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. Author manuscript; available in PMC: 2009 Jul 1.
Published in final edited form as: Dev Biol. 2008 Apr 15;319(1):68–77. doi: 10.1016/j.ydbio.2008.04.004

Three genes control the timing, the site and the size of blastema formation in Drosophila

Kimberly D McClure 1,2, Anne Sustar 2, Gerold Schubiger 2
PMCID: PMC2483308  NIHMSID: NIHMS56258  PMID: 18485344

Abstract

Regeneration is a vital process to maintain and repair tissues. Despite the importance of regeneration, the genes responsible for regenerative growth remain largely unknown. In Drosophila, imaginal disc regeneration can be induced either by fragmentation and in vivo culture or in situ by ubiquitous expression of wingless (wg/wnt1). Imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing and proliferation at the wound site, forming a regeneration blastema. Most blastema cells maintain their disc-specific identity during regeneration; a few cells however, exhibit stem-cell like properties and switch to a different fate, in a phenomenon known as transdetermination. We identified three genes, regeneration (rgn), augmenter of liver regeneration (alr) and Matrix metalloproteinase-1 (Mmp1) expressed specifically in blastema cells during disc regeneration. Mutations in these genes affect both fragmentation- and wg-induced regeneration by either delaying, reducing or positioning the regeneration blastema. In addition to the modifications of blastema homeostasis, mutations in the three genes alter the rate of regeneration-induced transdetermination. We propose that these genes function in regenerative proliferation, growth and regulate cellular plasticity.

INTRODUCTION

Regeneration and homeostatic tissue renewal are essential for normal physiology in most organisms, including humans. Cellular turnover and regeneration are mediated by tissue-specific resident stem cells, progenitor cells or normally quiescent differentiated cells (Gargioli and Slack, 2004). These cells are capable of reactivating specific developmental programs and genes, including those helping to maintain a pluripotent state, in order to replace lost or damaged tissues (Beck et al., 2003). However, the identification of gene products necessary to achieve regeneration is still in its infancy. Given the physiological differences between embryos and adults, it is conceivable that regeneration-specific genes or pathways exist. Such genes may play a role in the timing of regeneration, the recruitment of stem cells (or progenitor cells) to form the regenerate and in the regulation of their developmental plasticity.

Drosophila imaginal discs, precursors of the adult fly appendages, are an excellent model system to study regeneration. Disc cells are rigidly determined to differentiate specific adult structures during larval development (Gehring, 1978). Despite this, disc fragments can regenerate. Disc regeneration can be conventionally induced by fragmentation and in vivo culture or molecularly by ubiquitous expression of wingless (wg) in mid-second and early third instar larvae (Maves and Schubiger, 1998). In both experimental protocols, regeneration is accomplished by a localized region of cell division, known as the regeneration blastema, reminiscent of epimorphic limb/fin regeneration in lower vertebrates. In addition, both perturbations result in identical pattern deviations and can lead to disc fate changes (i.e., leg cells change to wing cells) in a process known as transdetermination (Hadorn, 1978; Johnston and Schubiger, 1996; Maves and Schubiger, 1995; Maves and Schubiger, 1998; Struhl and Basler, 1993). Transdetermination is initiated in a small number of proliferating blastema cells, approximately 3–5 founder cells, located at specific regions within each imaginal disc known as the weak point (Gehring, 1967; Gibson and Schubiger, 1999). These fate changes are the consequence of regenerative plasticity and altered homeotic or selector gene expression (Maves and Schubiger, 2003). In the leg imaginal disc, the weak point is in the proximodorsal cells, where the blastema forms.

One of the earliest signals involved in epimorphic limb/fin regeneration is the canonical Wnt signaling pathway (Kawakami et al., 2006; Stoick-Cooper et al., 2007b). This pathway is also used during regeneration of mammalian muscle, liver, bone and possibly heart (Gehring, 1978; Polesskaya et al., 2003; Sodhi et al., 2005; Zhong et al., 2006). Studies in lower vertebrates have demonstrated that blastema formation is impaired by loss of Wnt/β-catenin signaling (Stoick-Cooper et al., 2007b). The downstream targets of Wnt/β-catenin signaling during regeneration are largely unknown.

Previously, we performed a genome wide search using DNA microarray to identify Wg target genes that potentially function in regeneration (Klebes et al., 2005). Here we focus on three of those genes: regeneration (rgn) and augmenter of liver regeneration (alr), both of which are uncharacterized genes in Drosophila, and Matrix metalloproteinase-1 (Mmp1) (Page-McCaw et al., 2003). All three genes share homology with mammalian genes that have a characterized function in regeneration. Alr, for example, an enzyme with sulfhydryl oxidase activity, has been shown to function as a growth factor in mammalian liver and pancreatic regeneration (Gatzidou et al., 2006; Pawlowski and Jura, 2006). Based on the results of in vitro experiments, it is thought that Alr promotes liver regeneration by activating the MAPK cascade via the EGFR, one of the few signals that stimulates hepatocyte proliferation (Li et al., 2005; Pawlowski and Jura, 2006). However, other studies indicate that Alr supports hepatocyte proliferation during liver regeneration by suppressing the activity of liver-resident natural killer cells immediately after injury (Tanigawa et al., 2000). Thus, how Alr promotes regeneration in vertebrates is still unclear and the situation remains unresolved by the lack of loss of function studies. Members of the Regenerating gene (Reg) protein family also act as growth factors in pancreatic and stomach regeneration (Okamoto, 1999; Ose et al., 2007). In vitro experiments indicate that Reg stimulates cell-cycle progression during pancreatic β-cell regeneration by activating the cyclin D1 gene (Takasawa et al., 2006). Consistent with these results, Reg-I knockout mice exhibit slower proliferation and migration of intestinal stem cells, while transgenic mice overexpressing Reg show increased proliferation of gastric progenitor cells (Miyoshi et al., 2002; Ose et al., 2007). How these Reg-I transgenic mice respond to injury and regenerate has not yet been assessed. Finally, numerous studies have documented the expression of several MMPs in regenerating amphibian and fish appendages (Tsubota et al., 2002; Yang et al., 1999). Functional analyses suggest that remodeling of the extracellular matrix by MMPs is important for proper wound healing and blastema formation (Bai et al., 2005). Vertebrates have over twenty MMPs whereas flies have only two, Mmp1 and Mmp2 (Page-McCaw et al., 2003). Drosophila Mmp1 is distinct from Mmp2 in that it lacks a predicted membrane anchor. In our previous studies we found that both Mmp1 and Mmp2 are upregulated during imaginal disc regeneration (Klebes et al., 2005).

Here, we use Drosophila leg imaginal discs to investigate how mutations in rgn, alr and Mmp1 affect regeneration, specifically the process of blastema formation and regeneration-induced cellular plasticity. We find that rgn is involved in the initiation of regeneration by affecting the timing of blastema formation; alr functions in blastema cell proliferation and the extent of regeneration. Finally, Mmp1 delimits regeneration by regulating the cell-cycle arrest of non-blastema cells. Our results demonstrate that each of these genes functions early in the regeneration process and this has direct effects on cellular plasticity.

MATERIAL AND METHODS

Fly Stocks

Flies were raised on standard media at 25°C. ‘Sevelen’ flies were wild-type controls. Act5c> y+ > wg flies were provided by K. Basler; the null alleles, Mmp1Q112, Mmp12 and Mmp2W307 were provided by A. Page-McCaw.

rgn alleles

DNA flanking the P-lacZ (PZ) insertion line l(3)0054 (rgn1) was cloned by plasmid rescue and sequenced. Searching genomic sequence indicated that l(3)00543 was inserted 8 bp upstream of the first untranslated exon in the predicted gene CG6014. A second P-element in CG6014, KG05449 (rgn2) was obtained from the Bloomington stock center. Homozygous rgn1 and rgn2 animals are semi-lethal. Mobilization of the rgn1 P-element recovered many precise excisions; all suppressed lethality and two (rgn4 and rgn11) were tested that reverted the transdetermination phenotype (data not shown).

alr alleles

To generate mutations in CG12534 (alr) we mobilized the P-element P{EPgy2}EY03908. Animals homozygous for this P-element are viable, and mobilization recovered three recessive lethal imprecise excision lines, alr1–3 (Fig. 3B). Precise excision of the P-element did not enhance transdetermination.

Figure 3. rgn (CG6014) and alr (CG12534) genes in Drosophila.

Figure 3

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(A and B) Genomic regions of rgn (CG6014) and alr (CG12534), including sequenced P-element insertions and excision-derived deletions are shown. All P-elements, indicated by triangles, are oriented 5′ to the left and 3′ to the right. Gray boxes designate noncoding and black boxes indicate protein-coding mRNA. (A) l(3)00543 is 8bp upstream of the first untranslated exon and KG05449 is 30bp into the first untranslated exon of rgn (CG6014). (B) EY03908 is 79bp upstream of the first exon in alr (CG12534). Mobilization of EY03908 recovered three deletion lines, alr1–3. Sequences deleted in alr1–3 are indicated by dashed lines in parentheses. (A′ and B′) Domain organization of Drosophila Rgn and Alr proteins. (A′) The predicted Rgn protein is 800 residues, with a signal sequence at residues 7–29 (red; red arrowhead) and C-type (Ca2+-dependent) lectin domain (CTLD, blue) at residues 61–194. (B′) The predicted Alr protein is 193 residues with an Essential for Respiration and Vegetative growth/Augmenter of Liver Regeneration (ERV1/ALR) domain at it C-terminus (in blue). (A″ and B″) Rgn and Alr share significant amino acid sequence homology with mammalian proteins with a characterized role in regeneration. Dashes indicate gaps for maximal alignment. Identical amino acids in the proteins are shaded in red. Strong conservation in amino acid function is shaded in green; similarities in amino acid function are shaded in yellow. Sequences for all the proteins are based on full-length cDNAs. Asterisks indicate conserved cysteine residues in the mature proteins. (A″) Blast searches (blastp) of Rgn/CG6014 identifies several members of the vertebrate Regeneration (Reg) protein family. Clustal W alignment shows that Rgn/CG6014 shares 21% identity with human RegIβ, 19% identity with human RegIα, 18% identity with rat RegI and 16% identity with mouse RegI. (B″) The Alr/CG12534 protein shares sequence homology with FAD-linked sulfhydryl oxidases of the ERV1/ALR protein family. Clustal W analysis shows the highest level of sequence similarity between Drosophila Alr/CG12534 and the ERV1/ALR proteins from human, mouse and yeast at the C-terminal end. Drosophila Alr/CG12534 shares 39% sequence homology to human Alr, 36% to mouse Alr and 32% to yeast scERV1.

Disc regeneration induced by fragmentation or ubiquitous wg expression

To minimize genetic background in the fragmentation experiments, mutant flies were crossed to the Sevelen wild-type stock. 1–2 hour egg collections were taken after a 1 hour pre-collection. Disc fragmentation and in vivo culture were performed as previously described (Gibson and Schubiger, 1999). Note that in in vivo culture, disc growth and cell-doubling time is not different from larval disc development (Bryant and Simpson, 1984; Gibson and Schubiger, 1999)

Larvae of the genotype y w hs-flp122; Act5c> y+ > wg were heat-shocked at 37°C for 75 minutes at 60h or 72h AED to induce ubiquitous wg expression in over 90% of cells. Only the 60h AED heat shock protocol produced a significant frequency of pharate adults (differentiated adults that do not eclose), and only such adults produced transdetermined structures.

These same heat-shock protocols were used when females of genotype y w hs-flp122; +; rgn1/TM6B, y w hs-flp122, alr1/FM7-GFP; + ; + and y w hs-flp122; Mmp1Q112/CyO-GFP were crossed to males of genotype w−; Act5c > y+ > wg. rgn2, alr2 and Mmp12 and were also tested.

As previously reported, ubiquitous wg expression at 60h or 72h AED delays pupariation by up to three days (Maves and Schubiger, 1998). This delay is also observed when wg is expressed in heterozygous modifiers; however, rgn/+, alr/+ and Mmp1/+ alleles by themselves did not affect the timing of larval wandering, puparium formation or adult eclosion relative to wild-type controls.

In vivo culture was used to test if the loss of disc phenotype in alr mutants could be rescued. For this, anterior halves of first instar larvae were inverted and injected into wild-type adult females.

In situ hybridization

Discs were hybridized with digoxigenin-labeled antisense and sense RNA probes as previously described (O’Neill and Bier, 1994). Probes were made from full-length cDNAs (provided by BDGP).

Immunocytochemistry

Discs were fixed for 20 min in 4% formaldehyde in PBS. Rabbit anti-Vg (S. Carroll) at 1:200; mouse anti-BrdU (Becton-Dickinson) at 1:200; mouse or rabbit anti- β-gal (Promega and Cappel, respectively) at 1:1000; mouse anti-Mmp1 (A. Page-McCaw) at 1:100. BrdU (10 μg/ml) incorporation was performed for 20 minutes before a 30 minute fixation (Johnston and Schubiger, 1996).

RESULTS

Imaginal disc regeneration

We use Drosophila prothoracic leg discs to study the basic genetic mechanisms of organ regeneration. To induce regeneration, the leg disc is fragmented into a ‘3/4 lateral piece’ (Fig. 1A) and cultured in vivo. Disc regeneration, like epimorphic regeneration in lower vertebrates has three phases: (i) wound healing, which occurs 12–48 hours post-fragmentation (hpf) (ii) formation of the regeneration blastema (36–48 hpf), where cell division is limited to the wound site and (iii) blastema outgrowth and pattern reformation (48–72 hpf) (Fig. 1A–A‴) (Reinhardt and Bryant, 1981; Reinhardt et al., 1977). These three phases of regeneration closely parallel epimorphic regeneration in lower vertebrates (Brockes and Kumar, 2005; Poss et al., 2003). Moreover, the disc blastema, like its vertebrate counterpart, is able to complete regeneration when isolated from the remaining disc fragment (Brockes and Kumar, 2005; Karpen and Schubiger, 1981).

Figure 1. Fragmentation- and wg-induced disc regeneration and the activation of ectopic wg expression.

Figure 1

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(A–A‴) BrdU labeling (20 minutes) shows cell replication (S-phase) in uncut (A) and cut (A′–A‴) wild-type prothoracic leg discs during each stage of disc regeneration: wound healing (12–48 hpf), blastema formation (36–48 hpf) and blastema outgrowth and pattern reformation (48–72 hpf) (Reinhardt and Bryant, 1981). (B) Endogenous Wg expression (red asterisk) in an unfragmented leg disc. (B′–B‴) Ectopic Wg expression is found ventral to the cut site (indicated by arrows) during all stages of disc regeneration (89.5%, n=19 disc fragments). Red lines in (A) and (B) indicate vertical and horizontal cuts made in leg discs to induce blastema formation and regeneration. Arrowheads in (A′–A″) mark the site of wound healing. (C–C″) BrdU labeling shows formation of a regeneration blastema after ectopic wg expression. The regeneration blastema forms in the proximodorsal region of the leg disc (arrowheads) with a frequency of 75% (n=38 discs). Scale bar: 50 μm.

Wnt ligands and components of the β-catenin signaling pathway are key regulators of epimorphic regeneration in lower vertebrates (Kawakami et al., 2006; Stoick-Cooper et al., 2007a; Stoick-Cooper et al., 2007b). Therefore, we examined the temporal expression of Wingless (Wg/Wnt1) during fragmentation-induced leg disc regeneration. By 12h after fragmentation we find ectopic Wg expression just ventral to the disc cut site, the presumptive blastema site, prior to blastema formation and during the initiation of wound healing (data not shown) (Fig. 1B,B′, A″). The ectopic expression of Wg persists in the blastema throughout disc regeneration (Fig. 1B–B‴) (Gibson and Schubiger, 1999). Thus, in Drosophila, activation of Wg at the wound site is also implicated in the earliest stages of regeneration, before blastema formation. The importance of Wg signaling in the initiation of regeneration is supported by the fact that ubiquitous wg expression in unfragmented leg discs is sufficient to induce blastema formation and growth in situ, in a temporal pattern identical to fragmentation (Fig. 1C–C″) (Johnston and Schubiger, 1996; Sustar and Schubiger, 2005).

rgn, alr and Mmp1 are expressed in regenerating disc cells

Previously, DNA microarray analyses identified 143 genes whose expression is enriched in regenerating/transdetermining leg disc cells after ubiquitous wg expression (Klebes et al., 2005). Here we focused our functional studies on three of these candidate genes because (1) they have mammalian orthologs with a characterized function in stem cell biology and/or regeneration and (2) they have known connections to the Wnt signaling pathway. We made use of the two complementary approaches of disc regeneration (fragmentation and ubiquitous wg expression) to identify ‘regeneration’ genes. The three genes we focused on are: two uncharacterized genes in Drosophila, regeneration (rgn) and augmenter of liver regeneration (alr), and Matrix metalloproteinase-1 (Mmp1) (Mott and Werb, 2004; Page-McCaw et al., 2003).

We used in situ hybridization and antibody staining to determine whether rgn, alr and Mmp1 are expressed during normal imaginal disc development. At embryonic blastoderm stage, rgn and Mmp1 were not detected in the ectoderm (Page-McCaw et al., 2003; data not shown). In contrast, alr was abundantly expressed at this stage except at the anterior end (data not shown). Thus, the early expression of alr coincides with imaginal disc determination (Chan and Gehring, 1971). Later, during third instar larval development, we found that none of the genes are expressed in the prothoracic leg discs (Fig. 2A–C). Once regeneration was induced, however, either by fragmentation (Fig. 2A′–C′) or ubiquitous wg expression (Fig. 2A″–C″), all three genes were activated. For example, in fragmented leg discs, the three genes are expressed along the ventral cut site, where the blastema forms, as well as along the dorsal cut site, possibly owing to wound healing (Fig. 2A′–C′,) (Kiehle and Schubiger, 1985). After ubiquitous wg expression, we found ectopic rgn, alr and Mmp1 expression in the weak point of the disc, the approximate site of blastema formation (Fig. 2A″–C″). Thus, with both protocols, all three genes were specifically activated at sites of regeneration.

Figure 2. Regenerating blastema cells express rgn, alr and Mmp1.

Figure 2

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(A,B) In situ hybridization with rgn and alr probes, respectively; (C) antibody staining of Mmp1. (A–C) rgn, alr, Mmp1 expression is not detected in third instar prothoracic leg discs. (A′–C′) After fragmentation, rgn, alr, and Mmp1 expression is observed in cells dorsal and ventral to the cut site (2 days after in vivo culture). Black and white arrows indicate cut sites. (A″–C″) Two days after ectopic wg expression, rgn, alr and Mmp1 are expressed in the regeneration blastema at the weak point of the disc. Scale bar: 100 μm.

Characterization of two novel Drosophila genes: rgn (CG6014) and alr (CG12534)

Two of the three genes described here, rgn and alr, are novel in Drosophila. The rgn1 and rgn2 alleles are both P-element insertions in CG6014 (Fig. 3A), a predicted ortholog of the mammalian Reg gene (Fig. 3A″). The following observations indicated that rgn1 and rgn2 are hypomorphic alleles. First, animals homozygous for rgn1 and rgn2 were semi-lethal; only 41% (n=817) and 29% (n=300), respectively, survived to adulthood and have no detectable developmental delay, abnormalities or size differences. Second, rgn1 and rgn2 mutants failed to complement, indicating that they are alleles of the same gene (data not shown). Third, in situ hybridization with rgn probes on the homozygous mutants showed a reduction in transcript levels (data not shown).

A BLAST search of the predicted Rgn protein sequence revealed the presence of a signal sequence and a C-type (Ca2+-dependent) lectin domain (CTLD) (Fig. 3A′). The search also identified a series of related proteins present in invertebrates and vertebrates, of these are two mosquito proteins Aedes aegypti AET45628 and Anopheles gambiae EAA11599, with 41% and 36% identity, respectively (data not shown). In addition several members of the vertebrate Regeneration (Reg) protein family were identified (Fig 3A″). Members of this protein family function as growth factors in pancreatic and stomach regeneration (Okamoto, 1999; Ose et al., 2007).

alr1–3 are excision-derived deletions in CG12534, the Drosophila ortholog of mammalian Alr, for which there are no mutations (knock-outs) (Klebes et al., 2005; and Fig. 3B,B″). We found that homozygous alr1–3 larvae undergo developmental arrest very early in the third larval instar and had no imaginal discs (data not shown). To determine whether alr mutant animals contain imaginal disc primordia that failed to grow, we injected mutant and wild-type first instar tissues into wild-type adult hosts. After in vivo culture we found that wild-type diploid and polytenic tissues developed and their size was not different from larvae in the third larval instar. In contrast, in alr1 cultured transplants, all tissues developed, like controls, except the imaginal discs (n=10).

A BLAST search of the predicted Alr protein sequence revealed an Essential for Respiration and Vegetative growth/Augmentor of Liver Regeneration (ERV1/ALR) domain at the C-terminus (Fig. 3B′). Such domains are present in a family of flavin-linked sulfhydryl oxidases, where they are know to bind FAD and catalyze the formation of disulfide bonds (Pawlowski and Jura, 2006). Drosophila Alr shows significant amino acid sequence homology to these sulfhydryl oxidases, particularly in the C-terminal ERV1/ALR domain (Fig. 3B″). In mammals, Alr has been shown to function as a growth factor in liver and pancreatic regeneration (Gatzidou et al., 2006; Pawlowski and Jura, 2006).

Mmp1Q112 and Mmp12 are loss of function alleles that have been previously characterized (Page-McCaw et al., 2003). Mutant animals make imaginal discs but die with tracheal defects during the early third larval instar.

Mutations in rgn, alr and Mmp1 affect blastema formation and proliferation

We reason that when an increase in gene expression correlates with regeneration, reducing one gene copy allows us to test whether the gene functions in regeneration. Note that in rgn/+, alr/+ and Mmp1/+ larvae, disc size and morphology does not differ from wild-type controls (data not shown). Therefore, we examined blastema formation in animals heterozygous mutant for the rgn, alr and Mmp1 genes. In control discs, when wg is expressed during the early third larval instar, a regeneration blastema was detected in the proximodorsal region of the leg disc within 48h by a short pulse of 5-bromo-2′-deoxyuridine (BrdU) incorporation (Fig. 4A–A″) (Sustar and Schubiger, 2005). This phenotype is observed in 75% of leg discs analyzed (n=38). In the non-blastema part, or ventral aspect of the leg disc where the cell cycle is downregulated, almost no cells have BrdU labeling (Fig. 4A′–A″). Earlier studies using FACS analysis indicate that non-blastema cells are arrested in G1 and G2 (Sustar and Schubiger, 2005). Leg discs from rgn1/+ animals sharply deviate from this pattern, in that, after 48h of wg expression, a blastema failed to form. Instead, random cell division is maintained throughout the leg discs (70%, n=50) (Fig. 4A′,B′). A regeneration blastema subsequently appears at the correct location (Fig. 4B″), yet one day later than in the wg-expressing controls (63%, n=47). While ectopic wg expression delays entry into metamorphosis we did not observe a longer delay in rgn1/+ animals.

Figure 4. rgn, alr and Mmp1 heterozygous mutant animals modify wg-induced blastema formation or blastema growth.

Figure 4

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BrdU labeling shows cell replication (S-phase) in prothoracic leg discs 24, 48 and 72 hours after ubiquitous wg expression (induced at 72h after egg deposition, AED) from Act>wg, wg controls (A–A″), rgn1/+ animals (B–B″), alr1/+ animals (C–C″) and Mmp1Q112/+ animals (D–D″). (A) 24h after wg induction, disc size is bigger and replication is still observed throughout the disc. (A′) However, at 48h, replication is limited to the proximodorsal leg cells (the regeneration blastema). (A″) The blastema grows in size and expands ventrally at 72h. (B–B″) In Act>wg, rgn1/+ animals, the disc grows in size, like in controls, but formation of the wg-induced regeneration blastema is delayed. Act>wg, rgn1/+ leg discs exhibit random BrdU labeling at 24h and still at 48h after wg induction (70%, n=50 discs). (B″) A delayed blastema forms at 72h in 63% (n=47) of discs analyzed. (C–C″) In Act>wg, alr1/+ animals the wg-induced blastema forms with normal temporal progression, but is severely reduced in size. Note that after 24h of wg expression, Act>wg, alr1/+ discs are smaller than controls (Act>wg) indicating an early suppression of wg-induced overgrowth. At the time of wg induction, alr1/+ discs were of the same size as the control discs. (D–D″) Blastema formation is aberrant in Act>wg, Mmp1Q112/+ animals due to the incomplete cell-cycle arrest of non-blastema cells (ventral leg disc cells) (marked by white arrows). Scale bar: 50 μm. (E–H, E′–H′) BrdU labeling in fragmented leg discs 48h (E–H) and 72h after in vivo culture (E′–H′). Arrowheads indicate sites of wound healing. (E–E′) Fragmented wild-type discs show cell replication only in the regeneration blastema in >90% of leg discs analyzed (n=26, 48h after in vivo culture; n=19, 72h after in vivo culture). (F) After 48h of in vivo culture, regenerating discs from rgn1/+ animals exhibit random BrdU labeling (58%, n=22). (F′) A delayed blastema forms in rgn1/+ disc fragments 72h after in vivo culture (67%, n=16). (G–G′) Regenerating discs from alr1/+ animals form small blastemas. Blastema size (blastema area/disc area) was 14% in wild-type disc fragments (n= 18) and 9% in alr1/+ fragments after 48h of in vivo culture (n=16) (P=0.0021, t-test). (H–H′) Regenerating discs from Mmp1Q112/+ animals feature BrdU labeling at the wound site, indicating blastema formation, and failure of cell cycle arrest in non-blastema cells (58%, n=21 and 67%, n=10 after 48h or 72h of in vivo culture, respectively). Scale bar: 50 μm.

To test whether the rgn blastema phenotype is protocol-independent, we turned to fragmentation-induced regeneration. rgn1/+ discs were cut and then cultured in vivo for 48h, a period sufficient for blastema formation in wild-type leg disc fragments (Fig. 4E). After 48h of in vivo culture, more than half of rgn1/+ leg disc fragments still display asynchronous cell divisions (58%, n=22 discs) (Fig. 4F). However, if the fragments are cultured for 72h, a blastema is formed in most of the discs analyzed (68%, n=16 discs) (Fig. 4F′). This again indicates a disc-autonomous delay in blastema formation. We conclude that two gene copies of rgn are required for proper timing of blastema formation and to initiate blastema cell division and growth.

When wg was ubiquitously expressed in controls, tremendous leg disc overgrowth was observed (Fig. 4A′,A″). Strikingly, this overgrowth phenotype was strongly suppressed after wg overexpression in alr1/+ and alr2/+ animals, and the difference was noticeable as early as 24h after wg induction (Fig. 4C–C″ and data not shown). Importantly, leg disc growth was not different between wild-type and in alr1/+ or alr2/+ animals, indicating that mutations in alr, by itself, did not have a dominant growth phenotype (data not shown). In addition, we observed that over-expression of wg in alr1/+ and alr2/+ animals resulted in a significantly smaller blastema that formed at the normal time (Fig. 4C′,C″). The smaller blastemas in alr1/+ leg discs contained far fewer cells in S phase (106 ± 83 cells, n=10) than the wg-expressing control blastemas (1100 ± 247 cells, n=10) (P=0.0001, t-test). Cell death did not account for this phenotype because Acridine orange staining was not different between regenerating alr1/+ and control discs (data not shown).

Fragmented alr1/+ leg discs, after 48h of in vivo culture, also showed significantly smaller blastemas compared to wild-type controls (Fig. 4G). The ratio of blastema area to disc area was 14% in wild-type disc fragments (n= 18) and 9% in alr1/+ fragments after 48h of in vivo culture (n=16) (P=0.0021, t-test). These results indicate that two wild-type gene doses of alr are necessary for proper blastema size, regardless of how regeneration is induced.

This observation prompted us to test the blastema phenotype after wg is ectopically expressed in Mmp1Q112/+ animals. We found that a blastema formed at the correct time, with an area and cell density that were not significantly different from wg-expressing control blastemas (Fig. 4D–D″). However, Mmp1Q112/+ leg discs featured a subtle but consistent phenotype: non-blastema cells (ventral leg cells) did not undergo cell-cycle arrest (Fig. 4D′ and Fig. 4A′) and replication was still seen in the ventral aspect of these discs, even at 72h after wg induction (Fig. 4D″). The number of non-blastema cells in S phase in Mmp1Q112/+ leg discs was seven-fold higher than in wg-overexpressing control discs.

When Mmp1Q112/+ disc fragments were cultured in vivo for 48h and 72h, they formed a blastema near the wound site, yet cell division still occurred in non-blastema cells (58%, n=21 and 67%, n=10 after 48h or 72h of in vivo culture, respectively) (Fig. 4H, H′). These findings indicate that during blastema formation and outgrowth (either after fragmentation or overexpression of wg), Mmp1 protein regulates the site-specific cell-cycle arrest of non-blastema cells.

Interestingly, we found that in the center of the disc, the primordium of the four distal-most tarsal segments, the cell-cycle arrest occurs on time with all genotypes. Therefore, this arrest is independent of rgn, alr and Mmp1 gene function (Fig. 4A′–D′). One possible explanation for this phenotype is that these cells are developmentally more advanced, both in terms of completion to terminal differentiation and achieving the G1 cell-cycle arrest (Graves and Schubiger, 1981; Graves and Schubiger, 1982; Schubiger, 1973).

In summary, we provide evidence that rgn, alr and Mmp1 gene products modify regenerative proliferation and growth, independent of the protocols we use to induce regeneration. These observations are of general interest, because they suggest that activation of these three genes during epimorphic regeneration may regulate (directly or indirectly) the temporal dynamics and extent of the regeneration response. The observation that these genes function in non-wounding induced regeneration indicates that like wg itself, these genes may function in homeostatic reconstitution (e.g., blood or skin renewal).

rgn, alr and Mmp1 affect regeneration-induced transdetermination

Klebes et al., (2005) observed that rgn, alr and Mmp1 transcripts are up-regulated in leg cells that have transdetermined to wing identity. In discs, transdetermination is identified by the expression of the selector gene vestigial (vg). To determine whether vg-expressing cells also express the modifier genes, we double-labeled leg discs after over-expression of wg. We observed 100% overlap between the expression of an rgn-lacZ reporter and the ectopic expression of Vg (Fig. S1). Mmp1 expression was seen more broadly, in both Vg-expressing cells and also adjacent cells (Fig. S1). This was also observed in the fragmentation experiment (data not shown). Therefore, we asked whether heterozygous mutations in the three genes altered regeneration-induced transdetermination. Leg-to-wing transdetermination was induced by ubiquitous expression of wg during the early third larval instar (72h AED), and scored in the discs two days later for ectopic expression of Vg (Fig. 5B) and in the adult leg cuticle for the formation of wing-specific structures. Transdetermination in the adult was only observed in differentiated animals that did not eclose (pharate adults). In control animals, ubiquitous wg expression led to Vg expression in 51% of leg discs (Fig. 5B) and wing structures in 25% of adult legs (n=69). In rgn1/+ animals, Vg frequency decreased significantly to 36% (n=126; P=0.02, chi-square) and the relative area of the leg disc that expresses ectopic Vg also decreased (Fig. 5C). Moreover, rgn1/+ animals showed a significant reduction of transdetermination in the adult cuticle with a frequency of 7% (n=40 legs; P<0.05, chi square). Since rgn1/+ leg discs feature a delay in blastema formation (Fig. 4B–B″), we speculate that transdetermination frequency declines in these discs because blastema cells are slower to regenerate and thus slower to acquire pluripotency. Consistent with this idea, all previous work has shown that blastema cells become competent to transdetermine only after they have regenerated the missing disc structures (Gehring, 1967; Wildermuth, 1968).

Figure 5. Transdetermination events are modified in rgn, alr and Mmp1 heterozygous mutant animals.

Figure 5

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The wg-induced transdetermination rate is strongly suppressed in rgn1 heterozygous mutant animals and enhanced in alr1 and Mmp1Q112 heterozygous mutant animals. (A) Vg is not normally expressed in leg imaginal discs. (B–E) For all genotypes, transdetermination was induced by ubiquitous wg expression at 72h AED. The frequency of leg-to-wing transdetermination was scored two days later by the ectopic expression of Vg in prothoracic leg discs. Note that heat shock and ectopic wg expression delay the time to pupariation. The modifiers did not affect the timing of this delay. The transdetermination rate was 51% in wg-expressing control flies, and 36%, 73% and 92% in rgn1 (126 discs; P=0.02, chi-square), alr1 (n=39 discs; P=0.001, chi-square) and Mmp1Q112 (n=37 discs; P=0.0001, chi-square) heterozygous mutant flies, respectively. Compared to the wg-expressing controls, the area of transdetermination differed significantly in rgn1 and Mmp1Q112 heterozygous mutant animals. The transdetermination area (% of vg-expressing wing area/leg area) was 3% in wg-expressing control larva (n=14), and 1%, 3% and 6% in rgn1 (n=17; P=0.03, t-test), alr (n=18) and Mmp1Q112 (n=12; P=0.01, t-test) heterozygous mutant larva, respectively. When wg is induced earlier, at 60h AED, 15% of Mmp1Q112/+ leg discs show Vg expression on both the dorsal and ventral side of the disc (arrows). Scale bar: 50 μm.

There is strong evidence that enhanced cell division during regeneration stimulates transdetermination events (Schubiger, 1973; Tobler, 1966). Thus, we expected a lower frequency of transdetermination in alr1/+ animals, where wg-induced regenerative proliferation was inhibited (Fig. 4C–C″). Surprisingly, we found the opposite to occur; transdetermination frequency increased significantly in alr1/+ discs (73%, n=39 discs; P=0.001, chi-square) (Fig. 5D) and in the leg cuticle of pharate adults (53%, n=34 legs; P=0.004, chi-square). These results can only be explained by Alr playing a dual role in regeneration. It is clear that Alr augments blastema cell proliferation during regeneration. However, the increase in transdetermination frequency in alr1/+ animals suggests that the gene product also maintains the appendage-specific identity of blastema cells, and thus limits regenerative cellular plasticity.

Over-expression of wg in Mmp1Q112/+ animals also significantly elevated the rate of transdetermination in both the discs (92%, n=37 discs; P=0.0001, chi-square) (Fig. 5E) and adult cuticle (42% n=65 legs; P<0.05, chi-square). The area of Vg expression in the discs increased as well and many of these discs displayed transdetermination at more than two sites (Fig. 5E′).

When wg was induced earlier, at 60h AED, each of the modifiers displayed a transdetermination frequency and area more divergent from the wg-expressing control animals. For example, using this protocol leg discs from control animals exhibited leg-towing transdetermination at a rate of 32% (n=104). In rgn1/+ and rgn2/+ animals, transdetermination frequency in the leg discs decreased significantly to 5% and 12%, respectively (n=89, P=0.0001, chi-square; n=54, P=0.008), and when compared to controls, the relative fraction of the leg disc which expressed ectopic Vg also decreased from 5% to 2% (P=0.03, t-test). Transdetermination frequency increased significantly in alr1/+ and alr2/+ animals (84%, n=39; P=0.001, chi-square; 65%, n=34; P=0.006, chi square, respectively), and in Mmp1Q112/+ and Mmp12/+ animals (82%, n=40; P=0.001, chi-square; 63%, n=71; P=0.00003, chi square, respectively). Strikingly, 15% (n=40) of the Mmp1Q112/+ discs showed two sites of Vg expression, one on the ventral and one on the dorsal side of the disc (Fig. 5E′). This phenotype is only observed in leg discs that over-express both wg and decapentapelagic (BMP4/6) (Maves and Schubiger, 1998). Our findings indicate that transdetermination at multiple sites in Mmp1Q112/+ and Mmp12/+ leg discs may be a consequence of a failure to shut off the cell cycle in non-blastema cells (Fig. 4D–D″) and suggests that Mmp1 functions to limit regenerative proliferation as well as regenerative cellular plasticity.

Testing transdetermination frequency of the three genes in the fragmentation experiments resulted in no difference between heterozygous mutants of the three genes (data not shown). This observation clearly demonstrates that developmental plasticity is dependent on the doses of these three genes only when wg overexpression is continuous. Thus, these genes might play a significant role in homeostatic reconstitution.

DISCUSSION

The nature of regeneration genes

We have identified three genes, rgn, alr and Mmp1 that are activated (either directly or indirectly) by Wg signaling during disc regeneration. rgn and Mmp1 are not expressed in the prothoracic leg discs during development, and therefore, these genes are not necessary for the development of these discs. However, during leg disc regeneration, both genes are activated in the blastema and mutations in these two genes affect blastema formation and transdetermination. alr, in contrast, is expressed at cellular blastoderm stage, when disc cells become determined, but is not expressed in the imaginal discs during larval development, when disc cells grow and divide. In addition, alr mutant larvae lack imaginal discs. Together, these observations suggest that alr is required only during the earliest stages of disc development, possibly for their determination, but not for the growth and proliferation of the disc primordia. Activation of alr in the leg disc blastema, and in transdetermining cells (Klebes et al., 2005), could indicate that this gene product is necessary to establish a new primordium, like in early development. Previously we reported that members of the Polycomb and trithorax Group genes are also involved in regeneration, specifically, several of these genes are up or down regulated during leg disc regeneration (Klebes et al., 2005). These genes are abundantly expressed throughout development and are necessary to maintain the on or off state of homeotic selector gene expression (Ringrose and Paro, 2004). Thus, we have found at least three different classes of genes acting during regeneration: (1) genes, like alr, that are re-activated. This observation supports the notion that regeneration reactivates genes that function in early disc development. (2) Genes that are only expressed in the leg disc during regeneration, like rgn and Mmp1, and thus constitute the group of true regeneration genes, and (3) genes that modify their expression level, such as members of the Polycomb and trithorax group genes.

Here we have identified genes that modify when, where and how much of a blastema is formed. Since fragmentation of wild-type discs produces the same blastema phenotype as wg overexpression, the phenotypes we observe are not an artifact of overexpression. Such phenotypes provide us with valuable information to dissect the process of blastema formation into different steps of gene function. For the understanding of such steps, multiple genes have to be identified that generate the same phenotype. Indeed, previously we found a failure of cell-cycle arrest in non-blastema cells not only in Mmp1Q112/+ animals, but also jing/+ animals (McClure and Schubiger, 2008). In addition, we reported a delayed blastema phenotype and reduced transdetermination frequency, similar to the one observed in rgn1/+ animals, when blastema formation was induced in combgap (cg) and Aly heterozygous animals (McClure and Schubiger, 2008). The observation that loss of rgn, cg and Aly gene function led to a delay in blastema formation indicates that these and further studies will provide us with genetic tools to unravel the mystery of how gene products measure time.

Why do mutations of these genes have a dominant phenotype that is revealed only in regeneration? Many studies in Drosophila indicate that genetic buffering breaks down under environmentally-induced stress (Rutherford, 2000). For example, (Petersen and Mitchell, 1987) have shown that heat stress uncovers a dominant mutant phenotype in heterozygous recessive multiple wing hairs. More relevant to our work is that heat shock can phenocopy the bithorax mutant phenotype (Santamaria, 1979). Furthermore, human epidemiological studies most often correlate increased susceptibility to common diseases, such as cancer, with genetic heterozygosity. We propose that regeneration generates a stress situation, resulting in a dominant phenotype. For example, while cell-cycle dynamics are extremely well-buffered during normal development, our studies clearly indicate that genetic heterozygosity plays a noteworthy role in modulating the timing, extent and position of cell division in the blastema.

Rgn and Wg signaling

Overexpression of wg in rgn1/+ animals causes a delay in blastema formation and decreases regeneration-induced transdetermination (Fig. 4 and 5). We speculate for several reasons that these phenotypes may be caused by elevated Wg signaling in rgn mutant animals. First, ubiquitous expression of wg in rgn1/+ animals leads to the maintenance of cell replication throughout the leg disc instead of blastema formation in the proximodorsal region (Fig. 4); a similar phenotype is observed when ectopic Wg signaling activity is markedly increased in the leg disc (Johnston and Schubiger, 1996). Second, previous studies have shown that elevated Wg signaling decreases leg-to-wing transdetermination, which is the phenotype observed in rgn1/+ animals with wg overexpression (Fig. 5) (Johnston and Schubiger, 1996). Thus, Rgn may normally attenuate wg signaling, and the loss of one gene copy of rgn could increase Wg activity, leading to a delay in blastema formation and suppression of transdetermination. Preliminary experiments in the wing imaginal disc indicate that overexpression of rgn causes an abnormal accumulation of Wg protein in wg-expressing cells, yet diminishes the extracellular gradient of Wg leading to the loss of wg target genes (unpublished results). As a result, the adult wing blades of these animals are significantly smaller in size. These experiments establish a tentative molecular link between rgn and the Wg signaling pathway.

It is interesting to note that the Drosophila Rgn protein contains a CTLD at its N-terminus (Fig. 3A′). Such domains have been reported to bind a variety of ligands, principally carbohydrates, but also glycoproteins, lipids, receptors and inorganic surfaces (Zelensky and Gready, 2005). Wg, as a glycoprotein, could potentially bind Rgn. In addition, a receptor for the mammalian Reg protein has been identified and it shares significant sequence homology to the Exostoses-like-3 (EXTL3) gene, which in Drosophila is brother of tout-velu (botv) (Han et al., 2004; Kobayashi et al., 2000; Takei et al., 2004). Drosophila Botv functions as a glycosyltransferase required for the biosynthesis of heparan sulfate (HS) glycosaminoglycan (GAG) chains and for Wg signaling and distribution (Han et al., 2004; Takei et al., 2004). Taken together, these observations support the notion that rgn may play a role in Wg signaling.

Mmp1 and Dpp signaling

Blastema formation in Mmp1Q112/+ animals differs from controls in that cell-cycle exit in the ventral aspect of the disc is incomplete (Fig. 4). We hypothesize that continued proliferation of cells at the ventral site may lead to ectopic Vg expression, and thus transdetermination, in the ventral half of Mmp1Q112/+ discs (Fig. 5E′). Together, these phenotypes suggest that normally Mmp1 limits regeneration as well as cellular plasticity. What is the possible cause for Vg expression at ventral sites of Mmp1 leg discs? Ectopic Vg expression in both dorsal and ventral regions of leg discs is a phenotype previously observed when ectopic wg and dpp are coexpressed in the leg disc (Maves and Schubiger, 1998). Therefore, it is possible that during leg disc regeneration dpp expression and/or signaling is altered, and possibly elevated, by loss of one gene copy of Mmp1. Indeed, flies with hypomorphic mutations of Mmp1 exhibit clefts along the midline of the notum (Page-McCaw et al., 2003), a phenotype also observed by mutations in the dpp gene or interfering with dpp signaling (Martin-Blanco et al., 2000; Usui and Simpson, 2000). These studies indicate that loss of Mmp1 interferes with dpp expression and/or signaling, while our results suggest that during disc regeneration loss of Mmp1 may elevate dpp expression and/or signaling. Thus, it appears that the interaction between Mmp1 and Dpp signaling may be complex and different during regenerative processes.

Drosophila has two Mmps, Mmp1 and Mmp2, and by microarray analysis, we found that both were upregulated during imaginal disc regeneration (Klebes et al., 2005). However, we find that only Mmp1 functions in the regeneration and transdetermination process, while Mmp2 has no detectable effect (data not shown). Mmp1 differs from Mmp2 by lacking a membrane anchor; these observations may lend insight into the function of the broad vertebrate Mmp gene family in regeneration.

alr in disc regeneration

Overexpression of wg in animals heterozygous for alr presents the most striking phenotype: an obvious suppression of wg-induced disc overgrowth and blastema size as well as an enhancement of transdetermination frequency (Fig. 4). Drosophila alr shares homology with FAD-linked sulfhydryl oxidases (Fig. 3B′) (Klebes et al., 2005). In yeast these enzymes localize to the mitochondrial intermembrane space and are involved in respiration, vegetative growth and biogenesis of mitochondrial intermembrane space proteins (Pawlowski and Jura, 2006). In vertebrates, Alr is ubiquitously expressed and found in different subcellular localizations, including the cytosol. During liver regeneration, Alr is secreted from hepatocytes and functions as a comitogen with other growth-promoting factors such as HGF, EGF and TGFα (Pawlowski and Jura, 2006). Thus, Alr proteins have a diverse array of functions. In our study, we demonstrate that heterozygosity for alr effectively interferes with blastema growth as well as the growth-promoting effects of ectopic wg expression. This is the first time, to our knowledge, that loss of alr function and its consequences on regeneration have been examined. In addition, our study points to a new mechanism for how Alr functions in regeneration, by acting (directly or indirectly) downstream of Wg signaling.

Conclusion

Our results indicate that rgn, alr and Mmp1 function in the timing, extent and position of the regeneration blastema, as well as in the regulation of regenerative plasticity. rgn, alr and Mmp1 homologs have all been implicated in the process of regeneration in vertebrates, and now, invertebrates, which suggests that they play a broadly conserved role in organ and tissue regeneration (Bai et al., 2005; Okamoto, 1999; Pawlowski and Jura, 2006). Such findings indicate that the function of Wg signaling in epimorphic regeneration can be investigated using Drosophila imaginal discs. Without doubt, the genome-wide access in Drosophila will identify additional regeneration genes, and thus broaden our molecular and genetic understanding of the regenerative process. Ultimately, this information will aid in the development of therapies to replace or re-grow tissues lost to disease or injury.

Supplementary Material

01

Acknowledgments

We thank Margrit Schubiger, Celeste Berg and Lynn Riddiford for comments on the manuscript; the Bloomington Stock Center for fly stocks; S. Carroll for antibodies against Vestigial; A. Page-McCaw for antibodies against Mmp1. This work was supported by a grant and developmental biology training grant from the National Institutes of Health (to G.S. and K.D.M., respectively).

Footnotes

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