The Trim family of genes and the retina: Expression and functional characterization

Abstract

To better understand the mechanisms that govern the development of retinal neurons, it is critical to gain additional insight into the specific intrinsic factors that control cell fate decisions and neuronal maturation. In the developing mouse retina, Atoh7, a highly conserved transcription factor, is essential for retinal ganglion cell development. Moreover, Atoh7 expression in the developing retina occurs during a critical time period when progenitor cells are in the process of making cell fate decisions. We performed transcriptome profiling of Atoh7+ individual cells isolated from mouse retina. One of the genes that we found significantly correlated with Atoh7 in our transcriptomic data was the E3 ubiquitin ligase, Trim9. The correlation between Trim9 and Atoh7 coupled with the expression of Trim9 in the early mouse retina led us to hypothesize that this gene may play a role in the process of cell fate determination. To address the role of Trim9 in retinal development, we performed a functional analysis of Trim9 in the mouse and did not detect any morphological changes in the retina in the absence of Trim9. Thus, Trim9 alone does not appear to be involved in cell fate determination or early ganglion cell development in the mouse retina. We further hypothesize that the reason for this lack of phenotype may be compensation by one of the many additional TRIM family members we find expressed in the developing retina.

Fig 1. The TRIM family of genes is expressed in the developing mouse retina.

A Genesis produced heatmap representing the microarray expression of the TRIM family of genes in (A) single cycling retinal progenitor cells (Ccnd1+), (B) developing retinal ganglion cells (Nefl+) and amacrine cells (Tcfap2β+) isolated from the developing mouse retina. The intensities of Affymetrix signals have been scaled such that a signal of 0 corresponds to a black color and a signal of 10000 corresponds to a bright red color. (C-I”) In situ hybridization was performed using the following probes: Trim62 (C-C”), Trim2 (D-D”), Trim46 (E-E”), Trim32 (F-F”), Trim44 (G-G”), Trim28 (H-H”) and Trim9 (I-I”) on retinal sections at E12.5, E14.5, E16.5. Scale bars represent 100 μm.

Fig 2. Characterization of retinal neurons in adult WT and Trim9 deficient mouse retinas using in situ hybridization.

In situ hybridization was used to identify populations of retinal neurons in WT and Trim9 knockout littermates. Probes staining all RGCs (Syn-γ; A, A’), a subset of RGCs (Cartpt; B, B’), amacrine cells (Tcfap2α; C, C’), GABAergic amacrine cells (Gad1; D, D’), glycinergic amacrine cells (Slc6a9; E, E’), horizontal cells (Sept4; F, F’), short-wave cones (Opn1SW; G, G’), rod photoreceptors (Nrl; H, H’), bipolar cells (Vsx2; I, I’), and Müller glia (Clus; J, J’) were employed. Scale bars represent 100 μm.

Fig 3. Morphological characterization of adult WT and Trim9 deficient mouse retinas using immunohistochemistry on cryosections.

Immunohistochemistry for different retinal neurons was performed on adult cryosections from WT and Trim9 knockout littermates using antibodies for RGCs, amacrine cells and horizontal cells (anti-CALB and anti-CALR; A, A’), amacrine cells (anti-TCFAP2α; B, B’), cholinergic amacrine cells (anti-CHAT; C, C’), rod photoreceptors (anti- RHO4D2; D, D’), bipolar cells (anti-VSX2; E, E’ and anti-PKCα; F, F’). DAPI (blue) shows nuclear staining. Scale bars represent 50 μm.

Fig 4. Quantitative assessment of retinal neurons in adult WT and Trim9 deficient mouse retinas using immunohistochemistry on retinal flat-mounts.

Immunohistochemistry was performed on flat-mounted adult retinas from WT and Trim9 knockout littermates. Confocal scans were performed on four different quadrants from each retina and the number of stained cells counted. Representative quadrant-matched images are shown. Antibodies for RGCs, amacrine cells and horizontal cells (anti-CALB and anti-CALR; A, A’- C, C’), a subset of RGCs (anti-BRN3A; D, D’), intrinsically sensitive RGCs (anti-OPN4; E, E’) and amacrine cells (anti-TCFAP2α; F, F’). DAPI (blue) shows nuclear staining. Scale bars represent 50 μm.

Fig 5. Visualization of RGCs and their processes in adult WT and Trim9 deficient flat-mounted mouse retinas.

Retinas obtained from WT and Trim9 knockout mice were flat-mounted and immunohistochemistry was performed using the anti-SMI-32 antibody (A, A’). Scale bars represent 50 μm.

Fig 6. Examination of RGC subset genes in WT and Trim9 deficient mice using quantitative real time PCR (qPCR).

RNA was isolated from retinas of adult WT and Trim9 knockout mice and qPCR was performed using primers specific for RGC subsets marked by expression of the genes Brn3a, Drd2, Drd4, Tbr2, Scn4, Mmp17, Cdh6, Unc5d, Jam2 and Spig1. Error bars indicating standard deviation were calculated in Microsoft Excel.