A second major population of DSGCs, On-Off DSGCs, stratify their dendrites with both On and Off SACs in the IPL, project their axons to the dorsal lateral geniculate nucleus (dLGN) of the thalamus and the SC in the dorsal midbrain, and include RGCs that respond to higher-velocity image motion in all four cardinal directions (). Is Sema6A expressed in these On-Off DSGCs? We performed whole-mount Sema6A immunohistochemistry on retinas derived from DRD4-GFP and TRHR-GFP mice, two well-characterized BAC transgenic lines that genetically label subpopulations of On-Off DSGCs (). Sema6A immunoreactivity is not detected in DRD4-GFP Figures 1 E–1E″ and S1 I–S1L″) and is observed in only a very small fraction of postnatal day 1 (P1) TRHR-GFP Figures 1 F–1F″ and S1 M–S1P″) RGC cell bodies (quantified in Figure 1 I). In addition, Sema6Afibers are not present in the outer shell of the dLGN ( Figures 1 G–1G″) or the superficial layer of the SC ( Figures 1 H–1H″), two well-characterized On-Off DSGC retinorecipient targets (), in mice heterozygous for the PLAP-Trap Sema6A allele. Moreover, we found that at P10 Sema6A immunoreactivity does not co-localize with cocaine- and amphetamine-regulated transcript (CART) ( Figures S1 Q–S1Q″), a marker for the vast majority of On-Off DSGCs, but not for Hoxd10-GFPOn-Off DSGCs (). Therefore, Sema6A is a marker of AOS RGCs, but is not expressed by On-Off DSGCs involved in image formation.

To further investigate Sema6A protein distribution, we performed ocular injections of cholera toxin subunit B conjugated with Alexa555 (CTB-555) to label RGC axon projections, including those to the MTN (the major retinorecipient target of On DSGCs tuned to slow motion detection along the dorsal-ventral axis []), followed by Sema6A immunohistochemistry. We found that Sema6A immunoreactivity co-localizes with CTB-555 fluorescence ( Figures 1 C–1C″). We also utilized an alkaline phosphatase (AP) colorimetric reaction in the Sema6A heterozygous mouse line that harbors a “PLAP Trap” insertion in a Sema6A intron, and thus expresses AP robustly in axons extending from Sema6Aneurons (), to identify RGC axon projections that are Sema6A. We found that the AP reaction product is present in axons that include those which innervate the MTN ( Figures 1 D–1D″, white arrows). These results show that Sema6A is expressed in On DSGCs and strongly suggest that Sema6A protein is present in both cell bodies and axons of On DSGCs that innervate the MTN.

The transmembrane semaphorin Sema6A is expressed in On SACs and is required for certain DS responses to fast-moving objects in On-Off DSGCs (). Our analysis of Sema6A expression also revealed that it is expressed in a subset of RGCs (). To determine whether Sema6A might play additional roles in the perception of image motion, we identified the type(s) of RGCs that express Sema6A. By examining co-expression of Sema6A and GFP in whole-mount retinas from transgenic lines that express GFP in specific subtypes of RGCs, we found that Sema6A is expressed in all GFPOn DSGCs labeled in the ventral retina of the SPIG1::GFP knockin mouse line () ( Figures 1 A–1A″ and S1 A–S1D″). These On DSGCs have dendrites that stratify together with On SACs in the inner plexiform layer (IPL) of the retina, and they respond to relatively slow-velocity vertical image motion (). Furthermore, their axons project to the MTN, a central AOS retinorecipient target. SPIG1::GFPRGCs are Sema6A immune-positive throughout early postnatal retina development ( Figures S1 A–S1D″ and S1 E–S1H″; quantified in Figure 1 I). In addition, Sema6A immunoreactivity is detected in most Hoxd10-GFPRGCs ( Figures 1 B–1B″; quantified in Figure 1 I) in retinas from a BAC transgenic line (Hoxd10-GFP) in which GFP is expressed in all three populations of AOS On DSGCs: On DSGCs that respond to lower-velocity upward, downward, or forward motion, and also a small population of AOS On-Off DSGCs that respond to forward lower-velocity image motion (). Thus, RGCs that project to AOS structures in the brain express Sema6A.

(I) Quantification of the ratio of Sema6A and GFP double-immuno-positive cells to total GFP + cells in SPIG1::GFP, Hoxd10-GFP, DRD4-GFP, and TRHR-GFP retinas throughout early-postnatal retinal development (n ≥ 5 sample areas from 2–4 retinas of each genotype). For SPIG1::GFP retinas; 68 GFP + and Sema6A + cells/68 total cells at P1 (100%), 39/39 at P3 (100%), and 69/69 at P10 (100%). For Hoxd10-GFP retinas; 49/54 at P1 (90.74%), 96/127 at P3 (75.59%), and 123/137 at P10 (89.78%). For DRD4-GFP retinas; 9/215 at P1 (4.19%), 3/87 at P3 (3.45%), and 2/216 at P10 (0.93%). For TRHR-GFP retinas; 74/325 at P1 (22.77%), 7/213 at P3 (3.29%), and 3/159 at P10 (1.89%). Error bars indicate SEM. Scale bars, 10 μm in (A) for (A)–(B″) and (E)–(F″); 100 μm in (C) for (C)–(C″); 200 μm in (D) for (D)–(D″); 200 μm in (D″); and 200 μm in (G) for (G)–(H″).

(G–H″) RGC axon targeting to the dorsal lateral geniculate nucleus (dLGN, G) and the superior colliculus (SC, H) observed in coronal brain sections following labeling by ocular injection of CTB-555 and subsequent AP enzymatic reactions (G′ and H′), showing that RGC axons innervating the dLGN “shell” region and the superficial region of SC are AP − (G″ and H″).

(E–F″) Double immunostaining of DRD4-GFP + (E–E″) and TRHR-GFP + (F–F″) On-Off DSGCs with antibodies directed against GFP (E and F) and mouse Sema6A (E′ and F′), showing that Sema6A is not expressed by DRD4-GFP + or TRHR-GFP + cells (E″ and F″).

(D–D″) Colorimetric alkaline phosphatase (AP) enzymatic activity assay in adult Sema6A heterozygous mice (this line harbors a Sema6A AP “trap” allele and so expresses AP in Sema6Atissues []) reveals in coronal brain sections that retinal axons innervating the MTN (D, labeled by CTB-555 injection) express AP (D′ and D″), providing additional evidence that Sema6A is expressed by On DSGCs that target to the MTN (n = 7 animals). (D′) is the enlarged view of the white inset in (D″).

(C–C″) Postnatal day 2 (P2) coronal brain sections showing that the axons innervating the medial terminal nucleus (MTN), labeled here by ocular injection of CTB-555 (C), are Sema6A immuno-positive (C′ and C″).

(A–B″) Double immunostaining of SPIG1::GFP + (A–A″) and Hoxd10-GFP + (B–B″) RGCs by antibodies directed against GFP (green in A and B) and mouse Sema6A (red in A′ and B′), showing that both SPIG1::GFP + and Hoxd10-GFP + cells express Sema6A (merged in A″ and B″, respectively).

Sema6A Is Required for the Development of AOS Trajectories

−/− null mutants using ocular CTB injections. In WT adult mice, innervation of the MTN, a major On DSGC central target, can be visualized on the ventral brain surface in whole-mount preparations (white arrows in −/− mutants are greatly diminished (−/− mutants (n = 13 animals, phenotype observed with complete penetrance and expressivity). To further investigate On DSGC-MTN innervation, we used two mouse lines that genetically label On DSGCs: SPIG1::GFP, which labels a subset of MTN-innervating On DSGCs from embryonic stage e12.5 to postnatal developmental stages up to ∼P13 ( Yonehara et al., 2008 Yonehara K.

Shintani T.

Suzuki R.

Sakuta H.

Takeuchi Y.

Nakamura-Yonehara K.

Noda M. Expression of SPIG1 reveals development of a retinal ganglion cell subtype projecting to the medial terminal nucleus in the mouse. Yonehara et al., 2009 Yonehara K.

Ishikane H.

Sakuta H.

Shintani T.

Nakamura-Yonehara K.

Kamiji N.L.

Usui S.

Noda M. Identification of retinal ganglion cells and their projections involved in central transmission of information about upward and downward image motion. Dhande et al., 2013 Dhande O.S.

Estevez M.E.

Quattrochi L.E.

El-Danaf R.N.

Nguyen P.L.

Berson D.M.

Huberman A.D. Genetic dissection of retinal inputs to brainstem nuclei controlling image stabilization. Osterhout et al., 2014 Osterhout J.A.

El-Danaf R.N.

Nguyen P.L.

Huberman A.D. Birthdate and outgrowth timing predict cellular mechanisms of axon target matching in the developing visual pathway. −/− mutants and found that both SPIG1::GFP+ (+ (−/− mutants, providing additional support for the conclusion that Sema6A is required for On DSGC connectivity with the MTN. Figure 2 Sema6A Is Required for the Development of AOS Trajectories Show full caption (A and B) Whole-mount ventral view (A) and cross-sectional view (B, higher magnification to reveal the MTN) of adult WT mouse brains with ocular injections of CTB-488 and CTB-555 bilaterally. WT mice exhibit robust RGC-MTN innervation (white arrows in A and B) (n = 7 WT mice). (A′ and B′) RGC-MTN innervation in Sema6A−/− mutants is greatly diminished, as observed in a ventral whole-mount view (A′) and a cross-sectional view (B′) (n = 13 Sema6A−/− mutants, with phenotypes observed with full penetrance and expressivity). (C–D′) The On DSGC-MTN axon trajectory is illuminated by the SPIG1::GFP reporter in Sema6A+/− (C and D) and Sema6A−/− brains (C′ and D′). RGC-MTN innervation is prominent in P2 control animals (yellow arrows in C and D), whereas it is mostly abolished in Sema6A−/− mutants (C′ and D′) (n = 3 animals for both genotypes). (E–F′) Additional AOS trajectories are revealed by the Hoxd10-GFP reporter in Sema6A+/− (E and F) and Sema6A−/− mice (E′ and F′). Compared to control (white arrow in E), dorsal terminal nucleus (DTN) innervation is reduced in Sema6A−/− mutants (red arrowhead in E′). Innervation of the nucleus of optic tract (NOT) is apparently preserved in Sema6A−/− mutants (compare F and F′) (n ≥ 4 animals for both genotypes). (G–H′) TRHR-GFP+ On-Off DSGC axons project to the shell of dLGN (G) and the superficial layer of SC (H) in control animals. Sema6A−/− mutants (G′ and H′) exhibit similar innervation patterns compared to controls (G and H) (n = 3 animals for both genotypes). Scale bars, 1 mm in (A) for (A) and (A′); 200 μm in (B) for (B) and (B′); 1 mm in (C) for (C) and (C′); 200 μm in (D) for (D) and (D′); 200 μm in (F′) for (E)–(F′); 200 μm in (G′) for (G) and (G′); and 200 μm in (H′) for (H) and (H′). The selective expression of Sema6A in cell bodies and axons of On DSGCs raised the possibility that Sema6A directly participates in the development of these neurons. We first analyzed the central projections of On DSGCs in wild-type (WT) and Sema6Anull mutants using ocular CTB injections. In WT adult mice, innervation of the MTN, a major On DSGC central target, can be visualized on the ventral brain surface in whole-mount preparations (white arrows in Figure 2 A) and also in coronal brain sections (white arrow in Figure 2 B). Axon projections to the MTN in Sema6Amutants are greatly diminished ( Figures 2 A′ and 2B′). Although there is residual innervation of the dorsal-most and ventral-most regions of the MTN, axon projections to most of the MTN are absent in Sema6Amutants (n = 13 animals, phenotype observed with complete penetrance and expressivity). To further investigate On DSGC-MTN innervation, we used two mouse lines that genetically label On DSGCs: SPIG1::GFP, which labels a subset of MTN-innervating On DSGCs from embryonic stage e12.5 to postnatal developmental stages up to ∼P13 (), when the GFP signal becomes very weak; and Hoxd10-GFP, which labels all On DSGCs from late embryonic stages through adulthood (). We observed GFP expression following introduction of these GFP alleles into Sema6Amutants and found that both SPIG1::GFP Figures 2 C–2D′) and Hoxd10-GFP Figures S2 A and S2B′) projections that innervate the MTN are greatly diminished in Sema6Amutants, providing additional support for the conclusion that Sema6A is required for On DSGC connectivity with the MTN.

Simpson, 1984 Simpson J.I. The accessory optic system. −/− mutants. Compared to the controls, Sema6A−/− On DSGC axonal projections to the DTN are reduced, but still present ( In addition to the MTN, AOS On DSGCs also innervate two additional midbrain targets: the DTN and the NOT (). To characterize On DSGC axonal innervation of these AOS retinorecipient targets, we performed ocular CTB injections and genetic labeling experiments in control and Sema6Amutants. Compared to the controls, Sema6AOn DSGC axonal projections to the DTN are reduced, but still present ( Figures 2 E, 2E′, S2 G, and S2G′), whereas innervation of the NOT is unaffected ( Figures 2 F, 2F′, S2 H, and S2H′). Thus, Sema6A is partially required for On DSGC-DTN innervation, but is apparently dispensable for projections to the NOT.

−/− mutants raise the critical issue of whether On DSGC cell number is altered in these mutants. To address this question, we used the SPIG1::GFP allele to label On DSGC cell bodies throughout retina development. Sema6A−/−; SPIG1::GFP+ RGC cell number is the same as what we observe in Sema6A+/−; SPIG1::GFP+ RGCs at e14.5 (+ cell numbers at later embryonic stages, when the On DSGC-MTN innervation defects in Sema6A−/− mutants are already prominent (at e16.5, Pequignot et al., 2003 Pequignot M.O.

Provost A.C.

Salle S.

Taupin P.

Sainton K.M.

Marchant D.

Martinou J.C.

Ameisen J.C.

Jais J.P.

Abitbol M. Major role of BAX in apoptosis during retinal development and in establishment of a functional postnatal retina. + On DSGCs we observe in SPIG1::GFP; Sema6A+/− retinas from late embryogenesis to P10 (−/− retinas that GFP+ RGC cell number exhibits a greater decrease than in controls, starting at P1 and continuing throughout early postnatal retina development (−/− retinas exhibit 68% of the number of GFP+ RGCs observed in SPIG1::GFP; Sema6A+/− retinas (control GFP+ cell number in the ventral retina, 304.7 ± 9.4; Sema6A−/− GFP+ cell number in the ventral retina, 95.8 ± 7.1; mean ± SEM, p < 10−6). Cell apoptosis analysis also reveals a significant increase in SPIG1::GFP; Sema6A−/− retinas at e18.5 as compared to controls with respect to RGCs that are immuno-positive for both SPIG1::GFP and Cleaved-Caspase3 (−/−; SPIG1::GFP+ RGCs undergo cell apoptosis during late embryogenesis compared to apoptosis normally observed at this stage in Sema6A+/−; SPIG1::GFP+ retinas. Therefore, Sema6A+ On DSGC axons project to, and elaborate within, the MTN region during embryonic development. In the absence of Sema6A, On DSGCs exhibit aberrant MTN innervation that is apparent at e16.5 and at e18.5, developmental time points when On DSGC cell number in this mutant remains similar to controls. However, by P1, MTN innervation defects become apparent, as does enhanced On DSGC apoptosis. The On DSGC-MTN innervation defects observed in Sema6Amutants raise the critical issue of whether On DSGC cell number is altered in these mutants. To address this question, we used the SPIG1::GFP allele to label On DSGC cell bodies throughout retina development. Sema6A; SPIG1::GFPRGC cell number is the same as what we observe in Sema6A; SPIG1::GFPRGCs at e14.5 ( Figures 3 F, 3F′, S3 F, and S3F″). Importantly, loss of Sema6A does not affect SPIG1::GFPcell numbers at later embryonic stages, when the On DSGC-MTN innervation defects in Sema6Amutants are already prominent (at e16.5, Figures 3 G, 3G′, S3 G, and S3G′; and at e18.5, Figures 3 H, 3H′, S3 H, and S3H′). RGC apoptosis during retinal development is normally apparent by e18.5 (), and this is reflected in the reduction of GFPOn DSGCs we observe in SPIG1::GFP; Sema6Aretinas from late embryogenesis to P10 ( Figures 3 F–3I and S3 F–S3K, quantified in Figure 3 J). However, we observe in SPIG1::GFP; Sema6Aretinas that GFPRGC cell number exhibits a greater decrease than in controls, starting at P1 and continuing throughout early postnatal retina development ( Figures 3 F′–3I′ and S3 F′–S3K′, quantified in Figure 3 J). By P10, SPIG1::GFP; Sema6Aretinas exhibit 68% of the number of GFPRGCs observed in SPIG1::GFP; Sema6Aretinas (control GFPcell number in the ventral retina, 304.7 ± 9.4; Sema6AGFPcell number in the ventral retina, 95.8 ± 7.1; mean ± SEM, p < 10). Cell apoptosis analysis also reveals a significant increase in SPIG1::GFP; Sema6Aretinas at e18.5 as compared to controls with respect to RGCs that are immuno-positive for both SPIG1::GFP and Cleaved-Caspase3 ( Figures S4 G–S4I′, quantified in Figure S4 J); this shows that an increased number of Sema6A; SPIG1::GFPRGCs undergo cell apoptosis during late embryogenesis compared to apoptosis normally observed at this stage in Sema6A; SPIG1::GFPretinas. Therefore, Sema6AOn DSGC axons project to, and elaborate within, the MTN region during embryonic development. In the absence of Sema6A, On DSGCs exhibit aberrant MTN innervation that is apparent at e16.5 and at e18.5, developmental time points when On DSGC cell number in this mutant remains similar to controls. However, by P1, MTN innervation defects become apparent, as does enhanced On DSGC apoptosis.