The pioneer transcription factor Zelda controls the exit from regeneration and restoration of patterning in Drosophila

Abstract

Many animals can regenerate tissues after injury. While the initiation of regeneration has been studied extensively, how the damage response ends and normal gene expression returns is unclear. We found that in Drosophila wing imaginal discs, the pioneer transcription factor Zelda controls the exit from regeneration and return to normal gene expression. Optogenetic inactivation of Zelda during regeneration disrupted patterning, induced cell fate errors, and caused morphological defects yet had no effect on normal wing development. Using Cleavage Under Targets & Release Using Nuclease, we identified targets of Zelda important for the end of regeneration, including genes that control wing margin and vein specification, compartment identity, and cell adhesion. We also found that GAGA factor and Fork head similarly coordinate patterning after regeneration and that chromatin regions bound by Zelda increase in accessibility during regeneration. Thus, Zelda orchestrates the transition from regeneration to normal gene expression, highlighting a fundamental difference between developmental and regeneration patterning in the wing disc.

Fig. 1.. Gene expression during regeneration.

(A) Wing pouch of third instar wing discs with immunostaining for Nubbin (Nub) (gray) and rn > green fluorescent protein (GFP) (green) demarcating where rpr was expressed. (B) Adult wing with margin outlined in red. (C to F) Ct expression at the D/V boundary (red arrowheads) in undamaged (C), R24 (D), R48 (E), and R72 (F) wing discs. (G) Adult wing with some intervein regions marked by yellow arrowheads. (H to K) Bs-GFP expression in the intervein regions (yellow arrowheads) in undamaged (H), R24 (I), R48 (J), and R72 (K) wing discs. (L) Adult wing with the margin outlined in red and intervein regions marked with yellow arrowheads. (M to P) E(spl)Mb-CD2 (CD2) expression in the margin (red arrowhead) and intervein regions (yellow arrowheads) in undamaged (M), R24 (N), R48 (O), and R72 (P) wing discs. (Q) Adult wing with veins marked by arrowheads: L1 (red), L2 (yellow), L3 (magenta), L4 (white), and L5 (blue). (R to U) Dl immunostaining in provein cells with arrowheads the same colors as in (Q) in undamaged (R), R24 (S), R48 (T), and R72 (U) wing discs. (V) Adult wing with green lines marking the D/V boundary and approximate locations of the Wg IR and OR. (W to Z) Wg immunostaining in undamaged (W), R24 (X), R48 (Y), and R72 (Z) wing discs. Scale bar, 100 μm.

Fig. 2.. Zld is important for cell fate and patterning during regeneration.

(A to D) Undamaged (A), R24 (B), R48 (C), and R72 (D) w¹¹¹⁸ wing discs immunostained for Zld and Nubbin. (E) Quantification of Zld immunostaining fluorescence intensity in the Nubbin-expressing wing pouch. n = 18 discs each except for R24 (n = 16). Undamaged versus R24, P = 0.0565 (n.s.); undamaged versus R48, ****P < 0.0001; undamaged versus R72, ****P < 0.0001; R24 versus R48, ***P = 0.0002; R24 versus R72, **P = 0.0040; R48 versus R72, P = 0.7223 (n.s.); F = 22.53, df = 67, one-way analysis of variance (ANOVA) test. a.u., arbitrary units. (F) Quantification of zld mRNA by qPCR. Undamaged versus R24, P = 0.9977 (n.s.); undamaged versus R48, ****P < 0.0001; undamaged versus R72, ***P = 0.0002; one-way ANOVA test. (G and H) Adult wings from w¹¹¹⁸ animal raised without blue light. (G) Undamaged adult wing. (H) Adult wing from regenerated wing disc. (I and J) Adult wings from w¹¹¹⁸ animal raised with blue light. (I) Undamaged adult wing. (J) Adult wing from regenerated wing disc. (K) Adult wing from a CRY2::zld fly not exposed to blue light. (L) Adult wing after disc regeneration in a CRY2::zld fly not exposed to blue light having some defects such as incomplete L2 vein (yellow arrowhead) and incomplete posterior margin (red arrowhead). (M) Adult wing from a CRY2::zld fly without disc damage exposed to blue light between R0 and R72. (N) Adult wing after disc regeneration in a CRY2::zld fly exposed to blue light between R0 and R72 having missing L2 vein (yellow arrowhead), altered L3 vein thickness (magenta arrowhead), incomplete L5 vein (blue arrowhead), incomplete posterior margin (red arrowhead), blister formation (black arrowhead), and distal edge vein material (teal arrowhead). Scale bars, 100 μm (wing disc) and 500 μm (adult wing).

Fig. 3.. Zld binds near genes important for wing development and morphogenesis.

(A) Heatmap of Zld occupancy in undamaged and regenerating wing discs ± 3 kb from TSSs. Each line is an individual gene. Color indicates normalized read counts from high (blue) to low (red). (B) Distribution of genomic locations of annotated Zld binding sites relative to genes using ChIPseeker in undamaged and R48 discs. UTR, untranslated region. (C) Volcano plot showing differential binding in undamaged versus R48 discs. FDR, false discovery rate. (D) Biological processes enriched in the genes near Zld binding sites according to Gene Ontology (GO) analysis.

Fig. 4.. Zld regulates margin, vein, and sensory organ fate after regeneration.

(A) Undamaged adult wing showing L1 (red arrowhead), L2 (yellow arrowhead), L3 (magenta arrowhead), L4 (white arrowhead), and L5 (blue arrowhead) veins and sensory bristles (orange arrowhead; with higher magnification inset). (B) Adult wing after Zld inactivation during normal development, arrowheads as in (A). (C) Adult wing after disc regeneration, arrowheads as in (A). (D and E) Adult wings after Zld inactivation during regeneration, arrowheads as in (A). Asterisk (*) indicates missing posterior margin. (F) Quantification of missing posterior margin for control wings after regeneration (n = 207), Zld-inactivated wings after regeneration (n = 190), control pdm2-LexA;attP2/+ RNAi wings after regeneration (n = 131), and pdm2-LexA;zld-RNAi/+ wings after regeneration (n = 91). ****P < 0.0001, Student’s t test. Controls of the same genetic background were used for comparison due to variation in background error rates in patterning. (G to K) Ct immunostaining in an undamaged wing disc (G), undamaged disc with Zld inactivation (H), R72 wing disc (I), R72 wing disc with Zld inactivation (J), and quantification of cut expression along D/V boundary (K) for undamaged discs: control (n = 10), Zld inactivated (n = 10), and R72 discs for control (n = 14) and Zld inactivated (n = 17). Undamaged versus Zld-inactivated undamaged, P = 0.972 (n.s.); undamaged versus Zld-inactivated R72, ***P = 0.0003; Zld-inactivated undamaged versus Zld-inactivated R72, **P = 0.0017; R72 versus Zld-inactivated R72, **P = 0.0083; one-way ANOVA (K). (L to O) Dl immunostaining in an undamaged wing disc (L), undamaged wing disc with Zld inactivation (M), R72 regenerating wing disc (N), and R72 regenerating wing disc with Zld inactivation (O). Arrowheads mark L2 (yellow), L3 (magenta), L4 (white), and L5 (blue). (P to U) Ac immunostaining in an undamaged wing disc (P), undamaged wing disc with Zld inactivation (Q), R72 wing disc (R), and R72 wing disc with Zld inactivated (S to U). Scale bars, 500 μm (adult wings) and 100 μm (all discs).

Fig. 5.. Zld regulates timely patterning transitions during regeneration.

(A to F) Wg immunostaining in an undamaged disc (A), undamaged disc with Zld inactivated (B), regenerating disc at R48 (C), R48 regenerating disc with Zld inactivation (D), regenerating disc at R72 (E), and R72 regenerating disc with Zld inactivation (F). (G) Region bound by Zld near Wg from CUT&RUN data in undamaged and R48 discs. Note that the binding site is distinct from the damage-responsive BRV118 enhancer and the developmental wg¹ enhancer. (H to M) Bs immunostaining in an undamaged disc (H), undamaged disc with Zld inactivated (I), regenerating disc at R60 (J), R60 regenerating disc with Zld inactivation (K), regenerating disc at R72 (L), and regenerating disc at R72 with Zld inactivated (M). Scale bars, 100 μm (all discs).

Fig. 6.. Zld stabilizes posterior cell fate during late regeneration.

(A and B) Control (A) and Zld-inactivated (B) undamaged adult wings. Blue arrowhead, L1 vein; magenta arrowhead, sensory bristles; red arrowhead, anterior crossvein. (C and D) Adult wings after disc regeneration, with arrowheads as in (A). (C) Control and (D) Zld inactivated during regeneration. (E to G) Ptc expression in undamaged (E), R72 control (F), and R72 with Zld-inactivated (G) wing discs. (H to J) En expression in undamaged (H), R72 control (I), and R72 with Zld-inactivated (J) wing discs. (K to M) Osa expression in undamaged (K), R72 control (L), and R72 with Zld-inactivated (M) wing discs. (N) Quantification of Osa levels in regenerating tissue (n = 13 for all). R72 versus Zld-inactivated R72, **P = 0.0044; Student’s t test. (O to R) tara-lacZ expression in undamaged (O), R48 control (P), and R48 with Zld-inactivated (Q) wing discs. (R) Quantification of tara-lacZ in regenerating tissue (n = 14 for all). R48 versus Zld-inactivated R48, ***P = 0.0007; Student’s t test. (S) Ptc expression in an R72 disc with Tara overexpression and Zld inactivation. (T) En expression in an R72 disc with Tara overexpressed and Zld inactivated. (U) Adult wing from regenerated wing disc with Tara overexpression and Zld inactivation. (V) Quantification for En silencing in control R72 discs (n = 6), Zld-inactivated R72 discs (n = 11), and Zld-inactivated R72 discs with Tara overexpression (n = 11). R72 versus Zld-inactivated R72, *P = 0.0138. Zld-inactivated R72 versus Zld inactivated; UAS-tara R72, *P = 0.0111; one-way ANOVA. (W) Quantification of aberrant Ptc expression in control R72 discs (n = 18), Zld-inactivated R72 discs (n = 16), and Zld-inactivated R72 discs with Tara overexpression (n = 9). R72 versus Zld-inactivated R72, *P = 0.0136. Zld-inactivated R72 versus Zld inactivated; UAS-tara R72, *P = 0.0413; one-way ANOVA. Scale bars, 500 μm (adult wings) and 100 μm (all discs).

Fig. 7.. Zld prevents blisters in the adult wing after disc regeneration.

(A and B) Normal adult wing, control (A) and after Zld inactivation (B). (C and D) Adult wing after regeneration of a control disc (C) and from pdm2-LexA/+;attP2/+ control disc (D). (E) Adult fly after disc regeneration with Zld inactivation. Yellow arrowhead indicates blister. (F) Adult wing after disc regeneration with RNAi-mediated zld knockdown. Yellow arrowhead indicates blister. (G) Three-dimensional image of a fly with regenerated adult wing after larval damage with Zld inactivation. Black arrowhead indicates Bs wing. (H) Quantification of blisters in adult wings in control (n = 207), Zld-inactivated (n = 190), pdm2-LexA;attP2/+ RNAi control (n = 131), and pdm2-LexA;zld-RNAi/+ (n = 91) wings after disc regeneration, **P < 0.01; Student’s t test. (I and J) Regions bound by Zld near mys (I) and mew (J) from CUT&RUN analysis. (K and L) Mys expression in undamaged wing discs, with elevated Mys at the D/V boundary (yellow arrowhead). Control (K) and Zld inactivated (L). (M and N) Mys expression in R72 discs with yellow arrowhead pointing to the D/V boundary. Control (M), Zld inactivated (N). (O) Quantification of Mys expression, R72 versus Zld-inactivated R72, **P = 0.0045; exact P value, Student’s t test. (P to S) Mew expression in wing discs, with yellow arrowhead indicating expression in the ventral half. Undamaged control (P), undamaged with Zld inactivated (Q), R72 control (R), R72 with Zld inactivation (S). Scale bars, 500 μm (adult wings) and 100 μm (all discs).

Fig. 8.. GAF and Fkh also regulate patterning after regeneration.

(A) Motif discovery and enrichment analysis of sequences bound by Zld at R48. Enriched motifs were GAF (MEME, E = 5.3 × 10⁻²⁴), Fkh (MEME, E = 1.4 × 10⁻⁴⁶), and CA-rich repeats (MEME, E = 6.2 × 10⁻⁴). (B) Undamaged adult wings. (C to G) Adult wings after regeneration for RNAi attP2/+ control (C), w¹¹¹⁸/+ control (D), Trl^13C/+ (E), Fkh-RNAi #27072 (F), and Fkh-RNAi #33760 (G). Arrowheads indicate incomplete L2 vein (yellow), incomplete L5 vein (blue), anterior vein on posterior margin (gray), anterior bristles on posterior margin (pink), extra vein material on distal edge of wing (teal), and blister formation (black). (H and I) Confocal images taken with a 63× oil objective of the proximity labeling assay (PLA) using antibodies against Zld and GAF-GFP in undamaged (H) and R48 (I) wing discs. PLA is white and the GAF-GFP in the nuclei is blue. Scale bars, 50 μm. (J) Chart showing regions of relative chromatin accessibility increases between early (R0) and mid (R15) regeneration aligned with Zld mid-regeneration CUT&RUN data. Dots indicate the presence of GAF/Fkh/CA repeat/Zld motifs at the specified genomic regions. (K) Unchanged chromatin accessibility between early (R0) and mid (R15) regeneration aligned with Zld mid-regeneration (R48) CUT&RUN data. (L) Accessible regions from ATAC-seq data with nucleosome-length sequenced included (nucleosome) and with nucleosome-length sequences removed (nucleosome-free) near two alternative TSSs for ct, aligned with Zld CUT&RUN data. All data ranges for ATAC-seq data are from −48.0 to 120. Data range for Zld R48 track is 0 to 18. Scale bars, 500 μm (adult wings) and 50 μm (wing discs).