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

Abstract

For a damaged tissue to regenerate, the injured site must repair the wound, proliferate, and restore the correct patterning and cell types. We found that Zelda, a pioneer transcription factor largely known for its role in embryonic zygotic genome activation, is dispensable for normal wing development but crucial for wing disc patterning during regeneration. Impairing Zelda function during disc regeneration resulted in adult wings with a plethora of cell fate errors, affecting the veins, margins, and posterior compartment identity. Using CUT&RUN, we identified and validated targets of Zelda including the cell fate genes cut, Delta and achaete, which failed to return to their normal expression patterns upon loss of Zelda. In addition, Zelda controls expression of factors previously established to preserve cell fate during regeneration like taranis and osa, which stabilizes engrailed expression during regeneration, thereby preserving posterior identity. Finally, Zelda ensures proper expression of the integrins encoded by multiple edematous wings and myospheroid during regeneration to prevent blisters in the resuting adult wing. Thus, Zelda is crucial for maintaining cell fate and structural architecture of the regenerating tissue.

Figure 1:. Gene expression during regeneration.

A) Wing pouch of 3rd-instar wing discs with immunostaining for Nubbin (gray) and rn>GFP (green) demarcating where rpr was expressed. B) Adult wing with margin outlined in red. C-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-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-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-U) Dl immunostaining in pro-vein 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-Z) Wg immunostaining in undamaged (W), R24 (X), R48 (Y), and R72 (Z) wing discs. Scale bar: 100μM.

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

A-D) Undamaged, R24, R48 and R72 w¹¹¹⁸ wing discs immunostained for Zld (A-D), Nubbin (A’-D’). Merged (A”-D”). E) Quantification of Zld immunostaining fluorescence intensity. n=20 discs each. ns> 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001, One-way ANOVA test. F) Quantification of zld mRNA by qPCR. One-way ANOVA test, ***p<0.001, ****p<0.0001. G) Adult wing from a CRY2::zld fly not exposed to blue light. H) Adult wing from a zld::CRY2 fly without disc damage exposed to blue light between R0 and R72. I) Adult wing after disc regeneration in a CRY2::zld fly not exposed to blue light having some defects like incomplete L2 vein (yellow arrowhead) and incomplete posterior margin (red arrowhead) . J) 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). Wing disc scale bars: 100μm. Adult wing scale bar: 750μm.

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

A) Heatmap of Zld occupancy in undamaged and regenerating wing discs +/− 3kb from Transcription Start Sites (TSSs). B) Location of Zld binding sites relative to genes. C) Motif discovery and enrichment analysis of sequences bound by Zld at R48. Enriched motifs were Fkh (MEME, E-value: 1.5e-086), Trl (STREME, E-value: 2.7e-002), Rib (MEME, E-value: 1.3e-039), and CA-rich repeats (MEME, E-value: 3.1e-063). D) Biological processes enriched in the genes near Zld binding sites according to GO analysis.

Figure 4:. Zld is important for margin, vein, and sensory organ fate after regeneration.

A) Undamaged adult wing showing ACV (black), PCV (Grey), L1 (red), L2 (yellow), L3 (magenta), L4 (white), L5 (blue) veins and sensory bristles (orange, outlined box with higher magnification inset). B) Adult wing with Zld inactivated during normal development, arrowheads same as in (A). C) Adult wing after disc regeneration, arrowheads same as in (A). D-E) Adult wings after Zld inactivation during regeneration, arrowheads same as in (A). * denotes 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, students t-test. G-K) Ct immunostaining in an undamaged wing disc (G), undamaged disc with Zld inactivated (H), R72 wing disc (I), R72 wing disc with Zld inactivated (J) and quantification of Cut expression along D/V boundary for undamaged discs: control (n=10), Zld inactivated (n=10) and R72 discs for control (n=14) and Zld inactivated (n=17). ns p >0.5, **p<.01, ***p<0.001, One-way ANOVA (K). L-O) Dl immunostaining in an undamaged wing disc (L), undamaged wing disc with Zld inactivated (M), R72 regenerating wing disc (N), and R72 regenerating wing disc with Zld inactivated (O). Arrowheads mark L2 (yellow), L3 (magenta), L4 (white), and L5 (blue). P-U) Ac immunostaining in an undamaged wing disc (P), undamaged wing disc with Zld inactivated (Q), R72 wing disc (R), and R72 wing disc with Zld inactivated (S-U). Scale bar for adult wings: 750μM. Scale bar for all discs: 100μM.

Figure 5:. Zld regulates timely patterning transitions during regeneration.

A-D)Wg immunostaining in an undamaged disc (A), undamaged disc with Zld inactivated (B), regenerating disc at R48 (C), and regenerating disc at R48 with Zld inactivated (D). E) Zld peak near Wg locus at R48, showcasing wg1_BRV118 region near it. F-K) Bs immunostaining in an undamaged disc (F), undamaged disc with Zld inactivated (G), regenerating disc at R60 (H), regenerating disc at R60 with Zelda inactivated (I), regenerating disc at R72 (J), and regenerating disc at R72 with Zld inactivated (K). Scale Bar: 100μM for all discs.

Figure 6:. Zld stabilizes posterior cell fate during late regeneration.

A-B) Undamaged adult wings, with L1 vein (blue arrowhead), sensory bristles (magenta arrowhead), and anterior crossvein (red arrowhead) marked. Control (A), and with Zld inactivated (B). C-D) Adult wings after disc regeneration. with arrowheads same as in (A), C) control, D) Zld inactivated during regeneration. E-G) Ptc expression in undamaged (E), R72 control (F), and R72 with Zld inactivated (G) wing discs. H-J) En expression in undamaged (H), R72 control (I), and R72 with Zld inactivated (J) wing discs. K-M) Osa expression in an undamaged disc (K), a control R72 disc (L), and an R72 disc that had Zld inactivated (M). N) Quantification of Osa levels in regenerating tissue (n=13 for all comparisons), **p<0.01 Student’s t-test. O-R) tara-lacZ enhancer trap expression in an undamaged wing disc (O), an R48 control wing disc (P), and an R48 wing disc with Zld inactivated (Q). R) Quantification of tara-lacZ in regenerating tissue (n=14 for all comparisons), ***p<0.001, Student’s t-test. S) Ptc expression in an R72 disc with Tara overexpressed and Zld inactivated. T) En expression in an R72 disc with Tara overexpressed and Zld inactivated. U) Adult wing from regenerated wing disc with Tara overexpressed and Zld inactivated. V) Quantification for En silencing in control R72 discs (n=6), Zld inactivated R72 discs (n=11) and Zld inactivated R7 discs with Tara overexpression (n=11). *p<0.05 One-way ANOVA. X) 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=8). *p<0.05 One-way ANOVA. Scale bar for adult wings: 750μM except for Figure 6W (500 μM);. Scale bar for all discs: 100μM.

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

A-B) Normal adult wing, control (A) and Zld inactivated (B). C-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 inactivated, 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 inactivated. Black arrowhead indicates blistered 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-J) Regions bound by Zld near mys (I) and mew (J) from CUT&RUN analysis. K-L) Mys expression in undamaged wing discs, with elevated Mys at the D/V boundary (yellow arrowhead). Control (K) and Zld inactivated (L). M-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, **p<0.01, One-way ANOVA. P-Q) 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 inactivated (S). Scale bar for adult wings: 750μM. Scale bar for all discs: 100μM.