Developmental synchrony of retinal waves, apoptosis, and angiogenesis in postnatal retina
- Biosciences Institute, Faculty of Medical Sciences, Newcastle University, United Kingdom
- School of Geography and Natural Sciences, Northumbria University, United Kingdom
Figures
Figure 1 with 1 supplement
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 postnatal day (P)3–6. (B) Mouse retinal wholemounts (P3–6) displaying superficial vascular plexus (SVP) (labelled with isolectin B4 [IB4]). Scale bar is equal to 1 mm. (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 ganglion cell layer (GCL) level and at the inner nuclear layer (INL) level in a whole mount. Green: ChAT; magenta: RBPMS. The cluster cells are visible only at the GCL level and are much larger than starburst amacrine cells (SACs) or retinal ganglion cells (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 two-tailed test.
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Figure 1—source data 1
Number of blood vessel intersections in ACC+ and ACC- regions, ACC D1/2 values for P2-P9.
- https://cdn.elifesciences.org/articles/111419/elife-111419-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Peripherality and vascular relation metrics.
Method for calculating the relative position of anatomical markers between the optic nerve head (ONH) (small black circle in the middle), the periphery, and the vascular plexus. Anatomical markers such as auto-fluorescent cluster complexes (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/D2 value greater than 0.5 equates to the periphery of the retina. D1/D3 value greater than 1 equates to the non-vascularised area of the retina.
Figure 2
Genetic analysis of auto-fluorescent cluster complexes (ACCs) suggests that they are complexes of microglia and dying retinal ganglion cells (RGCs).
(A) Uniform Manifold Approximation and Projection (UMAP) plot of single-cell RNA sequencing (scRNA-seq) data derived from ACCs taken from postnatal day (P)5 retinas. 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 retinal progenitor cell (RPC) markers TOPA2 and CDK1. (F) Expression of neurogenic progenitor cell markers NEUROD1 and RORB. (G) Expression of retinal ganglion cell markers RBPMS and BAX.
Figure 3
Microglia associate with the auto-fluorescent cluster complexes (ACCs).
(A) Representative micrograph of ACCs (white) surrounded by microglial cells labelled with Iba-1 (green). Scale bar = 100 µm. (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 optic nerve head (ONH), between the ONH and clusters (behind) at clusters and ahead of clusters, further in the periphery, in the unvascularised part of the retina. The micrograph shows blood vessels labelled with isolectin B4 (IB4) (magenta), microglia labelled with Iba-1 (orange), and ACCs (white). Scale bar = 500 µm. (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).
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Figure 3—source data 1
Microglia cell densities across different regions of eccentricity in ACC+ and ACC- regions.
- https://cdn.elifesciences.org/articles/111419/elife-111419-fig3-data1-v1.xlsx
Figure 4
Auto-fluorescent cluster complexes (ACCs) are composed of dying retinal ganglion cells (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 ACC locations and the superficial vascular plexus (SVP). (B) Mouse retinal wholemounts displaying apoptotic cells labelled with YO-PRO-1 in the periphery and overlapping with the microglia annulus. Scale bar=1 mm. (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 RBPMS. (F) Apoptotic cells labelled with YO-PRO-1 show no co-localisation with starburst amacrine cell (SAC) marker ChAT. (G) PANX-1 hemichannels location tightly overlaps with a cluster of YO-PRO-1-positive cells. (H) Higher-magnification inset of G displaying the tight association between YO-PRO-1-positive cells and PANX-1 hemichannels. Scale bar=50 µm.
Figure 5 with 1 supplement
Purinergic signalling through PANX-1 hemichannels and P2Y12 receptors influences Hmox1 microglia activation at postnatal day (P)4.
Quantification of microglial morphology at P4 under control conditions (black bars) and following pharmacological inhibition using probenecid 1 mM (darkest grey), probenecid 2 mM (medium grey), and PSB0739 5 μM (light grey) for 24 hr. 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 1 mM = 5401, probenecid 2 mM = 4266, and PSB0739 5 μM = 3099. (A) Circularity was significantly reduced in probenecid 2 mM and PSB0739 5 μM 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 2 mM and PSB0739 5 μM relative to control. (D) Total skeleton length was significantly higher in probenecid and PSB0739 5 μM compared to control. (E) Total number of Hmox1-positive cells observed after incubation of probenecid (1 mM or 2 mM) or PSB0739 5 μM for 24 hr prior to fixation and immunolabelling. (F) Total number of YO-PRO-1-positive cells observed after incubation of probenecid (1 mM or 2 mM) or PSB0739 5 μM for 24 hr prior to fixation and immunolabelling. (G) Total number of double positive YO-PRO-1 and Hmox1 cells observed after incubation of probenecid (1 mM or 2 mM) or PSB0739 5 μM for 24 hr prior to fixation and immunolabelling. (H) Percentage of Hmox1+ cells that co-localise with YO-PRO-1 signal after incubation of probenecid (1 mM or 2 mM) or PSB0739 5 μM for 24 hr 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 (1 mM or 2 mM) or PSB0739 5 μM for 24 hr 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 24 hr of incubation with probenecid 2 mM 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 24 hr of incubation with PSB0739 5 μM, with higher-magnification inset indicated by asterisk. Scale bar = 100 µm. Inset scale bar = 20 µm. (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 24 hr (1 mM). Bottom: Retina incubated in PSB0739 (5 μM) for 24 hr. Scale bars = 100 μm.
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Figure 5—source data 1
Microglia morphometric parameters across different drug conditions.
- https://cdn.elifesciences.org/articles/111419/elife-111419-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
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.
Figure 6
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 region of interest (ROI) (10 µm × 10 µm) within the calcium imaging recording. Waves are indicated by synchronised activity across channels. The activity is shown against time (in seconds). (B) Probenecid 1 mM profoundly reduces retinal excitability, with significant decrease in wave frequency and number of channels recruited within waves. (C) Retinal wave frequency increases from postnatal day (P)3–6 while ATP release blockade with probenecid reduces frequency across the board. (D) Wave retinal area coverage is reduced significantly by probenecid treatment. (E) Retinal wave propagation speed is reduced by probenecid treatment in the later stage of first postnatal week. (F) Retinal wave path length increases from P3 to P5 and is significantly reduced by probenecid treatment. Statistical analysis was conducted using Mann-Whitney rank-sum test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (G) 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). (H) Retinal wave origination becomes increasingly more peripheral (D1/D2>0.5) over the P3–6 period. (I) Example propagation extent of a retinal wave. (J) Retinal waves originate in the non-vascularised peripheral regions (D1/D3>1) of the retina, regardless of age.
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Figure 6—source data 1
Calcium retinal wave metrics in control and probenecid conditions.
- https://cdn.elifesciences.org/articles/111419/elife-111419-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
Relative peripherality of multiple developmental processes is age-invariant.
(A) Box plot showing developmental changes in D1/D2 peripherality ratios for stage II retinal waves recorded by microelectrode array (MEA) and calcium imaging, SVP extent, YO-PRO-1 labelling, HMOX-1 microglia locations, and auto-fluorescent cluster complexes (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/D2>0.5) over the postnatal day (P)3–6 period, see Figure 1—figure supplement 1. (B) Mean and standard deviation plots for each functional or anatomical measures expressed in terms of D1/D2 peripherality metrics across the P3–6 time period.
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Figure 7—source data 1
D1/2 metrics for retinal waves, vascular plexus, apoptotic cells, microglia, and ACCs.
- https://cdn.elifesciences.org/articles/111419/elife-111419-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Multi-electrode array recording of spontaneous waves affected by PANX-1 blockade.
Raster plots of waves from a postnatal day (P)5 mouse in control conditions, and in the presence of probenecid 1 mM (recording started 30 min after adding the drug). Each row represents an active electrode on the microelectrode array (MEA). Waves are indicated by synchronised activity across channels. The activity is shown against time (in seconds). Waves are detected according to methods described in Maccione et al., 2014. Each detected wave is in a different colour. Probenecid profoundly reduces retinal excitability, with significant decrease in wave frequency and number of channels recruited within waves.
Figure 8
Overview of synchronised development in retina.
(A) Diagram of a retina at two different timepoints displaying the organisation of apoptotic retinal ganglion cells (RGCs), the superficial vascular plexus (SVP), the annulus of Hmox1 microglia, and auto-fluorescent cluster complexes (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.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/111419/elife-111419-mdarchecklist1-v1.docx
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Supplementary file 1
Single Cell RNA-Seq gene and cluster table.
- https://cdn.elifesciences.org/articles/111419/elife-111419-supp1-v1.docx
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Developmental synchrony of retinal waves, apoptosis, and angiogenesis in postnatal retina
eLife 15:RP111419.
https://doi.org/10.7554/eLife.111419.3
