Zac1 functions through TGFβII to negatively regulate cell number in the developing retina

<p>Abstract</p> <p>Background</p> <p>Organs are programmed to acquire a particular size during development, but the regulatory mechanisms that dictate when dividing progenitor cells should permanently exit the cell cycle and stop producing additional daughter cells are poorly understood. In differentiated tissues, tumor suppressor genes maintain a constant cell number and intact tissue architecture by controlling proliferation, apoptosis and cell dispersal. Here we report a similar role for two tumor suppressor genes, the <it>Zac1 </it>zinc finger transcription factor and that encoding the cytokine TGFβII, in the developing retina.</p> <p>Results</p> <p>Using loss and gain-of-function approaches, we show that <it>Zac1 </it>is an essential negative regulator of retinal size. <it>Zac1 </it>mutants develop hypercellular retinae due to increased progenitor cell proliferation and reduced apoptosis at late developmental stages. Consequently, supernumerary rod photoreceptors and amacrine cells are generated, the latter of which form an ectopic cellular layer, while other retinal cells are present in their normal number and location. Strikingly, <it>Zac1 </it>functions as a direct negative regulator of a rod fate, while acting cell non-autonomously to modulate amacrine cell number. We implicate TGFβII, another tumor suppressor and cytokine, as a <it>Zac1</it>-dependent amacrine cell negative feedback signal. TGFβII and phospho-Smad2/3, its downstream effector, are expressed at reduced levels in <it>Zac1 </it>mutant retinae, and exogenous TGFβII relieves the mutant amacrine cell phenotype. Moreover, treatment of wild-type retinae with a soluble TGFβ inhibitor and TGFβ receptor II (TGFβRII) conditional mutants generate excess amacrine cells, phenocopying the <it>Zac1 </it>mutant phenotype.</p> <p>Conclusion</p> <p>We show here that <it>Zac1 </it>has an essential role in cell number control during retinal development, akin to its role in tumor surveillance in mature tissues. Furthermore, we demonstrate that <it>Zac1 </it>employs a novel cell non-autonomous strategy to regulate amacrine cell number, acting in cooperation with a second tumor suppressor gene, <it>TGFβII</it>, through a negative feedback pathway. This raises the intriguing possibility that tumorigenicity may also be associated with the loss of feedback inhibition in mature tissues.</p

Cell-Type Specific Roles for PTEN in Establishing a Functional Retinal Architecture

BACKGROUND: The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture. METHODOLOGY/PRINCIPAL FINDINGS: In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam. CONCLUSIONS/SIGNIFICANCE: We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina

The role of the tumour suppressor gene 'Pten' during retinal development

The retina is central nervous system tissue in the back of the eye. It serves as the first level of sensory processing of the visual environment. All the cell types of the retina have been well characterized, and we understand its physiological functioning. Developmental neurobiologists are interested in factors that dictate events such as the proliferation of neural progenitors, the differentiation of neural cell types, the migration of cells to their final destination, and how new neurons form synaptic connections to generate the mature brain. The retina is an attractive tissue to study neurodevelopment as its accessibility and cellular makeup are advantageous to conduct genetic manipulations of these developmental processes. When this project was undertaken, little was understood about the retinal-specific role of the protein PTEN (phosphatase and tensin homologue), and the PI3-K (phosphatidylinositol 3-kinase) signaling pathway – known to be responsible for proliferation, differentiation, cell death, migration, and neuronal process growth/connectivity events in other parts of the developing nervous system. This thesis describes experiments where I deleted the Pten gene in the mouse retina using a conditional knockout (cKO) approach, and characterized many phenotypes that occurred both during early and late retinal development. During early retinal development, I found that Pten is required to regulate retinal progenitor cell proliferation and differentiation of a selective subset of retinal cells, specifically amacrine cells and photoreceptors. During later stages of retinal development, I describe other phenotypes in the Pten cKO retina. There is an expansion of the inner plexiform layer, with specific process disruptions. The spacing of Pten mutant horizontal and dopaminergic amacrine cells was also aberrant, as was the fasciculation patterns of dopaminergic amacrine and ganglion cell processes in the retinal tangential plane. In electroretinograms, irregular oscillatory potentials were observed in Pten mutants, suggesting asynchronous amacrine cell firing. In visual behaviour tests, some Pten mutant animals also displayed impairments. I also discovered a morphological abnormality in the Pten cKO retina, and an alteration in the subcellular distribution of the protein DSCAM within dopaminergic amacrine cells. These data reveal new insights into the role of Pten signaling in the regulation of retinal development.5 year

Hamartoma-like lesions in the mouse retina: an animal model of Pten hamartoma tumour syndrome

PTEN hamartoma tumour syndrome (PHTS) is a heterogeneous group of rare, autosomal dominant disorders associated with PTEN germline mutations. PHTS patients routinely develop hamartomas, which are benign tissue overgrowths comprised of disorganized ‘normal’ cells. Efforts to generate PHTS animal models have been largely unsuccessful due to the early lethality of homozygous germline mutations in Pten, together with the lack of hamartoma formation in most conditional mutants generated to date. We report herein a novel PHTS mouse model that reproducibly forms hamartoma-like lesions in the central retina by postnatal day 21. Specifically, we generated a Pten conditional knockout (cKO) using a retinal-specific Pax6::Cre driver that leads to a nearly complete deletion of Pten in the peripheral retina but produces a mosaic of ‘wild-type’ and Pten cKO cells centrally. Structural defects were only observed in the mosaic central retina, including in Müller glia and in the outer and inner limiting membranes, suggesting that defective mechanical integrity partly underlies the hamartoma-like pathology. Finally, we used this newly developed model to test whether rapamycin, an mTOR inhibitor that is currently the only PHTS therapy, can block hamartoma growth. When administered in the early postnatal period, prior to hamartoma formation, rapamycin reduces hamartoma size, but also induces new morphological abnormalities in the Pten cKO retinal periphery. In contrast, administration of rapamycin after hamartoma initiation fails to reduce lesion size. We have thus generated and used an animal model of retinal PHTS to show that, although current therapies can reduce hamartoma formation, they might also induce new retinal dysmorphologies. This article has an associated First Person interview with the first author of the paper

Retinae develop an ectopic amacrine cell layer and supernumerary rod photoreceptors

<p><b>Copyright information:</b></p><p>Taken from "functions through to negatively regulate cell number in the developing retina"</p><p>http://www.neuraldevelopment.com/content/2/1/11</p><p>Neural Development 2007;2():11-11.</p><p>Published online 8 Jun 2007</p><p>PMCID:PMC1913510.</p><p></p> E18.5→8DIV retinal explants. DAPI-stained wild-type and explants. Rhodopsin expression in wild-type and + ECL retinae. Pax6 and syntaxin expression in amacrine cells in wild-type (e,e',g,g') and +ECL (f,f',h,h') retinae. Asterisks mark the ECL. The duplicated IPL is labeled by ipl' in (h'). Blue is DAPI counterstain. Average of the absolute number of DAPInuclei/layer in a standard counting field in wild-type (black bar; total DAPInuclei counted in 30 fields; ONL: 23,700; INL: 9,870; GCL: 1,776), without an ECL (grey bar; total DAPInuclei counted in 9 fields; ONL: 6,615; INL: 2,826; GCL: 498 nuclei) and +ECL (white bar; total DAPInuclei counted in 27 fields; ONL: 26,175; INL: 11,968; GCL: 1,674). Percentage of each retinal cell type based on total cell counts in wild-type (black bar; HC: 56 calbindin/7,183 DAPI; AC: 1,832 Pax6/11,696 DAPI; BP: 819 Chx10/9,302 DAPI; MG: 1,003 p27/18,465 DAPInuclei; 537 CRALBP/9,169 DAPI), without an ECL (grey bar; HC: 64 calbindin/12,960 DAPI; AC: 1,558 Pax6/10,304 DAPI; BP: 1,077 Chx10/10,171 DAPI; MG: 430 p27/9,966 DAPI; 332 CRALBP/6,773 DAPI) and +ECL retinae (white bar; HC: 11 calbindin/1,924 DAPI; AC: 2,068 Pax6/11,302 DAPI; BP: 646 Chx10/9,157 DAPI; MG: 395 p27/9,921 DAPI; 240 CRALBP/3,319 DAPI). AC, amacrine cell; BP, bipolar cell; HC, horizontal cell; MG, Müller glia

Cell-type specific roles for PTEN in establishing a functional retinal architecture.

BackgroundThe retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.Methodology/principal findingsIn the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.Conclusions/significanceWe conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina