Pten regulates the development of a stereotyped dendritic architecture in starburst amacrine cells

Abstract

Purpose : It is unknown how the stereotyped morphology of neurons like the starburst amacrine cell (SAC) are controlled during retinal development. To better understand how intracellular signaling pathways regulate SAC morphology, we used an in vivo genetic approach to test the hypothesis that Pten is required for SACs to properly form their canonical radially symmetric dendritic arbor.

Methods : Pten was selectively deleted from mouse SACs early in their development using ChAT^Cre and Pten^flox alleles. Retinal tissue was collected at a range of developmental ages (P7, P14, P21, P28, and P60). To sparsely label SACs for morphometric reconstruction, P1-P4 pups were intravitreally injected with Cre-dependent AAV8-FLEX-tdTomato. For each condition, 8-10 SACs were reconstructed. ChAT^Cre;Pten^+/+ and ChAT^Cre;Pten^f/+ animals were grouped as controls once it was shown that Pten heterozygous SACs have no phenotype. SACs in the inner nuclear layer (INL) and ganglion cell layer (GCL) were analyzed separately to account for known morphological differences. Cells were reconstructed using the filaments tool in Imaris, and morphological metrics were analyzed for statistical significance using an ANOVA or student’s t-test depending on the number of conditions.

Results : At P21, SACs lacking Pten (ChAT^Cre;Pten^f/f) show a significant increase in dendritic branch number (p < 0.0001) and total dendritic length (p = 0.033), but no change in their dendritic field area (p = 0.756). This branching phenotype arises late in SAC development, as there are no significant change at P7 and P14 (p = 0.375). At P60, SACs maintain their increased branching phenotype (p < 0.0001), but show a decrease in dendritic field area (p = 0.013). Preliminary data in a ChAT^Cre;Tsc2 cKO line suggests that this branching phenotype is not primarily exerted from elevated mTORC1 activity (p = 0.604).

Conclusions : Selective deletion of Pten via ChAT^Cre in SACs resulted in a significant upregulation of dendritic branching while leaving their total dendritic length and field area unaffected. These effects appear to be developmentally time-locked and persist into adulthood. These findings begin to unravel how intracellular signaling pathways contribute to the regulation of specific morphological properties in developing neurons.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.