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.     

Proximal and distal transformation during intercalary regeneration in the planarianDugesia(S)mediterranea

Summary Studies on intercalary regeneration in several organisms have shown that a regenerate is formed when surfaces of different positional value along the proximo-distal axis are opposed. One of the main problems posed by this phenomenon is to know which piece contributes to the building of the regenerate. In the present work we have studied this problem in planarians using chimaeras made between pieces of different body levels, irradiated or not, of the sexual and asexual races ofDugesia(S)mediterranea that differ in a chromosomal marker.The results found show very clearly that intercalary regenerates in planarians are formed by cells coming from both pieces (stumps), and that irradiated pieces keep the positional values and interact with non-irradiated pieces to restore the missing parts. This means that distal and proximal transformation do actually occur at the same time during intercalary regeneration in planarians. The implications of these results as regards to the origin of cells in the regenerate and to present models of intercalary regeneration are discussed.     

Involvement of hedgehog,wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs,a verification of the modified boundary model

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. We first succeeded in observing expression patterns of hedgehog, wingless (wg), and decapentaplegic (dpp) during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns were essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, we concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM.     

Leg Regeneration in Insects: Cell Interactions and Lineage

SYNOPSIS. Developing insect legs have positional informationspecified down the length and around the leg circumference.After grafting or amputation of larval cockroach or cricketlegs healing confronts epidermal cells with different positionalvalues. This stimulates growth, the intercalary regenerationof intervening tissue, the regeneration of all more distal tissuefrom a complete leg circumference and often the formation ofan incomplete distal regenerate from a symmetrical part-circumference.These processes will lead to regeneration of missing structures,duplication of structures, or the formation of branched supernumerarylegs, depending on the situation. During regeneration, cellscannot cross lineage restrictions which divide the leg intoanterior and posterior compartments.     

中华真地鳖的断足再生

报道了中华真地鳖Eupolyphaga sinensis Walker的断足再生特征。研究结果表明,不同虫龄期的若虫都有断足再生能力;足的不同部位断足后均能再生;断掉不同数量的足后,只要能成活均可再生。断足再生后,继续断掉再生足的原位或其他部位也可以再生。再生足的跗节均比正常的少一节,具有再生不完整性。断足后,只要经1～2次蜕皮,均可再生。断掉一对足的腿节后,再生足出现大小不一的现象,小的一般发育不全,断足数量多容易出现再生足发育不全。再生足比正常足要小,但生长速度要快,断掉足的腿节或跗节后的再生足经过2次蜕皮后基本可恢复到正常足大小。     

Intercalary regeneration in legs of crayfish: Proximal segments

An arthropod leg represents a protuberance of the body segmental integument which bears distinctive markers in both the mediolateral and the anteroposterior axes. To clarify the biaxial organization of the body segmental morphogenetic field, and to study the relation among the whole-limb, limb segmental, and body segmental fields previously recognized in arthropods, we have grafted a proximal leg segment into the ventral midline in crayfish. After this operation the majority of animals regenerated a mirror-symmetric pair of supernumerary legs at the host site. Some of these legs had the most proximal segment, the coxa, partially fused to the adjacent body surface. Minority patterns of regeneration included one midline leg with a gill, three midline legs with a gill, and two normal legs with a third double-half leg. These results are compatible with the principle that intercalary regeneration restores the continuity of positional information.     

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.     

Retinoic acid proximalizes level-specific properties responsible for intercalary regeneration in axolotl limbs

The objective of this study was to determine whether retinoic acid (RA) coordinately proximalizes positional memory and the cellular recognition system that detects pattern discontinuity in regenerating amphibian limbs. The strategy was to test the capacity of RA-treated blastemas to evoke intercalary regeneration when grafted to an amputation level proximal to their level of origin. Control wrist and ankle, or elbow and knee blastemas treated with the retinoid solvent, dimethylsulphoxide, evoked intercalary regeneration as effectively as untreated blastemas, when grafted to the midstylopodial amputation surface of host limbs. RA-treated wrist and ankle or elbow and knee blastemas were proximalized and formed complete limbs that were at an angle to, or continuous with, the midstylopodium of the host limb. No intercalary regeneration, from either graft or host, was observed in these cases. The results indicate that the cellular mechanism that recognizes disparities between non-neighbouring cells and initiates intercalary regeneration is coordinately proximalized with positional memory. Thus the recognition mechanism and positional memory are directly related. Intercalary regeneration and corrective displacement (affinophoresis), both of which restore a pattern of normal cell neighbours by different means in regenerating axolotl limbs, appear to use the same mechanism to recognize pattern discontinuity.     

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.     

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.     

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.     

The influence of denervation on grafted hindlimb regeneration of larval Xenopus laevis

The aim of the present research is to ascertain whether in larval Xenopus laevis nerve-independence for the regeneration of early stage limbs and nerve-dependence of late stage limbs observed in a previous work (Filoni and Paglialunga, '90) is related to extrinsic (systemic) factors or to intrinsic changes taking place in the limb cells themselves during development. In this paper the regenerative capacity of early and late stage hindlimbs under the same extrinsic conditions, insofar as both are grafted onto the denervated hindlimbs of host larvae at the same developmental stage, is studied. All the grafted limbs are amputated after the host larvae have reached stage 57-58 (according to Nieuwkoop and Faber, '56). In experiment I, the grafted limb is amputated at stage 52, at the thigh level; in experiment II, the grafted limb is amputated at stage 54-55, at the tarsalia level; in experiment III the grafted limb is amputated at stage 57, at the tarsalia level. In all three experiments, together with the grafted limb, also the host limb is amputated at the tarsalia level. The results show that while grafted limbs amputated at stages 52 and 54-55 regenerate in the absence of nerves, grafted limbs amputated at stage 57 cannot. The failure of late stage grafted limbs to regenerate cannot be explained in terms of an immune-type inhibiting reaction since it has been observed also in denervated autografted limbs and in the host limbs. Since all the grafted limbs are in the same environmental conditions, the results show that in larval Xenopus laevis nerve-independence for regeneration of early stage limbs and nerve-dependence of late stage limbs are not related to factors extrinsic to the limb but to intrinsic changes taking place in the limb cells themselves during development.     

Temporal analysis of the role of growth hormone in the initiation and maintenance of limb regeneration in the hypophysectomized newt Notophthalmus viridescens

This study was designed to investigate and determine for how long, after either hypophysectomy or the third (last) growth hormone injection (to previously hypophysectomized newts), the circulating and now declining titers of endogenous or exogenous hormone remained at a sufficient concentration to permit a morphologically normal forelimb regeneration response in the adult newt Notophthalmus viridescens. To examine the declining levels of endogenous hormone (hormone withdrawal series [HW]), left forelimbs were amputated at specific times following hypophysectomy. Right forelimbs were amputated 5 days prior to hypophysectomy. The declining levels of exogenous hormone (hormone replacement series [HR] were examined in newts whose left forelimbs were amputated at specific times following the last of three consecutive alternate-day growth hormone injections that were initiated 5 days post hypophysectomy. Right forelimbs were amputated immediately following the first hormone injection. All experimental animals were sacrificed when their right forelimbs regenerated to an advanced digitiform regenerate. In both series right forelimbs regenerated normally. In the HW series normal regeneration resulted only when forelimbs were amputated within 48 hours post hypophysectomy, whereas in the HR series normal regeneration occurred in only those newts whose forelimbs were amputated within 12 hours of the last hormone injection. The regeneration response of left forelimbs in both series gradually declined with the time interval between either hypophysectomy or hormone injection and forelimb amputation. As the hormone titer declined, fewer limbs initiated a normal response; they became progressively more hypomorphic and eventually failed to undergo typical regeneration.     

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.     

Contribution of cholinesterase to developmental decreases in the time course of synaptic potentials at an amphibian neuromuscular junction

Urodele limbs are able to regulate for intercalary deletions created when distal regeneration blastemas are grafted to a more proximal level. Using several morphological markers, pigmentation differences between white and dark axolotls, and the difference in nucleolar number between diploid and triploid animals, we show that the entire intercalary regenerate is derived from the stump when wrist or tarsus blastemas are grafted to the midstylopodium of the fore- or hindlimb. The transplanted prospective autopodium forms no more than would be expected in situ. Thus, the rule of distal transformation is not violated during intercalary regeneration in salamanders. The advantages and disadvantages of the several marking techniques are discussed.     

Experiments and Observations on Regeneration in Myriothela cocksi

Myriothela cocksi. a solitary athecate hydropolyp showing many peculiarities and being well investigated concerning gonophore development and embryology, has up to now hardly been examined in respect to regeneration. The simple experiments performed here demonstrate the capacity of all three body regions to regenerate, in due time, previously amputated parts entirely. Further, they reveal a clearly two-phased regeneration process. The first phase leads to a repaired wound, the second results in a rebuilding of the missing part. Ecto- and endoderm are differently involved in regeneration. A greater importance of the endoderm for the first stages of regeneration has been found. Finally, the experiments prove an existing polarity in the polyp. On the basis of these findings it can be concluded that the polyp meets the major criteria of regeneration. This is an absolutely necessary presupposition for all further experiments.     

Morphological,cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2’-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.     

Retinoic acid modifies positional memory in the anteroposterior axis of regenerating axolotl limbs

The effects of retinoic acid (RA) on anteroposterior (AP) positional memory of regenerating axolotl limbs were tested after removing the anterior or posterior half from the zeugopodium (lower arm or leg). RA (150 micrograms/g body wt) was injected into groups of animals bearing the following types of limbs: (1) anterior and posterior half zeugopodia grafted to the eyesocket and amputated distally 7 days later; (2) unamputated anterior and posterior half zeugopodia in situ; (3) double anterior and double posterior half zeugopodia amputated distally 7 days after their construction; (4) sham-operated zeugopodia amputated distally 7 days after operation. Controls consisted of these four groups injected with the retinoid solvent, dimethyl sulfoxide, or not injected. Control half zeugopodia grafted to the eyesocket regenerated no more than one or two digits. Control unamputated half zeugopodia in situ underwent partial or complete regeneration of the missing half from the proximal and midline wound surfaces exposed during construction of the half zeugopodia. Control double anterior and posterior zeugopodia both regenerated symmetrical, hypomorphic regenerates with 1-3 digits in the double anteriors and 1-6 digits in the double posteriors. Sham-operated controls regenerated normally. Regenerating anterior and posterior halves responded differently to RA. RA-treated anterior half zeugopodia in the eyesocket, and anterior half stumps adjacent to the unamputated posterior half zeugopodia in situ both produced regenerates that duplicated stump structures in the proximodistal axis and formed a complete and normal AP pattern. RA-treated double anterior zeugopodia regenerated proximodistal-duplicated pairs of mirror-imaged limbs, each with a complete and normal AP pattern. In contrast, half posterior zeugopodia in the eyesocket, the posterior half stumps of unamputated half anterior zeugopodia in situ, and double posterior zeugopodia all failed to regenerate. These results suggest that RA modifies positional memory in only one direction in the AP axis, posterior.