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Leg regeneration in Drosophila abridges the normal developmental program
Abstract and Figures
Regeneration of lost body parts has traditionally been seen as a redeployment of embryonic development. However, whether regeneration and embryonic development are controlled by identical, similar or different genetic programmes has not been fully tested. Here, we analyse proximal-distal regeneration in Drosophila leg imaginal discs using the expression of positional markers, and by cell-lineage experiments, and we compare it with the pattern already known in normal development. During regeneration, the first proximal-distal positional markers reappear in overlapping patterns. As the regenerate expands, they segregate and further markers appear until the normal pattern is produced, following a proximal to distal sequence that is in fact the reverse of normal leg imaginal disc development. The results of lineage tracing support this interpretation and show that regenerated structures derive from cells near the wound edge. Although leg development and leg regeneration are served by a set of identical genes, the ways their proximal-distal patterns are achieved are distinct from each other. Such differences can result from similar developmental gene interactions acting under different starting conditions.
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... The degree of regenerative capacity varies among different species, ranging from whole-body regeneration in hydra and planaria to limited tissue regeneration in mammals. Work in several model organisms has identified signaling pathways and molecular mechanisms that are important for initiating and executing regenerative growth after tissue damage, including JNK signaling [1][2][3][4][5], JAK/STAT signaling [6][7][8], EGFR signaling [9][10][11][12], Hippo signaling [13][14][15][16][17], Wnt signaling [18][19][20][21][22][23][24], and Myc [23,25]. Many of these mechanisms are also important during normal development, and the process of regeneration was traditionally thought to be a redeployment of earlier developmental steps [3,9,[25][26][27][28][29]. ...
... Work in several model organisms has identified signaling pathways and molecular mechanisms that are important for initiating and executing regenerative growth after tissue damage, including JNK signaling [1][2][3][4][5], JAK/STAT signaling [6][7][8], EGFR signaling [9][10][11][12], Hippo signaling [13][14][15][16][17], Wnt signaling [18][19][20][21][22][23][24], and Myc [23,25]. Many of these mechanisms are also important during normal development, and the process of regeneration was traditionally thought to be a redeployment of earlier developmental steps [3,9,[25][26][27][28][29]. However, recent evidence suggests that regeneration is not a simple reiteration of development but can employ regeneration-specific regulatory mechanisms [3,25,[30][31][32][33][34]. ...
... Many of these mechanisms are also important during normal development, and the process of regeneration was traditionally thought to be a redeployment of earlier developmental steps [3,9,[25][26][27][28][29]. However, recent evidence suggests that regeneration is not a simple reiteration of development but can employ regeneration-specific regulatory mechanisms [3,25,[30][31][32][33][34]. Indeed, faithful regeneration likely requires additional mechanisms, since regrowth happens in the presence of wound-response signaling and in a developed juvenile or adult organism. ...
Some animals respond to injury by inducing new growth to regenerate the lost structures. This regenerative growth must be carefully controlled and constrained to prevent aberrant growth and to allow correct organization of the regenerating tissue. However, the factors that restrict regenerative growth have not been identified. Using a genetic ablation system in the Drosophila wing imaginal disc, we have identified one mechanism that constrains regenerative growth, impairment of which also leads to erroneous patterning of the final appendage. Regenerating discs with reduced levels of the RNA-regulator Brain tumor (Brat) exhibit enhanced regeneration, but produce adult wings with disrupted margins that are missing extensive tracts of sensory bristles. In these mutants, aberrantly high expression of the pro-growth factor Myc and its downstream targets likely contributes to this loss of cell-fate specification. Thus, Brat constrains the expression of pro-regeneration genes and ensures that the regenerating tissue forms the proper final structure.
... For example, during normal leg imaginal disc development in D. melanogaster, PD patterning is established in a distal to proximal direction. However, when distal leg segments are removed from the centre of the imaginal discs, lineage tracing and gene expression studies demonstrated that the regenerating tissue underwent patterning intermediates distinct from those observed during normal development, and re-established patterning in a proximal to distal sequence (Bosch et al., 2010) (Figure 3b). Such deviation from the normal sequence of developmental patterning likely occurs because regenerating tissue begins repatterning from a starting point not seen during normal development. ...
... During normal development PD patterning is established from the distal-most region to the proximal parts. During regeneration after removal of the central (distal) portion of the disc, patterning is established in the proximal to distal direction (Bosch et al., 2010). (c) The patterning gene Taranis (Tara) protects cells from cell fate changes that can occur during regenerative growth in the Drosophila wing imaginal disc. ...
Many animal species have the capability to regenerate lost body parts. How regeneration takes place and why animals have varying potentials for regeneration remain active questions for biologists. The field of regenerative biology has witnessed unprecedented advances in the last several years owing to the availability of molecular and genomics tools and the establishment of many animal models. Regeneration research in arthropods has a long history, with extensive insights achieved from using model organisms from the taxa Crustacea and Insecta. Studies in animals ranging from fiddler crabs to crickets have revealed much about the different stages of regeneration, such as wound healing, blastema formation, growth, proliferation and patterning, as well as how hormonal control and systemic signalling impact regenerative capacity. The molecular and genetic insights achieved from studying these simpler model organisms have the potential to impact the field of regenerative biology by identifying conserved mechanisms of regeneration.
Key Concepts
Regeneration studies use the fiddler crab, crayfish, sand hopper, red flour beetle, fruit fly, cockroach, cricket and silverfish.
For amputated limbs, wounds heal by a combination of rapid closure of the wound with a scab or autotomy membrane, and migration of cells into the wound.
Imaginal disc wound closure involves cytoskeletal‐driven cell shape changes and zippering together of the epithelium, without cell migration.
A regeneration blastema, or zone of proliferating cells, forms after both external limb amputation and imaginal disc damage.
Growth of the blastema requires similar signals in multiple model organisms, including growth factor signalling in response to FGFs and EGFR activity, Wg/WNT signalling and Hippo signalling.
Many developmental patterning genes are also required for patterning during regeneration. However, knockdown of these patterning genes revealed additional roles in regeneration beyond those observed during normal development.
Some plasticity in cell fate enables replacement of lost cell types.
Signals at the wound can alter pattern and cell fate, generating ectopic eye spots in butterflies and requiring the activity of a protective factor that stabilises cell fate gene expression during regeneration in fruit flies.
Hormonal signalling, which controls moulting and metamorphosis, limits regenerative capacity. In some model organisms, tissue damage can influence hormone production and the timing of moults and metamorphosis.
... For example, a classic study used reciprocal tissue grafts between developing limb buds and regenerating blastemas in axolotls to reveal similar patterning activities in those tissues (9). Later studies contributing to this debate have compared the roles played by specific regulatory genes (10,11), the deployment of positional markers (12,13), and the transcriptional profiles of regenerating tissues (14)(15)(16) during development and regeneration, reaching different conclusions. ...
Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis . We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.
... Several studies provide evidence of significant similarities in the patterning mechanisms that operate during development and regeneration, in axolotl limbs (e.g. Muneoka andBryant 1982, Roensch et al. 2013;reviewed in Nacu and Tanaka 2011) and in other systems (Aztekin et al. 2021, Czarkwiani et al. 2021, while others point to significant differences (Fan et al. 2012, Bosch et al. 2010, Warner et al. 2019, Khan and Crawford 2020. ...
Regenerating animals have the ability to reproduce organs that were originally generated in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis . We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned.
HIGHLIGHTS
Single-limb data on transcriptional dynamics of leg development and regeneration
Developing embryonic legs show stereotypic transcriptional profiles
Regenerating leg transcriptomes show a high degree on individual variation
Regenerating leg transcriptomes are influenced by adult physiology, especially molting
Regenerating leg transcriptomes reveal distinct phases of leg regeneration
Leg development and regeneration use overlapping sets of genes in different temporal patterns
... There is a distinction between hemimetabolous insects whose regenerative capabilities seem very similar to that of other arthropods, such as crabs, and holometabolous insects whose regeneration is complicated by a pupal life stage required for complete metamorphosis (Das, 2015). In holometabolous insects, such as Coleoptera (Shah et al., 2011;Abdelwahab et al., 2018), Lepidoptera (Yang et al., 2016), and Diptera (Bosch et al., 2010), it is likely that imaginal cells or discs play an important role in their regenerative abilities (Lee et al., 2013;Das, 2015). In hemimetabolous insects, such as Ephemeroptera (Almudi et al., 2019), Odonata, Orthoptera (Yang et al., 2016), Phasmatodea (Maginnis, 2006b), Blattodea (Tan et al., 2013), and Hemiptera (Knobloch and Steel, 1988), there is a "breakage point" on the limb that results in the limb easily falling off when subject to pressure, a mechanism called autotomy (Maruzzo et al., 2005;Maginnis, 2006b). ...
The leg regeneration capabilities of damselflies are understudied. Here we present the first data of regenerated limbs across a genus of damselfly based on adult specimens collected in the field to illustrate the prevalence of limb loss among nymphs. We show that this phenomenon is much more prevalent than previously thought, as 42 percent of individuals were found with regenerated limbs. Furthermore, we test for patterns within these data to begin to unravel the potential causes of limb loss in nymphal damselflies, showing that intrinsic factors such as sex and species cannot explain the patterns of limb loss pointing to environmental factors as the probable cause. We argue that Odonata limb regeneration provides a potentially unique perspective into the nymphal stage of these organisms.
... Despite the diversity of species that are able to regenerate and the varying modes, mechanisms and degrees of their regeneration capabilities, only a small number of organisms have been used to investigate how regeneration occurs. This is particular evident for insects, where only a handful of species have been used as models for regeneration studies [77], namely Drosophila melanogaster, which only regenerates undifferentiated primordia-the imaginal discs-and the gut [78][79][80][81][82][83][84][85][86][87][88][89], and two hemimetabolous insects, the cockroach Blattella germanica [90,91] and the cricket Gryllus bimaculatus, which can take from 1 month to 18 weeks to regenerate an amputated limb [91][92][93]. ...
The great capability of insects to adapt to new environments promoted their extraordinary diversification, resulting in the group of Metazoa with the largest number of species distributed worldwide. To understand this enormous diversity, it is essential to investigate lineages that would allow the reconstruction of the early events in the evolution of insects. However, research on insect ecology, physiology, development and evolution has mostly focused on few well-established model species. The key phylogenetic position of mayflies within Paleoptera as the sister group of the rest of winged insects and life history traits of mayflies make them an essential order to understand insect evolution. Here, we describe the establishment of a continuous culture system of the mayfly Cloeon dipterum and a series of experimental protocols and omics resources that allow the study of its development and its great regenerative capability. Thus, the establishment of Cloeon as an experimental platform paves the way to understand genomic and morphogenetic events that occurred at the origin of winged insects.
Electronic supplementary material
The online version of this article (10.1186/s13227-019-0120-y) contains supplementary material, which is available to authorized users.
... Despite the diversity of species that are able to regenerate and the varying modes, mechanisms and degrees of their regeneration capabilities, only a small number of organisms have been used to investigate how regeneration occurs. This is particular evident for insects, where only a handful of species have been used as models for regeneration studies [77], namely Drosophila melanogaster, which only regenerates undifferentiated primordia-the imaginal discs-and the gut [78][79][80][81][82][83][84][85][86][87][88][89], and two hemimetabolous insects, the cockroach Blattella germanica [90,91] and the cricket Gryllus bimaculatus, which can take from 1 month to 18 weeks to regenerate an amputated limb [91][92][93]. ...
The great capability of insects to adapt to new environments promoted their extraordinary diversification, resulting in the group of Metazoa with the largest number of species distributed worldwide. To understand this enormous diversity, it is essential to investigate lineages that would allow the reconstruction of the early events in the evolution of insects. However, research on insect ecology, physiology, development and evolution has mostly focused on few well-established model species. The key phylogenetic position of mayflies within Paleoptera, as the sister group of the rest of winged insects and life history traits of mayflies make them an essential order to understand insect evolution. Here, we describe the established of a continuous culture system of the mayfly Cloeon dipterum and a series of experimental protocols and -omics resources that allow the study of its development and its great regenerative capability. Thus, the establishment of Cloeon as an experimental platform paves the way to understand genomic and morphogenetic events that occurred at the origin of winged insects.
... However, some differences have been identifi ed. For example, in Drosophila , genes of the leg patterning network exhibit some regeneration-specifi c interactions (Bosch et al. 2010 ). In terms of the function of wg and canonical Wnt signaling, the networks governing appendage specifi cation and patterning may be more similar between development and regeneration in Drosophila and other holometabolous insects than they are in hemimetabolous insects. ...
Arthropods are the most species-rich phylum. Within arthropods, species diversity is concentrated in the Hexapoda, which includes on the order of one million described species. The ancestor of hexapods was among the first metazoan lineages to move into a terrestrial environment. Hexapods were also the first lineage to evolve powered flight and remain the only invertebrate lineage to have done so. Hexapods are both exceptionally abundant in many habitats and exceptionally diverse ecologically, with lifestyles ranging from parasitic to agricultural. They also show extensive coevolutionary histories with other taxa, especially flowering plants, which hexapods both pollinate and consume. All of this diversity is achieved within a highly conserved body plan consisting of a segmented head, thorax, and abdomen, which bear an assortment of jointed appendages.
The mystery of appendage regeneration has fascinated humans for centuries, while the regulatory mechanisms remain unclear. In this study, a transcriptional landscape of regenerating leg was established in the American cockroach, Periplaneta americana, an ideal model for appendage regeneration with remarkable regeneration capacity. Through a large-scale in vivo screening, we identified multiple signaling pathways and transcription factors (TFs) controlling leg regeneration. Specifically, zfh-2 and bowl, which have not been previously implicated in appendage regeneration, contributes to blastema proliferation and morphogenesis in two novel transcriptional cascades BMP/JAK-STAT-zfh-2-bab1/B-H2/Lim1 and Notch-drm/bowl-bab1. Notably, zfh-2 was found working as a direct target of BMP signaling to promote cell proliferation in the blastema. These mechanisms might be conserved in the appendage regeneration of vertebrates from an evolutionary perspective. Overall, our findings reveal that two crucial transcriptional cascades orchestrate distinct cockroach leg regeneration processes, significantly advancing the comprehension of molecular mechanism in appendage regeneration.
This review summarizes recent advances in leg regeneration research, focusing on the cricket Gryllus bimaculatus. Recent studies have revealed molecular mechanisms on blastema formation, establishment of positional information, and epigenetic regulation during leg regeneration. Especially, these studies have provided molecular bases in classical conceptual models such as the polar coordinate model, the intercalation model, the boundary model, the steepness model, etc., which were proposed to interpret regeneration processes of the cockroach legs. When a leg is amputated, a blastema is formed through the activation of the Janus-kinase (Jak)/Signal-Transduction-and-Activator-of-Transcription (STAT) pathway. Subsequently, the Hedgehog/Wingless/Decapentaplegic/Epidermal-growth-factor pathways instruct distalization in the blastema, designated as the molecular boundary model. Downstream targets of this pathway are transcription factors Distal-less (Dll) and dachshund (dac), functioning as key regulators of proximodistal pattern formation. Dll and dac specify the distal and proximal regions in the blastema, respectively, through the regulation of tarsal patterning genes. The expression of leg patterning genes during regeneration may be epigenetically controlled by histone H3K27 methylation via Enhancer-of-zeste and Ubiquitously-transcribed-tetratricopeptide-repeat-gene-X-chromosome. For the molecular mechanism of intercalation of the missing structures between the amputated position and the most distal one, Dachsous/Fat (Ds/Ft) steepness model has been proposed, in which the Ds/Ft pathway maintains positional information and determines leg size through dac expression. This model was theoretically verified to interpret the experimental results obtained with cricket legs. Availability of whole-genome sequence information, regeneration-dependent RNA interference, and genome editing technique will have the cricket be an ideal model system to reveal gene functions in leg regeneration.
Modeling biological pattern formation with reaction-diffusion equations.
Much of the cell-cell communication that controls assign - ment of cell fates during animal development appears to be mediated by extracellular signaling molecules. The formation of the proximodistal (P/D) axis of the legs of flies is controlled by at least two such molecules, a Wnt and a TGFβ, encoded by the wingless (wg) and decapentaplegic (dpp) genes, respectively. The P/D axis appears to be initiated from the site where cells expressing wg are in close association with those expressing dpp. Support for this hypothesis comes from two sources: classical grafting experiments in cockroaches and ectopic protein expression in Drosophila. SUMMARY
Nymphs of hemimetabolous insects such as cockroaches and crickets exhibit a remarkable capacity for regenerating complex structures
from damaged legs. Until recent years, however, approaches to elucidate the molecular mechanisms underlying the leg regeneration
process have been lacking. Taking the cricket Gryllus bimaculatus as a model, we found that a phenotype related to regeneration frequently appears during leg regeneration, even though no
phenotype is induced by RNA interference (RNAi) in the cricket nymph, designated as regeneration-dependent RNAi. Since then,
we have investigated the functions of various genes encoding signaling factors and cellular adhesion proteins like Fat and
Dachsous during leg regeneration. In this review, we summarize the classical knowledge about insect leg regeneration and introduce
recent advances concerning the signaling cascades required for regenerating a leg. Our results provide clues to the mechanisms
of regeneration which are relevant to vertebrate systems.
Susan Bryant is one of the leading researchers in regeneration and pattern formation. Born in England in 1943, she studied biology at Kings College, London (UK). After a Ph.D. with Angus Bellairs on caudal autotomy and regeneration in lizards, she researched urodele regeneration in Marcus Singer's lab at Case Western Reserve University. Then, at the University of California, Irvine, she adopted the axolotl as a research model for limb regeneration and pattern formation. Her work supported models involving the intercalation of positional values in a polar coordinate system. Fibroblasts, often regarded as "junk" cells, are seen by Susan Bryant as central to patterning. She argues that fibroblasts express positional values needed for regeneration. She also argues that vertebrate species capable of regeneration have evolved steps to plug back into developmental programmes. Susan Bryant thinks that regeneration is essential for a full understanding of development, and believes that developmental biology has suffered though not embracing regeneration. She also believes that deeper knowledge of pattern formation will bring advances in emerging field of tissue engineering. Since 2000, she has served as Dean of Biological Sciences and more recently, as Vice Chancellor for Research, at UC Irvine (USA). She is an advocate of equal opportunities for women and other under-represented groups in academia. She lives in California with husband David Gardiner, her scientific partner for over 20 years. They have two children. We interviewed Susan Bryant in her office in Irvine on October 5th, 2007.
The proximodistal epidermal organization of the re-generated insect leg has been studied by grafting between corresponding and noncorresponding levels of the pro-and metathoracic femur and tibia. The results have been studied quantitatively (growth rates of the associated parts and of unsegmented intercalary structures) and qualitatively (nature, length, polarity of intercalary structures). In grafts between equival-ent levels, no intercalary structure is formed, but a differential growth has been observed, the distal one fifth of segments growing about 1-5 to 2 times more than the proximal one. In grafts between different levels, unsegmented intercalary structures are formed from the distal part which thereby acquires proximal characteristics (proximalization). However, distal tibial cells do not form femur in this process under an hypothetical femoral influence: there is no 'domi-nance' of femur over tibia. Some segmented interca-lary structures have also been observed, but their formation cannot be related clearly to differences in the healing process. To explain proximalization, an hypothesis is presented suggesting that distal cells, which grow faster, would be the first to enter a period when positional value becomes labile and would then adapt to the proximal cells' value.
A model is proposed for pattern formation in secondary embryonic fields. It is stipulated that the boundaries, resulting from the primary embryonic organization of a developing organism, act as organizing regions for secondary embryonic fields, e.g., imaginal discs in insects. This boundary mechanism would allow very reliable pattern formation in the course of development: Primary positional information leads to cells of different determination, separated by sharp borders. At these borders, in turn, positional information would be generated for the next finer subdivision, and so on. This occurs if two or more differently determined cell types (e.g., compartments) cooperate for the production of a morphogenetic substance. A high concentration of the morphogen would appear at the common boundary of the cell types involved. Many experiments reported in the literature, for instance, the formation of duplicated and triplicated insect legs and the regeneration-duplication phenomenon of imaginal disc fragments can be explained under this assumption. The proposed boundary mechanism provides a molecularly feasible basis for the polar coordinate model.
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.
In 1969, Lewis Wolpert published a paper outlining his new concepts of "pattern formation" and "positional information". He had already published research on the mechanics of cell membranes in amoebae, and a series of classic studies of sea urchin gastrulation with Trygve Gustavson. Wolpert had presented his 1969 paper a year earlier at a Woods Hole conference, where it received a very hostile reception: "I wasnt asked back to America for many years!". But with Francis Crick lining up in support of diffusible morphogen gradients, positional information eventually became established as a guiding principle for research into biological pattern formation. It is now clear that pattern formation is much more complex than could possibly have been imagined in 1969. But Wolpert still believes in positional information, and regards intercalation during regeneration as its best supporting evidence. However, he and others doubt that diffusible morphogen gradients are a plausible mechanism: "Diffusible gradients are too messy", he says. Since his retirement, Lewis Wolpert has remained active as a theoretical biologist and continues to publish in leading journals. He has also campaigned for a greater public understanding of the stigma of depression. He was interviewed at home in London on July 26th, 2007 by Michael Richardson.