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.     

EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph

Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation mode.     

Regenerative ability of double-half and half upper arms in the newt, Notophthalmus viridescens

The upper arms of adult newts (Notophthalmus viridescens) were surgically manipulated to create double-half dorsal, double-half ventral, double-half anterior, and double-half posterior upper arms, and longitudinal half-dorsal, half-ventral, half-anterior, and half-posterior upper arms. Amputation through the double-half upper arms usually failed to elicit normal distal regeneration, despite the fact that an apparently normal regeneration blastema was initially formed. Instead, regeneration in these cases was limited to the formation of a variable number of small cartilage elements. On the basis of these results it is concluded that a complete limb circumference is required for distal transformation in newts, in addition to the well-established requirements for a wound epidermis, adequate innervation and dedifferentiation leading to blastema formation. A model for the sequential generation of new parts of the limb pattern during distal transformation from a complete circumference is presented. This model can also account for the occurrence of normal early stages of regeneration in double-half upper arms. Half upper arms which were amputated immediately were shown to develop single, complete regenerates. If amputation of half upper arms was delayed three or more weeks to permit complete wound healing, a supernumerary limb from the lateral wound surface sometimes developed in addition to a complete, single limb from the distal amputation surface.     

Regenerative interactions between Drosophila imaginal discs of different types.

We have tested the ability of fragments of one type of imaginal disc to stimulate regeneration of another type. It has been shown by others that, when extreme proximal and distal fragments of the wing disc are combined, intercalary regeneration of the missing tissue ensues. Each fragment, if cultured alone, will merely duplicate its structures. We now find that distal fragments of other thoracic discs, haltere and leg, while retaining their autonomy for differentiation, also interact with proximal wing tissue to promote regeneration of more distal wing structures. The proximal wing tissue used in these experiments was the wingless abnormal wing disc which, in the absence of interaction, yields only proximal wing structures. These results suggest that spatial organization is controlled by similar systems in the various thoracic discs. In contrast, head and genital disc material provided no regenerative stimulus to the mutant wing disc tissue.     

Leg regeneration in insects. An experimental analysis in Drosophila and a new interpretation.

Fragments from prospective distal regions of Drosophila male foreleg imaginal discs failed to undergo proximal intercalary regeneration across leg segment borders when mechanically intermixed and cultured for 8 days with various fragments from prospective proximal disc regions. The failure of the distal cells to regenerate proximal leg segments was not due to a general restriction in their developmental potentials: Distal fragments, when deprived of their distal-most tips, regenerated in the distal direction at a high frequency. It is concluded that there exist in Drosophila leg discs the same restrictions with respect to regeneration along the proximodistal leg axis as had been previously observed in legs of several hemimetabolous insect species: Intersegmental discontinuities between grafted tissue pieces are not eliminated by intercalation. Based on the available evidence in hemimetabolous insects and in Drosophila, a new interpretation of the different aspects of regeneration in insect legs is offered. It is proposed that the two categories of regulative fields observed in insect legs, the leg segment fields and the whole leg field, represent the units of regulation for two fundamentally different regulative pathways that a cell at a wound edge can follow, the intercalative pathway and the terminal pathway, respectively. It is suggested that the criterion used by cells at healing wounds to choose between the two pathways is the difference in circumferential positional information between juxtaposed cells. The intercalative regulative pathway is switched on when cells with disparities in their axial positional information, or cells with less than maximal disparities in their circumferential information, contact one another. The terminal regulative pathway is triggered whenever cells with maximal circumferential disparities come into contact.     

Limb regeneration and apolysis in the tick, Ornithodoros tartakovskyi.

Limb regeneration potential and the apolysis process were investigated in the argasid tick, Ornithodoros tartakovskyi. Developmental instars received single or multiple amputations and were subsequently allowed to undergo single or multiple apolyses. Amputated ticks regenerated complete normal limbs but only after four successive apolyses. Following a single apolysis, the majority of regenerated limbs were essentially miniature duplicates of normal legs but commonly lacked normal chaetotaxy and/or tarsal hump(s). The site of amputation distal to the coxa-trochanter joint, number of limbs removed from an individual, and instar amputated did not consistently influence the extent of regeneration. Coagulation and clot formation were observed.The limbs of the tick apolysed within the old leg hulls. Larvae and nymphs amputated relatively early during the period of apolyses regenerated limbs; late amputations precluded regeneration. The process of apolysis was irreversible and not obviously affected by amputations.     

Intercalary leg regeneration in the large milkweed bug Oncopeltus fasciatus.

Grafting operations were performed on the metathoracic legs of fourth instar Oncopeltus fasciatus within 24 hr after ecdysis. Different levels along the tibia were combined so that a lengthened tibia, approximately 1.5 times the normal length, or a shortened tibia, about half the normal length, were created. Intercalary regeneration occurred between the graft and stump in both combinations and the extra tissue was visible on the adult leg. The intercalary regenerate produced by the lengthened tibia showed reversed bristle polarity, while that produced in the shortened segment showed normal polarity. It is suggested that a regenerate with reversed polarity represents a mirror image duplication of the graft and might originate from the graft, whereas a regenerate with normal polarity may originate, as in normal regeneration, from the stump. It appears that each level in the appendage has the developmental capacity to produce only more distal structures. This conclusion is supported by the results of a grafting operation in which a portion of the tibia was grafted back on to the stump with its proximo-distal axis reversed. Regeneration of appropriate distal structures proceeded from the free proximal surface of the grafted tibia.     

Fgfs control homeostatic regeneration in adult zebrafish fins

Adult teleost fish and urodele amphibians possess a spectacular ability to regenerate amputated appendages, based on formation and maintenance of progenitor tissue called a blastema. Although injury-induced, or facultative, appendage regeneration has been studied extensively, the extent to which homeostatic regeneration maintains these structures has not been examined. Here, we found that transgenic inhibition of Fgf receptors in uninjured zebrafish caused severe atrophy of all fin types within 2 months, revealing a requirement for Fgfs to preserve dermal bone, joint structures and supporting tissues. Appendage maintenance involved low-level expression of markers of blastema-based regeneration, focused in distal structures displaying recurrent cell death and proliferation. Conditional mutations in the ligand Fgf20a and the kinase Mps1, factors crucial for regeneration of amputated fins, also caused rapid, progressive loss of fin structures in otherwise uninjured animals. Our experiments reveal that the facultative machinery that regenerates amputated teleost fins also has a surprisingly vigorous role in homeostatic regeneration.     

Brista: a gene involved in the specification and differentiation of distal cephalic and thoracic structures in Drosophila melanogaster

Summary A gene Brista has been identified in chromosome 2R, in the region 60D11-E4, in which mutations cause homoeotic transformation of distal antennal structures to distal leg derivatives, and in which certain alleles also lead to upsets in the formation of distal elements of the legs. This gene is haploinsufficient for the homoeotic phenotype. Several putative null and two hypomorphic alleles have been recovered. The effects of exposure to the non-permissive temperature of a temperature-sensitive allele are cummulative and depend upon the length of the exposure during the period of antennal cell proliferation. It is suggested that this gene contributes to the stability of the state of determination in distal domain of the antennal and leg discs, and its relationship to other genes with similar mutant phenotype is discussed.     

Direct control of antennal identity by the spineless-aristapedia gene of Drosophila

Loss-of-function mutations in the spineless-aristapedia gene of Drosophila (ssa mutants) cause transformations of the distal antenna to distal second leg, deletions or fusions of the tarsi from all three legs, a general reduction in bristle size, and sterility. Because ssa mutants are pleiotropic, it has been suggested that ss+ has some rather general function and that the ssa antennal transformation is an indirect consequence of perturbations in the expression of other genes that more directly control antennal or second leg identity. Here we test whether the ssa transformation results from aberrant expression of Antennapedia (Antp), a homeotic gene thought to specify directly the identity of the second thoracic segment. We find that Antp-ssa mitotic recombination clones in the distal antenna behave identically to Antp+ ssa clones, and are transformed to second leg. This demonstrates that the ssa antennal transformation is independent of Antp+, and suggests that ss+ may itself directly define distal antennal identity. The results also reveal that Antp+ is not required for the development of distal second leg structures, as these develop apparently normally in Antp- ssa antennal clones. Because Antp- mutations cause deletions or transformations that are restricted to proximal structures, whereas ssa alleles cause similar defects that are distally restricted, we suggest that ss+ and Antp+ may play similar, but complementary, roles in the distal and proximal portions of appendages, respectively.     

Formation of cartilage in organ-cultured leg buds of chick embryo

When the leg buds from chick embryos at stages 17-23 were cultured as organ cultures in F12 medium, which contained chick serum and an extract of chick embryos, leg cartilage structures developed that included pelvic girdle, femur, tibiofibula, and tarsometatarsus. The proportion of explants with definite distal elements increased when the leg buds from later embryos were used. Supplementation with chick serum was essential. These results suggest that the pattern of formation of limbs in chick embryos can be studied in organ culture.     

Limb type-specific regulation of bric a brac contributes to morphological diversity

The insect antenna and leg are considered homologous structures, likely to have arisen via duplication and divergence from an ancestral limb. Consistent with this, the antenna and leg are derived from primordia with similar developmental potentials. Nonetheless, the adult structures differ in both form and function. In Drosophila, one conspicuous morphological difference is that the antenna has fewer distal segments than the leg. We propose that this is due in part to the variations in the regulation of bric a brac. bric a brac is required for joint formation, and loss of bric a brac function leads to fusion of distal antennal and leg segments, resulting in fewer total segments. Here, we address how bric a brac is regulated to generate the mature expression patterns of two concentric rings in the antenna versus four concentric rings in the leg. We find that bric a brac expression is activated early throughout most of the Distal-less domain in both antenna and leg and subsequently is restricted to the distal portion and into rings. Although bric a brac expression in the antenna and in all four tarsal rings of the leg requires Distal-less, only the proximal three tarsal rings are Spineless-dependent. Thus bric a brac is regulated differentially even within a single appendage type. The restriction of bric a brac expression to the distal portion of the Distal-less domain is a consequence of negative regulation by distinct sets of genes in different limb types. In the leg, the proximal boundary of bric a brac is established by the medial-patterning gene dachshund, but dachshund alone is insufficient to repress bric a brac, and the expression of the two genes overlaps. In the antenna, the proximal boundary of bric a brac is established by an antenna-specifying gene, homothorax, in conjunction with dachshund and spalt, and there is much less overlap between the bric a brac and the dachshund domains. Thus tissue-specific expression of other patterning genes that differentially repress bric a brac accounts for antenna-leg differences in bric a brac pattern. We propose that the limb type-specific variations in expression of bric a brac repressors contribute to morphological variations by controlling distal limb segment number.     

The failure of double-half forelimbs to undergo distal transformation following amputation in the axolotl, Ambystoma mexicanum

Although capable of initiating early regenerative responses, axolotl forelimb stumps which are composed of double-half limb tissues fail to undergo the events that normally lead to the replacement of missing parts. In the present study, the posterior halves of right forelimbs were exchanged with the anterior halves of left forelimbs, or the dorsal halves of right forelimbs were exchanged with the ventral halves of left forelimbs. Forelimbs were amputated through the graft region 30 days after grafting. Limb stumps bearing double-dorsal, double-ventral or double-posterior tissues either produced hypomorphic regenerates or failed to form any externally visible outgrowth. When the limb stump bore double-anterior tissues, no externally visible structures were formed. Normal and multiple regenerates were never formed by double-half limbs. These results are discussed in terms of the polar coordinate model and suggest that the regeneration blastema requires a complete circumference of positional values in order to complete distal transformation.     

A complex role for distal-less in crustacean appendage development.

The developing leg of Drosophila is initially patterned by subdivision of the leg into proximal and distal domains by the activity of the homeodomain proteins Extradenticle (Exd) and Distal-less (Dll). These early domains of gene expression are postulated to reflect a scenario of limb evolution in which an undifferentiated appendage outgrowth was subdivided into two functional parts, the coxapodite and telopodite. The legs of most arthropods have a more complex morphology than the simple rod-shaped leg of Drosophila. We document the expression of Dll and Exd in two crustacean species with complex branched limbs. We show that in these highly modified limbs there is a Dll domain exclusive of Exd but there is also extensive overlap in Exd and Dll expression. While arthropod limbs all appear to have distinct proximal and distal domains, those domains do not define homologous structures throughout arthropods. In addition, we find a striking correlation throughout the proximal/distal extent of the leg between setal-forming cells and Dll expression. We postulate that this may reflect a pleisiomorphic function of Dll in development of the peripheral nervous system. In addition, our results confirm previous observations that branch formation in multiramous arthropod limbs is not regulated by a simple iteration of the proximal/distal patterning module employed in Drosophila limb development.     

Dependence of transdetermination frequency on the developmental stage of cultured imaginal discs of Drosophila melanogaster

Male foreleg tissue from prepupal stages of Drosophila melanogaster was tested for its capacity to grow when cultured in the adult fly hemocoel and for its capacity, after culture, to produce adult cuticular structures when differentiated in a metamorphosing larva. Evaginated, segmented leg tissue from 8-hr-old prepupae (at 25°C), still retained the capacity to grow well in culture. Growth was, however, restricted to cells of the proximal half of the leg. Tissue from 11- and 24-hr stages (pupal ecdysis at 11 hr) was not successfully cultured. Cultured proximal halves of 8 hr prepupal legs frequently differentiated not only proximal structures, but also distal structures, such as sex combs and claws, indicating regeneration of missing leg structures during the culture period. Transdetermination to wing tissue occurred only rarely (once in 90 implants) whereas third-instar leg tissue in culture transdetermined frequently (50% of the implants) to wing, even though growth of tissue of the two stages was equivalent. Identical results were obtained with third-instar foreleg discs evaginated in vitro with β-ecdysone. This is the first in vitro treatment reported to reduce transdetermination frequency, without affecting growth proportionately. These results indicate that cell proliferation in culture, while probably a necessary condition for transdetermination, is not a sufficient condition. The developmental stage of the cultured tissue strongly affects the frequency of transdetermination.     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints.     

Clathrin self-assembly involves coordinated weak interactions favorable for cellular regulation

The clathrin triskelion self-assembles into a polyhedral coat surrounding membrane vesicles that sort receptor cargo to the endocytic pathway. A triskelion comprises three clathrin heavy chains joined at their C-termini, extending into proximal and distal leg segments ending in a globular N-terminal domain. In the clathrin coat, leg segments entwine into parallel and anti-parallel interactions. Here we define the contributions of segmental interactions to the clathrin assembly reaction and measure the strength of their interactions. Proximal and distal leg segments were found to lack sufficient affinity to form stable homo- or heterodimers under assembly conditions. However, chimeric constructs of proximal or distal leg segments, trimerized by replacement of the clathrin trimerization domain with that of the invariant chain protein, were able to self-assemble in reversible reactions. Thus clathrin assembly occurs because weak leg segment affinities are coordinated through trimerization, sharing a dependence on multiple weak interactions with other biopolymers. Such polymerization is sensitive to small environmental changes and is therefore compatible with cellular regulation of assembly, disassembly and curvature during formation of clathrin-coated vesicles.     

Leg regeneration in the cockroach,Blattella germanica

The interactions occuring between graft and host leg epidermis at a congruent junction (non-rotated, homopleural combination of components cut perpendicular to the proximal-distal axis) were studied at the tibia level in the cockroach,Blattella germanica. Grafts were made between dark (Bl) and light (br) cuticle colour mutants.