Cerebellar primordium immunostained with NAA and SNCA shows that a subset of neurons is present at the rostral end of the cerebellum at E9

A: Dorsal view of the schematic illustration of the cerebellar primordium, mesencephalon, isthmus, and 4th ventricle. The red line indicates the sagittal plane about which the sections shown in B-D were taken. B-D: Sagittal section through the cerebellar primordium at early E9 immunoperoxidase-labeled with NAA 3A10 shows the presence of neurons in the cerebellar primordium that cross the isthmus (i) and continue to the mesencephalon. C. A higher magnification of B. D. Differentiated neurons at E9.5 are visible; a higher magnification is shown in the inset, d.

E: Sagittal section through the cerebellar primordium at late E9. Immunofluorescence labeling of SNCA shows SNCA+ (green) expressing CN neurons in the mesencephalon, at the isthmus (i) and in the rostral part of the cerebellar primordium (cb). F. A higher magnification of E.

Abbreviations: 4thv, 4th ventricle; cb, cerebellum; c, caudal; d, dorsal; i, isthmus; m, mesencephalon; r, rostral; v, ventral

Scale bar = 100 µm in B; 50 µm in C and E; 20 µm in D

Sagittal section through the cerebellar primordium at E12.5: double immunofluorescence labeling with SNCA and LMX1A antibodies of medial (A–D) and lateral (E–H) sections

A–G: SNCA (green, A and E) and LMX1A (red, B and F) immunopositive cells are located at the NTZ. SNCA+ cells continue to the mesencephalon, and LMX1A+ cells toward the rhombic lip. Merged images show that the SNCA+ cells form a population of CN neurons distinct from the rhombic lip-derived cells (LMX1A+) in the NTZ (C and G). D and H show a higher magnification of C and G, respectively.

Abbreviations: 4thV, 4th ventricle; cb, cerebellum; c, caudal; d, dorsal; i, isthmus; m, mesencephalon; rl, rhombic lip; NTZ, nuclear transitory zone; r, rostral; v, ventral.

Scale bar = 100 µm.

Double immunofluorescence labeling with SNCA and OTX2 antibodies in sagittal cerebellar primordium sections (E12.5)

A–E: Double immunolabeling of SNCA (green) and OTX2 (red) of a sagittal section of the cerebellar primordium at E12.5 A. High OTX2 immunoreactivity was detected in the mesencephalon. SNCA+ cells in the mesencephalon accompany OTX2+ cells across the isthmus (i) and in the NTZ. B. higher magnification of A. C-E. Immunolabeling with SNCA (green, C) and OTX2 (red, D) and merged (E) shows co-expression in the NTZ.

F-G: Cerebellar cells at E12 and E14 were dissociated and immunolabeled for SNCA + OTX2, SNCA + LMX1A, or each antibody individually (F). Quantitative analysis was performed by flow cytometry to detect SNCA+, OTX2+, LMX1A+, SNCA+/ LMX1A+ and SNCA+/ OTX2+ cells (G). No significant differences were observed for the number of LMX1A+ cells between E12 and E14. An increase in SNCA and OTX2 positive cells was shown at E14 in comparison to E12 (*P<0.05). The number of cells positive for SNCA/LMX1A or SNCA/OTX2 was very low at E12 and E14.

Abbreviations: 4thv, 4th ventricle; cb, cerebellum; i, isthmus; m, mesencephalon; rl, rhombic lip; NTZ, nuclear transitory zone.

Scale bar = 200 µm in A; 50 µm in B,; 20 µm in C, D, and E.

Sagittal sections through the cerebellar primordia at E12, 13, 14, and 15 were studied for peroxidase immunoreactivity of OTX2

A–F: Sagittal section through cerebellar primordium at E12 (A; higher magnification shown in B), E13 (C; higher magnification shown in D), E14 (E), and E15 (F). High OTX2 immunoreactivity at the mesencephalon is evident, and a few OTX2+ cells cross the isthmus and position at the rostral part of the cerebellar primordium at the NTZ.

G: Comparison of the number of OTX2 positive cells in the cerebellar primordium from E12 to E15. Results indicate a slight increase in the number of OTX2 positive cells over time. Significant differences were observed between E12 and E14 (**P<0.01), E12 and E15 (***P<0.001), as well as E13 and E15 (#P<0.05). Data were analyzed by One-Way ANOVA followed by a Tukey’s multiple comparison test.

Abbreviations: 4thv, 4th ventricle; i, isthmus; m, mesencephalon; rl, rhombic lip

Scale bar = 200 µm in A and C; 50 µm in F (applies for B, D, E, and F).

Sagittal section through cerebellar primordium of Otx2-GFP mice at E13 (A-D), and in situ hybridization of Otx2 at E12 (E-J).

A-D: DAPI labels the outline of the cerebellar primordium and mesencephalon (A, blue). GFP expression, which is enhanced by immunofluorescence labeling with anti-GFP (B, green), and immunofluorescence labeling for OTX2 (C, red), reveal co-labeled cells (D, merged) in the mesencephalon and NTZ at the rostral end of the cerebellar primordium. Arrows indicate the isthmus.

E-G: Merged channels of the in situ hybridization of Otx2 mRNA probe counterstained with DAPI at low (E) and high (F and G) magnification captured by confocal microscopy.

H-J: The Otx2 mRNA signal was strong in the mesencephalon and extended as a tail through the isthmus to the rostral cerebellar primordium in the NTZ. The isthmus is indicated by arrows in (E, F, H, I) and a line in (J).

Abbreviations: cb, cerebellum; m, mesencephalon; rl, rhombic lip; ntz, nuclear transitory zone.

Scale bar = 200 µm in D (applies to A-D); 100 µm in H (applies to E and H); 20 µm in J (applies to F - J).

Sagittal section through cerebellar primordium at E10.5: double immunofluorescence labeling for SNCA and p75NTR (NGFR)

A–D: Double immunofluorescence labeling with SNCA (A, green) and p75NTR (B, red) antibodies reveals co-labeled cells (C, merged) in the NTZ; a higher magnification of the NTZ is shown in D.

d. Western blot analysis of SNCA and p75NTR expression during cerebellar development. Immunoblots of total cerebellar lysates from embryos at E11, E13 and E15 indicate an increase in expression of SNCA and p75NTR from E11 to E15. Equal protein loading was confirmed by β-actin expression.

E–H: Primary dissociated cultures of cerebellum obtained from an E10 mouse embryo (DIV 4), double immunofluorescence stained for SNCA (E: green) and p75NTR (F: red) (merged image shown in G). H is a higher magnification of G; punctuate cellular p75NTR immunoreactivity is marked by arrow heads. I-L: Primary dissociated cultures of cerebellum from E10 mouse embryo (DIV 21), double immunofluorescence stained for NAA (green, I) and p75NTR (red, J). Neuronal somata and axons were immunopositive for NAA 3A10 (I, L), and p75NTr (J, L) immunoreactivity was localized in the cell membrane. Merged images are shown in panel K (higher magnification in L).

Abbreviations: cb, cerebellum; NTZ, nuclear transitory zone

Scale bar = 50 µm in A–C and D–F; 20 µm in H; 10 µm in G.

OTX2 immunoperoxidase staining in the cerebellar primordium of Pap-/- ; Snca-/- mice at E12. Results show OTX2+ cell are present in the NTZ and terminate in the rostral cerebellar primordium.

Abbreviations: cb, cerebellum; NTZ, nuclear transitory zone

Scale bar = 50 µm

Sagittal sections through the cerebellar primordia of Atoh1+/+ and Atoh1-/- embryos at E12.5; double immunofluorescence labeling with SNCA + OTX2 and SNCA + MEIS2 antibodies

A–C: SNCA (green, A) and OTX2 (red, B) immunopositive cells are present in the mesencephalon and NTZ of Atoh1 KO mouse (merged channels, C).

D-F: In E12.5 Atoh1+/+ sagittal sections, SNCA+ (green, D) cells are observed in the mesencephalon and NTZ. MEIS2+ (red, E) cells are present in the mesencephalon and NTZ in addition to another population of MEIS2+ cells in the dorsal region of the NTZ which extends to the rhombic lip. Merged image (F) confirms the presence of two distinct sets of MEIS2+ cells in the NTZ: rhombic lip-derived CN neurons located in the caudodorsal (cd) region, which do not express SNCA (MEIS2+/SNCA-), and a subset of MEIS2+/SNCA+ CN neurons situated in the rostroventral (rv) region of the NTZ.

G-I: Higher magnification of D-F.

J-L: In E12.5 Atoh1-/- sagittal sections, the expression of SNCA (green, J) shows no change as compared to Atoh1+/+, whereas Meis2 abundance (red, K) exhibited a different pattern. Thus, MEIS2+/SNCA+ cells were still present in the rostroventral region of the NTZ. However, the subpopulation of MEIS2+/SNCA- rhombic lip-derived CN neurons were absent in the caudodorsal region of the NTZ.

M-O: Higher magnification of J-I.

Abbreviations: 4thV, 4th ventricle; cb, cerebellum; cd, caudodorsal; c, caudal; d, dorsal; i, isthmus; m, mesencephalon; rl, rhombic lip; NTZ, nuclear transitory zone; r, rostral; rv, rostroventral; v, ventral Scale bar = 100 µm

Fast DiI applied to an embryo at E9 where it was kept for 4 days (DIV 4, A-C) or removed after 24 hours (DIV1, D-E).

A, a: Fast DiI was inserted in the mesencephalon at E9 (DIV 0), arrow shows insertion location of DiI crystal in the mesencephalon. The arrowhead indicates the isthmus.

B-C: 4 days post DiI insertion, DiI positive cells directed both rostrally to the mesencephalon (B) and caudally to the rostral cerebellar primordium (C).

D, d: Fast DiI was inserted in the mesencephalon at E9 (DIV 0) (indicated by arrowhead) and removed at DIV1, arrow shows the isthmus.

E, e: DiI positive cells present in cerebellar primordium at DIV 6 after removal of Fast DiI at DIV1. Abbreviations: cb, cerebellum; m, mesencephalon; NTZ, nuclear transitory zone

Scale bar = 500 µm in a,d; 200 µm in A,D; 100 µm in B-C, E.

Fast DiI applied to an embryo at E9 where it was kept for 4 days in vitro (DIV 4).

A, a: Fast DiI was inserted in the mesencephalon at E9 (DIV 0), arrow shows insertion location of DiI crystal in the mesencephalon. The arrowhead indicates the isthmus.

B-C: 4 days post DiI insertion, DiI positive cells directed both rostrally to the mesencephalon (B) and caudally to the rostral cerebellar primordium (C).

D-F: Low and high magnifications show only a few positive cells in the rostral cerebellar primordium in the NTZ at the level of the medial cerebellar section.

G-I: Low and high magnifications indicate the presence of just a few positive cells in the rostral cerebellar primordium in the NTZ at the level of the lateral cerebellar section.

Abbreviations: cb, cerebellum; m, mesencephalon; NTZ, nuclear transitory zone

Scale bar = 500 µm in a; 200 µm in A; 100 µm in B-D and G; 50 µm in E and H; 20 µm in F and I.

Fast DiI applied to the embryo at E9 and removed after 24 hrs.

A, a: Fast DiI was inserted in the mesencephalon at E9 (DIV 0) (indicated by arrowhead) and removed at DIV1, arrow shows the isthmus.

B: DiI positive cells present in cerebellar primordium at DIV 6 after removal of Fast DiI at DIV1 (indicated by arrowhead), arrow points to the isthmus.

C-D, d: A higher magnification from the caudal to mesencephalon and rostral rhombencephalon shows DiI positive cells in the cerebellar primordium, arrow points to the isthmus.

E-G: Low and high magnifications clearly show Dil stained cells in the rostral cerebellar primordium in the NTZ after whole mount IHC with NAA and sectioning.

Abbreviations: cb, cerebellum; m, mesencephalon

Scale bar = 500 µm in a and B; 250 µm in A; 200 µm in E; 100 µm in C, D, and F; 50 µm in G.

In situ hybridization images show distribution of Otx2, Snca, C-Ret and Tlx3 in the cerebellar primordium at E11.5, E13.5 and E15.5; all markers are present in the rostroventral region of the NTZ. All images show cerebellar primordia. Image credit: Allen Institute. © 2008 Allen Institute for Brain Science. Allen Developing Mouse Brain Atlas. Available online at: https://developingmouse.brain-map.org/.