Auto-fluorescent Cluster Complexes (ACCs) display centrifugal development pattern during the first postnatal week in C57/BL6 and align with vasculature development.

A. Mouse retinal wholemounts displaying ACCs (green) across P3-P6. B. Mouse retinal wholemounts (P3-P6) displaying SVP (labelled with Isolectin B4 (IB4)). Scale bar is equal to 1mm. C. Zoom into one auto-fluorescent cluster complex (P5) visualised at three different wavelengths without any additional immunolabeling, indicating intrinsic auto-fluorescent signals. D. Retinal sections showing VAChT (red) expression in a cluster area (upper panel, P4 retina) and in an area devoid of clusters (lower panel, P5 retina). E. Scatter plot of blood vessel branch intersections (Sholl analysis). There are significantly more branches in ACC positive areas (ACC +, green) than in ACC negative areas (ACC -, purple). Median with interquartile range. F. P5 cluster viewed at the GCL level and at the inner nuclear layer (INL) level in a whole-mount. Green: ChAT; red: RBPMS. The cluster cells are visible only at the GCL level and are much larger than SACs or RGCs (in red). At the INL level, there are only SACs and no ACCs. Right side of panel is magnified version of box on left. G. Box plot showing developmental changes in ACCs D1/D2 ratios. Each box illustrates the median (horizontal line) and interquartile range, with minimum and maximum values (whiskers). Asterisks indicate significant changes between consecutive days (One-way ANOVA with post-hoc Tukey test). The red dotted line illustrates the percentage difference in values between consecutive days, showing peak difference between P3 and P4 and no further changes from P6 onwards. ****: P<0.0001, Mann Whitney 2-tailed test.

Genetic analysis of ACCs suggests they are complexes of microglia and dying RGCs.

A. UMAP plot of scRNA-Seq data derived from ACCs. Each cluster was identified based on the expression of retinal specific cell markers. Highly expressed markers for each cluster are shown in subsequent panels. B. Expression of microglia cell markers SPARC and AIF1. C. Expression of endothelial cell markers DAB2 and F13A1. D. Expression of proliferating cell markers STMN1 and TUBB3. E. Expression of RPC markers TOPA2 and CDK1. F. Expression of neurogenic progenitor cell markers NEUROD1 and RORB. G. Expression of retinal ganglion cell markers RPBMS and BAX.

Microglia associate with the ACCs

A. Representative micrograph of ACCs (white) surrounded by microglial cells labelled with iba1 (green) Scale = 100µm. B. Box plot (median and interquartile ranges) comparing microglia density within cluster areas (C+) versus cluster negative areas (C-). P<0.0138. Mann Whitney two-tailed test. C. Strong positive correlation between the densities of microglial cells and auto-fluorescent cluster cells (p<0.0001). D. Method for measuring microglia density at different retinal eccentricities, with measures taken near the ONH, between the ONH and clusters (behind) at clusters and ahead of clusters, further in the periphery, in the unvascularized part of the retina. The micrograph shows blood vessels lableled with IB4 (magenta), microglia labelled with iba1 (orange) and ACCs (white) Scale bar = 500µm. E. Box plot (with medians and interquartile ranges) showing that the density of microglia is significantly higher within clusters (p<0.01, Mann Whitney, two-tailed test).

ACCs are composed of dying RGCs engulfed by Hmox1 expressing microglia

A. Mouse retinal wholemounts displaying a subtype of microglia labelled by Hmox1 which sit as an annulus astride the ACCs locations and the SVP. B. Mouse retinal wholemounts displaying apoptotic cells labelled with YO-PRO-1 in the periphery and overlapping with the microglia annulus. Scale bar 1mm. C. Example Hmox-1 positive microglia with ACCs present inside intracellular vacuoles responsible for breaking down and storing apoptotic cells. D. Apoptotic cells labelled with YO-PRO-1 are surrounded by Hmox-1 ramified microglia which become activated and engulf dying cells storing them in intracellular vacuoles similar to AAC storage. E. Apoptotic cells labelled with YO-PRO-1 show complete overlap with RGC marker RPBMS. F. Apoptotic cells labelled with YO-PRO-1 show no colocalisation with SAC marker ChAT. G. PANX-1 hemichannels location tightly overlaps with a cluster of YO-PRO-1 positive cells. H. Higher magnification inset of E displaying the tight association between YO-PRO-1 positive cells and PANX-1 hemichannels. Scale bar 50µm.

Purinergic signalling through PANX-1 hemichannels and P2Y12 receptors influences Hmox1 microglia activation at P4.

Quantification of microglial morphology at P4 under control conditions (black bars) and following pharmacological inhibition using Probenecid 1 mM (darkest grey), Probenecid 2mM (medium grey), and PSB0739 5μM (light grey) for 24 hours. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s multiple comparisons test. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n= 8-10 retinas per group. Cells per group: control= 6829, Probenecid 1mM= 5401, Probenecid 2mM= 4266, and PSB0739 5μM= 3099. A. Circularity was significantly reduced in Probenecid 2 mM, and PSB0739 5μM compared to control. B. Probenecid and PSB0739 in general significantly increased the number of branch points compared to control. C. Cell perimeter was significantly increased by Probenecid 2mM and PSB0739 5μM relative to Control. D. Total skeleton length was significantly higher in Probenecid and PSB0739 5μM compared to Control. E. Total number of Hmox1 positive cells observed after incubation of probenecid (1mM or 2mM), or PSB0739 5μM for 24 hours prior to fixation and immunolabelling. F. Total number of YO-PRO-1 positive cells observed after incubation of probenecid (1mM or 2mM), or PSB0739 5μM for 24 hours prior to fixation and immunolabelling. G. Total number of double positive YO-PRO-1 and Hmox1 cells observed after incubation of probenecid (1mM or 2mM), or PSB0739 5μM for 24 hours prior to fixation and immunolabelling. H. Percentage of Hmox1+ cells that co-localise with YO-PRO-1 signal, after incubation of probenecid (1mM or 2mM), or PSB0739 5μM for 24 hours prior to fixation and immunolabelling. I. Percentage of YO-PRO-1 positive cells that express Hmox1, representing the proportion of apoptotic cells associated with Hmox1 positive microglia after incubation of probenecid (1mM or 2mM), or PSB0739 5μM for 24 hours prior to fixation and immunolabelling J. Micrograph displaying overlap of apoptotic cells labelled with YO-PRO-1 and HMOX-1 positive microglia with higher magnification inset indicated by asterisk. K. Micrograph displaying reduced overlap of apoptotic cells labelled with YO-PRO-1 and HMOX-1 positive microglia after 24hr incubation with Probenecid 2mM with higher magnification inset indicated by asterisk. L. Micrograph displaying reduced overlap of apoptotic cells labelled with YO-PRO-1 and HMOX-1 positive microglia after 24hr incubation with PSB0739 5μM with higher magnification inset indicated by asterisk. Scale bar = 100µm. Inset scale bar = 20µm M. Top- Cumulative frequency distributions of apoptotic cells D1/D2 ratio. N=3344. Bottom- Cumulative frequency distributions of apoptotic cells: D1/D3 ratio Key: Control (black), Probenecid-treated (red), PSB0739-treated (blue). N=3344. N. Top- Representative image of P4 mouse retinal whole-mounts showing the SVP (IB4, red) and apoptotic cells (YO-PRO-1, green) across the centre-peripheral axis gradient from left (ONH region) to right (vascular leading edge). Middle- Retina incubated in probenecid for 24hrs (1mM). Bottom- Retina incubated in PSB0739 (5μM) for 24hrs. Scale bars = 100μm.

Centrifugal Progression of Retinal Wave Origination is Driven by PANX1-Mediated Purinergic Signalling.

A. Calcium retinal waves plots of waves in control conditions. Each row represents a pixel ROI (10µm x 10µm) within the calcium imaging recording. Waves are indicated by synchronised activity across channels. The activity is shown against time (in seconds). B. Probenecid 1mM profoundly reduces retinal excitability, with significant decrease in wave frequency and number of channels recruited within waves. C. Mouse retinal wholemount (P4) displaying superior vascular plexus (SVP) (labelled with isolectin B4 (IB4)) and the origin points of spontaneous retinal waves recorded with calcium imaging (green spots). D. Example propagation extent of a retinal wave. E. Retinal wave origination becomes increasingly more peripheral (D1/2 > 0.5) over the P3-P6 period. F. Retinal waves originate in the non-vascularised more peripheral regions (D1/3 > 1) of the retina regardless of age. G. Retinal wave frequency increases from P3-P6 while ATP release blockade with probenecid reduces frequency across the board. H. Wave retinal area coverage is reduced significantly by probenecid treatment. I. Retinal wave propagation speed is reduced by probenecid treatment in the later stage of first postnatal week. J. Retina wave path length increases from P3-5 and is significantly reduced by probenecid treatment. Statistical analysis was conducted using Man-Whitney rank sum test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Relative Peripherality of Multiple Developmental Processes Is Age Invariant.

Box plot showing developmental changes in D1/D2 peripherality ratios for stage II retinal waves recorded by MEA, and calcium imaging, SVP extent, YO-PRO-1 labelling, HMOX-1 microglia locations, and ACCs. Each box illustrates the median (horizontal line) and interquartile range, with minimum and maximum values (whiskers). The distribution of each marker of interest increases in peripherality (D1/2 > 0.5) over the P3-P6 period, see Figure S2.

Overview of Synchronised Development in Retina

A. Diagram of a retina at two different timepoints displaying the organisation of apoptotic RGCs, the SVP, the annulus of Hmox1 microglia, and ACCs. Arrows indicate areas explored in higher detail in B,C. B. Pro-angiogenic and spontaneous activity phase of the apoptotic RGCs releasing purinergic markers through PANX-1 hemichannels. C. Phagocytic phase where Hmox1 microglia sense apoptotic RGCs and engulf them for destruction, creating ACCs.

Microglial Morphometric Parameter Measurements.

A. Microglial cell shape example, B. Area of the cell is illustrated by red stripes covering the cell. C. Convex Area illustrated by area covered in red stripes, D. Length of individual branches- shown by a red line and red dot at the end point of each measurement, E. Number of branch points shown by a red dot covering every branch intersection, F. Geodesic diameter highlighted by a black line along the shortest path through the maximum distance of 2 points of the microglia cell, G. Perimeter of the cell show by a dashed red line around the cell outline, H. The geometric centre of the cell is represented by a yellow circle.

Peripherality and Vascular Relation Metrics

Method for calculating the relative position of anatomical markers between the ONH (small black circle in the middle) the periphery, and the vascular plexus. Anatomical markers such as ACCs are represented by a green dot. D1: distance from centre of ONH to the object of interest. D2: distance between the object of interest to periphery. D3: The distance from the ONH through the line of the object of interest to the vascularised border. D1/2 value greater that 0.5 equates to the periphery of the retina. D1/3 value greater than 1 equates to the non-vascularised area of the retina.