmentation cascade. Ultimately, Wingless, Hedgehog, Veinlet (an indirect activator of the Egfr) and Serrate (an activator of Notch) are expressed in non-overlapping narrow stripes. The interface between any two stripes allows a reliable prediction of individual denticle types and polarity, suggesting that contact-dependent signaling modulates individual cell fates. Attributes of a morphogen can be ascribed to Hedgehog in this system. However, no single morphogen organizes the whole denticle pattern (Alexandre, 1999).

Both Wingless and Hedgehog signaling pathways repress Serrate expression. Since both pathways are believed to activate transcription, it is imagined that they activate the expression of a repressor of Serrate. In addition, Serrate may also be negatively regulated by the transcriptional repressor Engrailed. In contrast to Serrate, veinlet is regulated both positively and negatively: it is repressed by Wingless and activated by Hedgehog. In addition to this vertical flow of information, regulatory interactions also exist between veinlet and Serrate. At the least, Serrate activates veinlet expression by way of the Notch pathway. This effect is purely non-cell autonomous. In contrast, Serrate appears to repress veinlet in a cell autonomous manner (indeed, in cells where it is expressed, Serrate represses the Notch pathway). However, it is also possible that whichever mechanism activates Serrate expression also represses veinlet expression. This would explain why the expression of Serrate and veinlet is always mutually exclusive (Alexandre, 1999).

The regulatory interactions summarized above are sufficient to explain the spatial pattern of both Serrate and veinlet expression. Non-autonomous repression of Serrate by Wingless and Hedgehog ensures that Serrate is expressed in stripes. The spread of Wingless toward the anterior defines the posterior edge of the domain of Serrate expression. Similarly, the anterior edge of the Serrate domain appears to be specified over three cell diameters by Hedgehog slightly further than expected since Hedgehog is thought to act only over 1-2 cells in Drosophila embryos. Expression of veinlet is activated by two different signals, Hedgehog at the anterior and Serrate at the posterior. Although Hedgehog signaling is symmetrical, it does not activate veinlet (rhom) expression anteriorly because there, Wingless represses veinlet expression. Likewise, Serrate activates veinlet expression but only on one side because of unilateral repression by Wingless (Alexandre, 1999).

These interactions display a clear temporal hierarchy. The secreted molecules Hedgehog and Wingless are expressed first and where they do not reach, Serrate expression is subsequently allowed. At stage 11, Hedgehog and Serrate activates veinlet expression in separate cells. Ultimately, this chain of interactions results in detailed patterns of gene expression (Alexandre, 1999).

Mapping the expression pattern of various genes onto the denticle pattern suggests simple correlations. These correlations have allowed the visualization of pattern where it was previously thought there was none, as in wingless mutants. It is now believed that wingless mutants make denticle type 3, 4 and 5 and not exclusively type 5 as has been suggested. The correlations provide a guide to understand various phenotypes such as those of patched mutants and wg-en-double mutants. In wg-en-double mutants, the correlation between gene expression and denticle type/polarity is particularly evident. Expression of veinlet is in circles surrounded by Serrate expression; this correlates with polarity reversal in the cuticle. Non-uniform gene expression shows that these embryos have more pattern than previously noted. For such embryos to be truly unpatterned, they would have to express Serrate uniformly as well as not express veinlet (rhom). This may occur in wg-en-hh- triple mutants since they may not contain any repressor of Serrate. It is presumed that the converse situation (Serrate 'off'and veinlet 'on' everywhere) would also lead to unpatterned embryos. This situation would prevail in wg-ptc-en-triple mutants. Although the correlations have good predictive value, they suffer from several limitations. (1) Denticle shape does not necessarily reflect an integer value. Indeed, unambiguous typing is not always possible and exact denticle shapes vary from segment to segment. (2) Causal relationships between the activation of a particular signaling pathway and a given denticle type still remain to be investigated. The various signaling pathways are predicted to control cytoskeletal behavior, which in turn affects denticle shape and cell polarity. Local polarity reversals indicate that individual cells are able to locate the source of a particular signal, suggesting that subcellular signaling complexes control the cytoskeleton directly. (3) The involvement of additional regulators cannot be excluded. In particular, it is possible that redundant regulators of the Notch and Egfr pathway contribute to the choice of denticle type. These could include Vein (another Egfr ligand), Delta (a Notch ligand) or possibly Fringe. vein is not required for embryogenesis suggesting that it does not play an important role if any. Possible contributions from Delta to denticle patterning are not readily assessed because of Delta's earlier action in neurogenesis (Alexandre, 1999).

These results show that no single morphogen organizes the denticle pattern: patterning arises, at least initially, from the combined actions of Wingless and Hedgehog. Wingless is clearly not involved in the specification of denticle types (or diversity) across each belt since it does not act in this region of the epidermis. If it did, veinlet and Serrate would not be expressed because, as has been shown, they are both repressed by Wingless. Nevertheless, Wingless acts at a distance, over 3- to 5-cell diameters to set the boundaries of the Serrate expression domain and thus establishes conditions for subsequent juxtacrine signaling. Long-range Wingless action is also required for the asymmetric action of Serrate: Serrate does not activate veinlet (rhom) expression posteriorly because of the presence of Wingless there, 3- to 5-cell diameters from the site of wingless transcription. In this sense, Wingless modulates, at a distance, the outcome of local signaling. In neither of these activities is there evidence for concentration-dependent signaling. However, one cannot formally exclude the possibility that the specification of type 6 denticle requires low-level Wingless. Furthermore, the suggestion that Wingless is not a morphogen in the embryonic epidermis is at odds with studies of the first thoracic segment where various levels of Wingless signaling lead to the specification of distinct cuticular structures. Re-assessment of these phenotypes with early molecular markers might tell whether or not Wingless acts directly in a concentration-dependent manner in the embryonic epidermis (Alexandre, 1999).

The situation with Hedgehog is clearer since it has qualitatively distinct effects over a narrow strip of cells. It activates veinlet expression in adjoining posterior cells while its repressive effect on Serrate expression extends over three cell diameters. This suggests that, at high level, Hedgehog activates veinlet (near the source) while at both low and high levels it repress Serrate expression (further away from the source). In this sense, Hedgehog qualifies as a morphogen. Whether differential responses at different distances from the Hedgehog source reflect true concentration dependence remains to be assessed. It is noted that the repressive effect of Hedgehog on Serrate expression might take place early in development since, in wingless mutants, hedgehog expression decays around stage 10 and yet Serrate expression is still confined at the anterior. It is suggested that early Hedgehog has a repressive effect on Serrate expression that lasts at least until stage 11, when veinlet expression commences. It is therefore conceivable that the 3-cell-wide domain where Serrate is repressed at stage 11 originates by cell proliferation from a single row of cells that abut the Hedgehog source at early embryonic stages. According to this scenario, the effects of Hedgehog on Serrate and veinlet expression would both be occurring over one cell diameter. The apparent difference in range would reflect the difference in timing between these two effects and the intervening proliferation. This model is being tested by assessing the activity of a membrane-tethered form of Hedgehog (Alexandre, 1999).

To sum up, in the bald area of abdominal segments, one cell type forms in response to one signaling pathway while within denticle belts, a rich pattern of cell types arise from juxtacrine cell interactions initiated by the activation of distinct signaling pathways. Some of these pathways are controlled by the localized expression of segment polarity genes such as wingless and hedgehog, while others are regulated by downstream genes like veinlet and Serrate. Because wingless and hedgehog are expressed first, they are effectively at the top of the hierarchy and the knock-on effects of losing hedgehog or wingless function explain the 'organizer activity' of the parasegment boundary. Interestingly, the denticle Hedgehog originating from the parasegment boundaries of adjacent segments (and therefore, two parasegment boundaries) are needed to provide the signals that pattern a single denticle belt (Alexandre, 1999).

Wnt genes are often expressed in overlapping patterns, where they affect a wide array of developmental processes. To address the way in which various Wnt signals elicit distinct effects, the activities of two Wnt genes in Drosophila, DWnt-4, and wingless, were compared. These Wnt signals produce distinct responses in cells of the dorsal embryonic epidermis. Whereas wingless acts independently of hedgehog signaling in these cells, DWnt-4 requires Hh to elicit its effects. Expression of Wg signal transduction components does not mimic expression of DWnt-4, suggesting that DWnt-4 signaling proceeds through a distinct pathway. The dorsal epidermis may therefore be useful in the identification of novel Wnt signaling components (Buratovich, 2000).

DWnt-4 and wg are expressed in many of the same cells during Drosophila embryogenesis, including the ventral epidermis. However, in the cells of the dorsal epidermis each gene is expressed in distinct groups of cells. Whereas wg is expressed in the most posterior row of cells in each parasegment throughout most of embryogenesis, DWnt-4 is expressed in the anterior region of the parasegment. This expression is transient, beginning at stage 10 and fading by the end of stage 12. The wg-like ventral expression of DWnt-4 is dependent on hh, which may be due to shared regulatory elements between the two genes. However, since the dorsal expression of the two genes is nonoverlapping, this aspect of DWnt-4 expression appears to be regulated differently. Since the dorsal stripes of DWnt-4 lie in between the hh and wg stripes, the effect these genes have on dorsal DWnt-4 expression was examined. In wg mutants DWnt-4 is expressed normally, indicating that its expression in the epidermis is not dependent on the activity of wg. However, in hh mutants, both dorsal and ventral expression is eliminated. The regulation of DWnt-4 by hh within the anterior half of the dorsal parasegment suggests that it acts in concert with hh to pattern these cells (Buratovich, 2000).

To determine whether DWnt-4 is able to modulate the patterning of the dorsal epidermis, and whether it mimics or otherwise regulates wg signaling in these cells, it was ubiquitously expressed using the GAL4 system. The results of ubiquitous expression of wg or DWnt-4 were compared. Ubiquitous expression of wg driven by a GAL4 insertion under the control of a daughterless enhancer (daGAL4) results in a uniform lawn of 4^o cells. Thus the hh-dependent cell types are deleted or transformed to 4^o fates. Ubiquitous expression of DWnt-4 elicits a distinct response in the hh-dependent cells, while having no effect on the wg-dependent cells. The phenotype produced by ectopic DWnt-4 is variable and dependent on levels of ectopic expression. With one copy of ectopic DWnt-4 expressed at 29^oC, 21% (23/108) of the segments exhibit a 2^o-3^o-4^o pattern, in which 1^o cells are missing and 3^o cells are expanded. In contrast, 62% of the segments exhibit either a 1^o-3^o-4^o or a 3^o-4^o pattern; it was found that 1^o and 3^o cells are difficult to distinguish. Lower levels of expression produced by rearing the flies at a lower temperature produces a higher percentage of embryos with a pattern that is more clearly 1^o-3^o-4^o along the dorsal midline, since the 2^o cell fate is still apparent laterally. Nevertheless, the 2^o-3^o-4^o phenotype shows that DWnt-4 can abolish 1^o cells, and indicates that the primary effect of DWnt-4 is to expand 3^o cells at the expense of the other two cell types (Buratovich, 2000).

These data show that cells in the anterior half of each parasegment have the ability to respond to both Wnt genes, but that each gene elicits a distinct response. Whereas Wg transforms these cells to 4^o cells or deletes them, DWnt-4 appears to modulate the specification of cell fate within the hh-dependent domain but has no effect on cell fate specification by wg. The phenotypes produced by ectopic DWnt-4 and wg therefore appear to be qualitatively distinct, in that each gene induces ectopic specification of different cell types (Buratovich, 2000).

The alteration in pattern by DWnt-4 suggests three possible interactions with hh. (1) DWnt-4 might affect the anterior half of the parasegment through modification of hh expression. However, analysis of hh transcripts following ectopic DWnt-4 expression has revealed that hh expression is not affected. (2) Since DWnt-4 expression requires hh activity, it could be a downstream effector of hh in pattern specification. (3) DWnt-4 could act in concert with hh to alter pattern. To address these possibilities, DWnt-4 was ectopically expressed in a hh temperature sensitive mutant shifted to the restrictive temperature at 6 h. Under these conditions the entire anterior half of the parasegment is missing in hh mutants. When DWnt-4 is ectopically produced in this background, anterior cell fates still fail to be specified, indicating that DWnt-4 does not simply act downstream of hh but requires hh for its activity after 6 h of development. If hh ts mutants are shifted to the restrictive temperature at 7 h, one row of 3^o denticles typically forms, while 1^o and 2^o fates are still missing. If DWnt-4 is ectopically expressed under these conditions, the number of 3^o rows increases, supporting the conclusion that DWnt-4 acts in concert with hh to specify 3^o cell fates (Buratovich, 2000).

Ectodermal and mesodermal muscle segment homeobox expression depend on wingless and hedgehog. The intricate pattern of msh expression in segmentally arranged clusters during stages 10 and 11, is altered in segment polarity mutants. Mutation of hh affects the intermediate column of msh expressing clusters. In hh mutant embryos, ectodermal msh expression is absent at these positions and the mesodermal expression in fat body precursors is strongly reduced. In contrast, in mutants for wingless the intermediate clusters of msh are normal, whereas the dorsal clusters, both from ectoderm and the mesoderm are completely absent. As a consequence, later stage embryos lack msh expression both in dorsal muscles and around chordotonal organs (D'Alessio, 1996).

Body structures of Drosophila develop through transient developmental units, termed parasegments, with boundaries lying between the adjacent expression domains of wingless and engrailed. Parasegments are transformed into the morphologically distinct segments that remain fixed. Segment borders are established adjacent and posterior to each engrailed domain. They are marked by single rows of stripe expressing cells that develop into epidermal muscle attachment sites. The positioning of these cells is achieved through repression of Hedgehog signal transduction by Wingless signaling at the parasegment boundary. The nuclear mediators of the two signaling pathways, Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively (Piepenburg, 2000).

A cis-acting element of stripe (sr) has been identified that specifically directs gene expression in segment border cells during embryogenesis. This element was used to illuminate the molecular mechanism underlying segment border selection. The results show that Hedgehog (Hh) signaling can activate gene expression in two rows of cells, one on each side of the engrailed (en) expression domain. However, anterior Hh signaling causes the maintainance of wingless expression anterior to the PS boundary. Wg in turn antagonizes Hh-dependent gene expression and thereby prevents the formation of segment border cells anterior to the en domain. Hh and Wg activities relevant for the selection of segment border cells are mediated by functional binding sites of their nuclear mediators, Cubitus interruptus (Ci) and Pangolin (Pan), respectively within the sr cis-acing element. The data suggest that the segment border is established in response to the asymmetry of Wg signaling at the PS boundary (Piepenburg, 2000).

How repeating striped patterns arise across cellular fields is unclear. To address this the repeating pattern of Stripe expression across the parasegment (PS) was examined in Drosophila. This pattern is generated in two steps. Initially, the ligands Hedgehog (Hh) and Wingless (Wg) subdivide the PS into smaller territories. Next, the ligands Hh, Spitz (Spi), and Wg each emanate from a specific territory and induce Sr expression in an adjacent territory. The width of Sr expression is determined by signaling strength. Finally, an enhancer trap in the sr gene detects the response to Spi and Wg, but not to Hh, implying the existence of separable control elements in the sr gene. Thus, a distinct inductive event is used to initiate each element of the repeating striped pattern (Hatini, 2001).

The repeating pattern of Stripe (Sr) expression across the parasegment (PS) is generated by inductive inputs from three spatially localized ligand sources. The ligands, Hh, Spi, and Wg, emitted by En, Ve, and Wg territories, respectively, control Sr expression in cells adjacent to each ligand source. There are three notable features to this regulation: (1) each ligand-producing territory induces Sr expression in the adjacent territory; (2) the induction is asymmetric, either anterior or posterior to each source; (3) the induction is initiated at the high level of signaling achieved near the source, limiting expression of Sr to a narrow row of cells. Because these same ligands act more broadly in cuticle cell fate specification, these results also suggest that the ligands and signaling territories operate in a fundamentally distinct way in order to construct a repeating striped pattern. These observations reveal a strategy used to generate a repeating striped pattern across a cellular field that may be used generally (Hatini, 2001).

Each Sr row is initiated adjacent to a different ligand source. The induction of Sr was limited to a narrow row of cells at each position. Manipulating either the ligand level, or the sensitivity of cells to a specific signaling pathway, leads to a broadened territory of Sr induction. Thus, local activity gradients of Hh, Spi, and Wg are each generated, and a threshold for activation of Sr is only surpassed in cells adjacent to each source. The gradient landscape of Spi and Wg is sculpted using the inducible antagonists Argos and Naked, respectively. Although how the activity landscape for Hh is sculpted was not specifically addressed, the Hh pathway also makes use of an inducible antagonist. It is likely that Hh spread is limited by binding to the Hh receptor Ptc, which is upregulated by Hh input (Hatini, 2001).

To generate the repeating striped tendon pattern, the Sr gene must be able to respond to each of three different ligands. To account for this, it is expected that the Sr promoter is modular, and each Sr row is induced via a separable, cis-acting response element. An enhancer trap P-insertion in the sr gene (sr⁰³⁹⁹⁹) provides evidence for this since it detects the response to Spi and Wg, but not to Hh, implying that the P-insertion separates response elements in the sr gene. A separable Sr promoter element controlling Hh-dependent expression has been identified. Although this element operates only in dorsal and lateral epidermis, and not ventrally where Sr is expressed in repeating striped pattern, this observation strongly suggests that the control elements will be modular. Furthermore, in this dorsal/lateral element, the presence of functional, consensus Cubitus interruptus (Ci) DNA binding sites suggests direct regulation of Sr by the Hh signaling pathway. The obvious analogy is to the modularity of regulatory regions of certain pair-rule genes, which are able to integrate non-periodic information in order to generate periodicity. Note that the induction of the sr gene is limited to cells bordering each ligand source, even though each of the signals can act across several cell diameters. It is predicted that a given Sr response-element is configured to sense and respond only to a particularly high threshold level of each ligand (Hatini, 2001).

The ligands controlling Sr expression emanate from each of three territories across the PS. These territories are established by the primary organizing signals, Wg and Hh. In the earliest step, cross regulation between Wg and En/Hh-expressing cells stabilizes each ligand's expression and consolidates these two territories. In addition, through negative regulation, both Wg and Hh limit the expression of Ser to a central territory within the PS. Finally, signals from the En/Hh territory induce Ve expression in two cell rows just posterior to the En/Hh territory. The exact width of the Ve-expressing territory is adjusted as local input from the Ser territory induces a third Ve-expressing cell row. Thus, Hh and Wg act indirectly by defining and limiting each other's expression territory, as well as that of downstream ligands. All of these ligands then organize the repeating pattern. Three ligands induce Sr expression at specific positions across the PS, while the role of Ser reveals a particularly interesting spatial cue. Although the second row of Sr is induced in the anterior-most row of Ser-expressing cells, Ser expression is not necessary for this. Rather, Ser dictates the spacing between Sr row 1 and 2, because it defines the breadth of the Ve territory and thereby the position of the first non-Ve cell that can induce Sr in response to Spi-Egfr signaling (Hatini, 2001).

Sr expression is induced asymmetrically relative to each ligand source. For instance, Hh induces Sr posterior to the En territory, but not in the En territory or anterior to it. Wg imparts asymmetry to Hh/En signaling, and thereby prevents Ve expression anterior to the En/Hh territory. In exactly the same way, via antagonism of Hh signaling, Wg appears to block Sr expression anterior to the En/Hh territory, because the removal of Wg function allows Sr expression anterior to the En/Hh territory. The Wg signaling pathway imposes asymmetry to the dorsal/lateral Sr regulatory element via consensus Pangolin DNA binding sites. This principle is likely to extend to the ventral control of Sr for the generation of one element of the repeating striped pattern (Hatini, 2001).

One reason why Hh signaling cannot induce Sr expression in the En cells is that En represses expression of the Hh signal transducer, Ci. Nevertheless, it is still necessary to explain why signals from both the Ve and Wg territories cannot induce Sr in the En cells, even though each signal definitely acts on these cells to specify cuticle fate. A clue comes from the observation that when the En territory is not maintained Sr is induced symmetrically relative to the Wg or to the Ve sources. Thus, it is proposed that the En protein prevents Sr induction by Wg or Egfr inputs by repressing Sr expression. This is supported by the observation that activating Wg signaling at high levels in En cells still does not lead to Sr expression. To explain why Sr is not induced by Spi in the Ve territory, nor by Wg in the Wg territory, it is inferred that there is a specific block to autocrine signaling in each territory. Interestingly, this block is specific to Sr induction, and not to other outcomes of signaling, such as cuticle fate specification. It suggests a lack of an activator essential to induce Sr expression or expression of a repressor that blocks such a response in the Ve and Wg territories (Hatini, 2001).

The same ligands establish strikingly distinct patterns across the same cellular field. While the cuticle pattern comprises a diversity of cell types, the Sr expression pattern reflects the near-periodic specification of the same cell type. These distinct outcomes arise because the same ligands act in a fundamentally different manner in these two processes. As an example, Wg specifies smooth cuticle in a broad region anterior to the Wg territory, in the Wg territory, and posterior to the Wg territory (in anterior En/Hh cells). However, as is shown in this study, Wg induces Sr only anterior to the Wg territory in a narrow region, and not in the Wg territory or posterior to it. Also, Spi, through Egfr function, induces denticles over a broad region, both in the Ve territory and anterior to it in a subset of En/Hh cells. However, Egfr function induces Sr only in a narrow region posterior to the Ve territory. Thus, despite the broad effects of Wg and Egfr on cuticle pattern, the effect of Wg and Egfr in building the repeating striped pattern is constrained to a narrow region of cells. As a final example of the distinction between control of denticle pattern and control of repeating striped pattern, in the same row of cells, cuticle fate is specified by Spi while tendon fate is specified by Hh input. The unique effects of these ligands on Sr expression are crucial for the establishment of the repeating striped pattern. Thus, the information encoded in the signaling territories is decoded in different ways to achieve both repeating pattern and cell-type diversity across the same field (Hatini, 2001).

The generation of near-periodic Sr pattern across the PS is conceptually similar to the two-step process that is used in establishing the periodic body plan through pair-rule gene expression in syncitial embryos. Initially, primary pattern organizing centers are established at the boundaries of a field of naive nuclei or cells. In the first step, these centers establish patterned expression of secondary organizing genes across the field, subdividing the field into distinct gene expression territories. In the second step, the information encoded in these territories is used to initiate a repeating striped gene expression pattern. In the embryo, Bicoid together with Hunchback and Nanos organize expression of the gap genes. Territories of gap gene expression are then used to establish the periodic pattern of primary pair-rule gene expression. In the PS, Wg and Hh organize overall parasegmental pattern by first defining each other's territory and then the territories of secondary regulatory genes, Ser and Ve. The signaling territories are then used to establish near-periodic expression of Sr. Note that although the conceptual similarity is striking, the mechanisms generating these two repeating patterns are distinct. The pair-rule gene expression pattern is established in a unique, syncitial system by diffusion of transcriptional regulators in a common cytoplasm, whereas Sr expression is established across an epithelial monolayer by communication between cells via inter-cellular signaling systems. In addition, while a balance between diffusible activators and repressors determines pair-rule gene expression at any point along the syncitial embryo, the unique properties of the signaling territories across the PS determine Sr expression. In particular, the juxtaposition of pairs of territories, one that sends a signal with one that can initiate Sr expression in response to the signal, is utilized to initiate Sr expression adjacent to the boundaries between these territories (Hatini, 2001).

A near-periodic striped pattern of veins is produced in the developing wing disc. Emerging evidence suggests that the two-step strategy may also apply to this system, and that unique properties of putative signaling territories are used to initiate wing veins adjacent to at least two territories across the wing blade. In the first step, combined action of Hh and Dpp establishes different territories of downstream regulatory genes across the A/P axis of the future wing blade. While Hh establishes a territory of Ptc expression adjacent to the compartment boundary, Dpp acts more broadly across the wing and establishes two nested territories of Spalt and Optomotor-blind expression. In the second step, veins are induced adjacent to at least two territories, suggesting that an unknown ligand emanates from one territory and induces the vein in the adjacent territory. The possibility that the two-step process described here for the PS is used across the wing blade suggests that this may be a general strategy for creating repeating striped patterns across other cellular fields (Hatini, 2001).

Hedgehog function during neurogenesis

How is neuroblast-specific gene expression established? This paper's focus was on the huckebein gene, because it is expressed in a subset of neuroblasts and is required for aspects of neuronal and glial determination. hkb is required within the neuroblast 1-1, 2-2 and 4-2 lineages for proper axon pathfinding of interneurons and motoneurons and for proper muscle target recognition by motoneurons. The secreted Wingless and Hedgehog proteins activate huckebein expression in distinct but overlapping clusters of neuroectodermal cells and neuroblasts, whereas the nuclear Engrailed and Gooseberry proteins repress huckebein expression in specific regions of neuroectoderm or neuroblasts. Hedgehog activates hkb in cells that give rise to the 5HT expressing lineage), while Wingless activates hkb in cells that give rise to an eve expressing motorneuron lineage). Wingless and Hedgehog activate hkb in the neuroectoderm of hemisegment row 5 neuroblast precursors. Early-forming neuroblasts of rows 5 and 6 never express hkb even though they develop from Hkb+ neuroectoderm (row 5) (McDonald, 1997).

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. This study shows that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia (Bossing, 2006).

The determination of midline cells appears to take place during germband elongation, since by germband retraction most midline subsets can be identified by the expression of unique molecules. The anteroposterior position of midline siblings was determined during germband elongation. Midline precursors were labelled with the lipophilic dye DiD or DiI in embryos expressing GFP in the Engrailed domain (en-GAL4/UAS-tauGFP). After division of the precursors, the daughter cells were followed throughout development, recording their segmental position at stage 10 and stage 11. MP1 interneurons, UMI and MNB neurons each arise from one precursor, and their daughter cells occupy fixed anteroposterior positions during germband elongation. The four daughter cells of the two glial precursors can be located either in the middle of the segment or just anterior to the Engrailed domain. VUM neurons arise from three midline precursors, and the six daughter cells of these precursors are located inside the Engrailed domain and immediately posterior to the domain, in the anterior of the next segment (Bossing, 2006).

In summary, the midline glia and MP1 interneurons are the most anterior midline subsets, followed by a second pair of midline glia and a pair of UMIs, and, finally, the VUM and MNB neurons. DiI labelling cannot resolve whether the MP1 interneurons or the midline glia are the most anterior cells. Since determination of the MP1 interneurons depends on Notch/Delta signalling, it is possible that the anteroposterior position of the most anterior midline cells, the midline glia and MP1 interneurons, is random. Interestingly, four VUM neurons and the MNB neurons seem to arise from the anterior compartment of the next posterior segment. These cells initiate Engrailed expression half-way through germband elongation, and, during germband retraction, they join the adjacent anterior segment to become the most posterior midline subsets (Bossing, 2006).

The separation of midline cells into two compartments is an early and crucial step in midline cell determination. During germband elongation, a second phase of Engrailed expression is initiated at the midline in the anterior cells of the next posterior segment. During germband retraction, these cells join the anterior segment where they develop into posterior midline cells. Expression of late Engrailed depends on Hedgehog signalling and the proneural gene lethal of scute. Lethal of scute precedes Engrailed expression and is also activated by Hedgehog. Hedgehog and Wingless signalling counteract each other to define the position of the Lethal of scute cluster, and to divide the 16 midline daughter cells into eight non-Engrailed- and eight Engrailed-expressing cells (Bossing, 2006).

It has generally been believed that the determination of the different subsets of midline cells occurs before the precursors undergo their simultaneous division at stage 8. This view is challenged by the observation that expression of the proneural gene lethal of scute, and the subsequent expression of Engrailed, is initiated in midline daughter cells at stage 10, about one hour after the precursors divide. In the neuroectoderm, proneural genes confer neural competence to a cluster of ectodermal cells. Lateral inhibition by Notch/Delta signalling then limits the expression of proneural genes to a single cell, which delaminates from the ectoderm and becomes a neural precursor (neuroblast). Because the only neuroblast at the ventral midline (median neuroblast, MNB) originates from the proneural Lethal of scute cluster, it seems likely that the MNB is selected by lateral inhibition from a cluster of midline daughter cells. However, the process of lateral inhibition in the midline differs from that in the adjacent neuroectoderm. In the neuroectoderm, a single cell delaminates and the remaining cells of the cluster cease proneural expression and give rise to the epidermis. The proneural cluster in the midline consists of three pairs of siblings generated by the division of three separate precursors. Labelling of single precursors shows that, during the selection of the MNB, only one of the two labelled siblings enlarges, but both delaminate from the embryo. In contrast to the neuroectoderm, the remaining cells of the midline cluster continue to express Lethal of scute after delamination of the MNB. This extended proneural expression might be necessary to maintain neural competence in the non-delaminating cells that develop into VUM neurons (Bossing, 2006).

The results cannot exclude the possibility that some of the midline subsets are determined as precursors, but at least two of the five midline subsets, the VUM neurons and the MNB, are determined after precursor cell division. There are striking similarities between the development of the ventral midline of Drosophila and grasshopper embryos. In grasshopper, Engrailed expression can be detected in the MNB, its progeny and the midline precursors MP4 to MP6, which each give rise to two neurons with projection patterns comparable to the Drosophila VUM neurons. Hence, the same types of midline cells express Engrailed in grasshopper and Drosophila, but in grasshopper Engrailed expression is initiated in all midline precursors prior to division (Bossing, 2006).

In the ectoderm from stage 10 onwards, Wingless, Engrailed and Hedgehog maintain the expression of one another by a feedback loop: Wingless maintains Engrailed expression, Engrailed is needed for the expression of Hedgehog and Hedgehog maintains Wingless expression. In the developing CNS, Wingless and Hedgehog expression seem to be independent of each other. At the ventral midline there are two separate stages of Engrailed expression: the early phase is maintained by Wingless; the late phase does not require Wingless and is instead activated at stage 10 by Hedgehog signalling and Lethal of scute. In the ectoderm, Wingless and Hedgehog act in concert to maintain Engrailed expression, but at the midline Wingless and Hedgehog act in opposition: Wingless represses and Hedgehog activates Lethal of scute expression (Bossing, 2006).

Wingless may repress Lethal of scute expression indirectly, via its maintenance of early Engrailed. As in the ectoderm, midline Engrailed represses expression of the Hedgehog receptor Patched and the Hedgehog signal transducer Cubitus interruptus. It is possible that early Engrailed-expressing midline cells are not able to receive the Hedgehog signal. However, ectopic expression of Hedgehog is able to induce Lethal of scute in all midline cells, suggesting that Wingless may repress Lethal of scute by a yet unknown mechanism. Recently it has been reported that a vertebrate wingless orthologue, Wnt2b, can maintain the naïve state of retinal progenitors by attenuating the expression of proneural and neurogenic genes (Bossing, 2006).

The differentiation of midline cells was studyed in wingless and hedgehog mutants. Consistent with earlier reports, many midline cells become apoptotic in both mutants. The surviving midline cells are not integrated into the CNS and show no morphological differentiation. The reduction in the number of Engrailed-positive midline cells in hedgehog mutant embryos may be mainly due to the loss of midline cell identity. In hedgehog mutants, midline cells lose the expression of Sim, the master regulator of midline development. As described for sim mutants, the loss of midline identity results in increased cell death and misspecification of the surviving midline cells as ectoderm (Bossing, 2006).

Ectopic expression of Hedgehog in the neuroectoderm and the developing CNS induces the expression of Lethal of scute and, approximately 40 minutes later, the expression of late Engrailed in all midline cells. It seems likely that Lethal of scute is an early target of Hedgehog signalling, and its activation may only require release from repression by the short form of Cubitus interruptus. By contrast, the delay in induction of late Engrailed in the same midline cells indicates that Engrailed activation may not only require release from repression, but also activation by the long form of Cubitus interruptus (Bossing, 2006).

Uniformly high levels of ectopic Hedgehog prevent the differentiation of most midline subsets and cause increased cell death. A single source of ectopic Hedgehog, achieved by cell transplantation, does not result in midline cell death, but reveals that the differentiation of the MP1 interneurons is more sensitive to Hedgehog levels than is the differentiation of midline glia. No other midline subsets are affected. It seems likely that Hedgehog not only activates Lethal of scute and late Engrailed, but also acts as a morphogen to control the differentiation of the MP1 neurons and midline glia (Bossing, 2006).

The phenotypes caused by ectopic Hedgehog are due to the induction of Engrailed in all midline cells. Expression of ectopic Hedgehog and ectopic Engrailed blocks the differentiation of midline glia and MP1 interneurons, and also prevents the formation of the anterior commissure. Labelling single midline precursors enabled examination of cell fates in embryos expressing ectopic Engrailed in the midline. The frequency of clones obtained indicates that ectopic Engrailed expression does not transform non-Engrailed-expressing midline subsets (MP1 interneurons, midline glia and UMI) into Engrailed-expressing subsets (VUM and MNB). Instead, embryos expressing midline Engrailed show increased cell death. In particular, the MP1 interneurons seem to be affected and were never obtained during this analysis. The low frequency of midline glia also points to apoptosis caused by expression of Engrailed. Surviving midline glia are not able to differentiate properly and cannot enwrap the remaining, posterior, commissure. All other midline subsets, including the UMIs, are able to differentiate, but they show a variety of axonal pathfinding defects that may result from the loss of anterior midline subsets and the absence of the anterior commissure (Bossing, 2006).

It is likely that genes other than hedgehog and wingless are crucial for midline cell determination. In these experiments, non-Engrailed-expressing midline subsets are never transformed into Engrailed-expressing subsets, or vice versa. gooseberry-distal may be one of these genes. From the blastoderm stage, Gooseberry-distal is expressed by two midline precursors and their four daughter cells. During early embryogenesis Gooseberry-distal expression at the midline does not depend on Wingless and Hedgehog. The anterior Gooseberry-distal cells also express Wingless and most likely give rise to the UMIs. The posterior Gooseberry-distal pair also express early Engrailed and Hedgehog, and develop into the most anterior VUM neurons. At stage 10, Hedgehog activates the expression of Lethal of scute and Engrailed in midline cells posterior to the Gooseberry-distal domain. Lateral inhibition by Notch/Delta signalling selects one cell from the Lethal of scute cluster to become the MNB. The remaining cells become VUM neurons. At stage 10, the absence of Engrailed in the six midline cells anterior to the Gooseberry-distal domain defines a cell cluster that will give rise to midline glia and MP1 interneurons. Based on the expression of Odd, Delta mutants have an increased number of MP1 interneurons, up to six per segment. In Notch mutants, midline glial-specific markers are absent and the number of cells expressing a neuronal marker increases. Therefore, Notch/Delta signalling appears to determine midline glial versus MP1 interneuron cell fates in the anterior cluster. In the current model, midline cell determination takes place mainly after the division of the precursors. Although the initial determination of midline cells appears to be directed by a small number of genes, a far larger number is needed to control the differentiation of the various midline subsets. This work, and the recent identification of more than 200 genes expressed in midline cells, is the beginning of a comprehensive understanding of the differentiation of the ventral midline (Bossing, 2006).

Hedgehog targets in heart development

Expression of ladybird genes in the subset of cardioblast and pericardial cell precursors is critically dependent on mesodermal tinman function, epidermal Wingless signaling and the coordinate action of neurogenic genes. lb-expressing heart progenitors contribute to the increased number of cardiac precursor cells in Notch, Delta, Enhancer of split, mastermind, big brain and neuralized mutants. Negative regulation by hedgehog is required to restrict ladybird expression to two out of six cardioblasts in each hemisegment. Overexpression of ladybird causes a hyperplasia of heart precursors and alters the identity of even-skipped-positive pericardial cells (Jagla, 1997).

The Drosophila larval cardiac tube is composed of 104 cardiomyocytes that exhibit genetic and functional diversity. The tube is divided into the aorta and the heart proper that encompass the anterior and posterior parts of the tube, respectively. Differentiation into aorta and heart cardiomyocytes takes place during embryogenesis. Living embryos have been observed to correlate morphological changes occurring during the late phases of cardiogenesis with the acquisition of organ function, including functional inlets, or ostiae. Cardiac cell diversity originates in response to two types of spatial information such that cells differentiate according to their position, both within a segment and along the anteroposterior axis. Axial patterning is controlled by homeotic genes of the Bithorax Complex (BXC) that are regionally expressed within the cardiac tube in non-overlapping domains. The segmentally repeated expression of svp is regulated by a positive inductive effect of Hh secreted by cells from the overlying ectoderm. A role for hh signaling in Drosophila cardiogenesis has not previously been acknowledged. It has been observed that in hh mutant embryos heart progenitors are lacking, however this has been interpreted to be an indirect influence of hh upon wg signaling. The results reported here strongly favor the idea that hh has a direct and positive effect on the determination and specification of the sub-population of cardioblasts that expresses svp. It has been proposed that each segment of the trunk is sub-divided into two domains, A and P. The cells from the anterior domain (A domain) of the dorsal mesoderm would be directed towards a cardiogenic fate while cells from the posterior domain (P domain) would adopt a visceral mesoderm fate. The wg and hh signals released, respectively, from the anterior and posterior compartments of the ectodermal parasegments have been proposed to be the determinants in specification of the two domains. The observations in this study, however, provide strong evidence that a subtype of cardiac cells can originate from the mesodermal P domain. The P domain origin of some cardioblast progenitors has been suggested by the presence at stage 11-12, within the P domains, of bkh-expressing cells, which contribute to the cardiac epithelium later in development. It seems, therefore, that there is not a perfect superimposition between A domains within mesodermal segments and the capacity of the cardiac cells to be integrated into the cardiac tube (Ponzielli, 2002).

The hh signal secreted by cells belonging to the posterior compartments of the segmented ectoderm is sufficient to promote svp expression. The Hh morphogen needs to be secreted and to freely diffuse from the ectoderm to the underlying mesoderm, as judged from the loss of svp expression in the cardioblasts when a membrane-bound form of Hh is expressed in the same genetic background in place of endogenous Hh. The existence of a specific mechanism to constrain diffusion of the secreted morphogen to the cardioblasts of the P-domain can thus be postulated. Further investigation of this mechanism will provide insight into how specificity of morphogen signaling is achieved across embryonic germ layers (Ponzielli, 2002).

Based on gene expression patterns, Hh signaling is likely to be instrumental in the specification of tin- and svp-cardioblasts by inducing the expression of svp in cardioblasts which, in turn, leads to the repression of tin. Such a repressive action of svp has already been reported, although a direct interaction between tin regulatory sequences and svp has not been demonstrated (Ponzielli, 2002).

Similar relationships between homologs of hh, svp and tin have been described in vertebrate cardiogenesis. A homolog of svp, COUP-TFII is expressed in the posterior region of the mouse primitive heart tube where it is required for heart development; furthermore the expression of COUP-TFII is induced by Sonic hedgehog. Shh (and Indian hedgehog) participates in mouse cardiac morphogenesis but, in contrast to the situation in Drosophila, induces rather than represses the expression of the tin homolog, NKx2.5. It must be concluded, from these remarks, that the genetic networks can be differently interpreted and utilized in invertebrates and vertebrates. Further studies should give better insights into the conservation of the genetic programs at work in heart development (Ponzielli, 2002).

Hedgehog and mesoderm development

How does Drosophila mesoderm become subdivided? The process may be illustrated by Bagpipe expression, which is restricted to metameric clusters of cells in the dorsal mesoderm. Under the control of bap, cell clusters develop into midgut visceral mesoderm, whereas cells in segmental portions lacking bap form other mesodermal derivatives. Among the segment polarity genes, both hedgehog and engrailed are required for full activation of bap. These results suggest that ectodermal hh and en participate in the establishment of the mesodermal posterior (P) domains opposite the posterior domains of the ectoderm. Ectodermal Wingless is synthesized adjacent to the anterior (A) domains. Wingless appears to act negatively on bap and serpent, because bap and srp expression is expanded in wg mutant embryos. Thus it appears that ectodermal WG and HH have opposing roles in establishing mesodermal A and P domains (Azpiazu, 1996).

The Drosophila visceral mesoderm (VM) is a favorite system for studying the regulation of target genes by Hox proteins. The VM is formed by cells from only the anterior subdivision of each mesodermal parasegment (PS). The VM itself acquires modular anterior-posterior subdivisions similar to those found in the ectoderm. Mesodemal cells located just under the engrailed-expressing cells in the posterior ectodermal compartment have been called the mesodermal "P domain." The dorsal-most cells of the mesodermal P domain in each PS express the homeobox gene bagpipe (bap); they detach from the mesodermal fold and move inward toward the center of the embryo. These bap-expressing cells form the VM progenitor groups. The VM cells initiate expression of Fasciclin III (FasIII) as they migrate to join each other and form a continuous band of VM running along each side of the embryo. Thus all the VM derive from the posterior parts of the initial mesoderm metameres. As VM progenitors merge to form a continuous band running anterior to posterior along the embryo, expression of connectin (con) occurs in 11 metameric patches within the VM, revealing VM subdivisions analogous to ectodermal compartments (Bilder, 1998).

The VM subdivisions, and the metameric expression of con, form in response to ectodermal production of secreted signals encoded by the segment polarity genes hedgehog (hh) and wingless (wg) and are independent of Hox gene activity. A cascade of induction from ectoderm to mesoderm to endoderm thus subdivides the gut tissues along the A-P axis. Induction of VM subdivisions may converge with Hox-mediated information to refine spatial patterning in the VM. Con patches align with ectodermal engrailed stripes, so the VM subdivisions correspond to PS 2-12 boundaries in the VM. The PS boundaries demarcated by Con in the VM can be used to map expression domains of Hox genes and their targets with high resolution. The resultant map suggests a model for the origins of VM-specific Hox expression in which Hox domains clonally inherited from blastoderm ancestors are modified by diffusible signals acting on VM-specific enhancers (Bilder, 1998).

Since Con expression marks the imprint of ectodermal PS boundaries on the VM, Con patches can be used to precisely map the domains of Hox gene transcription in relation to Con patches. teashirt is expressed in two domains. The anterior midgut domain extends from visceral mesoderm segment (VS) 4 to mid-VS 6, where it shares a posterior boundary with Antennapedia; the central midgut domain extends several cells to either side of the VS 8 boundary. dpp is also expressed in two domains: at the gastric caeca, it is found in the A domain of VS2 and the P domain of VS 3, while in the central midgut it extends from the A domain of VS 6 to terminate just anterior to the VS 8 boundary. wg is expressed just anterior to the VS 8 boundary, with some cells after stage 12 lying in VS 8. pnt is expressed throughout VS 8, although expression is not seen until early stage 13. At stage 13, the two domains of odd paired (opa) expression extend from the P domain of VS 4 to the VS 6 boundary and from VS 9 through VS 11 (Bilder, 1998).

Several Hox targets appear to respect the PS subdivision organization of the VM. The initial VM expression of opa is seen only adjacent to Con patches, in A domains of VS 3-5 and 8-11. Similarly, wg is limited to a subset of abdA-expressing cells: those at the border of VS 8. wg is activated by abdA and dpp. Ectopic expression of abdA leads to induction of wg in a single posterior patch. Strikingly, the sites of ectopic wg induction in both genotypes align with the VS boundaries: in cells just anterior to VS 3, 5, and 6 in ectopic AbdA embryos and anterior to VS 9 in ectopic Dpp embryos. these results suggest that metameric subdivisions in the VM limit Hox gene activation of VM targets (such as wg) to restricted areas. It is suggested that divergent Hox expression in the VM has its basis in tissue-specific regulation of Hox expression in the VM and this expression is governed by unknown regulators that control VM-specific Hox enhancers (Bilder, 1998).

In Drosophila, trunk visceral mesoderm (VM), a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. These findings have been extended by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided in the A/P axis into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts a feedback effect on trachea and ectodermal derivatives (Hosono, 2003).

Enhancer analysis of hh has demonstrated that one hh enhancer fragment (Sph-hh) is capable of inducing reporter gene expression segmentally in VM. lacZ driven by Sph-hh and also hh RNA and protein are expressed as ten VM patches, well aligned with overlaying ectodermal hh stripes. Expression of hh RNA in late stage 11 embryos almost entirely overlaps with that of lacZ driven by Sph-hh. Consequently, hh is expressed in VM in a metameric fashion. hh expressed in VM is hereafter referred to as VM-hh. VM-hh RNA expression, which is initially detected at mid-stage 11, diminished at stage 12, but was weakly detectable until stage 15. Sph-hh-driven lacZ (VM-hh-lacZ) signals are detected up to stage 16. ptc, a general target gene of Hh signaling, is expressed in or around hh-expressing VM cells at mid stage 12, suggesting autocrine and paracrine functions of VM-Hh. Even-numbered VM-PSs in early mesoderm is marked by ftz-lacZ expression. hh RNA staining of late stage 11 ftz-lacZ embryos disclosed VM-hh expression in the anterior terminal region of each VM-PS; VM-hh expression is absent from VM-PS2 (Hosono, 2003).

VM is presently considered to develop in two steps under the control of ectodermal Hh and Wg signals. First, by stage 10 (when four mesodermal primordia have become specified), VM competent or bap expression regions are promoted by hh but repressed by wg, via a direct targetor, slp. The second surge of hh and wg activity at stages 10-11 is responsible for subdividing VM-PSs into two regions: con positive and negative. These results indicate that the expression of four other VM-metameric genes, hh, tin, bnl and bap, is also regulated by the second surge of hh and wg activity at stages 10-11 (Hosono, 2003).

To examine the regulation of VM-metameric genes with changing the activity of hh and/or wg, it may be necessary to evaluate the effects of possible change in cell number on VM-PS subdivision or VM-PS cell specification. In temperature-sensitive mutants of hh and wg shifted-up from stage 10, the number of VM cells positive to FAS3 at mid stage 11 on is essentially identical to that of wild type, indicating that VM-cell number change is negligible under the conditions used, while the expression of some VM metameric genes appear compromised. In hh^ts mutants, VM-hh and con are not expressed, though tin, bnl and bap are expressed. In wg^ts mutants, VM-hh and tin are not expressed, but con is expressed. All these observations are totally in agreement with those found in simple loss-of-function and overexpression experiments; under these conditions, the formation of a VM competent region should be hindered. Thus, these results may indicate that ectodermal Hh and Wg regulate directly, but in different ways, the expression of metameric genes in VM; VM-hh expression requires both Hh and Wg. tin, bnl and bap are positively regulated by Wg alone, and con is activated by Hh and repressed by Wg (Hosono, 2003).

In view of morphological changes in a VM competent region and consideration of these findings on VM gene regulation, the following model for VM-PS cell specification is proposed. At stage 10 to early stage 11, anterior terminal cells of VM-PSs are presumed to be situated near an ectodermal AP border, where they are capable of continuously receiving Wg and Hh signals, and Wg confers competence on these cells to express tin/bnl/bap. Wg and Hh are responsible for inducing VM-hh, and Hh, for con expression. In the anterior-most cells, con expression is reduced, which would be expected in view of repression by high Wg signal. The different thresholds of hh for con and VM-hh expression may explain why the con area expands more posteriorly compared with that of VM-hh. Posterior terminal VM cells, when formed, are situated far from Wg expressed on the ectodermal PS border. But as they migrate posteriorly and close to the posteriorly neighboring AP border by early stage 11, they become capable of receiving Wg and acquire competence to express tin/bnl/bap. Thus, the tin/bnl/bap domain would appear regulated by spatially and temporally distinct Wg signals. The two-step induction of tin/bnl/bap expression is supported by experiments using the wg^ts mutant, where, either posterior or anterior expression within one patch can be differentially turned off. Indeed, a stepwise activation of tin/bnl expression is seen in VM-PSs around stage 11. tin and bnl metameric expression became apparent almost simultaneously at mid-stage 11, and preliminary experiments have shown that neither tin nor bnl misexpression can induce the ectopic expression of any other metameric genes examined here. Thus, tin and bnl expression might be initiated in a mutually independent manner (Hosono, 2003).

This VM-PS subdivision model should be modified when applied to thoracic segments, where hh may not be the sole determinant of con expression (Hosono, 2003).

This study strongly suggests that metameric VM-hh is required for the maintenance of its own as well as metameric con expression, although the latter becomes independent of VM-hh at late stages. That Ptc, a direct target of hh, is upregulated in each VM-hh expression domain at stage 12, at that time VM is far away from the epidermis or ectodermal Hh sources, is additional evidence supporting the notion that hh signaling caused by metameric VM-hh is operative in VM (Hosono, 2003).

These results also show that Hh is required for gastric caecum development. Hh may operate in multiple steps in mesoderm and its source for the last step is VM-Hh emanating from VM-PS3, a part of the future gastric caecum region. Most, if not all, gastric caecum defects found in Hh signaling mutants may be due to the reduction or loss of VM-PS3 dpp, whose production is under the control of VM-PS3 Hh and Hh at stages prior to stage 11. vn expression, which is positively controlled by VM-PS3 dpp, is also significantly reduced in Hh signaling mutants, while Dwnt4 expression is not seriously affected even in hh null mutants. Thus, the effect of hh activity loss on gastric caecum formation may be due to partial changes in fate/transcription programs of VM-PS3 cell precursors (Hosono, 2003).

Reiterative bnl expression in VM is likely to be a determinant of the particular mode of visceral branches (VB) migration of the trachea, an ectodermal organ. The tip of VB first comes in touch with the vicinity of the posterior end of the tin/bnl/bap expressing alpha region, where all the five metameric genes examined are expressed. Bnl misexpression with VM-specific-GAL4 drivers induces VB misrouting and bifurcation, but neither hh misexpression nor transient loss of Hh activity during stage 11 has any effect on VB budding. BNL misexpression brings about no significant change in expression of tin, while restriction of the tin/bnl/bap expression domain using either dTCF-DeltaN or wg^ts causes a shift in the first VB/VM contact point. Furthermore, under a wg mutant condition, no change is detected in VM-hh or in con expression. Thus, only the bnl expression appears to be closely correlated with VB budding, strongly suggesting that BNL serves as a chemoattractant for initial VB migration (Hosono, 2003).

Hedgehog targets in the gut

Coordinated cell movements are critical for tissue and organ morphogenesis in animal development. The proventriculus is a multiply folded muscular organ of the foregut. Formed from a simple epithelial tube it grinds and masticates food. Epithelial morphogenesis during proventriculus development requires Drosophila genes hedgehog and wingless, which encode signaling molecules, and the gene myospheroid, which encodes a beta subunit of the integrins. In contrast, this morphogenetic process is suppressed by the decapentaplegic gene (Pankratz, 1995).

bagpipe expression in mesodermal tissue overlying foregut and hindgut, both considered to be ectodermal derivatives, is regulated by wingless and hedgehog activities in the underlying gut epithelium. The mesodermal layer of the fore- and hindgut is gradually assembled around the invaginating stomodeal and proctodeal tubes. bagpipe is strongly expressed in mesodermal cells on top of the proctodeum that will give rise later to the muscles of the hindgut. The expression domain then splits to give rise to two subdomains, one around the future small intestine and the other around the future rectum of the hindgut. Later bagpipe expression appears in a continuous expression domain. In both wg and hh mutants, bap expression is reduced or absent in the visceral mesoderm primordia of the developing hindgut. Similar results were obtained for the foregut (Hoch, 1996).

Hedgehog targets in the head, eye and antenna

Hedgehog acts upstream of glass, scabrous, hairy and decapentaplegic and is required for the progression of the morphogenetic furrow in the developing eye (Ma, 1993).

The dorsal head capsule, which lies between the compound eyes, contains three morphologically distinct domains. The medial domain includes the ocelli and their associated bristles, which lie on the triangular ocellar cuticle. The mediolateral region contains the frons cuticle, which consists of a series of closely spaced parallel ridges. The lateral region is occupied by the orbital cuticle, which contains a stereotypical pattern of bristles. The head capsule forms primarily from the two eye-antennal imaginal discs. Each half of the dorsal head derives from a primordium in the disc immediately adjacent to the anlage of the compound eye. During the pupal stage, the two discs fuse at what will form the midline of the dorsal head capsule (Amin, 1999).

hh is expressed within the medial domain of the dorsal head capsule. Specifically, it is expressed in the interocellar cuticle, which contains the small interocellar bristles. Using a vn-lacZ strain, vn is found to be expressed in the dorsal head capsule. vn expression lies primarily within the mediolateral frons cuticle, near but not immediately adjacent to the region of hh transcription. The regions of hh and vn expression in the dorsal head primordium of the eye-antennal disc are compared. Consistent with its expression on the adult head capsule, hh is expressed in the region of this primordium that lies between the precursor cells of the ocelli. vn is expressed in the wing and haltere discs, but its expression in the eye-antennal disc has not been described. Using both the vn-lacZ strain and in situ hybridization with a vn probe, vn is also found to be expressed in the dorsal head primordium. As on the adult head, vn expression lies near that of hh. Double-labeling shows that the domains of hh and vn expression are immediately adjacent to each other. In situ hybridization reveals that vn is also expressed at low levels in the morphogenetic furrow. Eliminating Hh function during head development results in the deletion of the entire medial domain, including the interocellar cuticle and bristles, and the ocelli and their associated bristles. This region is replaced by frons cuticle, which is normally confined to the mediolateral region of the head capsule. Ectopic hh expression generates ectopic medial structures at more lateral positions. Hh is therefore necessary for the specification of the medial domain and sufficient to direct more lateral regions of the dorsal head towards a medial fate (Amin, 1999).

Particular combinations of Egfr alleles cause a reduction in the size of the ocelli and the loss of the two ocellar bristles, which flank the medial ocellus. Since vn is expressed within the dorsal head primordium, the effects of eliminating either vn expression or Egfr-mediated signaling on head development were determined. Examination of vn mutant clones shows that Vn is required for the development of some, but not all, of the Hh-dependent medial head structures. The ocelli and ocellar bristles are deleted and the postvertical bristles, which lie near the lateral ocelli, are also lost. However, most of the interocellar cuticle is retained, indicating that the vn dorsal head phenotype is less global than that caused by loss of Hh function. Since vn encodes a ligand for Egfr, the effects of eliminating Egfr-mediated signaling on head development were examined. To do so, the GAL4/UAS system was used to express a dominant negative form of the Egfr (DN-DER) across the entire dorsal head primordium. DN-DER expression eliminates the same structures deleted in vn clones, suggesting that vn is primarily responsible for activating Egfr signaling in this region. As was the case for Vn, the interocellar cuticle is retained in the absence of Egfr signaling (Amin, 1999).

Since the hh mutant phenotype is more extensive than either the vn or Egfr phenotypes, a test was made to determine whether Hh acts upstream of the Egfr pathway. Using a temperature-sensitive hh allele (hh^ts2), Hh function was eliminated during the third instar larval stage. Loss of Hh eliminates or strongly reduces vn expression in both the dorsal head primordium and the morphogenetic furrow. To determine whether Hh can induce vn expression outside the dorsal head primordium, ectopic hh expression was induced using the Flp recombinase technique. Hh is found to be capable of activating vn in other regions of the eye disc. A disc-specific enhancer from the dpp gene was used to induce ectopic hh expression using the GAL4/UAS system. This enhancer drives reporter gene expression at the posterior and lateral margins of the third instar eye disc as well as in a portion of the antennal anlage. Ectopic hh expression induced by this enhancer severely disrupts eye-antennal disc morphology. It also induces a band of ectopic vn expression anterior to the region of hh transcription. Combined with the previous results, these experiments demonstrate that Hh is necessary for vn expression in the dorsal head primordium, and is sufficient to induce ectopic vn expression in other regions of the disc (Amin, 1999).

To test whether Hh is also required to activate Egfr-mediated signaling, a monoclonal antibody that specifically recognizes the active, dually phosphorylated form of mitogen-activated protein kinase (dp-ERK) was used. dp-ERK is expressed at high levels in the morphogenetic furrow, and at lower levels in ommatidia posterior to the furrow. When the anti-dp-ERK signal is allowed to develop for longer periods, weaker expression appears in cells within the dorsal head primordium. Eliminating Hh function reduces or eliminates dp-ERK expression both in these cells and in the furrow. Hh mediated induction of the Egfr pathway has been shown to be medated by Cubitus interruptus. Expression of an N-terminal fragment of Ci with repressor activity reduces or eliminates vn expression in the dorsal head analage. On the contrary, expression of the Ci155 activator increases the intensity and extent of vn expression and causes the ocelli to increase in size and fuse (Amin, 1999).

wingless is broadly expressed throughout the early eye-antennal disc, where it confers a default state of head cuticle. Later, wg expression becomes restricted to the primordia of the orbital cuticle and ptilinium, and to a portion of the antennal anlage. Just as hh expression is medially adjacent to that of vn on the adult head capsule, wg expression abuts vn in the frons both laterally and anteriorly. Loss of Wg signaling causes the deletion of both the frons and orbital cuticles. To determine whether Wg participates in vn regulation, a temperature-sensitive allele was used to eliminate Wg function during second instar development. In contrast to Hh, Wg negatively regulates vn. Loss of Wg activity during this time window expands the domain of vn expression in the dorsal head primordium and induces ectopic vn expression in other regions of the eye-antennal disc (Amin, 1999).

decapentaplegic mediates the effects of hedgehog in tissue patterning by regulating the expression of tissue-specific genes. In the eye disc, the transcription factors eyeless, eyes absent, sine oculis and dachshund participate with these signaling molecules in a complex regulatory network that results in the initiation of eye development. Analysis of functional relationships in the early eye disc indicates that hh and dpp play no role in regulating ey, but are required for eya, so and dac expression. Ey is expressed throughout the eye portion of the wild-type eye disc during early larval stages, prior to MF initiation. Eya and Dac are expressed throughout the posterior half of the eye imaginal disc, with stronger expression at the posterior margin. Ey is expressed normally in homozygous Mad^1-2 clones that touch the posterior margin and in clones that are positioned internally in the disc, indicating that Dpp signaling is not required for Ey expression prior to MF initiation. In contrast, neither Eya nor Dac is expressed in homozygous Mad^1-2 clones that touch the margin of the eye disc. In addition, Eya and Dac are not expressed, or are expressed weakly, in internal clones that lie well anterior of the posterior margin. However, strong Eya and Dac expression is observed in internal clones that lie within a few cell diameters of the posterior margin. Like Eya and Dac protein, SO mRNA is expressed in the posterior region of the eye disc prior to MF initiation. Mad^1-2 posterior margin clones fail to express so. These results suggest that dpp function is required to induce or maintain Eya, SO and Dac expression, but not Ey expression, at the posterior margin prior to MF initiation. This function is consistent with the pattern of DPP mRNA expression along the posterior and lateral margins at this stage of eye disc development. Whereas dpp is not necessary for Eya and Dac expression in internal, posterior regions of the early eye disc, it does play a role in regulating Eya and Dac expression in internal, anterior regions of the disc. Although DPP mRNA expression does not extend to the very center of the eye disc, it is expressed in a significant proportion of the interior of the disc. The possibility that dpp may regulate gene expression in more central regions may be attributed to the fact that it encodes a diffusible molecule (Curtiss, 2000).

Restoring expression of eya in loss-of-function dpp mutant backgrounds is sufficient to induce so and dac expression and to rescue eye development. Thus, once expressed, eya can carry out its functions in the absence of dpp. These experiments indicate that dpp functions downstream of or in parallel with ey, but upstream of eya, so and dac. Additional control is provided by a feedback loop that maintains expression of eya and so and includes dpp. The fact that exogenous overexpression of ey, eya, so and dac interferes with wild-type eye development demonstrates the importance of such a complicated mechanism for maintaining proper levels of these factors during early eye development. Whereas initiation of eye development fails in either Hh or Dpp signaling mutants, the subsequent progression of the morphogenetic furrow is only slowed down. However, clones that are simultaneously mutant for Hh and Dpp signaling components completely block furrow progression and eye differentiation, suggesting that Hh and Dpp serve partially redundant functions in this process. Interestingly, furrow-associated expression of eya, so and dac is not affected by double mutant tissue, suggesting that some other factor(s) regulates their expression during furrow progression (Curtiss, 2000).

The Drosophila eye is patterned by a dorsal-ventral organizing center mechanistically similar to those in the fly wing and the vertebrate limb bud. Here it is shown how this organizing center in the eye is initiated -- the first event in retinal patterning. Early in development, the eye primordium is divided into dorsal and ventral compartments. The dorsally expressed homeodomain Iroquois genes are true selector genes for the dorsal compartment; their expression is regulated by Hedgehog and Wingless. The organizing center is then induced at the interface between the Iroquois-expressing and non-expressing cells at the eye midline. It was previously thought that the eye develops by a mechanism distinct from that operating in other imaginal discs, but this work establishes the importance of lineage compartments in the eye and thus supports their global role as fundamental units of patterning (Cavodeassi, 1999).

Hedgehog is required for IRO-C. Similar to wg, hh is expressed in a dorsally restricted domain at late first/early second larval instar. Regulation of IRO-C by the Hh pathway was assayed in clones of cells deficient for the Hh receptor complex formed by Smoothened (Smo) and Patched (Ptc). In ptc mutant cells, a situation equivalent to constitutive activation of the Hh pathway in the receiving cells, mirror-lacZ and araucan/caupolican expression are ectopically activated within the mutant cells and in some wild-type adjacent cells. Late induced ptc clones (at 72-96 hours AEL) do not derepress mirr-lacZ. In smo clones, where Hh reception is blocked, ara/caup expression is absent in the center of the clone and strongly decreased in its periphery. This result, and the non-autonomous effect of ptc clones, suggest that a secreted signal, induced by Hh, rescues the loss of hh in the smo mutant cells. This factor could be Wg, as wg is derepressed in ptc clones in the anterior region of the eye disc (Cavodeassi, 1999).

Early generalized ectopic expression of hh dorsalizes the eye, severely reducing its size. Similar effects have been reported for early misexpression of wg. Together, these observations and the previous data support a model in which both Wg and Hh signaling organize DV patterning by directing IRO-C expression. However, Wg and Hh do not meet the complete requirement for the postulated gradient model: (1) their expression is already asymmetric in the early disc; (2) ubiquitous and high expression of Wg or Hh should prevent the formation of the straight DV boundary, but this is not the case (Cavodeassi, 1999).

Retinal differentiation is associated with the passage of the morphogenetic furrow, which normally begins at the intersection of the DV midline with the posterior margin. The site of furrow initiation is widely assumed to be specified at the lowest point of concentration of Wg activity. IRO-C expression borders can non-autonomously recruit mutant and wild-type cells to form an eye provided they are located close to the disc margin. Thus, IRO-C may induce retinal differentiation through the local repression of wg at the disc margin, causing a sink of the Wg gradient. Therefore the expression of wg was examined in relation to IRO-C borders. At late second/early third instar, wg is expressed around the anterior dorsal and ventral disc margins. wg expression is not impeded within marginal IRO-C mutant clones. Thus, it is concluded that an IRO-C expression border is sufficient to promote furrow initiation, even in the presence of wg. In the wild-type eye, this process requires the positive action of Decapentaplegic (Dpp) and Hh. dpp is expressed around the posterior and posterior-lateral disc margin, symmetrically across the IRO-C expression border. Similarly, dpp-lacZ is activated straddling the border of an IRO-C clone abutting the disc margin. hh is expressed along the dorsolateral and posterior margin of the early third instar eye disc. Just before morphogenetic furrow initiation, hh expression increases at the posteriormost region, which is the site where the IRO-C border intersects with the disc margin. This modulation of hh expression was investigated in eye discs where the IRO-C border has been eliminated (by generalized expression of ara using the ey-GAL4 driver). hh-lacZ expression initiates normally, but its levels fail to increase at the posteriormost domain. At mid/late third instar, hh-lacZ expression is completely eliminated from the posterior disc margin, a loss not due to generalized cell death, since wg expression around the posterior margin is not impeded in the mutant late third instar discs. Nor is the failure to maintain hh expression a consequence of the absence of ommatidial differentiation, because hh-lacZ posterior expression is not eliminated in atonal mutant eye discs, where eye neurogenesis fails to initiate. Thus, an IRO-C expression border is needed to maintain and upregulate hh expression at the posteriormost margin, which is necessary for furrow initiation (Cavodeassi, 1999).

These analyses demonstrate that an IRO-C border is essential and instructive for growth, DV polarity, and initiation of eye morphogenesis at both sides of the border. Nevertheless, the IRO-C is only expressed at the dorsal half of the eye disc and encodes transcription factors. Consequently, their non-autonomous effects should be mediated through a signaling pathway with long-range activity. It has been proposed that fringe acts downstream of the IRO-C in the formation of the DV organizer. Consistently, dorsal IRO-C mutant cells exhibit autonomous derepression of fng expression. Thus, eye patterning requires a dorsal expression of IRO-C that establishes a fng expression border. This leads to the localized activation of Notch along the DV midline. Accordingly, the artificial elimination of the fng expression border or the block of Notch activation produces a loss-of-eye phenotype equivalent to that caused by misexpression of caup. This effect on eye development is likely caused by the failure to maintain hh. Here, the Fng/Notch pathway has been shown to act downstream of the IRO-C border (Cavodeassi, 1999).

The differentiation of cells in the Drosophila eye is precisely coordinated in time and space. Each ommatidium is founded by a photoreceptor (R8) cell. These R8 founder cells are added in consecutive rows. Within a row, the nascent R8 cells appear in precise locations that lie out of register with the R8 cells in the previous row. The bHLH protein Atonal determines the development of the R8 cells. The expression of atonal is induced shortly before the selection of a new row of R8 cells and is initially detected in a stripe. Subsequently, atonal expression resolves into regularly spaced clusters (proneural clusters) that prefigure the positions of the future R8 cells. The serial induction of atonal expression, and hence the increase in the number of rows of R8 cells, requires Hedgehog function. In addition to this role, Hedgehog signaling is also required to repress atonal expression between the nascent proneural clusters. This repression has not been previously described and appears to be critical for the positioning of Atonal proneural clusters and, therefore, the position of R8 cells. The two temporal responses to Hedgehog are due to direct stimulation of the responding cells by Hedgehog itself (Dominguez, 1999).

The initial expression of ato in the eye discs occurs in a strip of cells anterior to the morphogenetic furrow. The levels of Ato within this stripe vary, with enhanced Ato expression corresponding to the approximate position of proneural clusters. Behind the furrow, the only cells that express ato are the future R8 cells. In mature R8 cells, the expression of ato is repressed. When ato and hh expressions are compared, it appears that the refinement of ato expression occurs in cells close to the hh-expressing cells, whereas the continuous stripe of ato, which is believed to be induced by Hh, is 5-7 ommatidial rows in front of the first row of hh-expressing cells. This observation suggests that Hh acts at a distance to induce ato. Such a long-range action of Hh could either be direct or indirect (relay by a secondary signal) (Dominguez, 1999).

In the eye disc, the Ci protein is expressed dynamically, with the highest levels of Ci protein overlapping with Ato expression. Accordingly, misexpression of high levels of Ci in clones of cells showed that Ci is able to induce Ato. The Ci accumulation in cells ahead of the furrow depends on Hh, because cells lacking smo activity have low uniform levels of Ci. Loss of Hh reception in more posterior regions results in the failure to downregulate Ci levels and consequently mutant cells have inappropriately high Ci protein levels when compared to wild-type neighbors. This indicates that Hh stimulates (at long-range) and inhibits (at short-range) Ci accumulation (Dominguez, 1999).

The regulation of ato by the Hh-signaling pathway was studied further by generating clones of marked cells expressing a membrane-tethered Hh protein tagged with CD2 (Hh-CD2). ato expression in cells that have gained hh was examined. Misexpression of hh-CD2 can either activate (when clones are lying anteriorly) or repress (when they lie adjacent to the furrow) the expression of ato. Repression of ato is autonomous in the hh-CD2 cells, suggesting that Hh may repress ato directly. These observations suggest that Hh is secreted near the advancing furrow: close to the source ato expression is inhibited, further away it is induced. If hh-CD2 is misexpressed, naive cells begin to express ato prematurely and this ectopic ato initiates precocious ommatidial formation. However, slightly later (and within the region of influence of the endogenous hh), misexpression of hh-CD2 results in the premature repression of ato. Thus, cells experiencing the extra Hh exhibit no ato expression while the wild-type neighbors just begin to express ato. This model has been tested by manipulating the reception of the Hh signal using in vivo assays. Genetic evidence shows that Hh is required for both promoting and inhibiting ato expression (Dominguez, 1999).

In the proposed model, the induction of Hh has two effects in the responding cells: (1) as an ato inducing signal, through the activation (by upregulation) of the Zn-finger transcription factor Ci, and (2) as an inhibitory signal, through activation of Rough, to inhibit ato expression in the cells in and behind the furrow. The two responses occur in a cell sequentially, as monitored by ato and rough expression in the wild-type pattern and by analysis of their expression in marked clones. The expression domains of ato, Ci protein and rough and their relationship with Hh supports the model. Ci and rough are activated and expressed, respectively, by Hh in restricted spatial domains across the furrow and their expression either overlaps (in the case of Ci) or is complementary (in the case of rough) with ato, consistent with their respective roles in promoting or inhibiting ato expression (Dominguez, 1999).

ato expression is controlled by two enhancer elements located 5' or 3' to the coding sequences (Sun, 1998). A 3' enhancer directs initial expression in a stripe anterior to the furrow and a distinct 5' enhancer drives expression in the proneural clusters and R8 cells within and posterior to the furrow. The 5' enhancer, but not the 3' enhancer, depends on endogenous ato function. The identification of the factors that activate the 5' enhancer element will require refining the ato regulatory sequences followed by binding studies in vitro and in vivo. One of the factors binding to these ato promoters might be Ci. Preliminary results for the loss of ci in mitotic clones are consistent with Ci acting as a positive transcriptional regulator of ato (M. D. and E. Hafen, unpublished, cited in Dominguez, 1999). During furrow progression, Ci is upregulated in the cells anterior to the furrow and in groups of cells in the furrow that coincide with cells expressing ato. These high levels of Ci are then later downregulated to a low level behind the furrow. Ci is thought to act as a transcriptional factor activating or repressing target genes in a concentration-dependent manner. The transcriptional activator form of Ci is thought to correlate with high levels of full-length Ci protein induced by Hh. This upregulation of Ci proteins by Hh is a conserved feature of Hh signaling in all systems. Therefore it is surprising that in the eye Ci is not upregulated near to the Hh source but only in cells far away. The analysis of Ci distribution in smo³, hh ^AC and viable fused alleles (where the reception and transduction of the Hh signal is blocked or very reduced) suggests that high levels of Hh protein may inhibit Ci protein levels. Probably this regulation is required to restrict the domain of Ci activation and therefore, the cells that are competent to express ato. Thus, by combining a positive long-range inductive signal with short-range inhibition of Ci, Hh may act to pattern ato expression along the anteroposterior axis and refine the array of R8 cells (Dominguez, 1999 and references).

The bHLH transcription factor Atonal is sufficient for specification of one of the three subsets of olfactory sense organs on the Drosophila antenna. Misexpression of Atonal in all sensory precursors in the antennal disc results in their conversion to coeloconic sensilla. The mechanism by which specific sense organ fate is triggered remains unclear. The homeodomain transcription factor Cut, which acts in the choice of chordotonal-external sense organ does not play a role in olfactory sense organ development. The expression of atonal in specific domains of the antennal disc is regulated by an interplay of the patterning genes, Hedgehog and Wingless, and Drosophila epidermal growth factor receptor pathway (Jhaveri, 2000).

Pattern formation in the epidermis is regulated by a hierarchy of genes; the patterning genes -- engrailed, hh, dpp and wg -- specify co-ordinates of the disc and are expected to influence expression of prepatterning genes. Lz is a putative prepatterning gene in the antennal disc and has been shown to regulate expression of amos; genes regulating ato in the antenna are as yet unclear. The olfactory sense organs are located in a distinct pattern across the antenna, thus requiring co-ordinated control of the different proneural genes (Jhaveri, 2000).

During Drosophila eye development, Hh and Dpp are required to initiate photoreceptors at the furrow while Wg inhibits differentiation at the lateral margins. Wg appears to act by antagonizing signaling through the Egfr pathway. In contrast, Hh may directly regulate ato expression, its diffusion ahead of the morphogenetic furrow turns on Ato, while higher levels behind the furrow lead to its downregulation. There is however evidence that Hh can also influence Egfr signaling since Ci has been shown to activate Mapk through the Egfr ligand Vein (Jhaveri, 2000 and reference therein).

Loss-of-function experiments have shown that Hh function is required for ato expression; misexpression analysis has demonstrated that low Hh levels turn on Ato, while higher levels suppress it. However the normal expression pattern of Hh in the antennal disc makes it unlikely that it could directly act to induce Ato in all domains. Ectopic expression of wg in the antennal disc has been shown to lead to induction of ato. Hh appears to act non-autonomously to induce Ato in neighboring cells; UAS-hh transgene produces the secreted form of Hh protein. The data suggests a dosage sensitivity in the regulation of ato by Hh. High levels of Hh produced within cells of the clone suppress ato expression, while low levels resulting from diffusion of protein outside the clone induce it. It is thus proposed that both Hh and Wg together pattern ato expression domains in the disc. The diffusible nature of Hh could allow its action at a long range to induce expression of ato as well as wg. Since Wg is also a secreted molecule, and can regulate ato through the Egfr cascade, it could serve to extend the range of Hh effect across the disc (Jhaveri, 2000).

Hedgehog and Dpp signaling induce cadherin Cad86C expression in the morphogenetic furrow during Drosophila eye development

During Drosophila eye development, cell differentiation is preceded by the formation of a morphogenetic furrow, which progresses across the epithelium from posterior to anterior. Cells within the morphogenetic furrow are apically constricted and shortened along their apical-basal axis. However, how these cell shape changes and, thus, the progression of the morphogenetic furrow are controlled is not well understood. This study shows that cells simultaneously lacking Hedgehog and Dpp signal transduction fail to shorten and do not enter the morphogenetic furrow. Moreover, a gene, cadherin Cad86C, has been identified that is highly expressed in cells of the leading flank of the morphogenetic furrow. Ectopic activation of either the Hedgehog or Dpp signal transduction pathway results in elevated Cad86C expression. Conversely, simultaneous loss of both Hedgehog and Dpp signal transduction leads to decreased Cad86C expression. Finally, ectopic expression of the extracellular region and transmembrane domain of Cad86C in either eye-antennal imaginal discs or wing imaginal discs results in apical constriction and shortening of cells. It is concluded that Hedgehog and Dpp signaling promote the shortening of cells within the morphogenetic furrow. Induction of Cad86C expression might be one mechanism through which Hedgehog and Dpp promote these cell shape changes (Schlichting, 2008).

The progression of the morphogenetic furrow provides an example of a developmentally regulated cell shape change. This paper studied the signaling pathways that regulate this cell shape change and has identified a transcriptional target of these pathways. The Hedgehog and Dpp signaling pathways both promote the shape change of cells that normally occurs in the morphogenetic furrow. Moreover, Cad86C, which is expressed in cells of the morphogenetic furrow was identified and evidence is provided that expression of this gene is regulated by both Hedgehog and Dpp signaling. Finally, Cad86C possesses, among known cadherins, an unique activity to organize elongated epithelial folds. The data suggest that Cad86C is a transcriptional target gene of Hedgehog and Dpp in the morphogenetic furrow. Furthermore, the data are consistent with the notion that Cad86C might be one effector that acts downstream of Hedgehog and Dpp signaling to help execute the cell shape changes associated with the progression of the morphogenetic furrow (Schlichting, 2008).

The conclusion that Cad86C expression in the morphogenetic furrow is regulated by Hedgehog and Dpp signal transduction is derived from the analysis of loss-of-function mutants in these signaling pathways and from the ectopic activation of these signaling pathways through expression of activated components. The level of Cad86C protein is highly reduced in smo³ tkv^a12 clones straddling the normal position of the morphogenetic furrow, whereas Cad86C protein is still detectable in smo³ or tkv^a12 single mutant clones. Conversely, expression of a constitutively active form of the Hedgehog-regulated transcription factor Ci, Ci^PKA4, or a constitutively active form of the Dpp receptor Thickveins, Tkv^Q253D, resulted in increased levels of Cad86C protein. Two observations indicate that Hedgehog and Dpp signal transduction regulate the expression of Cad86C mainly at a transcriptional level. First, the abundance of Cad86C RNA is highly increased in the morphogenetic furrow of wild-type eye imaginal discs and, second, Cad86C RNA is highly reduced when hedgehog activity (and Dpp expression) is impaired in hh^ts2 mutant eye imaginal discs. In the first intron of Cad86C, a cluster of three putative Ci binding sites have been identified based on their sequence similarity to the Gli/Ci consensus binding sequence. This provides a first indication that Cad86C might be a direct transcriptional target of the Hedgehog signaling pathway. In Cad86C^71C mutants, in which these putative Ci binding sites are deleted, Cad86C RNA appears to be normally expressed in the morphogenetic furrow, demonstrating that these sites are not essential for Cad86C expression in the morphogenetic furrow. This is consistent with the finding that the Hedgehog signal transduction pathway is not essential for Cad86C expression in cells of the morphogenetic furrow and, that in its absence, the Dpp signaling pathway can promote expression of Cad86C. Cad86C expression, in addition, might be controlled also at a posttranscriptional level, since Cad86C RNA, but not Cad86C protein, is detected in some cells posterior to the morphogenetic furrow (Schlichting, 2008).

In contrast to other known cadherins, Cad86C possesses an unique activity to organize elongated folds in epithelia. This activity appears to be mediated by the cadherin repeats, since expression of a deletion variant of Cad86C, Cad86C-EXTRA-HA, in which the intracellular region is missing, still induces epithelial folds. Since cadherin repeats mediate the binding between cadherin molecules, it is speculated that expression of Cad86C-HA promotes epithelial folding through its interaction with a cadherin. No evidence is found that Cad86C interacts homophilically in cells of the morphogenetic furrow. Cad86C might, therefore, interact with a different kind of cadherin to promote epithelial folding. Candidates for Cad86C-interacting cadherins include the non-classical cadherins Cad74A, Cad88C, and Cad96Cb, which, during embryonic spiracle development, are expressed in sub-sets of cells adjacent to Cad86C (Lovegrove, 2006). Among these three cadherins, it was found that Cad88C is expressed in a complementary pattern to Cad86C. However, adult flies homozygous mutant for Cad86C and Cad88C had an apparently normal eye size, indicating that Cad88C is, if at all, not an essential interacting partner for Cad86C during morphogenetic furrow progression (Schlichting, 2008).

Cad86C-HA induces epithelial folding non-cell-autonomously, indicating that an imbalance in the expression level of Cad86C between neighboring cells might result in cell shortening. It is noted, however, that Cad86C^184A mutant clones in the wing imaginal disc and eye imaginal disc are not associated with epithelial folds, perhaps because the absolute difference in Cad86C expression between mutant cells and neighboring control cells is only modest (Schlichting, 2008).

Similar to the expression of Cad86C-HA, expression of an activated form of the regulatory light chain of non-muscle Myosin II has recently been shown to promote epithelial folding in the eye imaginal disc (Corrigall, 2007; Escudero, 2007). The finding that Cad86C-EXTRA-HA promotes epithelial folding indicates that Cad86C does not directly interact with non-muscle Myosin II to bring about cell shape changes. Future studies will need to examine the relationship between Cad86C and non-muscle Myosin II (Schlichting, 2008).

This study found that both the Hedgehog and Dpp signaling pathways operate to promote the cell shape changes that normally occur in the morphogenetic furrow. It is tempting to speculate that Cad86C acts downstream of Hedgehog and Dpp signal transduction to promote the progression of the morphogenetic furrow. This speculation is mainly based on three observations. First, Cad86C protein is present at high levels in cells of the leading flank of the morphogenetic furrow, the cells that undergo apical constriction and shortening first. Second, Cad86C expression in the eye imaginal disc is regulated by Hedgehog and Dpp signal transduction, the two signal transduction pathways that promote the progression of the morphogenetic furrow. And third, ectopic expression of Cad86C-HA in clones of cells results in apical cell constriction and cell shortening, cell shape changes typically associated with the progression of the morphogenetic furrow. However, since attempsts to detect a genetic requirement for Cad86C in morphogenetic furrow progression failed, it remains possible that Cad86C may play a role during eye development that is unrelated to morphogenetic furrow progression (Schlichting, 2008).

The morphogenetic furrow moves at a speed of one ommatidial cluster in approximately two hours. Based on these results, the following model is proposed of how the morphogenetic furrow progresses. Cells leaving the morphogenetic furrow start to differentiate and express Hedgehog. Hedgehog signals anteriorly to induce the expression of dpp in cells of the morphogenetic furrow. In response to Hedgehog and Dpp signaling, several target genes, including Cad86C, are induced in cells of the leading flank of the morphogenetic furrow. The resulting proteins promote the apical constriction and shortening of the leading edge cells, a process recently shown to require non-muscle Myosin II. The apical constriction and shortening of the leading edge cells then moves the leading flank of the furrow anteriorly. As cells proceed through the center of the morphogenetic furrow, Hedgehog signal transduction is switched off and target gene expression ceases. Downregulation of Cad86C expression in the center of the morphogenetic furrow appears to be important, since sustained expression of Cad86C-HA prevents cells from elongating. The cessation of target gene expression, therefore, might allow cells to extend to their normal length and shape and, thus, to leave the morphogenetic furrow. These cells will then start to differentiate and express Hedgehog (Schlichting, 2008).

Cad86C possesses an unique activity to induce elongated folds in epithelia. The identification of Cad86C interacting proteins will be important to elucidate the mechanisms by which Cad86C promotes epithelial fold formation. Identification of Cad86C interacting proteins, as well as the identification of additional Hedgehog and Dpp target genes, promises to shed further light on the cell biological mechanisms underlying morphogenetic furrow progression (Schlichting, 2008).

Odd-skipped genes function downstream of hedgehog to specify the signaling center that triggers retinogenesis in Drosophila

Although many of the factors responsible for conferring identity to the eye field in Drosophila have been identified, much less is known about how the expression of the retinal 'trigger', the signaling molecule Hedgehog, is controlled. This study shows that the co-expression of the conserved odd-skipped family genes at the posterior margin of the eye field is required to activate hedgehog expression and thereby the onset of retinogenesis. The fly Wnt1 homologue wingless represses the odd-skipped genes drm and odd along the anterior margin and, in this manner, spatially restricts the extent of retinal differentiation within the eye field (Bras-Pereira, 2006).

The eye disc is a flat epithelial sac. By early third larval stage (L3), columnar cells in the bottom (disc proper: Dp) layer are separated by a crease from the surrounding rim of cuboidal margin cells. Margin cells continue seamlessly into the upper (peripodial; Pe) layer of squamous cells. The Dp will differentiate into the eye, while the margin and Pe will form the head capsule. In addition, the posterior margin produces retinal-inducing signals (Bras-Pereira, 2006).

By examining gene reporters it was found that the zinc-finger gene odd is expressed restricted to the posterior margin and Pe of L3 eye discs. Since the odd family members drumstick (drm), brother of odd with entrails limited (bowl) and sister of odd and bowl (sob) are similarly expressed in leg discs, they were examined in eye discs. In L2, before retinogenesis has started, odd and drm are transcribed in the posterior Pe-margin, and this continues within the posterior margin after MF initiation. bowl is transcribed in all eye disc Pe-margin cells of L2 discs, but retracts anteriorly along the margins and Pe after the MF passes. In addition, bowl is expressed weakly in the Dp anterior to the furrow. sob expression in L2 and L3 is mostly seen along the lateral disc margins. Therefore drm, odd and bowl are co-expressed at the posterior margin prior to retinal differentiation initiation (Bras-Pereira, 2006).

Odd family genes regulate diverse embryonic processes, as well as imaginal leg segmentation. In embryos, the product of the gene lines binds to Bowl and represses its activity, while Drm relieves this repression in drm-expressing cells. Since drm/odd/bowl expression coincides along the posterior margin around the time retinal induction is triggered, it was asked whether they controlled this triggering. First, bowl function was removed in marked cell clones induced in L1. bowl^- clones spanning the margin, but not those in the DP, cause either a delay in, or the inhibition of, retinal initiation and the autonomous loss of hh-Z expression. Correspondingly, there is a reduction in expression of the hh-target patched (ptc). These effects on hh and ptc are not due to the loss of margin cells, since drm is still expressed in the bowl^- cells. The requirement of Bowl for hh expression is margin specific, since other hh-expressing domains within the disc are not affected by the loss of bowl (not shown). As expected from the bowl-repressing function of lines, the overexpression of lines along the margin phenocopies the loss of bowl. Nevertheless, the overexpression of bowl in other eye disc regions is not sufficient to induce hh. This suggests that, in regions other than the margin, either the levels of lines are too high to be overcome by bowl or bowl requires other factors to induce hh, or both (Bras-Pereira, 2006).

drm and odd are expressed together along the posterior disc margin-Pe, and drm (at least) is required for Bowl stabilization in leg discs. Nevertheless, the removal of neither drm nor odd function alone results in retinal defects. odd and drm may act redundantly during leg segmentation and this may also be the case in the eye margin. To test this, clones were induced of DfdrmP2, a deficiency that deletes drm, sob and odd, plus other genes. When DfdrmP2 clones affect the margin, the adjacent retina fails to differentiate, suggesting that drm and odd (and perhaps sob, for which no single mutation is available) act redundantly to promote bowl activity at the margin (although the possibility that other genes uncovered by this deficiency also contribute to the phenotype cannot be excluded). To test the function of each of these genes, drm, odd and sob were expressed in cell clones elsewhere in the eye disc. Only the overexpression of drm or odd induced ectopic retinogenesis, and this was restricted to the region immediately anterior to the MF, which is already eye committed. Interestingly, bowl is also expressed in this region of L3 discs. The retina-inducing ability of drm requires bowl, because retinogenesis is no longer induced in drm-expressing clones that simultaneously lack bowl function. Therefore, it seems that in the eye, drm (and very likely also odd) also promotes bowl function (Bras-Pereira, 2006).

The expression of hh or activation of its pathway anterior to the furrow is sufficient to generate ectopic retinal differentiation. Since (1) bowl is required for hh expression at the margin, (2) this hh expression is largely coincident with that of odd and drm, and (3) drm (and possibly odd) functionally interacts with bowl, whether drm- and odd-expressing clones induced the expression of hh was examined. In both types of clones hh expression is turned on autonomously, as detected with hh-Z, which would thus be responsible for the ectopic retinogenesis observed. That the normal drm/odd/bowl-expressing margin does not differentiate as eye could be explained if margin cells lack certain eye primordium-specific factors (Bras-Pereira, 2006).

These results indicate that the expression of odd and drm defines during L2 the region of the bowl-expressing margin that is competent to induce retinogenesis. How is their expression controlled? wingless (wg) is expressed in the anterior margin, where it prevents the start of retinal differentiation. drm/odd are complementary to wg (monitored by wgZ) during early L3, when retinal differentiation is about to start, and also during later stages. In addition, when wg expression is reduced during larval life in wgCX3 mutants, drm transcription is extended all the way anteriorly. This extension precedes and prefigures the ectopic retinal differentiation that, in these mutants, occurs along the dorsal margin. Therefore, wg could repress anterior retinal differentiation by blocking the expression of odd genes in the anterior disc margin, in addition to its known role in repressing dpp expression and signaling (Bras-Pereira, 2006).

Interestingly, the onset of retinogenesis in L3 is delayed relative to the initiation of the expression of drm/odd and hh in L1-2. This delay can be explained in three, not mutually exclusive, ways. (1) The relevant margin factors (i.e. drm/odd, hh) might be in place early, but the eye primordium might become competent to respond to them later. In fact, wg expression domain has to retract anteriorly as the eye disc grows, under Notch signaling influence, to allow the expression of eye-competence factors. (2) Building up a concentration of margin factors sufficient to trigger retinogenesis might require some time. In fact, the activity of the Notch pathway along the prospective dorsoventral border is required to reinforce hh transcription at the firing point. (3) Other limiting factors might exist whose activity becomes available only during L3. Such a factor might be the EGF receptor pathway, which is involved in the triggering and reincarnation of the furrow along the margins during L3 (Bras-Pereira, 2006).

Hedgehog function in Bolwig's organ

Bolwig's organ formation and atonal expression are controlled by the concerted function of hedgehog, eyes absent and sine oculis. Bolwig's organ primordium is first detected as a cluster of about 14 Atonal-positive cells at the posterior edge of the ocular segment in embryos and hence, atonal expression may define the region from which a few Atonal-positive founder cells (future primary photoreceptor cells) are generated by lateral specification. In Bolwig's organ development, neural differentiation precedes photoreceptor specification, since Elav, a neuron-specific antigen, whose expression is under the control of atonal, is expressed in virtually all early-Atonal-positive cells prior to the establishment of founder cells. Neither Atonal expression nor Bolwig's organ formation occurs in the absence of hedgehog, eyes absent or sine oculis activity. Genetic and histochemical analyses indicates that (1) the required Hedgehog signals derive from the ocular segment, (2) Eyes absent and Sine oculis act downstream of or in parallel with Hedgehog signaling and (3) the Hedgehog signaling pathway required for Bolwig's organ development is a new type and lacks Fused kinase and Cubitus interruptus as downstream components (Suzuki, 2000).

Prior to the establishment of Bolwig's organ founder cells, virtually all Bolwig's organ precursor (BOP) cells acquire neural fate. The earliest event of Bolwig's organ development may be ato expression at mid stage 10: this early ato expression defines the area of BOP. Early ato expression is regulated by the concerted action of Eya, So and Hh signals. During late stage 10 and early stage 11, Elav, a neuron-specific antigen, begins to be expressed in almost all BOP cells. This elav expression is likely to be regulated by Ato activity, since (1) BOP elav expression is reduced extensively in ato mutants and (2) the number of Elav-positive cells at stage 11 and Kr-positive Bolwig's organ neurons at stage 16 considerably increases upon ato misexpression. As with ato expression, eya, so and hh activity is essential for elav expression in BOP cells. In contrast to elav expression, ato expression is restricted to three founder cells at stage 12: this late ato expression disappears by the end of stage 12. Photoreceptor specification of putative founder cells may start during stage 11, since at late stage 11, 2-3 cells in a cluster start expressing Kr and/or Glass, which are specific markers for larval photoreceptors. Cells expressing Kr and/or Glass increase during stages 12-13 and all 12 photoreceptors express both Kr and Glass by stage 16. Similarly, a peripheral nervous system-specific signal recognized by mAb22C10 appears in a few BOP cells at stage 12 and becomes recognizable in all Bolwig's neurons by stage 16. Late ato expression may also be essential for normal photoreceptor formation. In ato mutants, neither Kr-positive nor mAb22C10-positive cells can be seen in stage-16 future larval eyes (Suzuki, 2000).

Hh signaling in Drosophila has been extensively analyzed in embryonic trunk segments and imaginal discs, and many common downstream components have been identified. In both systems, Ci activates target genes in response to hh signal. The pathway lying above Ci is thought to be bifurcated. Although the mechanism by which Smo passes signals to PKA or Fu remains unclear, PKA and Fu act under the direction of the putative Ptc/Smo receptor complex in parallel with each other. Ci is directly phosphorylated by PKA and cleaved to become a repressor, while Fu phosphorylates full-length Ci to make it a labile activator. Bolwig's organ development is regulated through the concerted action of Eya, So and Hh signaling. Although these three factors are essential for ato expression at stage 10, the earliest event in Bolwig's organ development so far identified, whether or not, they directly regulate other events of Bolwig's organ development remains to be clarified. Defects in stage-10 ato expression in BOP mutant for eya, so or hh are partially rescued by misexpression of the corresponding gene at late stage 9 and stage 10, suggesting that ato is a direct target of the putative Eya/So complex and an activator downstream of Hh signaling involved in Bolwig's organ development. Ci is expressed in BOP cells at stage 10. Fu is also ubiquitously expressed in the ectodermal head at stage 10. However, Fu and Ci are not involved in Hh signaling for Bolwig's organ development. Epistasis analysis has indicated that Eya and So act either downstream of or in parallel with Hh/Ptc signaling. Should the latter be the case, Hh signal must activate an unknown transcription activator (X) to positively regulate ato. This is the first demonstration of Hh signaling independent of both Fu and Ci. Hh signaling required for ocular-segment hh expression lacks Ci but not Fu, and this would imply the presence of another type of Hh signaling. The Hh signaling pathway required for ptc expression in cells posteroventral to Hh expression domains in the trunk has recently been shown to lack Fu but not Ci and consequently there must be considerable diversity in the downstream pathway of Hh signaling in Drosophila (Suzuki, 2000).

Hedghog targets in oogenesis

Throughout Drosophila oogenesis, specialized somatic follicle cells perform crucial functions in egg chamber formation and in signaling between somatic and germline cells. In the ovary, at least three types of somatic follicle cells, polar cells, stalk cells and main body epithelial follicle cells, can be distinguished when egg chambers bud from the germarium. Although specification of these three somatic cell types is important for normal oogenesis and subsequent embryogenesis, the molecular basis for establishment of their cell fates is not completely understood. Studies reveal the gene eyes absent (eya) to be a key repressor of polar cell fate. Eya is a nuclear protein that is normally excluded from polar and stalk cells, and the absence of Eya is sufficient to cause epithelial follicle cells to develop as polar cells. Furthermore, ectopic expression of Eya is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration. Finally, it has been shown that ectopic Hedgehog signaling, which is known to cause ectopic polar cell formation, does so by repressing eya expression in epithelial follicle cells (Bai, 2002).

Drosophila oogenesis provides an excellent system with which to study the mechanisms underlying specification of different cell fates. The Drosophila ovary is made up of germline cells and somatic follicle cells. Germline and somatic stem cells can be found at the anterior end of the ovary in a structure called the germarium. Germline stem cells divide asymmetrically and produce cystoblasts, which undergo four rounds of incomplete cell division and give rise to 16-cell germline cysts. One of the cyst cells becomes the oocyte and the remaining 15 cells differentiate as nurse cells. In the germarium, somatic follicle cells surround the 16-cell cysts. As the nascent egg chamber buds off from the germarium, at least three types of somatic cells can be distinguished by their morphologies and locations: polar cells, stalk cells and epithelial follicle cells. Polar cells are pairs of specialized follicle cells at each pole of the egg chamber, whereas the five to eight stalk cells separate adjacent egg chambers. Stalk and polar cells may descend from a common precursor. They differentiate and cease division soon after egg chambers form. The remaining somatic follicle cells, referred to here as epithelial follicle cells, proliferate until stage 6 of oogenesis and form a continuous epithelium around the sixteen germ cells. Subsequently, further differentiation of epithelial follicle cells occurs (Bai, 2002).

In wild-type egg chambers, the anterior polar cells recruit four to eight follicle cells to surround them and become migratory border cells at early stage 9. They migrate through the nurse cell cluster during stage 9 and arrive at the border between the oocyte and the nurse cells at stage 10. Ectopic HH signaling, e.g., caused by loss of cos2, results in the formation of ectopic polar cells and recruitment of extra border cells, which frequently migrate (Bai, 2002).

Since loss of eya in follicle cells leads to ectopic polar cells in the ovary, it was postulated that expression of Eya might normally be repressed in the polar cells. Alternatively, Eya might be repressed via a post-translational modification in polar cells. To distinguish between these possibilities, the expression pattern of the Eya protein was examined in the ovary. Egg chambers were double stained with antibodies against Eya and anti-ß-galactosidase antibodies in order to identify either polar cells, in the A101 enhancer trap line, or stalk cells in the enhancer trap line 93F. The earliest expression of Eya was observed in follicle cells in region 2b of the germarium. Eya continues to be expressed in all follicle cells with the exception of polar and stalk cells until late stage 8. After stage 8, Eya protein is restricted to the anterior follicle cells, including border cells, squamous cells and centripetal cells. Eya is not expressed detectably in the germ cells of any stage. Thus, the absence of Eya protein in the polar cells is consistent with a role as a repressor of polar cell fate (Bai, 2002).

Thus, the data demonstrate that eya is required to suppress polar cell fate in the epithelial follicle cells. The evidence for this is that Eya protein is absent from polar cells in wild-type egg chambers as soon as the polar cells express markers such as A101. Furthermore, loss of Eya can transform other epithelial follicle cells into polar cells in a cell autonomous fashion. Finally, ectopic expression of eya is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration (Bai, 2002).

Loss of Eya results in the production of ectopic polar cells virtually anywhere in the egg chamber. At first glance, this phenotype looks very similar to that of ectopic activation of the HH pathway, either by overexpression of Hh or by loss of the negative regulators Ptc, Pka or Cos2. Indeed the ectopic polar cells that form in ptc, Pka or cos2 mutant clones lack Eya. However, ectopic Hh signaling has additional effects besides ectopic polar cell formation, whereas loss of Eya does not. Several different cell types are observed in the ptc, Pka or cos2 mutant clones. There are Eya-positive but Fas3-negative cells, which may correspond to differentiated epithelial follicle cells. There are also cells expressing both Eya and Fas3, which could be immature, undifferentiated precursor cells. Finally, there are the Eya-negative but Fas3-postive polar cells. In this study, it has been shown that the production of ectopic polar cells caused by ectopic activation of the Hh pathway occurs by repression of Eya (Bai, 2002).

But what is the normal relationship between Hh signaling and polar cell formation, and why does excessive Hh signaling generate ectopic polar cells as well as other cell types? To address these questions, the normal role of Hh signaling in the ovary has to be considered. Expression of Hh protein has been observed only in the terminal filament and cap cells at the extreme anterior tip of the germarium. The normal function of Hh appears to be to regulate somatic stem cell fate and proliferation. Loss of Hh signaling in somatic stem cells results in the loss of stem cell fate. Conversely, overexpression of Hh leads to overproduction of stem-cells. Despite the fact that ectopic expression of Hh leads to ectopic polar cells, Hh signaling does not appear to specify polar cell fate normally. The best direct evidence for that is that smo mutant cells, which cannot transduce Hh signals, are still capable of generating normal polar cells at normal positions. In addition, normal polar cells can develop in the absence of ci (Bai, 2002).

Why, then, does ectopic Hh signaling produce ectopic polar cells? It has been argued that excessive Hh signaling might maintain follicle cells, and the polar/stalk cell lineage in particular, in a precursor state for an abnormally long period of time. Thus, delayed specification of polar cells would permit more proliferation than usual in this lineage. This model might explain the presence of extra polar cells at the two poles of the egg chamber, where the polar cells normally reside. However, it does not explain the presence of ectopic polar cells elsewhere in the egg chamber, or why there are three different cell types present in the ptc, Pka and cos2 mutant clones. Based on the normal role of Hh in regulating proliferation and maintenance of stem cells and their immediate progeny, the prefollicle cells, it is proposed that ectopic Hh signaling might cause ectopic prefollicle cell fates within the epithelial follicle layer of early egg chambers. As these cells undergo further proliferation, and then differentiation, they produce the various follicle cell types observed in the ptc, Pka and cos2 clones. Additional, as yet unknown, signals might determine which specific fates the differentiating cells adopt. However, the normal mechanisms that function to coordinate follicle cell fates spatially are obviously lacking in the mutant clones, since the three types of cells appear in random locations relative to each other. This provides an explanation for how ectopic Hh signaling might produce polar cells all over the egg chamber, rather than only at the two poles of the egg chamber, where the polar/stalk precursors normally reside (Bai, 2002).

Ectopic Hh signaling produces numerous effects in the Drosophila ovary, which include regulating proliferation of somatic cells as well as specification of polar cells. Both of these effects appear to be achieved through the cell autonomous action of Ci. This raises the question of how different effects are elicited by the same signal. The data presented here indicate that ectopic Hh activates polar cell fate by repressing eya expression, the function of which is required to repress polar cell fate. Since loss of eya does not mimic ectopic Hedgehog in causing extra proliferation, it is not yet clear what factors act downstream of ectopic Hh to affect proliferation (Bai, 2002).

The relationship between Eya and Ci is not a simple linear one. Although Eya expression is repressed by Ci^AC, mutations in eya also alter the balance between Ci^AC and Ci^R, without affecting overall ci expression. Ci^AC is upregulated in eya mutant follicle cells. In addition, some of the ectopic polar cells in eya mosaic egg chambers express ptc-lacZ, which is an indicator for activation of Ci. Thus, there appears to be mutual repression between Ci^AC and Eya. One place in the mammalian embryo where a similar relationship between Ci and Eya might exist is in patterning the eye field. Hh is normally expressed at the midline where it represses eye development. In the absence of Hh, a single cyclopic eye forms at the midline. The three mammalian homologs of Eya are all expressed in the eye primordium and therefore it may be that the antagonism between Hh and Eya revealed in this study is also employed in the mammalian embryo to repress midline eye development (Bai, 2002).

It is clear that the effect of the ectopic Hh signaling on the specification of the polar cell fate is through the repression of Eya. What still remains unknown is the spatially localized signal that normally represses Eya expression in polar and stalk cells. Since Notch signaling is necessary, but not sufficient, to define polar cells, it is likely that there is an additional, spatially localized signal required for specifying polar cell fate. Clearly, Eya is a key regulator that represses polar and stalk cell fates. Whatever the regulatory signal that normally specifies polar cell fate, it must regulate Eya expression to determine a polar versus non-polar cell fate in the follicular epithelium (Bai, 2002).

Continued Hedgehog Targets of Activity: part 2/2

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