13 research outputs found
Regulation of growth, patterning and cell fate-specification during imaginal disc regeneration in drosophila
The process of regeneration is immensely complicated and requires the exquisitely precise orchestration of growth, patterning and cell-fate specification. Drosophila melanogaster larval tissues have recently emerged as a useful model organism to study this process. These larval tissues are simple but experience complex gene expression to finally form the adult tissue. As such, Drosophila is an ideal model organism to study the complicated process of regeneration. In this study we have used the experimental tractability of Drosophila to investigate how growth, patterning, cell-fate specification and fate plasticity are regulated in regenerating imaginal discs.
Regenerative growth must be carefully controlled and constrained to prevent overgrowth and to allow correct organization of the regenerating tissue. However, the factors that restrict regenerative growth have not been identified. In this study, we have identified one mechanism that constrains regenerative growth, impairment of which 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. The loss of cell-fate specification is due to the aberrantly high expression of the pro-growth factor Myc and the self-renewal factor Chinmo. Thus, Brat ensures that the regenerating tissue forms the proper final structure by constraining expression of pro-regeneration genes.
Dissociation of imaginal disc cells has previously been carried out to enable flow cytometry and cell sorting to analyze cell cycle progression, cell size, gene expression, and other aspects of imaginal tissues. However, the lengthy dissociation protocols employed may alter gene expression, cell behavior and overall viability. In this study, we developed a new rapid and gentle method of dissociating the cells of wing imaginal discs that significantly enhanced cell viability and reduced the likelihood of gene expression changes. This method was successfully used to create a transcriptional profile of the regenerating tissue leading to the identification of many novel regulators of regeneration. We have also extended our investigation of regeneration to the antennal imaginal disc.
Drosophila imaginal discs have also been used to study cell-fate specification and plasticity, including homeotic changes and regeneration-induced transdetermination. In this study, we identified a change from antennal fate to eye fate induced by a Distal-less-GAL4 (DllGAL4) P-element insertion that is a mutant allele of Dll and expresses GAL4 in the antennal imaginal disc. While this fate change was not induced by tissue damage, it appeared to be a hybrid of transdetermination and homeosis. This plasticity appears to be unique to the DllGAL4 line, possibly due to cellular stress induced by the high GAL4 expression combined with the severity of the Dll mutation. Thus, we propose that even in the absence of tissue damage, other forms of cellular stress caused by high GAL4 expression can induce determined cell fates to change, and selector gene mutations can sensitize the tissue to these transformations.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste
Regulation of growth, patterning and cell fate-specification during imaginal disc regeneration in drosophila
The process of regeneration is immensely complicated and requires the exquisitely precise orchestration of growth, patterning and cell-fate specification. Drosophila melanogaster larval tissues have recently emerged as a useful model organism to study this process. These larval tissues are simple but experience complex gene expression to finally form the adult tissue. As such, Drosophila is an ideal model organism to study the complicated process of regeneration. In this study we have used the experimental tractability of Drosophila to investigate how growth, patterning, cell-fate specification and fate plasticity are regulated in regenerating imaginal discs.
Regenerative growth must be carefully controlled and constrained to prevent overgrowth and to allow correct organization of the regenerating tissue. However, the factors that restrict regenerative growth have not been identified. In this study, we have identified one mechanism that constrains regenerative growth, impairment of which 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. The loss of cell-fate specification is due to the aberrantly high expression of the pro-growth factor Myc and the self-renewal factor Chinmo. Thus, Brat ensures that the regenerating tissue forms the proper final structure by constraining expression of pro-regeneration genes.
Dissociation of imaginal disc cells has previously been carried out to enable flow cytometry and cell sorting to analyze cell cycle progression, cell size, gene expression, and other aspects of imaginal tissues. However, the lengthy dissociation protocols employed may alter gene expression, cell behavior and overall viability. In this study, we developed a new rapid and gentle method of dissociating the cells of wing imaginal discs that significantly enhanced cell viability and reduced the likelihood of gene expression changes. This method was successfully used to create a transcriptional profile of the regenerating tissue leading to the identification of many novel regulators of regeneration. We have also extended our investigation of regeneration to the antennal imaginal disc.
Drosophila imaginal discs have also been used to study cell-fate specification and plasticity, including homeotic changes and regeneration-induced transdetermination. In this study, we identified a change from antennal fate to eye fate induced by a Distal-less-GAL4 (DllGAL4) P-element insertion that is a mutant allele of Dll and expresses GAL4 in the antennal imaginal disc. While this fate change was not induced by tissue damage, it appeared to be a hybrid of transdetermination and homeosis. This plasticity appears to be unique to the DllGAL4 line, possibly due to cellular stress induced by the high GAL4 expression combined with the severity of the Dll mutation. Thus, we propose that even in the absence of tissue damage, other forms of cellular stress caused by high GAL4 expression can induce determined cell fates to change, and selector gene mutations can sensitize the tissue to these transformations.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste
Regenerative growth is constrained by brain tumor to ensure proper patterning in Drosophila.
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
The Drosophila Duox maturation factor is a key component of a positive feedback loop that sustains regeneration signaling.
Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth
Labeling and isolating regeneration blastema cells.
<p><b>(</b>A-B) Wing imaginal discs that are undamaged (A) or ablated and at 0 hrs recovery (R0) (B). Green = <i>rnGal4</i>, <i>UAS-EGFP</i>. Red = anti-Nub. Blue = DAPI. (C) Wing imaginal disc showing overlap of anti-Nub immunostaining (red) and expression of the <i>nub-GFP</i> MiMIC enhancer trap (green). (D-F) <i>nub-GFP</i> marks the wing pouch at 24 hrs (D) 48 hrs (E) and 72 hrs (F) after ablation. (G) <i>nub-GFP</i> (green) coincides with the regeneration blastema as defined by a zone of high EdU incorporation (red). (H) Schematic of the mRNA-seq procedure, from tissue ablation through cell dissociation and sort to sequencing and data analysis. Scale bars are100 μm.</p
Genetic assays demonstrating that differentially expressed genes have functional roles in regeneration.
<p><b>(</b>A) Representative examples of wings from damaged discs that are approximately <25%, 25%, 50%, 75%, and 100% of a normal wing. Scale bar is 1mm. (B-G) Sizes of adult wings after regeneration in control (<i>w</i><sup><i>1118</i></sup>) and heterozygous mutant animals. Three independent experiments each, error bars are SEM. (B) Sizes of adult wings after regeneration in <i>w</i><sup><i>1118</i></sup> and <i>Ets21C</i><sup><i>f03639</i></sup><i>/+</i> animals. <i>w</i><sup><i>1118</i></sup> n = 318 wings, <i>Ets21C</i><sup><i>f03639</i></sup><i>/+</i> n = 255 wings, p<0.0001 using a chi-squared test. (C) Sizes of adult wings after regeneration in <i>w</i><sup><i>1118</i></sup> and <i>CG9336</i><sup><i>MI03849</i></sup><i>/+</i> animals. <i>w</i><sup><i>1118</i></sup> n = 374 wings, <i>CG9336</i><sup><i>MI03849</i></sup><i>/+</i> n = 215 wings, p<0.0001 by a chi-squared test. (D) Sizes of adult wings after regeneration in <i>w</i><sup><i>1118</i></sup> and <i>Alp4</i><sup><i>07028</i></sup><i>/+</i> animals. <i>w</i><sup><i>1118</i></sup> n = 239 wings, <i>Alp4</i><sup><i>07028</i></sup><i>/+</i> n = 217 wings, p<0.0001 by a chi-squared test. (E) Sizes of adult wings after regeneration in <i>w</i><sup><i>1118</i></sup> and <i>Thor</i><sup><i>06270</i></sup><i>/+</i> animals. <i>w</i><sup><i>1118</i></sup> n = 224 wings, <i>Thor</i><sup><i>06270</i></sup><i>/+</i> n = 146 wings, p = 0.0021 by a chi-squared test. (F) Sizes of adult wings after regeneration in <i>w</i><sup><i>1118</i></sup> and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> animals. Three independent experiments, <i>w</i><sup><i>1118</i></sup> n = 356 wings, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 183 wings, p = 0.00001 by a chi-squared test. (G) Sizes of adult wings after regeneration in <i>w</i><sup><i>1118</i></sup>, <i>Col4a1</i><sup><i>k00405</i></sup><i>/+</i>, and <i>vkg</i><sup><i>k00236</i></sup><i>/+</i> animals. <i>w</i><sup><i>1118</i></sup> n = 320 wings, <i>Col4a1</i><sup><i>k00405</i></sup><i>/+</i> n = 71 wings, and <i>vkg</i><sup><i>k00236</i></sup><i>/+</i> n = 134 wings, p<0.0001 by a chi-squared test.</p
NIP is required to sustain JNK signaling during late regeneration.
<p><b>(</b>A-H) Confocal images of fluorescence from the <i>TRE-red</i> reporter for JNK signaling in <i>w</i><sup><i>1118</i></sup> (A-D) and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> (E-H) regenerating discs at R0 (A,B), R24 (B,F), R48 (C,G) and R72 (D,H). (I) Quantification of fluorescence intensity of the <i>TRE-red</i> reporter in max projections of the confocal images at R0, because at this time point the epithelium cannot be distinguished from the debris. <i>w</i><sup><i>1118</i></sup> n = 10 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 14 discs. (J) Quantification of fluorescence intensity of the <i>TRE-red</i> reporter in single slices of the confocal images through the regenerating epithelium at R24, R48, and R72. R24 <i>w</i><sup><i>1118</i></sup> n = 11 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 11 discs. R48 <i>w</i><sup><i>1118</i></sup> = 14 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 15 discs. R72 <i>w</i><sup><i>1118</i></sup> n = 11 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 11 discs. (K) Regeneration assays using adult wing size to assess extent of regenerative growth in the imaginal discs in <i>w</i><sup><i>1118</i></sup>, <i>mol</i><sup><i>e02670</i></sup><i>/+</i>, <i>puc</i><sup><i>E69</i></sup><i>/+</i>, and <i>mol</i><sup><i>e02670</i></sup><i>/+;puc</i><sup><i>E69</i></sup><i>/+</i> animals. Two independent experiments, thus error bars are SD. <i>w</i><sup><i>1118</i></sup> n = 26 wings, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 83 wings, <i>puc</i><sup><i>E69</i></sup><i>/+</i> n = 99 wings, and <i>mol</i><sup><i>e02670</i></sup><i>/+;puc</i><sup><i>E69</i></sup><i>/+</i> n = 95 wings. p<0.0001 for all comparisons using a chi-squared test. Dashed blue line outlines the wing primordium. Scale bars are 100 μm. Error bars are SEM unless otherwise noted. *p<0.05, **p<0.001, ***p<0.0001.</p
<i>moladietz</i> is required for wing disc regeneration.
<p><b>(</b>A-D) DHE fluorescence (red) indicates the presence of ROS. <i>nub-GFP</i> (green) marks the regenerating wing pouch. (A-B) Confocal slices of a <i>w</i><sup><i>1118</i></sup> regenerating disc through the debris field (A,A’) and the disc epithelium (B,B’). Asterisks mark cellular debris in the debris field and in a few folds in the epithelium. Arrow points to the position of the regenerating wing pouch. (C-D) Confocal slices of a <i>mol</i><sup><i>e02670</i></sup><i>/+</i> regenerating disc through the debris field (C,C’) and the regenerating epithelium (D,D’). Asterisk and arrow same as above. (E-F) Quantification of DHE fluorescence intensity in the debris fields of <i>w</i><sup><i>1118</i></sup> and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> regenerating discs (E) and in the regenerating epithelia of <i>w</i><sup><i>1118</i></sup> and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> regenerating discs and control undamaged discs (F). For R24, three independent experiments, for a total <i>w</i><sup><i>1118</i></sup> regenerating n = 12 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> regenerating n = 18 discs, <i>w</i><sup><i>1118</i></sup> undamaged n = 11 discs. For R48, three independent experiments for a total <i>w</i><sup><i>1118</i></sup> regenerating n = 30 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> regenerating n = 25 discs, <i>w</i><sup><i>1118</i></sup> undamaged n = 10 discs. (G,H) Quantification of GFP fluorescence from a <i>gstD-GFP</i> reporter for ROS-regulated transcription in regenerating <i>w</i><sup><i>1118</i></sup> and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> discs. For R24, <i>w</i><sup><i>1118</i></sup> n = 12 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 20 discs. For R48, <i>w</i><sup><i>1118</i></sup> n = 12 discs, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 10 discs. (I) Adult wing area in <i>w</i><sup><i>1118</i></sup> and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> male and female wings from undamaged discs and after disc regeneration. Three independent experiments. Undamaged: <i>w</i><sup><i>1118</i></sup> females n = 125 wings, <i>w</i><sup><i>1118</i></sup> males n = 132 wings, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> females n = 82 wings, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> males n = 73 wings. Regenerated: <i>w</i><sup><i>1118</i></sup> females n = 226 wings, <i>w</i><sup><i>1118</i></sup> males n = 134 wings, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> females n = 128 wings, <i>mol</i><sup><i>e02670</i></sup><i>/+</i> males n = 133 wings. (J-O) Anti-Nub marks the regenerating wing primordium at R0, R24 and R48 in <i>w</i><sup><i>1118</i></sup> and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> discs. (P) Quantification of the size of the regenerating wing primordium at R0, R24 and R48. R0 <i>w</i><sup><i>1118</i></sup> n = 26 and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 29, R24 <i>w</i><sup><i>1118</i></sup> n = 42 and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 41, R48 <i>w</i><sup><i>1118</i></sup> n = 29 and <i>mol</i><sup><i>e02670</i></sup><i>/+</i> n = 42. Scale bars are 100 μm. Error bars are SEM. **p<0.05, *p<0.005, ***p<0.0002, ****p<0.0001.</p
Validation of genes identified as upregulated in the regeneration blastema.
<p>Undamaged (A-I) and regenerating (R24) (A’-I’) wing discs. (A-A’) <i>Alp4-lacZ</i> enhancer trap. (B-B’) Atf3-GFP protein trap. (C-C’) <i>chinmo-lacZ</i> enhancer trap. (D-D’) Ets21C-GFP protein trap. (E-E’) <i>mol-lacZ</i> enhancer trap. (F-F’) <i>fru-lacZ</i> enhancer trap. (G-G’) Lamin-GFP protein trap. (H-H’) <i>pigs-GFP</i> enhancer trap. (I-I’) 10xSTAT92E-GFP reporter for STAT activity. Blue dashed line outlines the wing primordium. Scale bars are 100μm.</p