Article Figures & Data
Figures
- Fig. 1.
Cloning and embryonic expression of chickZic genes. A, Sequence alignment of the chick Zic-1 (cZic-1) andZic-3 (cZic-3) DNA fragments with the homologous zinc finger region of the murine Zic(mZic) genes (Aruga et al., 1996) and the humanGli (hGli) genes.B, C, In situhybridization of E4.5 chick embryos using the chickZic-1 (B) and Zic-3(C) probes. Expression was seen in overlapping and yet distinct patterns. Both genes were expressed in the dorsal neural tube and the developing somites, but only Zic-3was expressed in the developing limb at this time. The area shaded inblack in A represents sequences identical to the mouse Zic-1. Hyphens indicate gaps in the sequences.
- Fig. 2.
Expression of granule cell-specific markers in the developing chick cerebellum reveals parasagittal arrays of granule cells during the early period of inward migration. Dorsal views of E9 (A) and E11 (B, C,G) chick cerebellum hybridized with theZic-1 (A, B), thePax-6 probe (C), or thePax-2 probe (G), and coronal sections of an E13 chick cerebellum hybridized with theZic-1 probe (D, E) are shown. Anterior is at the top in A–C,F, and G. Dorsal is at thetop in D and E.A–C, Linear arrays running perpendicular to the transverse folia were detected by the granule cell-specific markersZic-1 and Pax-6. D,E, Zic-1 expression was detected in granule cells in the external granule cell layer (egl), the internal granule cell layer (igl), the linear arrays spanning across the molecular layer (ml), and some scattered cells in the ml. F, The original three-dimensional reconstruction of granule cell raphes by Feirabend (adopted fromFeirabend, 1990) shows a global pattern similar to the expression pattern of Zic-1 and Pax-6.G, Pax-2 is expressed most strongly in the posterior subset of granule cell raphes. Scale bars:A–C, G, 425 μm; D, 400 μm; E, 100 μm.
- Fig. 3.
Characterization of the inward migratory arrays of granule cells. Coronal sections of the chick cerebellum, all with dorsal at the top, are shown. A–C, The correspondence of DAPI staining and Zic-1 andPax-6 expression (arrows) in the E11 chick cerebellum is indicated. DAPI nuclear staining is shown inblue (A), and in situ hybridization for Zic-1(B) or Pax-6(C) appears as purple.D–G, Granule cell arrays are postmitotic. Adjacent coronal sections are shown of an E11.5 chick cerebellum 1 hr after BrdU injection at the rostral level (D, E) and the caudal level (F, G). Pax-6 (D, F) and BrdU (E,G) antibody staining are shown as brownnuclei. The superficial layer of the EGL contained many BrdU+ cells, whereas the deeper layer of the EGL and the granule cell arrays (arrows) contained relatively few BrdU+ cells, suggesting they are postmitotic. The BrdU+ cells scattered underneath the EGL most likely represent the glial cell precursors. Scale bars:A–C, 100 μm; D–G, 200 μm.
- Fig. 4.
Granule cell raphes are present in the developing cerebellum of duck and quail. Coronal sections of E15 duck (A) and E14 quail (B) cerebellum are shown. DAPI nulear staining is shown inblue. Arrows indicate the parasagittal arrays of DAPI-labeled cells, most likely the granule cell raphes. Scale bar, 200 μm.
- Fig. 5.
The lack of spatial correlation between granule cell raphes and Bergmann glial fibers. A, A coronal section of an E12 cerebellum, with anti-vimentin staining inred and DAPI staining in blue, showing the homogeneous distribution of vimentin+ glial fibers. Arrows indicate the granule cell raphes stained by DAPI. B, A schematic diagram of the dorsal view of granule cell raphes (in magenta) in E12 chick cerebellum. The roman numerals identify the transverse folia. The symbols P0–P4 denote the domains separated by the granule cell raphes. C, D,In situ hybridization with a chKBP probe, another marker for Bergmann glia, at E10 (C) and E12 (D) revealing a pattern distinct from that of the granule cell raphes shown in B.
- Fig. 6.
Complementary patterns of CaBP and Pax-6 expression and cresyl violet staining suggest that the continuity of the Purkinje cell layer is interrupted by the granule cell raphes. Adjacent coronal sections of an E12.5 chick cerebellum stained by anti-CaBP (A, B) or anti-Pax6 (C, D), both in brown, and another coronal section of an E12 chick cerebellum stained with cresyl violet (E, F) are shown. Dorsal is to the top in all panels.A–D, The lower-power views (A,C) show the positional correspondence between the Purkinje cell gaps and the granule cell raphes and that the width of the former correlated with that of the latter. The high-power views (B, D) show that the Purkinje cell layer curves inwardly as it juxtaposes the granule cell raphe (arrows in B), suggesting an interruption of the Purkinje cell layer by the granule cell raphe. Therectangular frames in A andC indicate the high-power fields shown inB and D, respectively. E,F, The relatively cell-poor zones (black arrowheads) adjacent to the granule cell raphes (white arrows), which have a high cell density similar to that of the EGL, are shown. The rectangular frame inE indicates the area of higher-power view shown inF. EGL, External granule layer;IGL, internal granule layer; PCL, Purkinje cell layer. Scale bars: A, C, 200 μm; E, 100 μm; B,D, F, 50 μm.
- Fig. 7.
Identification of Purkinje cell markers in the developing chick cerebellum. A–F, Coronal sections of an E14 chick cerebellum, with dorsal to the top. The standard Purkinje cell marker calbindin (A) was detected by anti-calbindin antibody in brown. The granule cell marker Zic-1 (D) does not show the granule cell raphes as well as at the earlier stages, even though some granule cell raphes are still evident in the more posterior folia at E14 (data not shown). In situ hybridization with Shh (B),EphA5/Cek-7 (C),Bmp-7 (E), andEphA4/Cek-8 (F) probes shows that they are expressed in Purkinje cells. Arrows inA–F indicate the gaps devoid of any Purkinje cell marker expression. G–L, The domains ofEphA5/Cek-7+ Purkinje cells that are complementary to Zic-1+ granule cell arrays. Adjacent coronal sections of E11.5 chick cerebellum were hybridized with the EphA5/Cek-7 probe (G–I) or the Zic-1 probe (J–L). The arrows point to the Purkinje cell gaps (G–I) and the corresponding granule cell arrays (J–L). It was also noted that beyond the developing cerebellar cortex, these genes were expressed in other areas of the CNS. For instance,EphA5/Cek-7 was also expressed in a subset of deep cerebellar nuclei (G) and in the hindbrain (G–I). Zic-1 was also expressed in the VZ of the cerebellum (J–L). Additional sites of expression were also found for the other genes described here (see Discussion). Scale bars: A–F, 200 μm; G–L, 400 μm.
- Fig. 8.
Parasagittal Purkinje cell domains of differential gene expression are often bordered by the granule cell arrays.A1, B1, C1, Ten millimeter coronal sections of E11.5 chick cerebellum first hybridized withBmp-7 (A1), En-1(B1), or Gli-2/4 (C1) (in situ signals in dark purple) and then stained with anti-Pax6 antibody (in brown). The plane of coronal section in A1, B1, andC1 is indicated by the black horizontalarrow in A,B, and C, respectively. In situ hybridization of Bmp-7 (A1),En-1 (B1), or Gli-2/4(C1) labeled the parasagittal domains of Purkinje cells, whereas anti-Pax6 antibody labeled the EGL and granule cell raphes (green arrows and arrowheads). Each of these genes was expressed at a higher level in some domains than in others, and the boundaries of gene expression tended to coincide with the granule cell raphes. A–D,F–I, Whole-mount in situ hybridization of E11.5 chick cerebellum with Bmp-7(A), En-1(B), Gli-2/4(C), Zic-1(D), Shh(F), EphA5/Cek-7(G), EphA4/Cek-8(H), and En-2(I). Dorsal views, all with anterior at the top, are shown. E, The schematic representation of granule cell migratory streams (inmagenta) as revealed by Zic-1 expression (E). The roman numerals identify the transverse folia. The symbols P0–P4 denote the domains separated by the granule cell raphes.Arrows and arrowheads indicate a subset of granule cell raphes defining Purkinje cell domainsP1–P4 at folium IXa+b. Domains P1–P4each had a distinct level of gene expression. See the text for a detailed description of differential gene expression in the parasagittal domains. Scale bars: A1, B1,C1, 200 μm; A–I, 400 μm.
- Fig. 9.
Granule cell raphes disappear at E15–E16. Ten millimeter coronal sections of E12 (A,B), E13.5 (C, D), and E15.5 (E, F) chick cerebellum at the level of posterior folia (IXa-c and X). Sections were first hybridized with EphA5/Cek-7 (in situsignals in dark purple) and then stained with anti-Pax6 antibody (in brown). In situhybridization of EphA5/Cek-7 labeled Purkinje cells, whereas anti-Pax6 antibody labeled the EGL, the IGL, and granule cell raphes (black arrows). The regular arrays of granule cell raphes connecting the EGL and IGL were very prominent at E12 (A, B) and E13.5 (C,D), but they became indistinct by E15.5 (E, F). At E12 and E13.5, the granule cell raphes corresponded to gaps in the Purkinje cell layer (black arrows in B,D). However, at E15.5, there were fewer gaps in the Purkinje cell layer (black arrowhead inF). The gaps persisting to E15.5 were filled with Pax6+ granule cells, but there was not an array of granule cells connecting the EGL and the IGL. EGL, External granule layer; IGL, internal granule layer;PCL, Purkinje cell layer. Scale bars: A,C, E, 400 μm; B,D, F, 100 μm. Therectangular frames in A, C, andE represent the areas shown in B, D, and F, respectively, at a higher magnification.
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