Higenell V , Han SM , Feldheim DA , Scalia F , Ruthazer ES .

MOP-77567 Canadian Institutes of Health Research , R01 EY014689 NEI NIH HHS , R01 EY014689-09 NEI NIH HHS , R01-EY014689 NEI NIH HHS

	Figure 1. Ephrin‐A expression. (A) Dorsal (Ai) and side (Aii) views of a stage 48 whole‐mount Xenopus brain reacted with the negative control AP‐only probe. The tissue is a pale uniform pink and has no discernible staining pattern. Rostral (R), caudal (C), dorsal (D), and ventral (V) axes are labeled, and this orientation is consistent in all other whole‐mount images. (B–C) Patterns of ephrin‐A expression (EphA3‐AP binding) in representative stage 45 (B) and 48 (C) brains (dorsal: Bi, Ci, side: Bii, Cii). Additional examples of ephrin‐A tectal expression patterns (Biii, Ciii) are shown next to examples of the bulk DiI‐labeled retinal projection (Biv, Civ) in animals of the same ages (RGCs, green; autofluorescence, red). Two‐photon optical section at stage 45 (Biv) and z‐projection at stage 48 (Civ) highlight the relative location of retinorecipient tectal neuropil. (D–E) Ephrin‐A expression and retinal innervation pattern in stage 59 (D) and 66 (E) brains; dorsal views are above (Di, Diii, Ei, Eiii), and side views are below (Dii, Div, Eii, Eiv). Ephrin‐As are intensely expressed at stage 45 and 48 in the hypothalamus and in a very clear high caudal to low rostral gradient in the tectum. The gradient of tectal ephrin‐A expression shifts slightly in late developmental stages to a low rostrolateral to high caudomedial gradient. Throughout development, retinorecipient tectal neuropil is located rostrolateral to the caudal pole of the tectum and to the peak of ephrin‐A expression but within the zone of graded ephrin‐A expression. Paired dorsal and side view images are of the same brains for A–E except Dii, for which a more representative side view image was chosen. Scale bars: A–C = 100 μm, D–E = 500 μm. aob: accessory olfactory bulb; ch: cerebral hemisphere; di: diencephalon; hyp: hypothalamus; ic: implantation cones of the olfactory bulb; ob: olfactory bulb; pr: preoptic area; tec: tectum; telc: caudal region of the telencephalon.
	Figure 2. EphA expression. (A,B) Dorsal views of stage 48 (A) and stage 59 (B) brains showing EphA expression. (A′,B′) Side views of the same brains. EphAs are highly expressed throughout development in the cerebral hemispheres. In younger stages, EphAs are expressed in several regions of the diencephalon and faintly in rostral tectum. From stage 59 onward, a strong high rostrolateral to low caudomedial gradient is seen in the tectum. Staining is also present in older stages in the pretectum, striatum, habenular commissure, habenular nuclei, precommissural area, and the optic tract, although is notably absent in the olfactory regions. (C) Composite image showing EphA expression (left) and ephrin‐A expression (right) in the stage 66 brain. The complementarity of the gradients of expression of EphAs and ephrin‐As in the tectal lobes is striking. Scale bars: A,A′ = 100 μm, B–C = 500 μm. aob: accessory olfactory bulb; ch: cerebral hemisphere; hb: habenular nuclei; hc: habenular commissure; ic: implantation cones of the olfactory bulb; pt: pretectum; ob: olfactory bulb; ot: optic tract; p: precommissural area; st: striatum; telc: caudal region of the telencephalon.
	Figure 3 EphB expression. (A–D) Dorsal views of stage 45 (A), 48 (B), 59 (C), and 66 (D) brains stained for EphB expression. (A′–D′) Side views of brains at the same age. Dorsal and side views are of the same brains, except at stage 45, where a more representative side view was chosen. At both stage 45 and 48, EphBs are expressed in a high caudomedial to low rostrolateral gradient in the tectum and at stage 48 also in the tegmentum and optic tract. (C–D′) At st 59 and 66, EphBs are expressed uniformly across the surface of the tectum, as well as in the central anterolateral nucleus and the outer margins of the optic tract. Scale bars: A–B′ = 100 μm, C–D′ = 500 μm. cal: central anterolateral nucleus; mot: marginal zone of the optic tract; on: optic nerve; ot: optic tract; teg: tegmentum.
	Figure 4 Dorsoventral gradient of EphB expression in young tadpoles. (A) A coronal section of a stage 40/41 tadpole rostral to the tectum shows DAPI (blue), Alexa Fluor 488 dextran iontophoresed into the eye (green) and a high ventral to low dorsal gradient of EphBs in the eyes (red, ephrin‐B1‐Fc). The white box indicates the region magnified in A′. (A′) Several labeled retinal axons (green) can be seen traversing the optic chiasm. (B) A coronal section of the brain of a stage 40–41 animal, showing Alexa Fluor 488 dextran (retinal axons, green), ephrin‐B1‐Fc (EphBs, red), and DAPI (cell bodies, blue). Each channel is represented in a separate panel below the composite image, and the terminations of RGC axons in the neuropil are indicated with white arrows. Dorsal (D), ventral (V), medial (M), and lateral (L) axes are labeled. (B′) One section caudal to B (B′) is the most caudal section containing retinal axons. A distinct high dorsal to low ventral gradient of EphBs can be seen across the retinorecipient neuropil (indicated by the white bracket). (C) A graph indicating the EphB reactivity (normalized to the dorsal‐most value) across the dorsal‐most 150 μm of the retinorecipient neuropil as defined by the presence of RGC axon terminals. The black line is the average intensity, and the red line indicates the intensity of the image shown in B′. (D,D′) The high caudomedial to low rostrolateral gradient of EphBs across the dorsal surface of the tectum (D) outlines the region of tectal innervation of retinal axons (D′), as seen in two different stage 48 brains. Scale bars: A = 100 μm, A′ = 50 μm, B,B′,D,D′ = 100 μm.
	Figure 5 Ephrin‐B expression. (A,B) Dorsal views of stage 48 (A) and 59 (B) brains stained for ephrin‐B expression. (A′,B′) Side views of the same brains. Ephrin‐Bs are expressed very strongly in the cerebral hemispheres and olfactory regions at all stages. In older animals specifically, strong expression can be seen in the olfactory bulb, accessory olfactory bulb, cerebral hemispheres, and at the midline of the optic tectum, although staining is undetectable in the striatum and the tectal lobes. (C) Composite image showing EphB (left) and ephrin‐B (right) expression in the stage 66 brain. The overall expression patterns of EphBs and ephrin‐Bs are complementary; the telencephalon stains for ephrin‐Bs, while the diencephalon and optic tectum show staining only for EphBs. Scale bars: A,A′ = 100 μm, B–C = 500 μm. aob: accessory olfactory bulb; cal: central anterolateral nucleus; ch: cerebral hemisphere; ob: olfactory bulb; st: striatum.
	Figure 6 Expression of Ephs and ephrins in adult Xenopus. (A,B) A composite image of tectal lobes from early (stage 66, left) and late (right) postmetamorphic animals stained for ephrin‐A (A) and EphA (B) expression. The very striking gradients present in early postmetamorphic animals are faint if not absent in the late postmetamorphic brains stained either for EphAs or ephrin‐As. (C,D) Side view of EphA expression in an early (stage 66; C) and late postmetamorphic brain (D). EphAs are expressed in the optic tract at stage 66 but do not persist in the late postmetamorphic animal. (E,F) Tectal lobes from early (stage 66, left) and late (right) postmetamorphic animals stained for ephrin‐B (E) and EphB (F) expression. The very low expression of ephrin‐Bs and very high expression of EphBs appear to continue unchanged into adulthood. (G, H) The side view of an early (stage 66; G) and late (H) postmetamorphic brain stained for expression of EphBs. EphBs are strongly expressed in the marginal zone of the optic tract in both froglet and fully mature animal, but ephrin‐Bs were not detected in the optic tract at any timepoint. Scale bars = 500 μm. mot: marginal zone of the optic tract; ot: optic tract.
	Figure 7 Ephrin‐A and EphA expression patterns are unbanded in binocularly innervated tecta. (A) A two‐photon montage of an early postmetamorphic (stage 66) frog tectum following left tectal lobe removal at a larval stage shows a binocular banded innervation pattern 48 h after left eye wheat germ agglutinin Alexa 488 (WGA 488) injection and right eye WGA 594 injection. The arrows in A indicate bands of high intensity (strong innervation) from the right eye. The smaller structure to the left is the remains of the left tectal lobe. (B,B′) The green channel (B) and red channel (B′) of the two‐photon image seen in A. The white arrows refer to the same bands of WGA 594 maxima. (C,C′) The WGA labeling persists faintly following the ephrin staining protocol, as seen in an epifluorescence image of the entire tectal lobe (green channel: C, red channel: C′). (D) The expression of ephrin‐As in the same tectum is strongest caudomedially and decreases in a gradient rostrolaterally, similar to the expression of ephrin‐As in normal adults. This pattern does not align with the pattern of bands seen in the WGA labeling. (D′) A thermal map of the image shown in D. The images in B were collected at an angle to best visualize the banding, whereas the angle for corresponding images in C and D was optimized for the ephrin‐A gradient. (E) A two‐photon montage of another optic tectum following left tectal lobe removal and right eye injection with WGA 488 shows patches of innervation as well as unlabeled patches presumably innervated by the contralateral eye. (F,G) The bands of WGA 488 labeling persist after the ephrin staining protocol (F) but do not align with the pattern of ephrin‐A expression (G). (H) A two‐photon montage of the innervation pattern in a tectum following left tectal lobe removal and right eye injection with WGA 488. (I) The EphA expression pattern in the same tectum is strong, uniform, and unbanded. Scale bars: A–B′ = 300 μm, C–D′ = 500 μm, E = 300 μm, F,G = 500 μm, H = 400 μm, I = 500 μm.

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