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Comparative Study
, 27 (7), 1607-14

Somal Positioning and Dendritic Growth of Horizontal Cells Are Regulated by Interactions With Homotypic Neighbors

Affiliations
Comparative Study

Somal Positioning and Dendritic Growth of Horizontal Cells Are Regulated by Interactions With Homotypic Neighbors

Ross A Poché et al. Eur J Neurosci.

Abstract

Retinal neurons extend their dendritic fields to achieve a degree of dendritic overlap with homotypic neighbors that is cell-type specific. How these neurons regulate their dendritic growth is unclear. The dendritic field of a retinal horizontal cell varies inversely with horizontal cell density across different strains of mice, suggesting that proximity to neighboring cells regulates dendritic growth. To test this directly, we have employed the Cre-loxP conditional gene targeting strategy to achieve inactivation of Lim1 function in developing horizontal cells. Through this approach, Lim1 function was prevented within a subset of horizontal cells that in turn fail to migrate to the horizontal cell layer and differentiate normally. For those remaining horizontal cells with Lim1 intact (about half of the normal population in these mice), we show that they spread themselves out tangentially and differentiate a dendritic morphology that is essentially normal but for the fact that it has nearly doubled in area. Such larger horizontal cells, sampling from an area of retina containing twice their normal afferent number, differentiate a dendritic field with nearly double the number of higher order branches and terminal clusters. These results demonstrate directly that positioning and dendritic growth are regulated by interactions with homotypic neighbors, whereas afferents instruct the differentiation of dendritic patterning.

Figures

Figure 1
Figure 1
Schematic diagram showing the effect of deleting Lim1 function in mosaic-conditional Lim1-knockout mice, examined at P-20. Lim1-deficient horizontal cells have become mis-positioned within the amacrine cell layer, while Lim1-intact horizontal cells correctly target their normal stratum at the OPL (a, b). Mis-positioned horizontal cells express both calbindin and the lacZ gene product, β-galactosidase (double-labeled cell indicated by white arrow in a Six3-Cre; Lim1-lacZ/flox retina in d), as do normally positioned horizontal cells in both conditional knockout retinas (d) as well as in control (Lim1-lacZ) retinas (c). Note the ectopic β-galactosidase staining within the IPL (green) that emerges from such ectopically positioned horizontal cells (e.g. from the cell indicated by the arrow in d). Retinas present a Amosaic-conditional@ knockout phenotype due to the mosaicism in the early expression of Cre recombinase, evidenced in an embryonic day 13.5 retinal section from a Six3-Cre; R26R reporter mouse showing variegated lacZ expression in the pattern of β-galactosidase staining (e, f). The expression pattern is consistent with the clonal organization of the retina at these early stages, indicating that Cre-mediated recombination should excise Lim1 in only a proportion of horizontal cells, being consistent with the phenotype of Lim1 conditional knockout retinas in maturity, described above. The present investigation has asked, what is the effect of partially depleting horizontal cells of their homotypic neighbors upon their subsequent differentiation (black arrows in b)?
Figure 2
Figure 2
Distribution of calbindin-positive horizontal cells adjacent to the outer plexiform layer in the four quadrants of a typical Lim1-lacZ retina (a) and in a Six3-Cre; Lim1-lacZ/flox retina (b). Calibration bar = 50 μm.
Figure 3
Figure 3
Horizontal cell density is significantly reduced in the Six3-Cre; Lim1-lacZ/flox retina (p < 0.001) (a) and in the Chx10-Cre; Lim1-lacZ/flox retina (p<0.001) (d). There are slight but non-significant changes in retinal area in these mice (b and e). Cone photoreceptor density appears slightly reduced in the Six3-Cre; Lim1-lacZ/flox mice (c), while it is slightly increased in the Chx10-Cre; Lim1-lacZ/flox mice (f). The latter comparison was significantly different (p<0.05). n = the number of retinas analyzed.
Figure 4
Figure 4
The regularity index of the horizontal cell mosaic in Chx10-Cre; Lim1-lacZ/flox depleted retinas is reduced relative to Lim1-lacZ littermate retinas (p < 0.001), yet this reduction is not as great as that observed in the Lim1-lacZ control mosaics simulated to have undergone a comparable, random, reduction in horizontal cell density (p < 0.001) (a). The effective radius of these genetically depleted mosaics is larger than that for the control mosaics (p < 0.01), while the effective radius of the randomly deleted mosaics is no different from the control mosaics. Cells in the genetically depleted retina therefore display a larger minimal spacing between neighboring cells than would be expected from random deletions achieving the same comparable overall reduction in density (p < 0.05). n = the number of retinas analyzed.
Figure 5
Figure 5
Morphology of four single horizontal cells from Lim1-lacZ retinas (a) and Six3-Cre; Lim1-lacZ/flox retinas (b). Horizontal cells in each case look qualitatively normal when compared with the morphological features of horizontal cells in C57BL/6 mice. Calibration bar = 50 μm.
Figure 6
Figure 6
Horizontal cells in the either the Six3-Cre; Lim1-lacZ/flox retinas or the Chx10-Cre; Lim1-lacZ/flox retinas have larger dendritic field areas when compared with those from the Lim1-lacZ retinas (a, e) (p < 0.005 and < 0.00001, respectively). Their number of primary dendrites is the same (b, f), but the frequency of higher order branches within their dendritic fields is greater in the conditional knockout retinas (c, g) (p < 0.05 for both). Horizontal cells in the conditional knockout retinas receive innervation from a larger number of cone pedicles, evidenced by their differentiating a significantly larger number of terminal clusters (d, h) (p < 0.001 and < 0.00001, respectively). n = the number of cells analyzed.
Figure 7
Figure 7
Summary diagram showing the effects of reducing the frequency of homotypic neighbors in Lim1 conditional knockout mice upon the intercellular spacing, dendritic field size and afferent connectivity of horizontal cells. Only a single horizontal cell is filled to show more clearly the difference in dendritic morphology within the OPL between the control and depleted conditions (specifically, the size of the dendritic field and number of terminal clusters). Also depicted is the increase in minimal spacing between neighboring horizontal cells in the depleted condition.

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