Effects of Mutation or Deletion (part 2/2)

A model for formation of the anterior and posterior compartments maintains that cells at the inter-compartment interface drive pattern formation by becoming the source of a morphogen. Does this model apply to the ventral embryonic epidermis of Drosophila? First, it is shown that interfaces between posterior (engrailed ON) and anterior (engrailed OFF) cells are required for pattern formation. Second, evidence is provided that Wingless could play the role of the morphogen, at least within part of the segmental pattern. The cuticular structures are examined that develop after different levels of uniform Wingless activity are added back to unsegmented embryos (wingless- engrailed-). Unsegmented embryos are small and spherical, carry a low number of unpolarised denticles and have no Keilin's organs, presumably because they have no functional parasegmental boundary. Because it is rich in landmarks, the T1 segment is a good region to analyse. Here, the cuticle formed depends on the amount of added Wingless activity. For example, a high concentration of Wingless gives the cuticle elements normally found near the top of the presumed gradient. Unsegmented embryos are much shorter than wild type. Alternation of cells expressing engrailed in the "on" and "off" state is necessary for segmentation. If Wingless is added in stripes, the embryos are longer than if it is added uniformly. The response to Wingless depends on whether engrailed is on or off. When en is on, Wingless determines an anterior segmental identity, and when en is off, WG determines a posterior identity. For example when en is off, the ventral abdomen makes naked cuticle instead of denticles. It is suggested that the Wingless gradient landscape affects the size of the embryo, so that steep slopes would allow cells to survive and divide, while an even distribution of morphogen would promote cell death. Supporting the hypothesis that Wingless acts as a morphogen, it is found that at a distance these stripes affect the type of cuticle formed and the planar polarity of the cells. There are two functions of engrailed: (1) the interface between en on and en off ensures sustained expression of the Wingless morphogen; (2) the presence or absence of en determines the cells as posterior or anterior and selects the responses to the morphogen (Lawrence, 1996).

It is thought that the posterior expression of the 'selector' genes engrailed and invected control the subdivision of the growing Drosophila wing imaginal disc into anterior and posterior lineage compartments. In the selector-affinity model, cells in one compartment do not adhere to or recognize cells in the adjacent comparment and minimize contact with them, creating a sharp, smooth boundary between selector-expressing and non-expressing cells. At present, the cellular mechanisms by which separate lineage compartments are maintained are not known. Most models have assumed that the presence or absence of selector gene expression autonomously drives the expression of compartment-specific adhesion or recognition molecules that inhibit intermixing between compartments. Nevertheless, adhesion assays have provided little evidence for gross adhesive differences between cells in different compartments and no good candidate compartment-specific adhesion or recognition molecules has yet been described. However, the present understanding of Hedgehog signaling from posterior to anterior cells raises some interesting alternative models based on a cell's response to signaling. Anterior cells that lack smoothened, and thus the ability to receive the Hedgehog signal, no longer obey a lineage restriction in the normal position of the anterior-posterior boundary. Rather, these clones extend into anatomically posterior territory, without any changes in engrailed/invected gene expression. These cells of anterior origin retain anterior-like features in the posterior territory such as their neuronal character. Such smo- cells of anterior origin do not associate normally with posterior cells in that the mutant cells have abnormally smooth boundaries and do not interdigitate normally with neighboring posterior cells. Clones lacking both en and inv were also examined; these too show complex behaviors near the normal site of the compartment boundary, and do not always cross entirely into anatomically anterior territory. These double mutant cells do not associate normally with anterior cells; instead, they exhibit abnormally rounded boundaries. These results suggest that compartmentalization is a complex process that cannot be explained by simple affinity models (Blair, 1997).

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).

The adult abdomen of Drosophila is a chain of anterior (A) and posterior (P) compartments. The engrailed gene is active in all P compartments and selects the P state. Hedgehog enters each A compartment across both its anterior and posterior edges; within A its concentration confers positional information. The A compartments are subdivided into an anterior and a posterior domain that each make different cell types in response to Hedgehog. The relationship between Hedgehog, engrailed and cell affinity was studied. Twin clones can be made in which one sister clone lacks smoothened (smo) a gene essential for a response to Hedgehog protein and the other is normal, apart from a marker. If these twins are generated in the posterior region of the A compartment, the smo minus clone frequently moves back and into territory normally occupied by P compartment cells, leaving its twin in A territory. This 'sorting back' may imply that the cells of the smo minus clone, which no longer see Hh, have more affinity with P than with the nearby A cells (Lawrence, 1999b and references).

Twin clones were made and the shape, size and displacement of the experimental clone, relative to its control twin, were tested. The perceived level of Hedgehog was varied in the experimental clone and it was found that, if this level is different from the surround, the clone fails to grow normally, rounds up and sometimes sorts out completely, becoming separated from the epithelium. Also, clones are displaced towards cells that are more like themselves: for example groups of cells in the middle of the A compartment that are persuaded to differentiate as if they were at the posterior limit of A, move posteriorly. Similarly, clones in the anterior domain of the A compartment that are forced to differentiate as if they were at the anterior limit of A, move anteriorly. Quantitation of these measures and the direction of displacement indicate that there is a U-shaped gradient of affinity in the A compartment that correlates with the U-shaped landscape of Hedgehog concentration. Since affinity changes are autonomous to the clone it is believed that, normally, each cell’s affinity is a direct response to Hedgehog. By removing engrailed in clones it is shown that A and P cells also differ in affinity from each other, in a manner that appears independent of Hedgehog. Within the P compartment, some evidence was found for a U-shaped gradient of affinity, but this cannot be due to Hedgehog which does not act in the P compartment (Lawrence, 1999b).

For experimental purposes, the abdomen has an advantage over the wing: even in small clones, the types of cuticle being made can be assessed. Thus smo minus clones of A provenance can make the type of cuticle (a3) found in the middle of the A compartment. Such clones made near the back end of the A compartment come to lie between two sorts of alien cells. Behind them are P cells, and in front of them are posterior A cells (a6, a5). In the abdomen the results are unequivocal -- the smo minus clones fail to mix with either type of alien cell, forming straight boundaries with both. P clones lacking both smo and en can also form epidermal cells of the a3 type, and at the front of the P compartment, these behave the same way as a3 cells of A provenance. By contrast, en clones of P provenance, those that form a5 cells, cross over the boundary into A and mix there with a5 cells. It is concluded that the A/P boundary in the abdomen (and presumably in the wing) depends on two independent factors: the difference between A and P due to en and the differences within A due to the Hh signal (Lawrence, 1999b).

The Hh signal enters each A compartment from two directions: the results suggest that it acts to set up two opposing gradients of cell affinity. The behaviors of twin clones were examined: one having a different identity from its neighbors and the other acting as a control. The most detailed results concern the posterior domain within the A compartment. (1) A spatial gradient of clone survival is found; clones of different positional identity sort out most readily when there is a large disparity between their positional value and that of the surrounding cells. For example, ptc;en minus clones sort out rapidly when they are induced anteriorly, while they survive well in the posterior part. The same type of clones when induced later survive further to the anterior, which suggests there is a continuous gradient of affinity. (2) The wiggliness of the boundary made between the clone and its surrounding is a measure of the degree of affinity between the two types of cells. It is noted that with ptc;en minus clones induced at a certain stage in the pupa, the clones are more circular the more anterior their location. This also suggests that affinities change continuously. (3) Further evidence is provided for polarity in the epidermis, because relative to its twin, the clone moves toward the level appropriate to its own differentiation: if the clone differentiates as a5 cuticle, then it moves towards the a5 region. This implies that a vector is present in the epithelium, for if the clone were simply uncomfortable being surrounded by a uniform field of a3 cells, it might round up or sort out, but it would not migrate in a specific direction. This vector is imagined to be defined by a gradient of cell affinity; one would expect cells to take whatever opportunity they have to move in the direction that maximizes their affinity with their neighbors (Lawrence, 1999b).

The adult abdominal epidermis develops as a fairly loose sheet and cells might be somewhat free to exchange neighbors, perhaps during mitosis. The results also indicate that the anterior domain of the A compartment correlates with an affinity gradient of the opposite polarity -- accordingly, while the a5 or a6 clones in the a3 region move back, the a1 clones in the a2 region move forward. Both these findings suggest that the prime agent responsible for affinity in the A compartment is Hh itself. The response to Hh is cell autonomous and it is imagined the affinity depends on how much Hh is perceived: it is a scalar output from the Hh gradients (Lawrence, 1999b).

Why would gradients of cell affinity be biologically efficacious? The general model is that morphogen gradients define basic aspects of pattern: positional information is encoded in the scalar of the primary gradient, information relating to size and growth in the steepness and polarity encoded in the vector of a secondary gradient. To this hypothesis is now added the notion that affinity is also encoded in the scalar, giving a graded readout, perhaps in the amount of a homophilic adhesion molecule such as a cadherin. It is thought that gradients of cell affinity will prove to be basic properties of all cell sheets in vivo, where they act to ensure the integrity and stability of the sheet by keeping the cells adherent to their neighbors and reducing any tendency to roam. Without this gradient, even if all cells tended to cohere to one another, their intrinsic motility could allow them to move around by exchanging equally adhesive neighbours. Mobility like this could compromise pattern formation; it might be problematic if cells were to receive information of position from, for example, the ambient level of Hh, begin to respond to it, and then migrate to a different position too late to readjust their response. The stripes of different types of cuticle in the A compartment are a consequence of threshold responses to a continuously varying Hh concentration. In general, once differentiation has begun in any group of cells (such as one of these stripes) they might acquire additional affinity label(s) that would reduce mixing with neighbors, thus sharpening the border line. Maybe this explains the straightness of the line between a5 and a6 cuticle, which seems straighter than one would expect if the a5 and a6 cells were mixing as much as cells do elsewhere. In the wing, the Hh gradient is responsible for pattern only close to the A/P boundary, with the differentiation of cells further away in thrall to a gradient of Decapentaplegic (Dpp). There is some evidence that the gradient of affinity extends into parts of the wing outside the Hh territory: for example, clones of activated receptor for Dpp take up a circular shape, showing that their cell affinities are different from the surround. Thus affinity changes may accompany positional information even when this information depends on more than one morphogen (Lawrence, 1999b).

back to engrailed Effects of Mutation part 1/2 |

engrailed: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Developmental Biology | References

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