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Kun-Che Chang, Catalina Sun, Xin Xia, Suqian Wu, Jeffrey L Goldberg; Growth differentiation factors regulate retinal ganglion cell development. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2596. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
What regulates neuronal differentiation and integration into mature circuits in the retinal ganglion cell (RGC)? This question has begun to be studied in neural development but also strongly impacts the adult nervous system. Here we evaluated the influence of growth and differentiation factor (GDF)-11 and -15 on RGC fate specification.
In vitro, immunofluorescence (IF) staining and flow cytometry were conducted in human embryonic stem cell (hESC). Ex vivo, quantitative polymerase chain reaction (qPCR) and Western blot (WB) were conducted in retinal tissues treating with GDF-11 and/or -15 and/or Smads inhibitors from embryonic day 14 (E14) wild type (WT) mice. In vivo, in situ hybridization (retinas dissected from E10, E12 and E18), retinal cryoseciton IF staining (postnatal day 0, P0), qPCR (P0) and WB (P0) were conducted in WT, Chx10-Cre/GDF-11 cKO, Chx10-Cre/Smad2 cKO and GDF-15 KO mice (N ≥ 6), respectively. This research was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Data were analyzed by ANOVA with Tukey’s test with P value of <0.05 considered statistically significant.
Ex vivo data of E14 retina showed that GDF-11 decreases but GDF-15 increases pro-RGC markers (Neurog2, Math5, Brn3a and RBPMS) gene expression. However, both GDFs induce Notch1, Hes5 and REST gene expression. GDF-15 induces only Smad1 but GDF-11 induces both Smad1 and Smad2 phosphorylation. Blockade of either Smad1or Smad2 phosphorylation promotes RGC markers expression. Interesting, we found that GDF-15 pretreatment attenuates GDF-11-induced Smad2 phosphorylation and RGC marker expression. In vivo, mice of GDF-15 KO showed less, but GDF-11 cKO and Smad2 cKO showed more Brn3a staining in P0 retinas. Inhibition of Smad2 signaling through drinking water to pregnant mice (E10-P0) resulted in more Brn3a staining in P0 retinas. In vitro, we confirmed the regulatory effects of GDFs on hESC-derived RGC like cell, which were similar to ex vivo studies. Epithelial-mesenchymal-transition was induced by GDF-11 and neutralized by Smad2 inhibitor or GDF-15.
This study extends our knowledge of the regulatory mechanism of GDF-11, GDF-15 and Smad2 in RGC development. Understanding the role of GDFs and their signaling pathways may provide an alternative protocol for stem cell-to-RGC differentiation when considering cell replacement therapies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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