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

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

Survey of Plk3 expression in the developing mouse retina.

<p>(A) A heatmap showing the expression of Plk family members in Math5-positive cells. The four E12 cells at the left are Math5-negative, but were previously identified as G2/M progenitor cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150878#pone.0150878.ref002" target="_blank">2</a>]. These cells are shown as a comparison. In the heatmap, the genes (in rows) expressed in isolated single retinal progenitor cells (in columns) at various stages of development from embryonic day (E)12.5 to E16.5 are shown. Higher levels of microarray signal of a given gene correspond to higher expression levels in a particular single cell and are indicated by the different shades of red (see the scale below [B]), while the absence of expression is indicated with a black square. (B) A heatmap showing G2/M cell cycle marker expression in Math5 and Plk3 expressing single cells. As in (A) the log transformed signal intensities have been scaled according to the intensity of red color, with black indicating the absence of expression signal. The eleven single cells shown on the left are included for comparison and are Plk3-negative cells that were previously identified as in the G2/M phase of the cell cycle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150878#pone.0150878.ref002" target="_blank">2</a>]. (C) <i>In situ</i> hybridization of Plk3 expression in the embryonic mouse retina. (D) <i>In situ</i> hybridization showing the expression of Tcfap2b, Tcfap2d, and Nhlh2, markers of developing amacrine interneurons, at E14.5. (E) Double fluorescent <i>in situ</i> hybridization of Math5 (green) and Plk3 (red) at E14.5. The results are quantified in (F). All scale bars represent 100 μm.</p

Survey of retinal cell types in P14 Plk3-KO retinas.

<p>Populations of retinal cells in P14 Plk3-KO retinas were compared to WT littermates using cell type-specific markers for rod photoreceptors (anti-Rhodopsin [Rho4d2], A,A’), bipolar cells (anti-Chx10, B,B’), retinal ganglion cells (anti-Brn3b, C,C’), ganglion and amacrine cells (anti-Isl1, D,D’; anti-Pax6, E,E’) and amacrine, horizontal and ganglion cells (anti-Calretinin [Calr], anti-Calbindin28k [Calb28k], F,F’). Scale bars indicate 100 μm.</p

Examination of Plk family member expression patterns and cell cycle at E14.5.

<p>The expression patterns of Plk3 (A, A’), Plk1 (B,B’), Plk2 (C,C’), and Plk4 (D,D’) in WT and Plk3-KO retinas were determined using <i>in situ</i> hybridization at E14.5. E14.5 developing WT and Plk3-KO retinas were also stained using anti-phospho-histone H3 (PH3) to mark mitotic cells (E,E’).</p

Morphological characterization of postnatal developing Plk3-KO retinas.

<p>Populations of retinal cells in postnatal day (P)7 Plk3-KO retinas were compared to WT littermates using cell type-specific markers. Specifically, populations of rod photoreceptors (anti-Rhodopsin [Rho4d2], A,A’), bipolar cells (anti-Chx10, B,B’), retinal ganglion cells (anti-Brn3b, C,C’), bipolar cells, amacrine cells and ganglion cells (anti-Isl1, D, D’), amacrine, horizontal and ganglion cells (anti-Pax6, E,E’; anti-Calretinin [Calr], anti-Calbindin28k [Calb28k], F,F’) were examined. Scale bars indicate 100 μm.</p

Assessment of cell numbers by flat mount antibody staining.

<p>To more quantitatively assess any gains/losses in cell number in the Plk3-KO retina, immunohistochemistry was performed on flat mounted retinas from adult (>P35) wildtype and Plk3-KO littermates. Confocal scans were performed on four different quadrants from each retina and representative quadrant-matched images are shown. DAPI, in blue, marks nuclei. Horizontal cells (anti-Hnf6, A,A’; anti-Calbindin28k [Calb28k], B,B’), amacrine interneurons (anti-Ap2a, C,C’), a combination of amacrine and ganglion cells (anti-Calretinin [Calr], D,D’) and retinal ganglion cells (anti-Brn3a, E,E’; anti-Opn4, F,F’) are shown. Scale bars represent 100 μm.</p

qPCR based examination of gene expression in Plk3-KO retinas.

<p>Retinas of Plk3-KO and WT littermates were isolated and hybridized to Affymetrix microarrays at various timepoints (n = 3 for each of four timepoints). Genes with significant differential expression at multiple timepoints were confirmed using qPCR at adult timepoints. (A) An examination of Plk3 expression in the Plk3 deficient mouse. An amplicon at the 3’ end of the gene showed upregulation (p<0.001), while one at the 5’ end of Plk3 was significantly downregulated (p<0.05). (B) Tac1 showed decreased expression (p<0.05), whereas retinitis pigmentosa 1 (Rp1) displayed increased expression (p<0.05).</p

Morphological characterization of adult Plk3-KO retinas.

<p>Populations of adult (>P35) retinal cells were identified using antibodies to neuron-specific markers. DAPI, in blue, marks nuclei. Rod photoreceptors (anti-Rhodopsin [Rho4d2], A,A’), bipolar interneurons (anti-Chx10, B,B’; anti-PKC-α, C,C’), Müller glia (anti-Glutamine synthetase [GS] D,D’), amacrine interneurons (anti-Pax6, E,E’; anti-Chat, F,F’), retinal ganglion cells (anti-Brn3b, G,G’), and horizontal, amacrine and ganglion cells (anti-Calretinin [Calr], anti-Calbindin28k [Calb28k], H,H’) are shown. Scale bars represent 100 μm.</p

Survival and Motor Phenotypes in FVB C9-500 ALS/FTD BAC Transgenic Mice Reproduced by Multiple Labs.

Mordes et al. (2020) did not detect the survival or motor phenotypes in C9orf72 BAC transgenic mice originally described by Liu et al. (2016). We discuss methodological differences between the Mordes and Liu studies, several additional studies in which survival and motor phenotypes were found, and possible environmental and genetic effects. First, Nguyen et al. (2020) showed robust ALS/FTD phenotypes in C9-BAC versus non-transgenic (NT) mice and that α-GA1 treatment improved survival, behavior, and neurodegeneration. The groups of Gelbard and Saxena also show decreased survival of C9-BAC versus NT mice and neuropathological and behavioral deficits similar to those shown by Liu et al. (2016). Although FVB/N mice can have seizures, increases in seizure severity and death of C9 and NT animals, which may mask C9 disease phenotypes, have been observed in recent C9-500 FVB/NJ-bred cohorts. In summary, we provide an update on phenotypes seen in FVB C9-BAC mice and additional details to successfully use this model. This Matters Arising Response paper addresses the Mordes et al. (2020) Matters Arising paper, published concurrently in Neuron