LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum

Abstract

Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum.

Fig. 1.

Expression of Lhx1 and Lhx5 genes in the developing mouse cerebellum. Lhx1 (A–D) and Lhx5 (E–H) mRNAs were detected in E12.5 (A and E), E14.5 (B and F), E16.5 (C and G), and E18.5 (D and H) embryos by in situ hybridization. β-Galactosidase expressed from a LacZ reporter gene inserted into the Lhx1 gene locus was analyzed in E12.5 (I) and E16.5 (J) Lhx1^LacZ/+ embryos by X-gal staining. (K) A section of the cerebellum from a BrdU-injected E12.5 Lhx1^LacZ/+ embryo double labeled with X-gal staining (blue) to detect the activity of the LacZ reporter gene and with anti-BrdU staining (brown) to monitor proliferating cells. Arrows in A–F and I–K point at the region where Lhx1 or Lhx5 was expressed. Arrowheads in A, E, and K show the ventricular zone of the cerebellum. (Scale bar: A–H, 300 μm; I and J, 170 μm; K, 100 μm.)

Fig. 2.

Defects of the cerebellum in Lhx1/Lhx5 double mutants (Lhx1^fx1/LacZ; Nes-Cre⁺; Lhx5^−/−). The cerebellum of mutant embryos (B, D, F, H, J, and L), as compared with that of control embryos (A, C, E, G, I, and K), was analyzed by hematoxylin and eosin staining (A–D) and immunostaining of calbindin (E–H) and Pax2 (I–L) at E14.5 (A, B, E, F, I, and J) and E18.5 (C, D, G, H, K, and L). Arrows in A and B point at the cells of the developing deep cerebellar nuclei. Arrowheads in C, E, and G point out the developing Purkinje cells in the cerebellum. The arrowhead in H shows residual calbindin-positive cells present in the Lhx1/Lhx5 double mutants. (Scale bar: 100 μm.)

Fig. 3.

Purkinje cell-derived cell–cell signaling defect in the cerebellum of Lhx1/Lhx5 double mutants. (A and B) At E18.5, the granule cells in the developing cerebellum of both control (A) and Lhx1/Lhx5 double-mutant (B) embryos were labeled by immunostaining of Math1 (arrowheads). (C and D) Gli1 mRNA was detected by in situ hybridization in the Purkinje (arrows) and granule (arrowheads) cells of control embryos (C) but was largely missing in Lhx1/Lhx5 double-mutant embryos (D). Arrowheads in D point at the perimeter of the cerebellum. (Scale bar: 100 μm.)

Fig. 4.

Gradual reduction of Purkinje cells expressing the Lhx1–LacZ reporter gene in the cerebellum of Lhx1/Lhx5 double mutants. Cells expressing the Lhx1–LacZ reporter gene in the cerebellum of control (A and C) and Lhx1/Lhx5 double-mutant (B and D) embryos were labeled by X-gal staining for β-galactosidase activity (arrows). (A and B) E12.5. (C and D) E14.5. Arrowheads in D point at the perimeter of the cerebellum. (Scale bar: 100 μm.)

Fig. 5.

Defects in the development of the cerebellum in Ldb1 conditional mutants. E18.5 conditional Ldb1 mutant (B and D) and control (A and C) embryos were analyzed by hematoxylin and eosin (A and B) and by immunostaining of calbindin (C and D). (Scale bar: 100 μm.)