Distal regeneration and symmetry.

A revision of the "polar coordinate model" shows how pattern formation in diverse regenerating systems can be understood in terms of strictly local cell interactions.

Pattern regulation in epimorphic fields.

We have described a formal model for pattern regulation in epimorphic fields in which positional information is specified in terms of polar coordinates in two dimensions. We propose that cells within epimorphic fields behave according to two simple rules, the shortest intercalation rule and the complete circle rule, for both of which there is direct experimental evidence. It is possible to understand a large number of different behaviors of epimorphic fields as a straight-forward consequence of these two rules, and the model therefore provides a context in which to view many of the results of experimental embryology. Although we have confined our discussion to cockroach legs, the imaginal disks of Drosophila, and regenerating and developing amphibian limbs, the fact that the model can explain regulative behavior in such evolutionarily diverse animals suggests that it may have general applicability to epimorphic fields. The predictions which the model makes should make it possible to assess its applicability to other developing systems, and to investigate the cellular mechanisms involved.

Pattern formation in colour on butterfly wings.

Butterflies have uniquely vivid and varied wing-colour patterns. Recent studies show that their formation involves novel expression of homologues of Drosophila appendage-patterning genes, notably Distal-less. Pattern diversity can arise at several developmental stages, either through mutation or in response to environmental conditions. Many loci are known (e.g. in Heliconius) that have major effects on the colour pattern and these invite molecular analysis.

Insect segmentation: Genes, stripes and segments in "Hoppers".

Recent work has revealed that orthologues of several segmentation genes are expressed in the grasshopper embryo, in patterns resembling those shown in Drosophila. This suggests that, despite great differences between the embryos, a hierarchy of gap/pair-rule/segment polarity gene function may be a shared and ancestral feature of insect segmentation.

Pattern formation. The beginning and the end of insect limbs.

Recent results shed light on the mechanisms underlying pattern formation in the development of Drosophila imaginal discs, which give rise to the appendages of the adult fly.

Insect wings. Patterns upon patterns.

Butterfly wing patterns may be generated by an extra coordinate system superimposed on a basic wing-patterning system similar to that of the fruit-fly Drosophila melanogaster.

The generation and diversification of butterfly eyespot color patterns.

BACKGROUND: A fundamental challenge of evolutionary and developmental biology is understanding how new characters arise and change. The recently derived eyespots on butterfly wings vary extensively in number and pattern between species and play important roles in predator avoidance. Eyespots form through the activity of inductive organizers (foci) at the center of developing eyespot fields. Foci are the proposed source of a morphogen, the levels of which determine the color of surrounding wing scale cells. However, it is unknown how reception of the focal signal translates into rings of different-colored scales, nor how different color schemes arise in different species. RESULTS: We have identified several transcription factors, including butterfly homologs of the Drosophila Engrailed/Invected and Spalt proteins, that are deployed in concentric territories corresponding to the future rings of pigmented scales that compose the adult eyespot. We have isolated a new Bicyclus anynana wing pattern mutant, Goldeneye, in which the scales of one inner color ring become the color of a different ring. These changes correlate with shifts in transcription factor expression, suggesting that Goldeneye affects an early regulatory step in eyespot color patterning. In different butterfly species, the same transcription factors are expressed in eyespot fields, but in different relative spatial domains that correlate with divergent eyespot color schemes. CONCLUSIONS: Our results suggest that signaling from the focus induces nested rings of regulatory gene expression that subsequently control the final color pattern. Furthermore, the remarkably plastic regulatory interactions downstream of focal signaling have facilitated the evolution of eyespot diversity.

The genetics and development of an eyespot pattern in the butterfly Bicyclus anynana: response to selection for eyespot shape.

The normally circular eyespots on the wing of the butterfly Bicyclus anynana were selected to become elliptical in two divergent lines, with antero-posterior elongation of the eyespots in one line and proximodistal elongation in the other. Selection was continued for nine generations, and symmetrical realized heritabilities of approximately 15% were achieved initially. The elliptical eyespot shapes characteristic of each line were still produced when the signaling center of the eyespot (the focus) was surgically rotated by 90 or 180 degrees or when an eyespot was induced ectopically by localized damage. We conclude that selection changed general properties of the epidermis that responds to signals emanating from the eyespot focus but did not affect the mechanism of focal signaling.

Thermal evolution of growth efficiency in Drosophila melanogaster.

Drosophila melanogaster shows geographic clines in body size, with genetically larger flies being found further from the equator and at higher altitudes. In the laboratory, evolution at lower temperatures results in genetically larger flies, and development at low temperature increases adult body size. This study demonstrates that when newly hatched larvae from laboratory temperature selection lines were raised on fixed amounts of food (yeast) at the same temperature, larvae from the lines with the cold evolutionary history required less food to produce a given size of adult. Larvae from both high- and low-temperature selection lines required more food, however, to make a given size of adult when grown in the cold than when grown in the hot. The opposite associations between growth efficiency and adult body size seen with evolution or development at low temperature are puzzling, and suggest that different mechanisms may underlie the size changes. Since environmental and evolutionary effects of temperature on body size seem to be widespread among ectotherms, some basic aspects of thermal physiology must be involved.

The antibacterial activity of honey against coagulase-negative staphylococci.

OBJECTIVES: Development of antibiotic-resistant strains of coagulase-negative staphylococci has complicated the management of infections associated with the use of invasive medical devices, and innovative treatment and prophylactic options are needed. Honey is increasingly being used to treat infected wounds, but little is known about its effectiveness against coagulase-negative staphylococci. The aim of this study was to determine the minimum active dilution of two standardized, representative honeys for 18 clinical isolates of coagulase-negative staphylococci. METHODS: An agar incorporation technique was used to determine the minimum active dilution, with dilution steps of 1% (v/v) honey [or steps of 5% (v/v) of a sugar syrup matching the osmotic effect of honey]. The plates were inoculated with 10 microL spots of cultures of the isolates. RESULTS: The honeys were inhibitory at dilutions down to 3.6 +/- 0.7% (v/v) for the pasture honey, 3.4 +/- 0.5% (v/v) for the manuka honey and 29.9 +/- 1.9% (v/v) for the sugar syrup. CONCLUSIONS: Typical honeys are about eight times more potent against coagulase-negative staphylococci than if bacterial inhibition were due to their osmolarity alone. Therefore, honey applied to skin at the insertion points of medical devices may have a role in the treatment or prevention of infections by coagulase-negative staphylococci.

The structure of supernumerary leg regenerates in the cricket.

In many insects, grafting a larval leg onto the contralateral leg stump reverses one transverse axis and leads to the regeneration of supernumerary legs at each of the two points of maximum discrepancy on the graft/host junction. These operations were performed on the cricket Acheta domesticus, grafting between tibiae of pro- and metathoracic legs, and between tibia and tarsus, in order to deduce the mode of origin of the supernumeraries from their graft type, host type or composite structure. Supernumerary legs formed after A/P axis reversal are always half-and-half in structure, being of host type on the host side and graft type on the graft side, while supernumeraries formed after M/L axis reversal are variable in structure. These results are generally consistent with the recent Polar Co-ordinate Model (whereby supernumeraries form because the pattern of intercalation between graft and host generates two complete circumferences at the junction), provided that intercalation is restricted by the borders between anterior and posterior leg compartments. However there are features of the structure of the M/L supernumeraries which the Polar Co-ordinate Model does not explain. Medial supernumeraries are often of pure graft type (and lateral ones of host type) or half-and-half with anterior graft type and posterior of host type (while lateral supernumeraries often have the converse structure).

Positional information around the segments of the cockroach leg.

Epidermal cells from different circumferential positions around the femur of Blabera craniifer can interact to form an intercalary regenerate consisting of that section of the circumference normally separating graft and host positions, by the shorter route. This results is extended to other leg segments; the tibia and coxa (TT and CC Grafts). Grafting strips of integument from the tibia (TF Grafts) or the coxa (CF Grafts) to a corresponding position on the host femur results in simple healing. Grafting to a non-corresponding position leads to intercalation of the shorter intermediate arc of circumference, composed partly of graft-segment and partly of host-segment structures. These results show that that same continuous sequence of positional values is distributed around the circumferences of the coxa, femur and tibia. Cellular interactions along the edges of strip-grafts obey the Shortest Intercalation Rule. At the ends of strip-grafts intercalation usually restores continuity of positional values where possible but, when a complete circumference is generated, a supernumerary distal regenerate is usually formed. This is in general agreement with the Complete Circle Rule and the exceptions are discussed. In intercalary regeneration following the intersegmental strip-grafts, the host femur cells seem unable to intercalate beyond two positions (posterior/internal and posterior/external). These lineage restrictions operating during regeneration indicate that the cockroach leg, like the Drosophila leg disc, may consist of an anterior and a (smaller) posterior 'compartment'.

Intercalary regeneration around the circumference of the cockroach leg.

Epidermal cells from different circumferential positions around the femur of Blabera craniifer can interact to form an intercalary regenerate. Removal of a longitudinal strip of integument (cuticle plus epidermis) from any position around the circumference leads to the cut edges healing, localized growth and intercalary regeneration of the missing section of the circumference, so that the resulting femur is approximately normal in size and pattern of cuticular structures. Grafting a longitudinal strip of femur integument into a different circumferential position on the host femur confronts epidermal cells from different positions along both the inner and outer longitudinal graft/host junctions. In numerous different situations this results in local growth and intercalary regeneration of that section of the circumference normally separating graft and host positions, by the shorter route around the circumference. Confrontation of opposite positions results in the intercalation of either of the intervening half circumferences. In one opposite confrontation, between mid-anterior and mid-posterior, there was also a third result where graft and host healed together, provoking no intercalary regeneration. Grafts made with reversed proximal/distal polarity show that a confrontation between different circumferential positions gives the same result, regardless of the proximal/distal levels involved, hence circumferential position is an independent aspect of position on the femur. These results strongly suggest that epidermal position is not specified with respect to two transverse axes running through the epidermis and internal tissue of the leg, but that there is a continuous circular sequence of positional values running around the circumference, in the epidermis. This is analogous to but independent of the sequence previously shown by Bohn (1967) and Bullière (1971) to run proximal/distal along a leg segment. Hence epidermal position on the femur is specified in two dimensions and can be represented in terms of the French, Bryant & Bryant (1976) polar co-ordinate model. Interactions along the edges of the strip-grafts conform to the Shortest Intercalation Rule (French et al. 1976). At the proximal and distal ends of strip-grafts intercalation restores normal sequences of positional values where possible. However, where the graft, together with the intercalary regenerates formed at the longitudinal graft/host junctions and the adjacent host tissue formed a complete sequence of circular values, then a supernumerary distal regenerate was formed, in agreement with the Complete Circle Rule of French et al. (1976). The problem of generating a continuous circular sequence of positional values by one or more circumferential gradients, is briefly discussed.

Pattern regulation and regeneration.

Insect legs develop from small regions of the embryonic thorax. In most insects they differentiate in the embryo, forming functional larval legs, which grow and moult through larval life. In Drosophila the presumptive legs invaginate to form imaginal discs, which grow through larval life but only differentiate in the pupal stage. Analysis of the structures formed after amputation, grafting and wounding experiments on larval legs and on mature and immature imaginal discs suggests that the same organization of positional information and cellular behaviour is involved in the response of tahe developing leg to disturbance at early stages (termed 'regulation') and at later stages (termed 'regeneration'). The results suggest that developing legs form pattern in accordance with positional information specified in two dimensions within the epidermis, along polar coordinates. A continuous sequence of positional values runs around the circumference and an independent sequence runs down the leg. Two rules govern cellular behaviour after a disturbance. The shortest intercalation rule: interaction between cells with different positional values provokes local growth, producing cells with intermediate values (by the shortest route in the case of the circumferential values). The distalization rule: if intercalated cells have positional values identical to those of adjacent pre-existing cells then the new cells adopt a more distal value. These rules will produce a complete distal regenerate from a complete circumference and may produce a symmetrical regenerate from a symmetrical wound surface. This regenerate may taper (converge) or widen (diverge) and branch into two distal tips, depending on the extent of the original wound and the way in which it heals. The polar coordinate model provides a simple and unified interpretation, in terms of only local interactions, of a wide range of experimentally produced and naturally occurring insect (and crustacean and amphibian) limbs showing regeneration of missing structures, duplication of structures, and the formation of complete, tapering or branching supernumeraries. It is not yet clear what molecular mechanisms could underlie a polar map of positional information, nor how such a map could be initially established at a particular site in the early embryo.

Interaction between the leg and surrounding thorax in the beetle.

Interactions between the insect leg and surrounding thoracic epidermis were studied in the beetle, Tenebrio, by grafting the entire larval prothoracic leg into the metathoracic leg site in various orientations. Control grafts simply heal, but A/P reversed grafts lead to regeneration of supernumerary legs of host orientation in A and P positions. M/L reversed grafts also give supernumeraries, again with host orientation but in M (or MP) and L (or LA) positions. The differences in structure between adult prothoracic and metathoracic legs allow the origin of these supernumeraries to be analysed at coxa and tarsus level. The A/P supernumeraries are consistent and complementary in structure, of host origin on the host side and graft origin on the graft side, and with the borders in apparently constant midmedial and midlateral positions. The M/L supernumeraries, however, are variable and often non-complementary in structure. The results of the A/P and M/L reversals are similar to those found at a more distal level in Tenebrio legs and the legs of several hemimetabolous insects, suggesting that the arrangement of positional values and A and P compartments extends from the epidermis of the leg onto the surrounding thorax. The results of a 180 degrees rotation of the entire leg, however, differ from those found at a more distal level in that the grafted leg rarely derotated and two (or occasionally one or three) supernumeraries are formed in a wide variety of positions, some with constant and others with variable orientation. These results are not readily explained by current models of insect leg formation and regeneration.

Segmentation (and eve) in very odd insect embryos.

The formation of segments in the Drosophila early embryo is understood in greater detail than any other complex developmental process. Now, by studying other types of insect embryo, we can hope to deduce something of the ancestral mechanism of segmentation and the ways in which it has been modified in evolution. The parasitic wasp, Copidosoma floridanum, is spectacularly atypical of insects in that the small egg cell divides extensively, with no initial syncytial phase, and forms eventually some 2000 embryos. This process raises intriguing questions about the control of embryonic polarity and segmentation.

Leg regeneration in the cockroach, Blatella germanica. II. Regeneration from a non-congruent tibial graft/host junction.

The interactions occurring between host and graft leg epidermis at a non-congruent junction were studied in the cockroach, Blatella germanica. Graft and host tibia were cut perpendicular to the proximal-distal axis and two heteropleural combinations were used to reverse separately the two transverse axes of the graft relative to the host. Use of dark and light cuticle colour mutants gave a good indication of the graft or host origin of regenerated structures. Graft/host junctions regenerated segmented structures in various spatial arrangements, always comprising two copies of all structures distal to the level of the junction. It is concluded that the categories--two separate laterals, double lateral, completely and partially autonomous regeneration--reflect two processes. (i) If the graft tarsus is removed, graft and host may not heal together and interact, but form autonomous regenerates lying in mirror-image symmetry separating original graft and host levels. (ii) If interaction occurs between graft and host (or their developing autonomous regenerates) two laterals of dual origin are produced, one from each point of transverse axis incongruity. These laterals may secondarily fuse together to form a double structure originating from a point of congruity. The orientation and composition of the component tarsi of the double structure depend on the site of origin and the extent to which the two laterals fuse. It is argued that the four 'faces' and two 'transverse axes' of the leg are merely descriptive terms. A new model is developed whereby lateral regeneration arises directly from the circumferential organisation of the leg epidermis. Previous work has shown that position is specified continuously around the circumference, and that intercalary regeneration occurs by the shortest route between confronted positions. After reversal of one 'transverse axis' the shortest route between confronted graft and host positions is different on the two sides of each of the two points of 'axis' incongruity, and at these points the two halves of a complete circumference are formed. These lateral circumferences, like the terminal circumference exposed by amputation, cannot heal over by intercalary regeneration, and this leads to regeneration of distal structures. The model accounts for lateral regeneration after reversal of both 'transverse axes' by 180 degrees rotation of a homopleural graft. The possibility is discussed that there may be clonal restrictions on the circumferential positions which the progeny of a cell may occupy.