Knockdown of Dpn, Elav, Pros, and SoxN result in dedifferentiation of medulla neurons.

(A) Schematic representation of Drosophila larval central nervous system (CNS). The medulla (pink) is located with the Optic Lobes (OLs) of the CNS. The superficial surface of the medulla (0μm) contains neuroblasts (NBs, pink), whereas the deep layers (8–12μm) contain mature neurons (green). A single NB in the superficial layer and its neuronal progeny in the deep layers are labelled in light green. (B) During neurogenesis NBs undergo asymmetric divisions to generate a NB and an intermediate progenitor (ganglion mother cell, GMC). The GMC produces 2 neurons. (C) During dedifferentiation, mature neurons dedifferentiate to give rise to ectopic NBs, which continue to divide and create tumours. (D) Experimental design to induce clones with manipulate gene expression. Clones induced via heat shock (red arrows) at 48 hours after egg lay (AEL) are dissected 72 hours after heat shock (AHS). (E-I’) In the deep layers of the medulla, control clones contained no stem cells (E, E’), clonal overexpression of UAS-dpn (deadpan), elav-Ri (embryonic lethal), pros-Ri (prospero), SoxN-Ri (SoxNeuro) resulted in stem cell induction (Miranda+, magenta) in the deeper mature neuron layers, indicative of dedifferentiation (J). (J) Quantification of % Mira+ cell volume vs. clone volume: Control (n = 9, 1.874 ±0.4897), UAS-dpn (n = 17, 33.62 ±3.243), elav-Ri (n = 22, 13.16 ±1.164), SoxN-Ri (n = 19, 21.74 ±2.926), pros-Ri (n = 11, 49.34 ±4.36). Data are represented as mean ± SEM. ****p < 0.0001. Scale bars: 50 μm. Deadpan data is adapted from [24].

Her6/Hes1 is reduced in regenerating progenitors.

(A, B) Schematic representation of retinal cells and observed regenerative processes and timeline. Rod (purple) and cone (blue) photoreceptors (PR) are located in the outer nuclear layer (ONL). Müller glia (MG) are located in the inner nuclear layer (INL) with processes spanning across the retina from the ONL through to the GCL (ganglion cell layer). Following PR ablation, MG are activated and dedifferentiate into progenitors, which proliferate and migrate prior to differentiation into photoreceptors by 96 hours post ablation (hpa). (C-H”) In adult retina, during the 96-hour regenerative time span following the ablation of PRs (red), proliferation is marked by proliferative cell nuclear antigen (PCNA, pink) starts at 24 hpa. Expression of Hairy-enhancer of split (Hes1, green) is seen both in the control and following the ablation in the inner half of the INL and in the GCL. Focusing on the proliferative cells, the expression of Hes1 is downregulated as PCNA expression increases from 24 hpa onwards. DAPI (cyan) marks the retinal layers. Scale bars: 50 μm.(I) UMAP plot of FACS MG 3 days after injury is subdivided into distinct MG derived cell populations based on stereotypical markers. (J) The feature plots show that her6 is expressed mainly in the quiescent and some proliferating MG (pcna, ascl1a expressing) subsets and downregulated in differentiating (pcna, insm1a expressing) cell populations. (K) Summary of Hes1/her6 expression during regeneration. Hes1/her6 is present in the initial progenitors and downregulated upon their proliferation.

Loss of Her6 increases PR production at 72 hours post ablation.

(A-D”) In adult retina, at 48 and 72 hours post ablation (hpa), the number of proliferating cells marked by PCNA (pink) and Müller glia (MG) cells marked by glial fibrillary acidic protein transgene expressing (GFAP, green) are not significantly altered between standard control (SC, 48hpa n = 9, 6.621 ±0.9034, 72hpa n = 8, m = 12.27 ±2.579) and her6 morpholino (MO, 48 hpa n = 14, m = 6.59 ±0.5219, 72 hpa n = 14, m = 9.299 ±1.1) electroporated samples quantified in (E). (F-G’) In her6 MO samples, there was a significantly increase in zpr1 (pink, n = 12, 13.97 ±2.202) labelled cone photoreceptors compared to SC (n = 7, 7.809 ±1.938), quantified in H. (I-L) There was also an increase in phospho-histone 3 (PH3, pink) marking mitotic cells in the her6 MO (48hpa, n = 11, 1.519 ±0.3166, 72hpa n = 12, 2.806 ±0.4442) compared to SC (48hpa n = 11, 0.8812 ±0.2671, 72hpa (SC n = 10, 1.102 ±0.4956) quantified in M. In all micrographs DAPI is used to label nuclei cyan. Data are represented as mean ± SEM. *p < 0.05. Scale bars: 50 μm. (N) Summary of regenerative phases and timeline in her6 MO electroporated fish showing an increase in mature photoreceptor regeneration.

Prox1a is expressed in the photoreceptors within the outer nuclear layer.

(A-F”) Within adult retinas in uninjured control, post ablation (hpa), Prospero homeobox1a (Prox1a, green) is expressed in the inner nuclear layer (solid white outline), but never in proliferating (proliferative cell nuclear antigen positive, PCNA, pink) cells (dotted outlines). At 72hpa and 96hpa Prox1a in addition expressed in the ONL. (G-I”) cone photoreceptor marker, zpr1 (green) is expressed in the ONL in the control and at all the time points post ablation. (J-J”’) is the magnified images of (I-I”’). At 72hpa and 96hpa, Prox1a is expressed as distinct puncta within zpr1 expressing photoreceptors (arrows and high-power inset). Scale bars: 50 μm. (K, L) UMAP plot of FACS MG 3 days after injury can be subdivided into distinct MG derived cell population based on stereotypical markers. The feature plots show that prox1a is strongly expressed in the neurod4 and otx5 positive early photoreceptors. (M) Summary of Prox1a/prox1a expression during regeneration.

Loss of Prox1a decreases PR production at 120 hours post-ablation.

(A-D”) Knockdown of Prox1a via morpholino (MO) in adult retina during regeneration does not significantly affect percentage of PCNA+ (pink) proliferative Müller glia (marked by GFAP, green) following prox1a MO (72hpa n = 13, 18.7 ±03.692, 120hpa n = 10, 29.84 ±4.805) compared to SC (72hpa n = 8, 26.82 ±7.14, 120 hpa n = 9, 29.37 ±4.408), quantified in E. (F-I’) Knockdown of Prox1a resulted in a significant reduction of cone photoreceptors as marked by zpr1 (72hpa n = 15, 7.312 ±1.806, 120hpa n = 12, 14.05 ±2.539) compared to SC (72hpa n = 8, 25.98 ±8.962, 120 hpa n = 9, 35.4 ±7.836) in the ONL, quantified in J. DAPI labels cell nuclei (cyan). Data are represented as mean ± SEM. *p < 0.05. Scale bars: 50 μm. (K) Summary of regenerative time course following Prox1 MO knockdown showing reduction in mature regenerated photoreceptors.

Prox1a puncta in liquid-liquid phase separates regulate PR differentiation.

(A) Schematic highlighting the retinal layers analysed in the zebrafish larvae. (B) Experimental timeline. Larvae were swum in metronidazole (MTZ) for 24 hours at 3dpf (days post-fertilisation), followed by a 2 min treatment with 1,6-Hexanediol (1,6-HD) or 2,5-HD at 6dpf, before processing at 7dpf. (C-D”) Following photoreceptor (PR) ablation, Tg(lws2:nfsb-mCherry, gfap:eGFP) retinas show Prospero homeobox1a (Prox1a, pink) puncta only in the ONL of 2,5-HD control treated retinas (n = 5, 17.86 ±2.3), but not when liquid-liquid phase separates have been disassembled in the 1,6-HD treated samples (n = 7, 10.94 ±1.681, quantified in H. Arrow indicates nuclear Prox1a staining outside of the photoreceptor layer. (E-G”’) zpr1 cone photoreceptors are observed in both control samples (2,5-HD treated ablated retinas and 1,6-HD treated, non-ablated retinas), but much reduced in the ONL of 1,6-HD treated ablated retinas, quantified in I. DAPI labels cell nuclei (blue). Scale bars: 50 μm.

Her6 reduction increases Prox1a expression.

(A-D’) Downregulation of hairy-related 6 (her6) by morpholino (MO) in adult retina resulted in a significant increase of Prospero homeobox 1a (Prox1a) in the ONL during early phases of regeneration at 48hpa (SC n = 15, 5.294 ±0.3032, her6 MO n = 12, 9.507±1.339), but not at 72hpa (SC n = 9, 16.97 ±3.096, her6 MO n = 9, 11.34 ±2.625), quantified in E. (F-I’) Knockdown of Prox1a using MO led to a significant downregulation of Hes1/Her6 expression at 120 hpa (SC n = 11, 9.355 ±1.792, prox1a MO n = 6, 3.255 ±0.4484), but not at 72 hpa (SC n = 13, 9.958 ±0.9212, prox1a MO n = 14, 6.929±1.044), quantified in J. (K) Summary of expression and cross-regulation between Her6 and Prox1a expression during regeneration. Her6 is expressed during progenitor formation while Prox1a is present in differentiating PRs. Her6 is a negative regulator of Prox1a and Prox1a is a positive regulator of Her6. Data are represented as mean ± SEM. *p < 0.05. Scale bars: 50 μm.