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First published online 14 June 2006
doi: 10.1242/dev.02437

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A key role for the HLH transcription factor EBF2^COE2,O/E-3 in Purkinje neuron migration and cerebellar cortical topography

Laura Croci¹, Seung-Hyuk Chung², Giacomo Masserdotti¹, Sara Gianola³, Antonella Bizzoca⁴, Gianfranco Gennarini⁴, Anna Corradi^1,*, Ferdinando Rossi³, Richard Hawkes² and G. Giacomo Consalez^1,

¹ San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
² Department of Cell Biology and Anatomy, Genes and Development Research Group, and Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
³ Rita Levi Montalcini Center for Brain Repair, Department of Neuroscience, Section of Physiology, University of Turin, Italy.
⁴ Department of Pharmacology and Human Physiology, University of Bari School of Medicine, Italy.

View larger version (113K): [in a new window]   Fig. 1. Morphological alterations in the Ebf2 null cerebellum. (A-F) Nissl staining of cerebellar sections at different postnatal stages. Roman numerals indicate lobule numbers. At P20 (A,B), P30 (C,D), and P60 (E,F) the median region of the mutant cerebellum is sharply reduced (arrowheads in D,F). In B, note misfolding in the hemispheres. Foliation defects are evident in the vermis: lobules I-II and III are smaller (solid arrowheads in F), lobules IV and V are fused (white arrowhead in F). Lobules VIb and VII are malformed (arrows in F). (G) Mean number of PCs calculated in P15 wt versus mutant cerebella (n=3, ±s.e.m.). The value expresses the mean number of PCs/section in each series. In the mutant the number of PCs is 38% reduced. For details on procedures used in G, see Materials and methods. PM: paramedian lobule; Sim: simple lobule; MedDL: medial dorsolateral nucleus; PFl: paraflocculus; Fl: flocculus; IC: inferior colliculus. Scale bar: 500 µm.

View larger version (115K): [in a new window]   Fig. 2. Ebf gene expression in the embryonic and postnatal wt cerebellum. Non-radioactive in situ hybridization on adjacent 7 µm frontal sections of the embryonic (A-G) and postnatal hemicerebellum (H-J). At E12.5 (A-C) Ebf2 expression is restricted to a thin subventricular portion of the cortical transitory zone (ctz), while Ebf1 and Ebf3 are expressed throughout the ctz. At this stage Ebf2 is the only member of the family to be strongly expressed in the median region of the cerebellar primordium (arrow in B) and in the nuclear transitory zone (ntz). At E17.5, the nested expression of Ebf2, Ebf3 and Ebf1 (D-F) is compared with the distribution of ROR (G), a global PC-specific marker. In the paramedian region, all Ebf genes are expressed, while more laterally some groups of cells are only positive for Ebf1 (white arrows) or for Ebf1 and Ebf3 (black arrows). At P3 the Ebf2 transcript is detectable in a subset of PCs while Ebf1 and Ebf3 are expressed in the majority of PCs. Ebf3 is also expressed in the external granular layer and in the DCN. f: fastigial nucleus; i: interpositus nucleus; d: dentate nucleus; ctz: cortical transitory zone; ntz: nuclear transitory zone; egl: external granular layer; pcl: PC layer. Scale bars: in G, 50 µm for A-C; 100 µm for D-G; in J, 200 µm for H-J.

View larger version (58K): [in a new window]   Fig. 3. No changes in PC proliferation and cell cycle exit in the Ebf2 null cerebellar primordium. Immunohistochemistry (A-F) and in situ hybridization (G,H) on the E12.5 embryonic cerebellum (frontal sections). The expression of markers of DNA synthesis (A,B: BrdU incorporation), cell division (C,D: phosphorylated histone H3), cell proliferation (E,F: Ki67) and cell-cycle control and exit (G,H: kip2) is not altered in the Ebf2 mutant cerebellar plate. The quantitation of BrdU-incorporating cells (I) and phosphorylated histone H3 positive cells (J) was obtained counting complete series of sections stained with the indicated antibodies. Statistical analysis of mean cell numbers and s.d. in +/+ versus null mutant embryos was conducted using a two-tailed t-test, equal variance. Scale bar: in H, 100 µm for A-H.

View larger version (148K): [in a new window]   Fig. 4. Defective migration of a subset of PCs in the Ebf2 null cerebellum. (A-D) Immunohistochemistry with anti-CaBP antibody on sagittal paraffin-embedded sections, anterior to the left. At E15.5 many mutant PCs are abnormally retained in the anterior cortical transitory zone (ctz). (C,D) Higher magnification of insets in A,B, respectively. The results are representative of three experiments, each conducted in a different litter, comparing one mutant and one wt littermate. hb: hindbrain; mes: mesencephalon; vz: VZ; egl: external granular layer; chd: choroid plexus. Scale bar: in D, 200 µm for A,B; 50 µm for C,D.

View larger version (115K): [in a new window]   Fig. 5. Purkinje cell death in the neonatal cerebellum. (A) Apoptotic cell counts in wt versus mutant cerebella at P0 (n=3, ±s.d.) immunostained for active Caspase 3, reveal a significant increase in cell death in the null. (B-D) Double immunofluorescence, as labeled (ovl: overlay). The majority of active Caspase 3-positive cells are ectopically positioned near the VZ and are CaBP-positive (arrows in B-D), while cell debris positive for active Caspase 3 is CaBP-negative (arrowheads in B-D). (F) In the null cerebellum, activated caspase-3-positive cells are clustered near the VZ (wt control in E). (G) Dying cells located ventrally are double-positive for active Caspase 3 and lacZ. (I,L,M) An increased number of active Caspase 3 and lacZ-double-positive cells are observed in the mutant cerebellar cortex (arrows, controls in H,J,K). Scale bars: in D, 100 µm for B-D; in M, 250 µm for E,F,H-M; in G, 150 µm for G.

View larger version (99K): [in a new window]   Fig. 6. In the Ebf2 null cerebellum, the cell loss affects specific subtypes of PCs. All sections are frontal. E18.5 (A,B) and P4 (C-H) sections were immunoperoxidase-stained for neurogranin (A-F) and HSP25 (G,H) antibodies. In the mutant, at E18.5, neurogranin-positive PCs are located mostly near the VZ (arrow in B), and are selectively lost during development. At P4 neither neurogranin-positive (D,F) nor HSP25-positive PCs (H) are present in the mutant cortex. cf: climbing fibers. Scale bars: in F, 150 µm for A,B; 100 µm for C-F; in H, 200 µm for G,H.

View larger version (108K): [in a new window]   Fig. 7. Abnormal expression of PC markers in the postnatal Ebf2 null cerebellum. Non-radioactive in situ hybridization (A-G,J-O) of postnatal (P3) frontal sections with markers of PCs or PC subsets, as labeled. (H,I) Immunostaining for EphA4, same postnatal stage. (A,D,G,J,M) Ebf2 expression in corresponding wt sections. (C,F) Loss of PCs expressing Ebf1, and Sema3A, respectively, in the mutant's anterior vermis. Arrows in B,E show corresponding territories in the wt cerebellum. (I) Changes in the striped pattern of EphA4 protein expression (asterisks in I, control in H) in the mutant vermis. (L,O) loss of a large number of PCs is revealed by the expression of Ebf3 (L) and by the striped marker Sema3A (arrows in O). Corresponding wt sections are shown in K and N (arrows), respectively. Scale bar: 200 µm for A-F,J-O; 230 µm for G-I.

View larger version (96K): [in a new window]   Fig. 8. Ebf2-lacZ transgene expression in the heterozygote. (A) Wholemount X-gal staining of the adult cerebellum, posterior, dorsal and anterior views. I-IX: vermian lobules; l.ans, l.para: ansiform and paramedian lobules, respectively. In the vermis, transgene expression is absent from the NZ (e.g. lobule X) and CZ (e.g. lobule VIb), expressed in stripes in the CZ (e.g. lobule VIII) and in the AZ (e.g. lobule III; arrows: lacZ negative stripes). (B-H) transverse 40 µm sections double-labeled for the transgene (blue) and ZII (brown). (B) posterior hemisphere (crus II). The PC stripes P4a+ - P7+ are labeled [for stripe terminology, see Eisenman and Hawkes (Eisenman and Hawkes, 1993) and Sillitoe and Hawkes (Sillitoe and Hawkes, 2002)]. Transgene expression is restricted to P-stripes. Higher magnifications of the regions indicated are shown in D and E. (C) Posterior vermis. Lobule X and ventral lobule IX (the NZ) are comprised entirely of ZII+ PCs and there is no transgene expression. The NZ and PZ interdigitate in dorsal lobule IX and by lobule VIII a clear alternation of ZII+ (P1+ to P4+) and ZII-stripes is apparent. Ebf2-lacZ expression is restricted to P-stripes. A higher magnification view of the outlined area is illustrated in F. (G) Crus I shows transgene expression in the ZII- PC stripes. (H) Anterior vermis (AZ). ZII is expressed in an array of narrow stripes, P1+ at the midline, P2+ and P3+ laterally on either side. Transgene expression is in the ZII-immunonegative stripes. Scale bars: in A, 2 mm for A; in H, 1 mm for B,C,G,H; in F, 500 µm for D-F.

View larger version (98K): [in a new window]   Fig. 9. Profound alterations in cerebellar topography in the Ebf2 null cerebellum. (A) posterior, dorsal and anterior views of a wholemount X-gal stained adult cerebellum. The lobules in the vermis are labeled (I-X). Expression of the transgene is similar to that in the heterozygote (see Fig. 8) except for a dramatic narrowing on the anterior lobes due to PC death (I-V). (B,B'-H,H') Matched pairs of immunoperoxidase-stained transverse sections from the +/- (B-G) and -/- (B'-G') vermis; ZII expression from lobule IX to lobule I; SPHK1a expression in lobule VIII (H,H'). Posterior lobules: alternating ZII+/- stripes in the heterozygote (IX, VIII, VII) have been replaced by uniform ZII+ expression domains (IX, VII) or by intermingled positive and negative PCs as in lobule VIII, where superimposable changes are observed with SPHK1a immunostaining (H,H'). In the AZ, alternating ZII+ stripes are preserved in the null (E-G: P1+ and P2+ are labeled in F) but stripe spacing is reduced due to loss of the ZII-immunonegative P1-stripe. The alterations in stripe patterning are evident in cerebella wholemount- immunostained for ZII. (I,J) Posterior hemispheres. (K,L) Posterior vermis: ZII-immunonegative stripes are absent in L. (M,N) anterior hemispheres. (O,P) anterior vermis: ZII-stripes in P are present but shrunk, due to perinatal PC apoptosis. Scale bars: in A, 2 mm for A; in H', 500 µm for B,B'-H,H'; in I, 1 mm for I,J,M,N; in L, 1 mm for K,L,O,P.

View larger version (104K): [in a new window]   Fig. 10. `Transdifferentiation' of ZII-negative to ZII-positive PCs. Transverse 40 µm cryostat sections of the paramedian lobule (PM) from the adult Ebf2 null mouse cerebellum were double immunofluorescence stained, as labeled. (A-C) Low-definition images of the paramedian lobule (PM) of the hemisphere reveal a high number of transdifferentiated ZII-positive/beta-gal-positive PCs. Higher magnification images (D-F) confirm colocalization (arrows) of beta-gal and ZII expression in the null mutant PM lobule. (G) Conversely, at the same paramedian level in the heterozygous cerebellum, the distributions of ZII (brown, arrows) and lacZ (blue, arrowheads) are strictly non-overlapping and mutually exclusive. Scale bars: in C, 150 µm for A-C; in F, 50 µm for D-F; in G, 100 µm.