Differential expression and subcellular localization of Copines in mouse retina

Abstract

Combinatorial expression of Brn3 transcription factors is required for the development of cell‐specific morphologies in retinal ganglion cells (RGCs). The molecular mechanisms by which Brn3s regulate RGC type specific features are largely unexplored. We previously identified several members of the Copine (Cpne) family of molecules as potential targets of Brn3 transcription factors in the retina. We now use in situ hybridization and immunohistochemistry to characterize Copine expression in the postnatal and adult mouse retina. We find that Cpne5, 6, and 9 are expressed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in both amacrine cells and RGCs. Cpne4 expression is restricted to one amacrine cell population of the INL, but is specifically expressed in RGCs in the GCL. Cpne4 expression in RGCs is regulated by Brn3b both cell autonomously (in Brn3b+ RGCs) and cell nonautonomously (in Brn3b− RGCs). Copines exhibit a variety of subcellular distributions when overexpressed in tissue culture cells (HEK293), and can induce the formation of elongated processes reminiscent of neurites in these non‐neuronal cells. Our results suggest that Copines might be involved in a combinatorial fashion in Brn3b‐dependent specification of RGC types. Given their expression profile and previously proven role as Ca2+ sensors, they may participate in the morphogenetic processes that shape RGC dendrite and axon formation at early postnatal ages.

Molecular correlates of muscle spindle and Golgi tendon organ afferents

“…Muscles were kept in PBS at 4 °C until mounted in Vectashied (Vector labs). Primary antibodies used in immunohistochemistry experiments were: Rb anti-Calbindin D-28k (1:2,000) (Swant), Rb anti-Calretinin (Calb2; 1:2000) (Swant), Gp anti-tdTomato (1:32,000) 28 , Rb anti-dsRED (1:1000) (Clontech), Rb anti-GFP (1:1000) (ThermoFisher), Shp anti-GFP (1:500) (AbD Serotec), Rt anti-Troma1 (1:100) (DSHB), Rt anti-Substance P (1:200) (Santa Cruz Biotechnology), Rb anti-Rx3 (1:50,000) 65 , Gp anti-Rx3 (1:16,000) 66 , Ck anti-PV (1:30,000) 27 , Gp anti-Islet 1 (1:20,000) 67 , Rb anti-vGlut1 (1:16,000) 68 , Rb anti-VGlut2 (1:500) (Synaptic Systems), Rb S-100 (1:400) (Agilent), Ck anti-Brn3c (Pou4f3) (1:50) 69 , Ck anti-b-galactosidase (1:5,000) (Abcam), Rb anti-PCDH8 (1:250) (Millipore). Fluorophore-conjugated secondary antibodies generated in donkey (Jackson Immuno Research Laboratories) were used at 1:1000 (FITC) or 1:500 (Cy3, Cy5) in PBS, 1% BSA, 0.1% Triton X-100.…”

Insights into Potential Targets for Therapeutic Intervention in Epilepsy

“…To date, there are nine copines (1–9) identified in mammals, ubiquitously expressed in diverse tissues such as heart, lungs, spleen, kidneys, prostate and brain [ 305 , 307 , 308 , 309 , 310 , 311 , 312 , 313 ]. Although the precise function of copines in mammalian cells remains unclear, evidence suggests that they are involved in regulating signaling pathways [ 306 ], in membrane trafficking [ 314 ] and in morphogenetic processes associated with the maturation of retinal synaptic architecture [ 315 ].…”

Molecular studies into Copine-4 function in Retinal Ganglion Cells

“…GST tagged Cpne4 protein was synthesized from bacteria as described before (23). GST-Cpne4-vWAdomain and GST proteins were also synthesized similarly.…”

“…In the retina, we have previously reported that Cpne4, 5, 6 and 9 are enriched in the inner retina and they are regulated by Brn3b and Brn3a (1,23). Whereas Cpne5, 6 and 9 were expressed in most of the GCL as well as INL, Cpne4 is the only Copine specifically expressed in RGCs (with the exception of one INL amacrine cell type) (23).…”

“…In the retina, we have previously reported that Cpne4, 5, 6 and 9 are enriched in the inner retina and they are regulated by Brn3b and Brn3a (1,23). Whereas Cpne5, 6 and 9 were expressed in most of the GCL as well as INL, Cpne4 is the only Copine specifically expressed in RGCs (with the exception of one INL amacrine cell type) (23). Using over-expression studies in HEK293 cells, we found that Copines can significantly alter cell morphology, inducing elongated membrane processes similar to neurites.…”