FACS purification of retrogradely labeled mouse retinal ganglion cells at E16.5. A, Schematic of retrograde labeling and cell purification methods for microarray analysis. Diagram of the ventral view of embryonic brain depicts application of rhodamine dextran 3000 MW dye (RD3000) to a unilateral transected optic tract. B, Whole-mount preparations of E16.5 retina. RD3000 fully labels axons and cell bodies of E16.5 RGCs within 2 hours of incubation after dye application to the optic tract. Extra-VT RGCs are labeled in retina contralateral to labeled optic tract and VT RGCs are labeled in the ipsilateral retina. C, Coronal vibratome sections of E16.5 retina retrogradely labeled with RD3000 show specific labeling of contralateral and ipsilateral RGCs in their respective retinal domains (VT domain marked with white bracket). D, Fresh E16.5 retinas are screened for appropriate RD3000 labeling of ipsilateral and contralateral RGCs before FACS. E, FACS purification of ipsilateral and contralateral RGC populations retrogradely labeled with RD3000 with DAPI exclusion of nonviable cells. ∼3000 ipsilateral and ∼20,000 contralateral RGCs (P4 gate) are purified from two litters of E16.5 embryos. F, Cells purified by FACS are enriched in RGC marker Islet1/2 compared with presorting. G, Zic2 and SERT are enriched in the ipsilateral RGC cell population isolated by FACS compared with the contralateral RGCs. D, dorsal, V, ventral, N, nasal, T, temporal. Scale bars, 250 μm.
Microarray analysis of ipsilateral and contralateral RGCs purified at E16 reveals distinct expression profiles. A, Microarray analysis reveals 298 and 40 unique genes at least two-fold increased in ipsilateral or contralateral RGCs, respectively (p ≤0.05, Benjamini–Hochberg correction). B, Distribution of differentially expressed genes shows that the majority are upregulated in ipsilateral RGCs.
Expression patterns of known ipsilateral and contralateral RGC genes. Combined ISH and IHC analysis at E15.5 shows colocalization of Zic2 protein expression with Zic2 (A) and SERT (B) mRNA in ipsilateral RGCs in the VT retina. C, In contrast, contralateral RGC marker Brn3a shows complementary expression to Zic2 in immunostained sections in which all RGCs are labeled with a pan-Brn3 antibody. These patterns of expression were used as standards for expression analysis of microarray gene candidates. rpe, retinal pigment epithelium; nb, neuroblast layer; rgc, retinal ganglion cell layer; DT, dorsotemporal retina; VT, ventrotemporal retina. Scale bars, 250 μm (A and B) and 100 μm (C).
Genes enriched in the contralateral RGC population. ISH analysis at E15.5 shows complementary expression of Tbx20 (A), Sema3e (B), and Igf1 mRNA (contralateral RGCs) with Zic2 (ipsilateral RGCs). Fgf12 is highly expressed in Zic2– RGCs, with only trace levels of expression in Zic2+ cells. All candidate genes are expressed in Islet1/2+ (differentiated) RGCs. Scale bars, 250 μm.
Igfbp5 is expressed in the VT RGC zone in Zic2+ cells. ISH analysis at E15.5. A, Igfbp5 mRNA is expressed in a subset of Zic2+ RGCs in VT retina (red arrows) and a few Zic2– RGCs in dorsal retina. Red brackets mark the ipsilateral RGC domain. B, Igfbp5 (red brackets) and Igf1 are expressed in a complementary pattern throughout the retina. C, Igfbp5 expression is concomitantly reduced in the Foxd1 KO mutant compared with WT littermates; this reduction correlates with the similar reduction in Zic2 expression in the VT retina of the Foxd1 KO mutant. Red brackets mark the expected ipsilateral RGC domain. Scale bars, 250 μm.
Sox2, Math5, and cyclin D2 are enriched in the ipsilateral RGC population in addition to progenitor cells throughout the retina. ISH analysis at E15.5. A, Sox2 mRNA is expressed in the neuroblastic layer and Zic2+ RGCs but not Zic2–/Islet1/2+ (differentiated) RGCs extra-VT retina. B, Similarly, Math5 mRNA is expressed in Zic2+ RGCs located at the periphery of the VT retina in the RGC layer as well as in RGC precursors in the neuroblastic layer (red arrows). C, Cyclin D2 mRNA is expressed in the basal process of cells within the peripheral margin of the retina, particularly in the ventral retina, and also at low levels in the Zic2+ RGC zone at E15.5. D, The asymmetric expression of cyclin D2, with higher levels in ventral retina, is more pronounced at E13.5. Red brackets mark the Zic2+ ipsilateral RGC domain. Scale bars, 250 μm.
Cyclin D2 is enriched in the ventral peripheral retina during the temporal window of ipsilateral RGC genesis. IHC in coronal cryosections of E11.5 (A), E12.5 (B), E14.5 (C), E15.5 (D), and E16.5 (E) retina. Cyclin D2 is expressed in the lens and the retinal marginal zone bordering RGCs (labeled with Brn3). Cyclin D2 is highly expressed in the ventral periphery compared with the dorsal periphery within neural retina (white arrows). The cyclin D2 and Brn3 domains are separated by a large gap at E11.5 (A). At E13.5 (B) and E14.5 (C), cyclin D2+ cells intermingle with Brn3+ RGCs only in ventral retina (yellow arrows point to boundary) and have sharp boundaries in dorsal retina. At E16.5 (E), cyclin D2 expression is reduced in the retinal periphery and is no longer asymmetric in dorsal and ventral retinal (white arrows). Scale bars, 100 μm.