PMC

Lmx1a is expressed in the cerebellar RL independently of Atoh1. (A) Summary of Lmx1a expression in rh1 at e10.5 (). Dorsal view of the anterior hindbrain (Left) and a sagittal section (Right) at the level of the dashed line in the dorsal view. (B–G) In situ and immunostained (C–G) sagittal sections of the cerebellar anlage at the indicated stages; b′–b′′, e′–e′′′ and g′–g′′′ show higher magnification of boxed regions in B, E, and G, respectively. Arrows point to Lmx1a⁺/Atoh1⁻ cells; arrowheads point to rare Lmx1a⁺/Atoh1⁺ cells in the RL. (H–K) Lmx1a-immunostained sagittal sections of wild-type (H and J) and Atoh1^−/− (I and K) embryos at the indicated stages. Lmx1a expression is present in the RL of Atoh1^−/− embryos. (Scale bar: B, 300 μm; b′ and b′′, 100 μm; C–E and F–G, 50 μm; e′–e′′ and g′–g′′′, 20 μm; H and I, 150 μm; J and K, 75 μm.)

Premature regression of the RL and posterior vermis hypoplasia in dreher mice. (A–H) Sagittal sections of medial cerebellar anlage of wild-type (A, C, E, and G) and dreher (B, D, F, and H) embryos stained with H&E (A–D) or with indicated antibodies (E–H) at the indicated stages. In dreher embryos, RL was present at e13.75, but virtually no RL was detected in these embryos at e18.0 (D, open arrowhead). (E–H) Only RL and adjacent CP are shown. Open arrowheads in F and H point to a few Lmx1a⁺ and Tbr2⁺ cells occasionally found in the area corresponding to the dreher RL. (I–L) Dorsal whole mount views (I and J) and midsagittal sections (K and L) of wild-type (I and K) and dreher (J and L) cerebella. (I and J) Vertical lines distinguish vermis (v) from hemispheres (h) in wild-type. In the dreher cerebellum (J) the posterior vermis is reduced (open arrowhead). (K and L) Vermis folia are indicated by roman numerals. The predominant reduction of the posterior vermis (marked by bracket) is obvious in the dreher cerebellum. (Scale bar: A–D, 100 μm; E–H, 50 μm; I and J, 2 mm; K and L, 1 mm.)

Lmx1a regulates cell-fate decisions in the dorsal telencephalon. (A and B) Coronal hemisections of e12.5 wild-type (A) and dreher (B) telencephalon, which correspond to the boxed region in the diagram to the left, stained with Wnt3a in situ probe to visualize CH. CH is normally induced in dreher embryos. (C–H) Tracing progeny of Lmx1a⁺ cells in wild-type (C, D, and G) and dreher (E, F, and H) mice at indicated stages using the Lmx1a-Cre/ROSA alleles. Coronal hemisections are stained with indicated antibodies (C–F) or stained for β-gal activity (G and H). HF, hippocampal field. Limit of the Lmx1a expressing CH is shown by dotted line. In d′ and f′, Arrows point to β-gal⁺ Cajal-Retzius cells on the pial surface of wild-type (d′) and dreher (f′) telencephalon. In dreher telencephalon, they are reduced in numbers. Arrowheads point to β-gal⁺ cells within the hippocampal field. They are increased in dreher embryos. (G and H) Many more β-gal⁺ cells were detected in all hippocampal domains in adult dreher mice than in wild-type littermates. Arrows point to β-gal⁺ cells in the dentate gyrus (dg). Arrowheads point to β-gal⁺ cells in CA1–3 domains. (Scale bar: A and B, 240 μm; C–F, 150 μm; d′ and f′, 80 μm; G and H, 200 μm; g′ and h′, 450 μm.)

Lmx1a acts intrinsically in the RL to regulate proper exit of granule cell progenitors. (A) Slice coculture experiments in which the RL of a wild-type nontransgenic embryo (host, white) was replaced by the RL of a wild-type or dreher embryo carrying the Lmx1a-Cre/ROSA alleles (donor, blue). (B–E) Sections of explants stained with indicated antibodies. In explants that contain dreher Lmx1a-Cre/ROSA RL, numerous β-gal⁺ cells migrate abnormally into the EGL (D, arrowheads). These cells coexpress Atoh1 (arrowheads in e′) and, therefore, are granule cell progenitors. (F) Quantification of the area of wild-type (n = 12) and dreher (dr/dr) (n = 9) RL before and after culturing with wild-type cerebellar slices. Although equally sized RL pieces were used at the beginning of culture experiments, dreher RL became smaller than wild-type RL after 2 days in culture. Error bars represent SD. *, P < 0.001. (G and H) No difference in proliferation or apoptosis was detected between wild-type and dreher RL explants after 2 days in culture. (G) Percent of BrdU⁺ cells in wild-type (n = 4) and dreher (n = 4) RL. (H) Number of TUNEL⁺ cells per section in wild-type (n = 9) and dreher (n = 9) RL. Error bars represent SD. (Scale bar: B–E, 220 μm; e′, 110 μm.)

A switch in cell fate causes RP reduction in dreher mice. (A–D) Wild-type (A and C) and dreher (dr/dr; B and D) embryos stained with RP markers. Arrows point to fourth ventricle RP, which is induced in dreher embryos but does not grow properly. (E–H) Lineage analysis of RP cells in wild-type (E and F) and dreher (G and H) embryos using the Lmx1a-Cre/ROSA alleles. Insets (F, H) show higher magnification of RLS. In dreher mutants, some RP cells lose their identity and migrate into the cerebellar anlage (G, arrow). These β-gal⁺ cells could be labeled by an anti-Lhx2/9 antibody (Inset in H, arrowheads), which specifically marks RLS cells (). (I and J) Lineage analysis of RP cells using the Gdf7-Cre/ROSA alleles. Sagittal sections of adult wild-type (I) and dreher (J) vermis stained for β-gal activity. In the wild-type Gdf7-Cre/ROSA cerebellum, β-gal staining is limited mostly to the CP (i′). In the dreher;Gdf7-Cre/ROSA cerebellum, ectopic β-gal⁺ cells are present in DCN (arrowheads) and IGL of the posterior vermis (arrow) (j′). (K) Diagram summarizing a switch in fate of RP cells in dreher mice. In wild-type mice, RP (red) produces CP. In dreher mice RP aberrantly produces neurons of DCN and granule cells and UBC of posterior vermis. (Scale bar: A and B, 800 μm; C and D, 1.75 mm; E–H, 180 μm; I and J, 870 μm; i′ and j′, 410 μm.)

Abnormal distribution of granule cells originating from Lmx1a⁺ progenitors in the dreher cerebellum. Midsagittal sections of wild-type (A, B, E, F, I, and K) and dreher (C, D, G, H, J, and L) cerebella containing the Lmx1a-Cre/ROSA fate-mapping system at the indicated stages. Sections were stained with indicated antibodies (A–J) or were stained for β-gal activity (K and L). (A–D) Arrows point to the anterior limit of the RL in wild-type (A) and dreher (C) embryos. Arrowheads point to numerous β-gal⁺ cells migrating along the dorsal surface of the dreher (C and D) but not wild-type (A abd B) cerebellar anlage at e13.5. (E–J) Arrows indicate the anterior limit of β-gal⁺ cells in wild-type and dreher EGL. At both e18.5 and P3, β-gal⁺ cells extend much more into the anterior cerebellum in dreher mice than in their wild-type littermates. At all developmental stages, β-gal⁺ cells in both wild-type and dreher EGL express Atoh1 (D, F, H, I, i′, J, j′, and j′′), indicating that they are granule progenitors. (K and L) In adult wild-type vermis, β-gal⁺ cells predominantly populate posterior lobes IX and X (K). In dreher mice, numerous β-gal⁺ cells abnormally populate the intermediate and anterior vermis (L). Arrowheads (l′) point to ectopic β-gal⁺ cells in anterior IGL in ′adult dreher mice. (M) Diagram summarizing distribution of granule cells originating from Lmx1a⁺ RL progenitors in wild-type and dreher mice. Normally these cells (blue circles) contribute to posterior vermis. In dreher mice they overmigrate into intermediate and anterior vermis. (RP derivatives are shown in red. gives a detailed description). (Scale bar: A–D, 290 μm; E–H, 530 μm; I and J, 420 μm; i′ and j′, 200 μm; K and L, 1.5 mm; k′ and l′, 750 μm.)