The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen–induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokine signaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.Yoshimoto T., Chaya T., Varner L.R., et al. The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina. Journal of Biological Chemistry 299, 105461 (2023); https://doi.org/10.1016/j.jbc.2023.105461

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control

<div><p>Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the <i>Npy2r-Cre</i> transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of <i>Npy2r</i> encoding a ciliary GPCR. By mating <i>Npy2r-Cre</i> mice with <i>Ift80</i> flox mice, we generated <i>Ift80</i> conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that <i>Ift80</i> CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.</p></div

A screen to identify ciliary GPCRs using NIH3T3 cells.

<p><b>A</b>) Strategy of the screen to identify ciliary GPCRs. We constructed plasmids expressing 138 non-odorant GPCRs fused with a FLAG or mCherry tags at their C-terminals. These constructs were transfected into NIH3T3 cells. At 24 hrs after transfection, ciliogenesis was induced by serum starvation. At 48 hrs after transfection, subcellular localization of GPCRs was analyzed by immunostaining using an anti-FLAG or anti-mCherry antibody. Cilia were marked with an anti-acetylated α-tubulin antibody. <b>B, C</b>) Localization of FLAG-tagged Sstr3 (B) and MCHR1 (C) to cilia in NIH3T3 cells. GPCRs were stained with an anti-FLAG antibody (red) and cilia were stained with the anti-acetylated α-tubulin antibody (green). Co-localization of FLAG (red) and acetylated α-tubulin (green) signals was observed in cilia. Nuclei were stained with DAPI (blue). Arrowheads indicate cilia. Scale bars, 10 μm (<b>B, C</b>) and 5 μm (insets in <b>B, C</b>).</p

Subcellular localization of Npy2r and BAC-Npy2r-Cre transgenic mouse.

<p><b>A)</b> The subcellular localization of Npy2r. FLAG-tagged Npy2r was expressed in the NIH3T3 cells. Cells were stained with the anti-acetylated α-tubulin (green) and anti-FLAG (red) antibodies. FLAG-tagged Npy2r signals were observed in NIH3T3 cells (red). Co-localization of Npy2r and acetylated α-tubulin signals was observed in cilia (arrowheads). Nuclei were stained with DAPI (blue). <b>B</b>) Diagram representing the BAC-Npy2r-Cre transgene construct. <b>C–L</b>) Expression of mTFP1 in the brains of <i>R26-CAG-LoxP-mTFP1</i>; <i>BAC-Npy2r-Cre</i> transgenic mice (C, D, G-I, K, L) and control mice (E, F). BAC-Npy2r-Cre transgenic mice were crossed with reporter mice, R26-CAG-LoxP-mTFP1, to detect the expression of the Cre recombinase in BAC-Npy2r-Cre transgenic mice. Dorsal (C, E), ventral (D, F, G) and coronal (H, I) views of the brain are shown. Broad expression of mTFP1 in the brain including the hippocampus, cerebral cortex, and hypothalamus (paraventricular nucleus, arrowheads in G, I) was observed. In the arcuate nucleus, Npy2r (green in J, arrowheads) colocalized with Adcy3 (a ciliary marker, red in J) in cilia. Npy2r-stained cilia (red) were often observed in mTFP1-positive cells (green in K). Preabsorption of the anti-Npy2r antibody with a synthetic Npy2r-peptide antigen abolished the Npy2r staining in the cilia (L). Scale bars, 10 μm (A, J), 5 μm (the inset in A), 5 mm (C–F), 1 mm (G–I), and 20 μm (K, L).</p

Newly identified GPCRs localized to cilia.

<p><b>A–C</b>) Three GPCRs, PRLHR (A), NMUR1 (B) and NPFFR1 (C), were identified to localize to primary cilia. GPCRs were stained with the anti-FLAG antibody (red) and cilia were stained with the anti-acetylated α-tubulin antibody (green). Co-localization of FLAG (red) and acetylated α-tubulin (green) signals was observed in the cilia. <b>D, E)</b> NMUR2 (D) and Npffr2 (E), highly conserved paralogs of NMUR1 and NPFFR1, respectively, did not localize to cilia. GPCRs were stained with the anti-FLAG antibody (red) and cilia were stained with the anti-acetylated α-tubulin antibody (green). FLAG (red) and acetylated α-tubulin (green) signals were non-overlapped. Nuclei were stained with DAPI (blue). Arrowheads indicate cilia. Scale bars, 10 μm (A-E) and 5 μm (insets in A-E).</p

Prlhr localizes to cilia in the hypothalamic third ventricle.

<p><b>A–D</b>) Prlhr was localized to the ventricular cilium in the adult mouse brain. Frozen sections containing the third ventricle of the adult mouse brain were immunostained with an anti-Prlhr antibody (green). The Prlhr signal was observed in cilia protruding from cells on the surface of the third ventricle (arrowheads in A, B, D). Preabsorption of the anti-Prlhr antibody with a synthetic Prlhr-peptide antigen abolished the Prlhr staining in the cilia (C). Cilia were coimmunostained with an anti-Arl13b antibody (D, red). Cilia with the Prlhr signal (arrowheads) were not clustered together, unlike the multiple cilia of ependymal cells that lacked the Prlhr signal (arrows). <b>E–H</b>) Ventricular Prlhr-positive cilia (red) were double stained with GFAP (green) which stains a subtype of tanycytes. A partial population of the GFAP-positive tanycytes (green) possesses Prlhr-positive cilia (arrowheads). Nuclei were stained with DAPI (blue). V: third ventricles. Scale bars, 20 μm (the left panel in A; B, C, F, G), 10 μm (the right panel in A; H), 5 μm (D), and 40 μm (E).</p

A short isoform of dopamine D2 receptor (DRD2S) efficiently localizes to cilia.

<p><b>A</b>) Schematic diagrams of DRD2 isoforms, DRD2S and DRD2L. DRD2L differs from DRD2S in the presence of a 29-amino-acid insertion in the third intracellular loop. <b>B–D</b>) Subcellular localization of DRD2 isoforms. FLAG-tagged DRD2L (B) or DRD2S (C) was expressed in the NIH3T3 cells. Cells were stained with the anti-acetylated α-tubulin (green) and anti-FLAG (red) antibodies. DRD2L was mainly accumulated in perinuclear Golgi apparatuses-like structures but was barely localized to the cilia (B). In contrast, DRD2S was clearly enriched in cilia (C). Ratios of localization of DRD2S and DRD2L to cilia in ciliated NIH3T3 cells were quantified (D). The average values from three independent transfection experiments are shown (*<i>p</i><0.03). Nuclei were stained with DAPI (blue). Arrowheads indicate cilia. Scale bars, 10 μm (B, C) and 5 μm (insets in B, C).</p