BIOLOGICAL OVERVIEW

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

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

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

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

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

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

Hedgehog organizes the pattern and polarity of epidermal cells in the Drosophila abdomen

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

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

Hedgehog acts by distinct gradient and signal relay mechanisms to organize cell type and cell polarity in the Drosophila abdomen

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

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

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

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

hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen

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

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

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

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

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

Progression of the morphogenetic furrow in the Drosophila eye: the roles of Hedgehog, Decapentaplegic and the Raf pathway

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

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

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

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

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

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

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

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

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

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

A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary

Tissue organization requires the interplay between biochemical signaling and cellular force generation. The formation of straight boundaries separating cells with different fates into compartments is important for growth and patterning during tissue development. In the developing Drosophila wing disc, maintenance of the straight anteroposterior (AP) compartment boundary involves a local increase in mechanical tension at cell bonds along the boundary. The biochemical signals that regulate mechanical tension along the AP boundary, however, remain unknown. This study shows that a local difference in Hedgehog signal transduction activity between anterior and posterior cells is necessary and sufficient to increase mechanical tension along the AP boundary. This difference in Hedgehog signal transduction is also required to bias cell rearrangements during cell intercalations to keep the characteristic straight shape of the AP boundary. Moreover, severing cell bonds along the AP boundary does not reduce tension at neighboring bonds, implying that active mechanical tension is upregulated, cell bond by cell bond. Finally, differences in the expression of the homeodomain-containing protein Engrailed also contribute to the straight shape of the AP boundary, independently of Hedgehog signal transduction and without modulating cell bond tension. The data reveal a novel link between local differences in Hedgehog signal transduction and a local increase in active mechanical tension of cell bonds that biases junctional rearrangements. The large-scale shape of the AP boundary thus emerges from biochemical signals inducing patterns of active tension on cell bonds (Rudolf, 2015).

This study has analyzed the links between the determination of cell fate and the physical and mechanical mechanisms shaping the AP boundary of larval Drosophila wing discs. Previous work has shown a role for the transcription factors Engrailed and Invected and the Hedgehog signal transduction pathway in organizing the segregation of anterior and posterior cells of the wing disc. This study now shows that a difference in Hedgehog signal transduction between anterior and posterior cells significantly contributes to the straight shape of the AP boundary by autonomously and locally increasing mechanical cell bond tension that in turn biases the asymmetry of cell rearrangements during cell intercalations. Furthermore, Engrailed and Invected also contribute to maintaining the characteristic straight shape of the AP boundary by mechanisms that are independent of Hedgehog signal transduction and do not appear to modulate cell bond tension (Rudolf, 2015).

In the wild-type wing disc, anterior cells transducing the Hedgehog signal are juxtaposed to posterior cells that do not transduce the Hedgehog signal. Three cases were genereated to test whether this difference in Hedgehog signal transduction is important for the straight shape of the AP boundary, the morphological and molecular signature of cells along the AP boundary, and the local increase in cell bond tension. In case I, Hedgehog signal transduction was low (or absent) in both A and P cells. In case II, Hedgehog signal transduction was high in both A and P cells. And in case III, Hedgehog signal transduction was high in P cells, but low in A cells, reversing the normal situation. In cases I and II the AP boundary was no longer as straight as in the wild-type situation. Moreover, the increased apical cross-section area of cells along the AP boundary that is characteristic for the wild type was no longer seen. Finally, the levels of F-actin and cell bond tension were no longer increased along the AP boundary. In case III, it was found that the difference in Hedgehog signal transduction is sufficient to maintain the characteristic straight shape of the AP boundary, to induce the morphological signatures of cells along the AP boundary and to increase F-actin and mechanical tension. Taken together, these experiments establish that the difference in Hedgehog signal transduction between anterior and posterior cells plays a key role in increasing cell bond tension along the AP boundary, in maintaining the characteristic shape of the AP boundary, and in defining the molecular and morphological signatures of cells along the AP boundary. These findings account for the observation that while Hedgehog signal transduction is active within the strip of anterior cells, the increase in mechanical tension is confined to cell bonds along the AP boundary, where cells with highly different Hedgehog signal transduction activities are apposed. The small differences in Hedgehog signal transduction activity that might exist between neighboring rows of anterior cells in the vicinity of the AP boundary appear to be insufficient to increase cell bond tension. Importantly, Hedgehog signal transduction per se does not increase cell bond tension along the AP boundary. The role of Hedgehog signal transduction along the AP boundary thus differs from its roles during other morphogenetic processes in which all cells that transduce the Hedgehog signal, for example, respond by accumulation of F-actin and a change in shape. It will be interesting to elucidate the molecular mechanisms by which cells perceive a difference in Hedgehog signal transduction, and how such a difference in Hedgehog signal transduction results in increased cell bond tension (Rudolf, 2015).

F-actin and Myosin II are enriched along the AP boundary. Based on similar observations, the existence of actomyosin cables has been proposed for several compartment boundaries, including the AP boundary in the Drosophila embryonic epidermis, the DV boundary of Drosophila wing discs and the rhombomeric boundaries in zebrafish embryos. Actomyosin cables have been proposed to maintain the straight shape of compartment boundaries by acting as barriers of cell mixing between cells of the adjacent compartments. Actomyosin cables are also characteristic of additional processes, e.g. dorsal closure and germband extension in the Drosophila embryo, tracheal tube invagination and neural plate bending and elongation. During Drosophila germ band extension, it has been shown that mechanical tension is higher at cell bonds that are part of an actomyosin cable compared with isolated cell bonds, indicating that cell bond tension is influenced by higher-order cellular organization during this process. The results, based on laser ablation experiments, show that the increased cell bond tension along the AP boundary can be induced by single cells and does not depend on the integrity of the actomyosin cable. Thus, these data instead indicate that increased cell bond tension is autonomously generated cell bond by cell bond along the AP boundary. This suggests that differences in Hedgehog signal transduction activity regulate the structure and mechanical properties of cell junctions between adjacent cells and in particular upregulate an active mechanical tension, mediated by actomyosin contractility (Rudolf, 2015).

The cell cortex is a thin layer of active material that is under mechanical tension. In addition to viscous and elastic stresses, active stresses generated by actomyosin contractility are an important contribution. Adherens junctions are adhesive structures that include elements of the cell cortices of the adhering cells. Locally generated active tension, therefore, can largely determine the cell bond tension as long as cell bonds do not change length or rearrange. As a consequence, locally generated active tension also sets the cell bond tension at the actomyosin cable along the AP boundary. This view is consistent with experiments in which cell bond tension remains high even if the integrity of the actomyosin cable is lost. These mechanical properties of cell junctions along the AP boundary are thus different from those of a conventional string or cable in which elastic stresses are associated with stretching deformations. Such elastic stresses relax and largely disappear when the cable is severed. Thus, this work suggests that the mechanical properties of the actomyosin cable along the AP boundary are very different from those of a conventional cable, but fit well in the concepts of active tension studied in the cell cortex, e.g., in Caenorhabditis elegans. This active tension is a local property that can be set by local signals irrespective of the local force balances. Force balances rather determine movements and rearrangements, e.g. upon laser ablation (Rudolf, 2015).

How does a local increase in actively generated cell bond tension contribute to the straight shape of the AP boundary? Previous work showed that cell intercalations promote irregularities in the shape of compartment boundaries. The local increase in active cell bond tension enters the force balances during cell rearrangements. During cell intercalation, differences in active cell bond tension between junctions along the AP boundary and neighboring junctions are balanced by frictional forces associated with vertex movements. As a result, vertex movements are biased such that the AP boundary remains straight and cell mixing between neighboring compartments is suppressed. The observation that a local difference in Hedgehog signal transduction upregulates active cell bond tension leads to the prediction that cell rearrangements along the AP boundary should not be biased if there is no difference in Hedgehog signal transduction. This is indeed what was found in case II (Rudolf, 2015).

It has been previously suggested that the engrailed and invected selector genes play a role in maintaining the separation of anterior and posterior cells that is independent of Hedgehog signal transduction. Quantitative analysis of clone shapes in this study supports this notion. It is speculated that this Hedgehog-independent pathway contributes to the remarkably straight shape of the AP boundary in cases I and II, in which Hedgehog signal transduction activities between anterior and posterior cells have been nearly equalized. Two lines of evidence indicate that the Hedgehog-independent pathway shapes the AP boundary without modulating cell bond tension. First,several cases have been generated in which neighboring cell populations differed in the expression of Engrailed and Invected, but not in Hedgehog signal transduction activity. In none of these cases was an increase in cell bond tension detected along the interface of these two cell populations. Second, in cases in which a difference was created in Hedgehog signal transduction between two cell populations in the absence of differences in Engrailed and Invected expression, the same increase was detected in cell bond tension between these cell populations compared with the wild-type compartment boundary (Rudolf, 2015).

Previously studies have described several physical mechanisms that shape the DV boundary of wing discs. In addition to a local increase in mechanical tension along the DV boundary, evidence was provided that oriented cell division and cell elongation created by anisotropic stress contribute to the characteristic shape of the DV boundary. It is therefore conceivable that the Hedgehog-independent pathway influences the shape of the AP boundary by one or more of these mechanisms (Rudolf, 2015).

It is proposed that the AP boundary is shaped by mechano-biochemical processes that integrate signaling pathways with patterns of cell mechanical properties. In tjos model, Engrailed and Invected shape the AP boundary with the help of two different mechanisms. (1) Engrailed and Invected result in a difference in Hedgehog signal transduction between anterior and posterior cells. This difference leads to a cell-autonomous increase in F-actin and active cell bond tension along the AP boundary. The local increase in active cell bond tension then biases the asymmetry of cell rearrangements during cell intercalations and thereby contributes to maintaining the straight shape of the AP boundary. (2) Engrailed and Invected contribute independently of Hedgehog signal transduction to the straight shape of the AP boundary by an as yet unknown mechanism not involving the modulation of cell bond tension. The first mechanism uses biochemical signals to create mechanical patterns that subsequently guide junctional dynamics to organize a straight compartment boundary. It is speculated that the second mechanism also involves a mechano-chemical process, even though the nature of this process is currently unknown. The current work suggests that the large-scale shape of the AP boundary thus emerges from the collective behavior of many cells that locally exchange biochemical signals and regulate active mechanical tension (Rudolf, 2015).

Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila

Pain signaling in vertebrates is modulated by neuropeptides like Substance P (SP). To determine whether such modulation is conserved and potentially uncover novel interactions between nociceptive signaling pathways SP/Tachykinin signaling was examined in a Drosophila model of tissue damage-induced nociceptive hypersensitivity. Tissue-specific knockdowns and genetic mutant analyses revealed that both Tachykinin and Tachykinin-like receptor (DTKR99D) are required for damage-induced thermal nociceptive sensitization. Electrophysiological recording showed that DTKR99D is required in nociceptive sensory neurons for temperature-dependent increases in firing frequency upon tissue damage. DTKR overexpression caused both behavioral and electrophysiological thermal nociceptive hypersensitivity. Hedgehog, another key regulator of nociceptive sensitization, was produced by nociceptive sensory neurons following tissue damage. Surprisingly, genetic epistasis analysis revealed that DTKR function was upstream of Hedgehog-dependent sensitization in nociceptive sensory neurons. These results highlight a conserved role for Tachykinin signaling in regulating nociception and the power of Drosophila for genetic dissection of nociception (Im, 2015).

This study establishes that Tachykinin signaling regulates UV-induced thermal allodynia in Drosophila larvae. It is envisioned that UV radiation either directly or indirectly activates Tachykinin expression and/or release from peptidergic neuronal projections - likely those within the CNS that express DTK and are located near class IV axonal tracts. Following release, it is speculated that Tachykinins diffuse to and ultimately bind DTKR on the plasma membrane of class IV neurons. This activates downstream signaling, which is mediated at least in part by a presumed heterotrimer of α G alpha (Gαq, CG17760), a G β (Gβ5), and a G γ (Gγ1) subunit. One likely downstream consequence of Tachykinin receptor activation is Dispatched-dependent autocrine release of Hh from these neurons. It is envisioned that Hh then binds to Patched within the same class IV neurons, leading to derepression of Smo and activation of downstream signaling through this pathway. One new aspect of the thermal allodynia response dissected in this study is that the transcription factors Cubitius interruptus and Engrailed act downstream of Smo, suggesting that, as in other Hh-responsive cells, activation of target genes is an essential component of thermal allodynia. Finally, activation of Smo impinges upon Painless through as yet undefined mechanisms to regulate thermal allodynia. Some of the implications of this model for Tachykinin signaling, Hh signaling, and their conserved regulation of nociceptive sensitization are discussed below (Im, 2015).

The results demonstrate that Tachykinin is required for UV-induced thermal allodynia. UV radiation may directly or indirectly trigger Tachykinin expression and/or release from the DTK-expressing neurons. Given the transparent epidermis and cuticle, direct induction mechanisms are certainly plausible. Indeed in mammals, UV radiation causes secretion of SP and CGRP from both unmyelinated c fibers and myelinated Aγ fibers nociceptive sensory afferents. Furthermore, in the Drosophila intestine Tachykinin release is induced by nutritional and oxidative stress, although the effect of UV has not been examined. The exact mechanism of UV-triggered neuropeptide release remains unclear; however, it is speculated that UV causes depolarization and activation of exocytosis of Tachykinin-containing vesicles (Im, 2015).

In heterologous cells synthetic Tachykinins (DTK1-5) can activate DTKR. Immunostaining analysis of dTk and genetic analysis of tissue-specific function of dtkr supports the model that Tachykinins from brain peptidergic neurons bind to DTKR expressed on class IV neurons. Pan-neuronal, but not class IV neuron- specific knockdown of dTk reduced allodynia, whereas modulation of DTKR function in class IV neurons could either decrease (RNAi) or enhance (overexpression) thermal allodynia. How do brain-derived Tachykinins reach DTKR expressed on the class IV neurons? The cell bodies and dendritic arbors of class IV neurons are located along the larval body wall, beneath the barrier epidermal cells. However, the axonal projection of each nociceptive neuron extends into the ventral nerve cord (VNC) of the CNS in close proximity to Tachykinin-expressing axons. Because neuropeptide transmission does not depend on specialized synaptic structures, it is speculated given their proximity that Tachykinin signaling could occur via perisynaptic or volume transmission. An alternative possibility is that Tachykinins are systemically released into the circulating hemolymph as neurohormones following UV irradiation, either from the neuronal projections near class IV axonal tracts or from others further afield within the brain (Im, 2015).

Indeed the gain-of-function behavioral response induced by overexpression of DTKR, a receptor that has not been reported to have ligand-independent activity, suggests that class IV neurons may be constitutively exposed to a low level of subthreshold DTK peptide in the absence of injury. The direct and indirect mechanisms of DTK release are not mutually exclusive and it will be interesting to determine the relative contribution of either mechanism to sensitization (Im, 2015).

Like most GPCRs, DTKR engages heterotrimeric G proteins to initiate downstream signaling. Gq/11 and calcium signaling are both required for acute nociception and nociceptive sensitization. The survey of G protein subunits identified a putative Gαq, CG17760. It was demonstrated that DTKR activation leads to an increase in Ca2+, strongly pointing to Gαq as a downstream signaling component. To date, CG17760 is one of three G alpha subunits encoded in the fly genome that has no annotated function in any biological process. For the G β and G γ classes, Gβ5 and Gγ1 were identified. Gβ5 was one of two G β subunits with no annotated physiological function. Gγ1 regulates asymmetric cell division and gastrulation, cell division, wound repair, and cell spreading dynamics. The combination of tissue-specific RNAi screening and specific biologic assays, as employed in this study, has allowed assignment of a function to this previously 'orphan' gene in thermal nociceptive sensitization. The findings raise a number of interesting questions about Tachykinin and GPCR signaling in general in Drosophila: Are these particular G protein subunits downstream of other neuropeptide receptors? Are they downstream of DTKR in biological contexts other than pain? Could RNAi screening be used this efficiently in other tissues/behaviors to identify the G protein trimers relevant to those processes (Im, 2015)?

To date three signaling pathways were found that regulate UV-induced thermal allodynia in Drosophila: TNF, Hedgehog, and Tachykinin. All are required for a full thermal allodynia response to UV but genetic epistasis tests reveal that TNF and Tachykinin act in parallel or independently, as do TNF and Hh. This could suggest that in the genetic epistasis contexts, which rely on class IV neuron-specific pathway activation in the absence of tissue damage, hyperactivation of one pathway (say TNF or Tachykinin) compensates for the lack of the function normally provided by the other parallel pathway following tissue damage. While TNF is independent of Hh and DTKR, analysis of DTKR versus Hh uncovered an unexpected interdependence (Im, 2015).

This study showed that Hh signaling is downstream of DTKR in the context of thermal allodynia. Two pieces of genetic evidence support this conclusion. First, flies transheterozygous for dTk and smo displayed attenuated UV-induced thermal allodynia. Thus, the pathways interact genetically. Second, and more important for ordering the pathways, loss of canonical downstream Hh signaling components blocked the ectopic sensitization induced by DTKR overexpression. It was previously shown that loss of these same components also blocks allodynia induced by either UV or Hh hyperactivation (Babcock, 2011), suggesting that these downstream Hh components are also downstream of DTKR. The fact that Smo is activated upon overexpression of DTKR within the same cell argues that class IV neurons may need to synthesize their own Hh following a nociceptive stimulus such as UV radiation. The data supporting an autocrine model of Hh production are three fold: (1) only class IV neuron-mediated overexpression of Hh caused thermal allodynia suggesting this tissue is fully capable of producing active Hh ligand; (2) expression of UAS-dispRNAi within class IV neurons blocked UV- and DTKR-induced thermal allodynia, implicating a role for Disp-driven Hh secretion in these cells, and (3) the combination of UAS-dispRNAi and UV irradiation caused accumulation of Hh punctae within class IV neurons. Disp is not canonically viewed as a downstream target of Smo and indeed, blocking disp did not attenuate UAS-PtcDN-induced or UAS-TNF-induced allodynia, indicating that Disp is specifically required for Hh production between DTKR and Smo. Thus, Tachykinin signaling leads to Hh expression, Disp-mediated Hh release, or both (Im, 2015).

Autocrine release of Hh has only been demonstrated in a few non-neuronal contexts to date. This signaling architecture differs from what has been found in Drosophila development in two main ways. One is that DTKR is not known to play a patterning role upstream of Smo. The second is that Hh-producing cells are generally not thought to be capable of responding to Hh during the formation of developmental compartment boundaries (Im, 2015).

What happens downstream of Smoothened activation to sensitize class IV neurons? Ultimately, a sensitized neuron needs to exhibit firing properties that are different from those seen in the naïve or resting state. Previously, sensitization was examined only at the behavioral level. This study also monitored changes through extracellular electrophysiological recordings. These turned out to correspond remarkably well to behavioral sensitization. In control UV-treated larvae, nearly every temperature in the low 'allodynic' range showed an increase in firing frequency in class IV neurons upon temperature ramping. Dtkr knockdown in class IV neurons abolished the UV-induced increase in firing frequency seen with increasing temperature and overexpression of DTKR increased the firing rate comparable to UV treatment. This latter finding provides a tidy explanation for DTKR-induced 'genetic allodynia.' The correspondence between behavior and electrophysiology argues strongly that Tachykinin directly modifies the firing properties of nociceptive sensory neurons in a manner consistent with behavioral thermal allodynia (Im, 2015).

Genetically, knockdown of painless blocks DTKR- or PtcDN-induced ectopic sensitization suggesting that, ultimately, thermal allodynia is mediated in part via this channel. Indeed, the SP receptor Neurokinin-1 enhances TRPV1 function in primary rat sensory neurons. Tachykinin/Hh activation could lead to increased Painless expression, altered Painless localization, or to post-translational modification of Painless increasing the probability of channel opening at lower temperatures. Because thermal allodynia evoked by UV and Hh-activation requires Ci and En, the possibility is favored that sensitization may involve a simple increase in the expression level of Painless, although the above mechanisms are not mutually exclusive. Altered localization has been observed with a different TRP channel downstream of Hh stimulation; Smo activation leads to PKD2LI recruitment to the primary cilium in fibroblasts, thus regulating local calcium dynamics of this compartment. The exact molecular mechanisms by which nociceptive sensitization occurs is the largest black box in the field and will take a concerted effort by many groups to precisely pin down (Im, 2015).

Tachykinin and Substance P as regulators of nociception: What is conserved and what is not? The results establish that Tachykinin/SP modulation of nociception is conserved across phyla. However, there are substantial differences in the architecture of this signaling axis between flies and mammals. In mammals, activation of TRP channels in the periphery leads to release of SP from the nerve termini of primary afferent C fibers in the dorsal horn. SP and spinal NK-1R have been reported to be required for moderate to intense baseline nociception and inflammatory hyperalgesia although some discrepancies exist between the pharmacological and genetic knockout data. The most profound difference of Drosophila Tachykinin signaling anatomically is that DTK is not expressed and does not function in primary nociceptive sensory neurons. Rather, DTK is expressed in brain neurons and the larval gut, and DTKR functions in class IV neurons to mediate thermal pain sensitization. Indeed, this raises an interesting possibility for mammalian SP studies, because nociceptive sensory neurons themselves express NK-1R and SP could conceivably activate the receptor in an autocrine fashion. A testable hypothesis that emerges from these studies is that NK-1R in vertebrates might play a sensory neuron-autonomous role in regulating nociception. This possibility, while suggested by electrophysiology and expression studies, has not been adequately tested by genetic analyses in mouse to date (Im, 2015).

In summary, this study discovered a conserved role for systemic Tachykinin signaling in the modulation of nociceptive sensitization in Drosophila. The sophisticated genetic tools available in Drosophila have allowed uncovering both a novel genetic interaction between Tachykinin and Hh signaling and an autocrine function of Hh in nociceptive sensitization. This work thus provides a deeper understanding of how neuropeptide signaling fine-tunes an essential behavioral response, aversive withdrawal, in response to tissue damage (Im, 2015).

Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode

The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. This study characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define photoreceptor subtype identity. This model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning (Mishra, 2018).

In the fruit fly Drosophila melanogaster, all parts of the visual system develop from an optic placode, which forms in the dorsolateral region of the embryonic head ectoderm. During embryogenesis, neuroepithelial cells of the optic placode are patterned to form two subdomains. The ventroposterior domain gives rise to the primordium of the larval eye and consists of two photoreceptor (PR) precursor types (primary and secondary precursors), whereas the dorsal domain harbors neuroepithelial precursors that generate the optic lobe of the adult visual system. The basic helix-loop-helix transcription factor Atonal (Ato) promotes PR precursor cell fate in the larval eye primordium. The orphan nuclear receptor Tailless (Tll) is confined to the optic lobe primordium and maintains non-PR cell fate. Hedgehog (Hh) and Notch (N) signaling are critical during the early phase of optic lobe patterning. The secreted Hh protein is required for the specification of various neuronal and non-neuronal cell types, while Notch acts as neurogenic factor preventing ectodermal cells from becoming neuronal precursors by a process termed lateral inhibition. In the optic placode Ato expression is promoted by Hh and the retinal determination genes sine oculis (so) and eyes absent (eya). Notch delimits the number of PR precursors and maintains a pool of non-PR precursors. Ato is initially expressed in all PR precursors in the placode and its expression gets progressively restricted to primary precursors. In a second step, primary precursors recruit secondary precursors via EGFR signaling: primary precursors express the EGFR ligand Spitz, which is required in secondary precursors to promote their survival. After this initial specification of primary and secondary PR precursors, the transcription factors Senseless (Sens), Spalt (Sal), Seven-up (Svp) and Orthodenticle (Otd) coordinate PR subtype specification. Sens and Spalt are expressed in primary PR precursors, while Svp contributes to the differentiation of secondary PR precursors. By the end of embryogenesis, primary PR precursors have fully differentiated into blue-tuned Rhodopsin5 PRs (Rh5), while secondary PR precursors have differentiated into green-tuned Rhodopsin6 PRs (Rh6). While the functional genetic interactions of transcription factors controlling PR subtype specification has been thoroughly studied, it remains unknown how the placode is initially patterned by the interplay of Hh and Notch signaling pathways. Similarly, the mechanisms of how ato and tll-expressing domains are set up to ensure the correct number of primary and secondary PR precursors as well as non-PR precursors of the optic lobe primordium remain unknown (Mishra, 2018).

This study describes the genetic mechanism of neuroepithelial patterning and acquisition of PR versus non-PR cell fate in the embryonic optic placode and provide the link to subsequent PR subtype identity specification. The non-overlapping expression patterns of ato and tll in the optic placode specifically mark domains giving rise to the larval eye precursors (marked by Ato) and the optic lobe primordium (marked by Tll). ato expression in the larval eye primordium is temporally dynamic and can be subdivided into an early ato expression domain, including all presumptive PR precursors and a late ato domain, restricted to presumptive primary PR precursors. The ato expression domain directly forms a boundary adjacent to tll expressing precursors of the optic lobe primordium. tll is both necessary and sufficient to delimit primary PR precursors by regulating ato expression. Hh signaling regulates the cell number in the optic placode and controls PR subtype specification in an ato- and sens-dependent manner. Finally, this study also shows that Notch has two temporally distinct roles in larval eye development. Initially, Notch represses ato expression by promoting tll expression and later, Notch controls the binary cell fate decision of primary versus secondary PR precursors by repressing sens expression. In summary, this study has identified a network of genetic interactions between cell-intrinsic and cell-extrinsic developmental cues patterning neuroepithelial cells of the optic placode and ensuring the timely specification of neuronal subtypes during development (Mishra, 2018).

Neurogenic placodes are transient structures that are formed by epithelial thickenings of the embryonic ectoderm and give rise to most neurons and other components of the sensory nervous system. In vertebrates, cranial placodes form essential components of the sensory organs and generate neuronal diversity in the peripheral nervous system. How neuronal diversity is generated varies from system to system, and different gene regulatory networks have been proposed for each particular type of neuron. Interestingly, some transcription factors, like Atonal, play an evolutionary conserved role during neurogenesis both in Drosophila and in vertebrates (Mishra, 2018).

Neuroepithelial patterning of the Drosophila optic placode exhibits unique segregation of larval eye and optic lobe precursors during embryogenesis. This study has identified genetic mechanisms that control early and late steps in specifying PR versus non-PR cell fate that ensure the expression of precursor cell fate determinants. During germband extension at stage 10, transcriptional regulators (so, eya, ato and tll) show complex and partially overlapping expression patterns in the optic placode. Their interactions with the Notch and Hh signaling pathways define distinct PR and non-PR domains of the larval eye and optic lobe primordium. Intriguingly, the results show a spatial organization of distinct precursor domains, supporting a new model of how the subdivision of precursor domains emerges. In agreement with previous studies initially the entire posterior ventral tip expresses Ato, defining the population of cells that give rise to PR precursors, while neuroepithelial precursors for the presumptive optic lobe are defined by Tll-expression in the anterior domain of the optic placode. Subsequently, Ato expression ceases in the ventral most cells and thus gets restricted to about four primary PR precursors that are located directly adjacent to the Tll expression domain. Hence, a few cell rows are between the primary PR precursors and the ventral most edge of the optic placode. This is in agreement with a recent observation on the transcriptional regulation of ato during larval eye formation. Thus, primary PR precursors are directly adjacent to the Tll-expressing cells while the Ato and Tll negative domain of secondary PR precursors is located at the posterior ventral most tip of the optic placode. Setting the Tll-Ato boundary is critical to define the number of putative secondary PR precursors, which can be recruited into the larval eye, probably via EGFR signaling. A model is proposed during which coordinated action of Hh, Notch and Tll restricts the initially broad expression of Ato to primary PR precursors (see Ato to primary PR precursors). Lack of Tll results in a de-repression of Ato and results in an increased number of primary PR precursors, which in turn recruit secondary PR precursors. Interestingly, while tll mutants show an increase in both primary and secondary PR precursors, the ratio between both subtypes is maintained. This notion further displays similarities of ommatidal formation in the adult eye-antennal imaginal disc, where Ato expressing R8-precursors recruit R1-R6. In the eye-antennal disc, specification of R8-precursors determines the total number of ommatida and therefore also the total number of PRs, the ratio of R8 to outer PRs however always remains the same. Thus, the initial specification of primary PR precursors defines the total number of PRs in the larval eye similarly to R8 PRs, and the ratio of founder versus recruited cells remains constant. Interestingly, the maintenance of primary versus secondary PR precursor ratio is also maintained in ptc mutants further supporting this model (Mishra, 2018).

During photoreceptor development in the eye-antennal imaginal disc hh is expressed in the posterior margin and is required for the initiation and progression of the morphogenetic furrow as well as the regulation of ato expression. During embryogenesis the loss of hh results in a complete loss of the larval eye, while increasing Hh signaling (by means of mutating ptc) generates supernumerary PRs in the larval eye. During early stages, an increase of Ato expression was found in ptc mutants suggesting that similarly to the eye-antennal disc Hh positively regulates ato expression. The observed increase of Ato-expressing cells is not due to a reduction of Tll but is likely due to increased cell proliferation in ptc mutants. Hh also controls proliferation during the formation of the Drosophila compound eye (Mishra, 2018).

During embryonic nervous system development Notch dependent lateral inhibition selects individual neuroectodermal cells to become neuroblasts. Notch represses neuroblast cell fate and promotes ectodermal cell fate. During compound eye development, Notch regulates Ato expression and acts through lateral inhibition to select Ato expressing R8 PR precursors. Similarly, during Drosophila larval eye development, Notch is required for regulating PR cell number by maintaining epithelial cell fate of the optic lobe primordium. Inhibiting Notch signaling leads to a complete transformation of the optic placode to PRs of the larval eye. In the absence of Notch signaling, Ato expression is expanded in the optic placode and as a result the total number of PRs is increased. Despite the increase of the overall PR-number the number of secondary PR precursors is significantly decreased or lost in the absence of Notch activity. In the compound eye Notch promotes R7 cell fate by repressing the R8-specific transcription factor Sens. It was also proposed that genetic interaction between Notch and Sens is required for sensory organ precursor (SOP) selection in the proneural field in a spatio-temporal manner. This study found that during PR subtype specification Notch represses Sens expression, thereby controlling the binary cell fate decision of primary versus secondary PR precursors. Therefore, in the absence of Notch signaling, Sens expression represses the secondary PR precursor fate. As a result, all PR precursors are transformed and acquire primary PR precursor identity. In conclusion, this study observed that Notch is essential for two aspects during optic placode patterning. First, Notch activity is critical for balancing neuroepithelial versus PR cell fate mediated through Tll-regulated Ato expression. Second, Notch regulates the binary cell fate decision of primary versus secondary PR precursor cell fate through the regulation of Sens expression (Mishra, 2018).

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. This study shows that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, it was shown that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh-dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary (Emmons-Bell, 2021).

Initially discovered for its role in regulating segment polarity in Drosophila, Hh signalling has since been implicated in a multitude of developmental processes. Among the best characterized is the signalling between two populations of cells that make up the Drosophila wing imaginal disc, the larval primordium of the adult wing and thorax. The wing disc consists of two compartments of lineage-restricted cells separated by a smooth boundary. Posterior (P) cells make the morphogen Hedgehog, which binds to its receptor Patched (Ptc), which is expressed exclusively in anterior (A) cells. Hh has a relatively short range either because of its limited diffusion, or because it is taken up by nearby target cells via filopodia-like protrusions known as cytonemes. Hh alleviates the repressive effect of Ptc on the seven-transmembrane protein Smoothened (Smo) in A cells near the boundary, initiating a signalling cascade that culminates in the stabilization of the activator form of the transcription factor Cubitus interruptus (Ci), and expression of target genes such as the long-range morphogen Dpp. In turn, Dpp regulates imaginal disc patterning and growth in both compartments (Emmons-Bell, 2021).

While the role of cell-cell interactions, diffusible morphogens and even mechanical forces have been studied in regulating the growth and patterning of the wing disc, relatively little attention has been paid to another cellular parameter, membrane potential or Vmem. Vmem is determined by the relative concentrations of different species of ions across the cell membrane, as well as the permeability of the membrane to each of these ions. These parameters are influenced by the abundance and permeability of ion channels, the activity of pumps, and gap junctions. While changes in Vmem have been studied most extensively in excitable cells, there is increasing evidence that the Vmem of all cells, including epithelial cells, can vary depending on cell-cycle status and differentiation status. Mutations in genes encoding ion channels in humans ('channelopathies') can result in congenital malformations. Similarly, experimental manipulation of ion channel permeability can cause developmental abnormalities in mice as well as in Drosophila. Only more recently has evidence emerged that Vmem can be patterned during normal development. Using fluorescent reporters of membrane potential, it has been shown that specific cells during Xenopus gastrulation and Drosophila oogenesis appear more depolarized than neighbouring cells. A recent study established that cells in the vertebrate limb mesenchyme become more depolarized as they differentiate into chondrocytes, and that this depolarization is essential for the expression of genes necessary for chondrocyte fate. However, in many of these cases, the relationship between changes in Vmem and specific pathways that regulate developmental patterning have not been established (Emmons-Bell, 2021).

This study investigated the patterning of Vmem during wing disc development and showed that the regulation of Vmem has an important role in regulating Hh signalling. The cells immediately anterior to the compartment boundary, a zone of active Hh signalling, are more depolarized than surrounding cells, and Hh signalling and depolarized Vmem mutually reinforce each other. This results in an abrupt change in Vmem at the compartment boundary (Emmons-Bell, 2021).

This study shows that Vmem is patterned in a spatiotemporal manner during development of the wing disc of Drosophila and that it regulates Hedgehog signalling at the compartment boundary. First, it was shown that cells immediately anterior to the compartment boundary are relatively more depolarized than cells elsewhere in the wing pouch. This region coincides with the A cells where Hh signalling is most active, as evidenced by upregulation of Ptc. Second, the expression of at least two regulators of Vmem, the ENaC channel Rpk and the alpha subunit of the Na+/K+ ATPase were shown to be expressed at higher levels in this same portion of the disc. Third, by altering Hh signalling, this study demonstrated that the expression of both Rpk and ATPα is increased in cells with increased Hh signalling. Fourth, by manipulating Hh signalling in the disc and using optogenetic methods, both in the salivary gland and wing disc, it was shown that membrane depolarization promotes Hh signalling as assessed by increased membrane localization of Smo, and expression of the target gene ptc. Thus, Hh-induced signalling and membrane depolarization appear to mutually reinforce each other and thus contribute to the mechanisms that maintain the segregation of A and P cells at the compartment boundary (Emmons-Bell, 2021).

Two regions of increased DiBAC fluorescence were observed in the wing imaginal disc. No obvious upregulation of Rpk and ATPα was observed in other discs, and therefore, these studies have focused on the region immediately anterior to the A-P compartment boundary in the wing disc. In the late L3 wing disc, a region of increased DiBAC fluorescence was observed in the A compartment in the vicinity of the D-V boundary. This corresponds to a 'zone of non-proliferating cells' (ZNC). Interestingly, the ZNC is different in the two compartments. In the A compartment, two rows of cells are arrested in G2 while in the P compartment, a single row of cells is arrested in G1. The observation of increased DiBAC fluorescence in the DV boundary of only the A compartment is consistent with previous reports that cells become increasingly depolarized as they traverse S-phase and enter G2. In contrast, cells in G1 are thought to be more hyperpolarized. Additionally, increased expression of the ENaC channel Rpk was observed in two rows of cells at the D-V boundary in the anterior compartment, indicating that increased expression of Rpk could contribute to the depolarization observed in those cells. It is noted, however, that the increased DiBAC fluorescence in these cells was not entirely eliminated by exposing discs to amiloride, indicating that other factors are also likely to contribute (Emmons-Bell, 2021).

These data are consistent with a model where membrane depolarization and Hh-induced signalling mutually reinforce each other in the cells immediately anterior to the compartment boundary. Both membrane depolarization and the presence of Hh seem necessary for normal levels of activation of the Hh signalling pathway in this region; neither alone is sufficient. First, it was shown that Hh signalling promotes membrane depolarization. It was also shown that the expression of Rpk just anterior to the A-P compartment boundary is dependent upon Hh signalling. Elevated Rpk expression is not observed when a hh^ts allele is shifted to the restrictive temperature, and cells become more depolarized when Hh signalling is constitutively activated through expression of the ci^3m allele. Previously published microarray data suggest that Rpk as well as another ENaC family channel Ppk29 are both enriched in cells that also express ptc. However, there is no antibody to assess Ppk29 expression currently. The sensitivity of the depolarization to amiloride indicates that these and other ENaC channels make an important contribution to the membrane depolarization (Emmons-Bell, 2021).

Second, this study has shown that the depolarization increases Hh signalling. The early stages of Hh signalling are still incompletely understood. Hh is thought to bind to a complex of proteins that includes Ptc together with either Ihog or Boi. This alleviates an inhibitory effect on Smo, possibly by enabling its access to specific membrane sterols. Interestingly, it has recently been proposed that Ptc might function in its inhibitory capacity by a chemiosmotic mechanism where it functions as a Na+ channel. An early outcome of Smo activation is its localization to the membrane where its C-terminal tail becomes phosphorylated and its ubiquitylation and internalization are prevented. By manipulating channel expression in the wing disc, and by optogenetic experiments in both the salivary gland and wing disc, this study has shown that membrane depolarization can promote Hh signalling as assessed by increased Smo membrane localization and increased expression of the target gene ptc. The time course of Smo activation is relatively rapid (over minutes) and is therefore unlikely to require new transcription and translation. In the P compartment, membrane Smo levels are elevated likely because of the complete absence of Ptc, and some downstream components of the Hh signalling pathway are known to be activated. However, since Ci is not expressed in P cells, target gene expression is not induced. In the cells just anterior to the boundary, the partial inhibition of Ptc by Hh together with membrane depolarization seem to combine to achieve similar levels of Smo membrane localization. More anteriorly, the absence of this mutually reinforcing mechanism appears to result in Smo internalization (Emmons-Bell, 2021).

The experiments do not point to a single mechanism by which depolarization promotes Hh signalling. It is possible that depolarization results in increased Ca2+ levels by opening Ca2+channels at the plasma membrane or by promoting release from intracellular sources (e.g. the ER or mitochondria). Indeed, there is evidence that Ca2+ entry into the primary cilium promotes Hh signalling, and recent work shows that targets of Sonic Hedgehog (Shh) signalling during mammalian development is augmented by Ca2+ influx. A second possibility is that membrane depolarization could, by a variety of mechanisms, activate the kinases that phosphorylate the C-terminal tail of Smo and maintain it at the plasma membrane in an activated state. Depolarization could also impact electrostatic interactions at the membrane that make the localization of Smo at the membrane more favourable. Since Rpk and ATPα are expressed at higher levels in the cells that receive Hh, which have been postulated to make synapse-like projections with cells that produce Hh, it is conceivable that these channels could modulate synapse function. Additionally, while this work was under review, it has been reported that reducing glycolysis depletes ATP levels and results in depolarization in the wing imaginal disc, reducing the uptake of Hh pathway inhibitors and stabilizing Smo at the cell membrane (Spannl, 2020). Importantly, all these mechanisms are not mutually exclusive and their roles in Hh signalling are avenues for future research (Emmons-Bell, 2021).

It is now generally accepted that both cell-cell signalling and mechanical forces have important roles in cell fate specification and morphogenesis. This work adds to a growing body of literature suggesting that changes in Vmem, a relatively understudied parameter, may also have important roles in development. Integrating such biophysical inputs with information about gene expression and gene regulation will lead to a more holistic understanding of development and morphogenesis (Emmons-Bell, 2021).

The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth

Hedgehog (Hh) and bone morphogenetic proteins (BMPs) pattern the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. This study shows that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the Drosophila wing imaginal discs, this study identified nord as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron Derived Neurotrophic Factor (NDNF) involved in Congenital Hypogonadotropic Hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, biochemical evidence ia presented that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass bottom boat (Gbb) to modulate their release and activity. Furthermore, this study demonstrates that Nord is a dosage-depend BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, it is proposed that Hh-induced Nord expression fine tunes both the range and strength of BMP signaling in the developing Drosophila wing (Yang, 2022).

In Drosophila, the short-range morphogen Hh and the long-range morphogen BMP function together to organize wing patterning. It has been previously shown that the Hh signal shapes the activity gradient of BMP by both inducing the expression of Dpp and simultaneously downregulating the Dpp receptor Tkv, resulting in lower responsiveness to Dpp in cells at the A/P compartment border. This study showed that the activity of BMP is further fine-tuned by another previously unknown Hh-dependent mechanism. Using a genome-wide expression profiling of the Drosophila wing imaginal discs, this study identfied nord as a novel target gene of the Hh signaling pathway. Nord and its homolog NDNF belong to a family of secreted proteins that can exist in two distinct pools: diffusible Nord/NDNF proteins that can reach a longer distance and membrane/matrix-associated Nord/NDNF proteins spreading within a short distance from the source cells. During larval and early pupal wing development, Nord is expressed together or in close proximity with the BMP ligand Dpp along the A/P compartment boundary. Elimination of nord caused a reduction of overall wing size and resulted in ectopic posterior crossvein (PCV) formation. Both of these phenotypes are attributable to alterations of BMP signaling activity as monitored by the level of Mad phosphorylation, yet in opposite directions: loss of nord led to decreased pMad in larval wing discs, whereas ectopic pMad surrounded the primordial PCV in nord mutant pupal wings. Moreover, expressing exogenous Nord at different levels and during different developmental stages and contexts showed that Nord is a dosage-dependent modulator of BMP signaling both in wing growth and crossvein patterning. At the molecular level, it was further demonstrated that Nord is a BMP-binding protein that directly enhances or inhibits BMP signaling in cultured S2 cells (Yang, 2022).

Combining the genetic and biochemical evidence, it is proposes that Nord mediates BMP signaling activity through binding of the BMP ligands Dpp and Dpp-Gbb. Depending on the levels of Nord proteins and the source/types of BMP ligands, Nord-mediated binding of Dpp and Dpp-Gbb may promote or repress BMP signaling activity. Additionally, the existence of two spatially distinct pools of diffusible and membrane/matrix-associated Nord proteins may introduce further complications in Nord-mediated BMP signaling regulation. In the wild-type wing discs, expressed in a subset of Dpp-secreting cells along the A/P boundary, Nord binds and enhances the local BMP signaling activity by augmenting ligand concentration near the Nord/Dpp-secreting cells. Meanwhile, Nord also impedes the mobilization of Dpp, especially the long-range BMP signaling mediator Dpp-Gbb heterodimer. Loss of nord simultaneously led to reduced local BMP and increased long-range BMP activities, and therefore gave rise to the seemingly opposite phenotypes of reduced wing size and ectopic PCV. In contrast, low levels of ectopic Nord in the P compartment autonomously increased BMP signaling activity, whereas high levels of Nord, either in the P compartment or throughout the wing pouch, inhibited BMP signaling activity likely through interfering with the normal BMP reception. Taken together, it is proposed that Hh-induced Nord expression provides an exquisite regulation of the strength and range of BMP signaling in the developing Drosophila wing (Yang, 2022).

The activity of TGF-β type factors, including the BMP subfamily, is modulated by a large variety of binding proteins that can either enhance or inhibit their signaling in a context-dependent manner. These modulator proteins vary broadly in structure, location, and mechanism of action. Well-known extracellular and freely diffusible proteins include Noggin, Tsg, Follistatin, the CR (cysteine-rich) domain containing proteins such as Chordin/Sog, and the Can family named after two founding members, Dan and Cerberus. With the exception of Tsg and Tsg/Sog or Tsg/Chordin complexes that in some cases can promote BMP signaling, all of these factors behave as antagonists, where BMP binding prevents association of the ligand with the receptor complex (Yang, 2022).

The other broad category of BMP-binding proteins includes membrane-bound or matrix-associated proteins and, in contrast to the highly diffusible class of BMP-binding factors, these proteins often act as either agonists or antagonists depending on context. These proteins are also structurally diverse, but to date, none contain FN3 or DUF2369 domains that are characteristic of Nord and NDNF, its vertebrate counterpart. From a mechanistic point of view, perhaps the two most instructive Drosophila members of this class of modulators are the heparan sulfate proteoglycan (HSPG) Dally and the CR-containing protein Cv-2. HSPGs are well characterized as modulators of growth factor signaling. In the case of FGFs, HSPGs act as true co-receptors in which they form a tripartite complex with ligand and FGFR, the signaling receptor. However, they can also mediate signaling in other ways. Analysis of dally loss-of-function clones in imaginal discs demonstrates that it has both cell-autonomous and non-autonomous effects with respect to BMP signaling. In general, low levels tend to promote signaling while high doses attenuate signaling. Many models have been put forth to explain these opposing effects and often come down to balancing ligand sequestration and diffusion properties. For instance, in the absence of HSPGs, Dpp may more freely diffuse away from the disc epithelial cell surface. In this case, HSPG acts to enhance signaling by keeping Dpp tethered to the cell surface where it can engage its signaling receptors. On the other hand, a high level of HSPG may compete with signaling receptors for BMP binding and thereby reduce signal (Yang, 2022).

The situation with respect to signal modulation becomes even more complex for factors such as Nord that bind both HSPGs and BMPs. An instructive example to consider is Cv-2, a secreted factor that, like Nord, binds both to HSPGs and BMPs and is also induced by BMP signaling. Like Dally, Cv-2 also has dose-dependent effects on signaling in wing imaginal discs, where low levels enhance while high levels inhibit BMP signaling. By virtue of being bound to HSPGs, it may simply function as an additional tethering molecule that keeps BMPs localized near the cell surface. However, Cv-2 has the unique property that it is also able to bind Tkv, a Drosophila BMPR type I receptor. This has led to speculation that it could act as an exchange factor that aids in handing off a BMP ligand from the HSPG pool to the type I receptor. Mathematical modeling showed that this mechanism can produce a biphasic signal depending on affinities of the various BMP-binding proteins involved and their concentrations (Yang, 2022).

In the case of Nord, its mechanism of action is likely compatible with a variety of these and/or alternative models. While this study has shown that Nord is a BMP-binding protein and Akiyama (2021) have shown that it also binds HSPGs, it is not clear whether the BMP and HSPG-binding sites overlap or are distinct and where they are positioned relative to the FN3 and DUF2369 domains. This is an important issue to consider with respect to the two CRISPR mutants that were generated that truncate Nord within the DUF2369 domain. Interestingly, the nord^3D allele appears to retain some function since it does not generate ectopic crossveins as do the nord^MI06414 or nord^22A alleles, yet nord^3D still produces small wings in transheterozygous combination with a deficiency or nord^22A, consistent with having lost the BMP growth-promoting ability. The discrepancy in crossvein patterning between the different nord alleles may be explained by a difference in residual function of the various truncated Nord protein products. Because the nord^MI06414 allele yields a much shorter predicted Nord peptide compared to the two CRISPR alleles, it is likely to behave as a protein null with a stronger phenotype. The two nord CRISPR alleles, although similar in the sequence deleted from the C-terminus, differ in how many non-nord encoded amino acids occur between the frameshift and the stop codon. The nord^22A allele has additional 14 amino acids relative to nord^3D. Perhaps this extension of the truncated fragment destabilizes or interferes with residual function found in the nord^3D allele. Additional biochemical studies defining the BMP and HSPG-binding sites, the stability of truncated Nord fragments, and whether Nord can also associate with either the type I or II receptors will aid in formulating a more precise mechanistic model (Yang, 2022).

Nord shows some sequence similarity to the NDNF family of proteins. Based on a very recent study, like many other neurotrophic factors, NDNF arose in the ancestor of bilaterians or even later. In agreement, by analyzing the genome and EST sequences from various organisms, this study found that nearly all bilaterian animals have either single or multiple orthologous genes for Nord/Ndnf. Of note, no Ndnf homologs were identiied in the flatworm Planarian, but these factors are highly conserved across vertebrates. All vertebrate family members contain a signal peptide, two FN3-like repeats, and a domain of unknown function (DUF2369) that is now referred to as the NDNF domain. The NDNF domain partially overlaps with the first FN3 but shows some additional conservation that extends between the two FN3 domains. The FN3 module is quite diverse in sequence but is thought to exhibit a common fold that is used as an interaction surface or spacer. The function of the NDNF domain is not clear, but it may also provide a protein interaction surface (Yang, 2022).

Although the vertebrate NDNFs are highly conserved throughout the entire protein length, the Caenorhabditis elegans and Drosophila relatives are quite divergent in primary sequence and show little conservation beyond a few key residues that define the second FN3 and NDNF domains. Notably, the Drosophila protein is missing the first FN3 domain, and therefore it is not clear the extent to which Nord and the vertebrate NDNFs may exhibit functional conservation. Ironically, the original human NDNF clone was identified on the basis of domain structure conservation with Drosophila Nord, which was identified via enhancer trapping to be a gene expressed in mushroom bodies and whose loss leads to defects in olfactory learning and memory (Dubnau et al., 2003). Unfortunately, that particular LacZ enhancer trap line that disrupted the nord locus is no longer available. The use of these new alleles should prove helpful for either confirming or eliminating the involvement of Nord as a modulator of learning and memory and/or other neuronal functions in larva and adult Drosophila (Yang, 2022).

In the mouse, NDNF is highly expressed in many neurons of the brain and spinal cord. Studies using cultured mouse hippocampal neurons revealed that it promotes neuron migration and neurite outgrowth, hence its name. In later studies, NDNF was also found to be upregulated in mouse endothelial cells in response to hindlimb ischemia, where it promotes endothelial cell and cardiomyocyte survival through integrin-mediated activation of AKT/endothelial NOS signaling. Additionally, recent studies have shown that NDNF expression is significantly downregulated in human lung adenocarcinoma (LUAD) and renal cell carcinoma (RCC), indicating that NDNF may also provide a beneficial function as a tumor suppressor (Yang, 2022).

Taken together, these studies have suggested some possible functions for vertebrate NDNF. However, they have primarily relied on in vitro cell culture models, and only recently have in vivo loss-of-function studies been reported. Remarkably, NDNF mutants were discovered in the genomes of several probands with congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder that is characterized by absence of puberty, infertility, and anosmia (loss of smell). This phenotype is very similar to that produced by loss of the anos¹, which also encodes an FN3 superfamily member and is responsible for Kallmann syndrome, a condition that similarly presents with CHH and anosmia due to lack of proper GnRH and olfactory neuron migration. Although in vitro studies indicated that NDNF modulates FGFR1 signaling after FGF8 stimulation, the in vivo molecular mechanism responsible for the neuronal migration defects is not clear. The results of the current study on the function of Drosophila Nord raise the issue of whether any of the ascribed vertebrate NDNF functions could involve alterations in BMP signaling. In the case of angiogenesis and EMT, BMPs, as well as other TGF-β family members, participate at many levels. At present, however, no involvement of BMP or TGF-β signaling has been implicated in migration of the GnRH neurons, although BMP signaling does define neurogenic permissive areas in which the olfactory placode forms. A clear objective for the future is to determine if the vertebrate NDNF factors bind BMPs and/or HSPG proteins such as Dally-like glypicans to modulate BMP signaling activity. On the Drosophila side, additional non-BMP-modulating roles for Nord should also be examined (Yang, 2022).

GENE STRUCTURE

cDNA clone length - 2.2 kb

Bases in 5' UTR - 385

Exons - three

Bases in 3' UTR - 570

PROTEIN STRUCTURE

Amino Acids - 471

Structural Domains

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

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

date revised:   10 June 2024 

 

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