Mosaics of islet-1-expressing amacrine cells assembled by short-range cellular interactions

Abstract

The nervous system has a modular architecture with neurons of the same type commonly organized in nonrandom arrays or mosaics. Modularity is essential to parallel processing of sensory information and has provided a key element for brain evolution, but we still know very little of the way neuronal mosaics form during development. Here we have identified the immature elements of two retinal mosaics, the choline acetyltransferase (ChAT) amacrine cells, by their early expression of the homeodomain protein Islet-1, and we show that spatial ordering is an intrinsic property of the two Islet-1 mosaics, dynamically maintained while new elements are inserted into the mosaics. Migrating Islet-1 cells do not show this spatial ordering, indicating that they must move tangentially as they enter the mosaic, under the action of local mechanisms. Clonal territory analysis in X-inactivation transgenic mice confirms the lateral displacement of ChAT amacrine cells away from their clonal columns of origin, and mathematical models show how short-range cellular interactions can guide the assemblage of these mosaics via a simple biological rule.

Fig. 1.

a, Islet-1 mosaic in the inner nuclear layer (INL) of a flat-mounted adult rat retina. Scale bar, 20 μm. b, Islet-1⁺(red) cells in the ganglion cell layer (GCL) of the same retina as in a. Ganglion cells, retrogradely labeled with Fluorogold (light yellow), in some cases reveal Islet-1 labeling. Blue patches are salt precipitates that fluoresce when viewed with the filter used for Fluorogold. Scale bar, 20 μm. c, Islet-1⁺ mosaics correspond to the mosaics of cholinergic amacrine cells, as demonstrated by double-immunofluorescence studies in adult rat retina. The figure shows a confocal image of a radial retinal section (perpendicular to the plane in a and b).Arrowheads point to the amacrine cells that are double-labeled for ChAT (green) and Islet-1 (red). All cholinergic amacrine cells are Islet-1⁺ immunoreactive amacrine cells and vice versa. Bipolar cells are also Islet-1⁺ in the adult retina but can easily be distinguished from other Islet-1⁺ cells by their different position in the retina. The retina illustrated has no ganglion cells; these had all been selectively removed by optic nerve section at birth. Scale bar, 20 μm. d, Expression of Islet-1 in the embryonic retina. On E17, Islet-1 (brown) was expressed by cells that were still in the proliferative zone (PZ), as well as by cells that had already migrated to the vitreal side of the retina (bottom), where different layers cannot yet be distinguished at this age. Scale bar, 10 μm. e, Islet-1⁺ cells are all postmitotic. Two hours after BrdU injection, no Islet-1⁺ cell (green, arrowheads) is labeled with BrdU (red), which had been incorporated by proliferating cells. Superimposition of eight optical sections was obtained with a confocal microscope across an overall depth of 10 μm in an E17 retinal section. The only apparent double-labeled cell (arrow) is due to the partial physical superimposition of two cells. PZ is the proliferative zone. Scale bar, 10 μm. f, Islet-1⁺ mosaic in the INL on E21.5. Note the similarity to the adult mosaic. Scale bar, 10 μm. Nomarski optics.

Fig. 2.

Islet-1 mosaics preserve their spatial organization while new cells are added. a, Voronoi domains and (b) nearest neighbor distances of the INL mosaic on E21.5 (triangles), P2 (circles), and P4 (squares). No change with time is observed in the normalized histograms, although ∼30% more cells enter the mosaic between E21.5 and P4. When experimental variation is considered (not shown), the P4 distribution represents a fit for the E21.5 distribution, with p > 0.99 for the nearest neighbor distance and p > 0.95 for the Voronoi distribution, as determined by χ² test.Red lines represent the distributions expected if the newly arrived cells were randomly added to the E21.5 mosaic. The difference between the P4 mosaic and the expected distribution in the case of random addition is statistically significant (χ² test; p < 0.001). No change with time was also observed in the distribution of Delaunay segments (not shown). c, Normalized histograms of Voronoi domains and (d) nearest neighbor distances for the GCL mosaic of Islet-1⁺ cells. P1.5 (triangles), P3 (circles), and P4 (squares). Approximately 20% more cells are added to the Islet-1 mosaic of the GCL between P1.5 and P4 (24,000 ± 2000 on P1.5; 29,000 ± 1300 on P4), but the two distributions remain more regular than the expected distribution (red lines) if the 20% additional cells were randomly inserted in the GCL mosaic between P1.5 and P4. Although the mean of the distributions obtained for the random addition of cells (red lines) is the same as that for the real data in the case of Voronoi areas, the SD is larger, and the difference between the real and simulated distribution is statistically significant (p< 0.00001, as determined by χ² analysis). Note that the two Islet-1 mosaics have the same minimal Voronoi area (compare a and c) and the same minimal intercellular spacing (compare b and d), although the regularity of the GCL mosaic is lower than that of the INL.

Fig. 3.

a, Islet-1⁺cells crossing the IPL have no minimal intercellular spacing. Cross section of a P1 retina. The spacing between Islet-1⁺amacrine cells migrating through the inner plexiform layer (IPL) was studied after P0 in the central retina, where the migration of ganglion cells that also express Islet-1 has ceased by this time. Scale bar, 10 μm. b, Lack of regular intercellular spacing between Islet-1 cells that are still outside the GCL mosaic. Normalized histogram of nearest neighbor distances between Islet-1⁺ cells in the IPL (filled squares) in P2 retinal sections. The histogram shows that the only limit to the spacing between Islet-1⁺ cells migrating through the IPL is the diameter of the cell, simply reflecting the fact that two cells cannot occupy the same physical space. The nearest neighbor distribution in the INL derived from the same sections (open circles) is shown as a comparison, because the minimal spacing is the same in both Islet-1 mosaics. As should be expected, the normalized distributions of nearest neighbor distances computed in sections and flat-mounted retina have the same minimal spacing but different tails, because the nearest neighbor in sections can be farther away (but, of course, never closer) than the real nearest neighbor in the mosaic. Counts were made in 30 randomly selected P2 sections from two retinae (n = 2).c, Retinal whole mount from an X-inactivation transgenic mouse showing a portion of the INL. Arrowheads point to four mosaic cells (brown; ChAT-immunoreactive) that are transgene-positive (blue) but are displaced in transgene-negative columns. These cells must be derived from neighboring transgene-positive clones. d, A portion of the ganglion cell layer showing one transgene-positive mosaic cell (arrowhead) and several transgene-positive ganglion cells (the largest cells in the field) in a transgene-negative column. These cells must have moved tangentially away from their clone of origin. Scale bar, 20 μm.

Fig. 4.

Islet-1 mosaics are indistinguishable from random distributions generated by imposing a minimal distance of 15 ± 2 μm between neighboring cells. a, A field of the INL Islet-1 mosaic is illustrated (dots represent cells) together with the tiling of the plane that it generates (Voronoi domains). b, Tiling of the plane arising from a random distribution of cells (random_dmin) that was generated, imposing a lower limit (dmin = 15 ± 2 μm) to the distance between neighboring cells. Cell density is identical to that of the Islet-1⁺ mosaic shown in a. c, Tiling generated by a random distribution of cells at the same cell density as ina. d, e, Fast Fourier Transform (FFT) of the Islet-1 mosaic (d) and the random_dmindistribution (e). FFTs are represented as amplitude maps and illustrated in the intervals ω_x ∈ (−60,60 μm⁻¹), ω_y ∈ (−40,40 μm⁻¹). The center of the figure is the center of the coordinates. In both cases the phase distributions are random. The FFTs of the real and simulated mosaic are nearly identical, which is also illustrated in f(top), where a horizontal section throughd (continuous line) and e(dotted line) at the midline are shown.Bottom, Difference between the amplitude of the two FFTs at the section level, illustrating that the two FFTs differ by no more than 10%.