PtenRegulates Retinal Amacrine Cell Number by Modulating Akt, Tgfβ, and Erk Signaling

Abstract

All tissues are genetically programmed to acquire an optimal size that is defined by total cell number and individual cellular dimensions. The retina contains stereotyped proportions of one glial and six neuronal cell types that are generated in overlapping waves. How multipotent retinal progenitors know when to switch from making one cell type to the next so that appropriate numbers of each cell type are generated is poorly understood. Pten is a phosphatase that controls progenitor cell proliferation and differentiation in several lineages. Here, using a conditional loss-of-function strategy, we found that Pten regulates retinal cell division and is required to produce the full complement of rod photoreceptors and amacrine cells in mouse. We focused on amacrine cell number control, identifying three downstream Pten effector pathways. First, phosphoinositide 3-kinase/Akt signaling is hyperactivated in Pten conditional knock-out (cKO) retinas, and misexpression of constitutively active Akt (Akt-CA) in retinal explants phenocopies the reduction in amacrine cell production observed in Pten cKOs. Second, Akt-CA activates Tgf␤ signaling in retinal explants, which is a negative feedback pathway for amacrine cell production. Accordingly, Tgf␤ signaling is elevated in Pten cKO retinas, and epistatic analyses placed Pten downstream of Tgf␤RII in amacrine cell number control. Finally, Pten regulates Raf/Mek/Erk signaling levels to promote the differentiation of all amacrine cell subtypes, which are each reduced in number in Pten cKOs. Pten is thus a positive regulator of amacrine cell production, acting via multiple downstream pathways, highlighting its diverse actions as a mediator of cell number control.

Effects of reduced β2 glycoprotein I on high glucose-induced cell death in HUVECs

“…activated the survival signaling pathway. PTEN is a negative regulator of AKT phosphorylation (27). The accumulation of PTEN attenuates AKT phosphorylation.…”

Generation of an animal model of Pten hamartoma tumour syndrome in the retina

“…It was previously shown that rapamycin upregulates Akt phosphorylation ( Leipert et al, 2016 ; O'Reilly et al, 2006 ; Schmid et al, 2014 ), which is already elevated in Pten cKO retinas ( Cantrup et al, 2012 ; Tachibana et al, 2016 ). We thus asked whether pAkt levels were further increased in animals treated with rapamycin.…”

“…We previously reported the generation of a Pten cKO using a Pax6::Cre driver that is more active in the peripheral versus central retina ( Cantrup et al, 2012 ; Marquardt et al, 2001 ; Tachibana et al, 2016 ). Strikingly, we noted that, in postnatal day (P)21 Pten cKO mice ( Pten fl/fl ;Pax6::Cre + ), a bulge-like ectopic mass developed in a site-specific manner on the dorsal-medial retinal surface in a completely penetrant manner ( n =49/49; Fig.…”

Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina

“…We also used the heterochronic pellet assay to examine how amacrine cell feedback signals are themselves regulated. We found that Pten is an essential positive regulator of amacrine cell differentiation, and using the pellet assay, we demonstrated that Pten acts in RPCs to control responsiveness to Tgfβ2 signaling (Tachibana et al, 2016). Understanding how amacrine cells and RPCs interact provides important new insights into how cell number is controlled in the retina.…”

“…We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997;Ringuette et al, 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al, 2007;Tachibana et al, 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different celltypes (heterotypic) in either an isochronic or heterochronic manner.…”