Article Figures & Data
Figures
- Fig. 1.
Complementary immunostaining of EphA4 (A, C, E) and ephrin-A5 (B, D, F) in sagittal frozen sections at stage 27 (E5) (A–D) and stage 32 (E7) (E, F).Arrowheads in B, D, andF point to the boundaries of reciprocal EphA4–ephrin-A5 expression within the cerebellar and isthmal regions. Sections inA, C, and E are adjacent to those in B, D, and F.C, D, High magnification of the isthmal region from sections lateral to A and Bshowing the complementarity of staining within the isthmal region. ca, Cerebellar anlage; cb, cerebellum;is, isthmus; tc, tectum; V, ventricle. Scale bars, 200 μm.
- Fig. 2.
EphA4 immunostaining in paraffin sections at various stages of early cerebellar development. A, Transverse section of a cerebellum at stage 27 (E5) with an adjacent nissl stain in B. Note EphA4 expression in the ventricular zone (vz), in the medial (M) and lateral (L) subdivisions of the mantle zone, and on a fiber tract (ft) separating the medial and lateral subdivisions. C, High magnification of the cerebellar anlage in A with an adjacent Tau staining inD showing the fibrous nature of the tract separating the medial and lateral subdivisions. E, Transverse section of stage 29 (E6) cerebellum with an adjacent nissl stain inF. The expression is similar to stage 27 except for downregulation in the vz and an area in the cerebellum that is devoid of EphA4 expression (E,egl). G, Transverse section through the caudal cerebellum at stage 29 (E6) with adjacent nissl stain in H. EphA4 expression appears more extensive in caudal sections as compared with rostral sections (E). egl, Future external granule cell layer as defined by Feirabend (1990); ft, fiber tract; hb, hindbrain; mz, mantle zone;M, medial subdivision; L, lateral subdivision; V, ventricle; vz, ventricular zone. Scale bars, 200 μm.
- Fig. 3.
EphA4 immunostaining in transverse (A, B), coronal (D–F), and sagittal (C) paraffin sections at stages 31 (E7, A), 34 (E8,B), 37 (E11, C, D), 45 (E20, E), and P7 (F). Parasagittal banded EphA4 expression is apparent at E8 (B) and persists to posthatch (F).Asterisk in A refers to the segment with the lower level of EphA4 expression. Roman numeralsrefer to cerebellar lobules. A–E in Band C refer to Purkinje cell parasagittal bands labeled alphabetically from the midline. cbn, Deep cerebellar nuclei; cc, ventral cerebellar commissure;eff, corticonuclear efferents; egl, external granular layer; L, lateral subdivision;M, medial subdivision; V, ventricle. Scale bar, 200 μm.
- Fig. 4.
Dorsal and ventral views of EphA4 (A, B), ephrin-A5 (C,D), EphA3 (E, F), and ephrin-A2 (G, H) whole-mount immunostaining on chick cerebella taken between stages 36 (E10) and 38 (E12). A, B, C,D, and E refer to Purkinje cell domains labeled alphabetically from the midline. Asterisks inB, D, and H point to EphA4 staining (B) or its corresponding location (D, H). Roman numeralsrefer to cerebellar lobules. Scale bar, 200 μm.
- Fig. 5.
Double immunostaining of EphA4 (Band D) and calbindin (A and C) in coronal plane of frozen sections of a stage 40 (E14) cerebellum. A, B, Purkinje cell band, C, is devoid of EphA4 labeling but is calbindin positive. C, D, In most areas, EphA4 and calbindin bands colocalize. B,C, and D in A andB refer to Purkinje cell parasagittal bands. Scale bar, 100 μm.
- Fig. 6.
Complementary immunostaining of ephrin-A5 (A, C, E) and EphA4 (B, D, F) in coronal frozen sections at stage 38 (E12). A,B, C, and D refer to Purkinje cell domains labeled alphabetically from the midline.Roman numerals refer to cerebellar lobules. Scale bar, 200 μm.
- Fig. 7.
Double immunostaining of EphA4 (redin B, C, E,F, H, I, K,L, N, O, Q, and R) and ephrin-A2 (green inA, C, D, F,G, I, J, L,M, O, P, andR) in coronal plane of frozen sections of a stage 37 (E11) chick cerebellum. Sections in A–O are within 20–40 μm from those in Figure 9. A, B,C, D, and E refer to Purkinje cell compartments labeled alphabetically from the midline.Roman numerals refer to cerebellar lobules.cc, Cerebellar commissure. Scale bar, 100 μm.
- Fig. 8.
A–D, Immunostaining on coronal (A, B) and sagittal (C,D) adjacent frozen sections of EphA3 (A,C) and ephrin-A5 (B, D) at stage 37 (E11). Complementary expression is observed in Purkinje cell domains in lobule VII (A, B) and in the external granule cell layer along the anterior–posterior axis (C, D). E,F, EphA3 immunostaining in a coronal section taken from lobule IX of a stage 37 (E11) chick cerebellum. F, High magnification of inset in E.egl, External granule cell layer; gcr, granule cell ribbon; V, ventricle. A,B, C, D, andE refer to Purkinje cell compartments labeled alphabetically from the midline. Roman numerals refer to cerebellar lobules. Scale bar, 200 μm.
- Fig. 9.
Double immunostaining of EphA4 (redin B, C, E,F, H, I, K,L, N, and O) and BrdU (green in A, C,D, F, G, I,J, L, M, andO) in coronal plane of frozen sections of a stage 37 (E11) cerebellum that was pulsed with BrdU on E5. In all lobules, late-born cells colocalize to the EphA4 negative band,C, E, and to band A (or medial portion of band A) where a lower level of EphA4 immunostaining is detected (see Results). For technical reasons (see Material and Methods, BrdU labeling), lower EphA4 expression inband A of lobule VII is obscured in H and is better observed in a 20 μm adjacent section in Figure7H. P–R, Double immunostaining of EphA4 (red in Q and R) and BrdU (green in P andR) on E11 chick cerebellum that was pulsed with BrdU on E3. Early-born cells colocalize with EphA4-positive bands.A, B, C, D, and E refer to Purkinje cell bands labeled alphabetically from the midline. Roman numerals refer to cerebellar lobules. Scale bar, 100 μm.
- Fig. 10.
Double immunostaining of the granule cell marker Pax6 (green in A,C, D, F, G, and I) with EphA4 (red inB and C) or ephrin-B1 (redin E and F) or EphB2 (red in H and I) in the coronal plane of a frozen section of a stage 39 (E13) cerebellum.A–C, Granule cells (arabic numerals inA) migrate as ribbons at the boundaries of EphA4 Purkinje cell bands (labeled alphabetically from the midline).D–I, From E9 to E14, both the EphB2 receptor and its ligand eprhin-B1 are expressed on the premigratory zone (pmz) of the external granular layer (egl), the migrating granule cell ribbons (gcr), and the parallel fibers (pf). L, Double immunostaining of ephrin-B1 (red) and the glial marker vimentin (green) showing a lack of colocalization between the two. Arrows in L point to glial cell bodies. J, K, Double immunostaining of the Purkinje cell marker, calbindin (green), with either eprhin-B1 (red in J) or EphB2 (red in K) at E17. By E15, both EphB2 and ephrin-B1 expression appear localized to the parallel fibers, and a lower level of expression is observed in the internal granule cell layer. gcr, Granule cell raphe; igl, internal granule cell layer; pf, parallel fibers;pmz, premigratory zone of the external granule cell layer. Scale bar, 100 μm.
Tables
- Table 1.
Distribution patterns of Eph receptors and ephrins within parasagittal domains of Purkinje cells during the middle period of cerebellar development
Zone/lobules EphA4 Ephrin-A5 EphA3 Ephrin-A2 Posterior IX A (lower), B, D Absent Homogeneous-EGL A (higher), B, D Figs. 4, 7,9 Fig. 4 Figs. 4, 8 Figs. 4, 7 VIII1-a A (Lm–Hl), B, D C, E Homogeneous-EGL A (Hm–Ll), B, D Figs. 4, 7, 9 Fig.4, 6 Figs. 4, 8 Figs. 4G, 7D Central VI, VII M, A (lateral), B (Hm–Lc–Hl), D A1-b (medial), C, E A (medial), B, D (low) M, A (medial), B (Lm–Hc–Hl), D Figs. 4,6, 7, 9 Figs. 4C, 6C Figs. 4, 8 Figs. 4, 7 Anterior I–V M, A (lateral), B (medial), D A (medial), B (lateral), C Absent (stops at lobule V) M, A (lateral), B (medial) Figs. 4, 6, 7 Figs. 4, 6 Fig. 4 Figs. 4,7 Gradients within bands are described only if relevant to the localization of late born cells or the expression of a cognate ligand. M refers to the thin zone bordering the midline in band A (Fig.4B, midline asterisk). EGL, external granule layer; Hm, high medial, Hl, high lateral, Lc, low central, Lm, low medial, Ll, low lateral.
↵F1-a Except for the expression of ephrin-A5, this lobule would be classified as a posterior lobule because the expression pattern of the other molecules is similar to that of lobule IX.
↵F1-b The expression of ephrin-A5 in band A of lobule VII is uncertain.
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