Molecular Markers of Neuronal Progenitors in the Embryonic Cerebellar Anlage

Abstract

The cerebellum, like the cerebrum, includes a nuclear structure and an overlying cortical structure. Experiments in the past decade have expanded knowledge beyond the traditional function of the cerebellum to include critical roles in motor learning and memory and sensory discrimination. The initial steps in cerebellar development depend on inductive signaling involving FGF and Wnt proteins produced at the mesencephalic/metencephalic boundary. To address the issue of how individual cerebellar cell fates within the cerebellar territory are specified, we examined the expression of transcription factors, including mammalian homologues of LIM homeodomaincontaining proteins, basic helix-loop-helix proteins, and three amino acid loop-containing proteins. The results of these studies show that combinatorial codes of transcription factors define precursors of the cerebellar nuclei, and both Purkinje cells and granule neurons of the cerebellar cortex. Examination of gene expression patterns in several hundred lines of Egfp-BAC (bacterial artificial chromosome) transgenic mice in the GENSAT Project revealed numerous genes with restricted expression in cerebellar progenitor populations, including genes specific for cerebellar nuclear precursors and Purkinje cell precursors. In addition, we identified patterns of gene expression that link granule and Purkinje cells to their precerebellar nuclei. These results identify molecular pathways that offer new insights on the development of the nuclear and cortical structures of the cerebellum, as well as components of the cerebellar circuitry.

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

“…1K). The expression patterns of Lhx1 and Lhx5 mRNAs and Lhx1-LacZ reporter shown here are identical to those of Lhx1 and Lhx5 proteins and Lhx1-and Lhx5-Egfp reporters that have been described elsewhere (27).…”

“…25 and 26 and data not shown). More recently, the expression of Lhx1 and Lhx5 proteins and of Lhx1-and Lhx5-directed Egfp reporters have been observed in developing Purkinje cells soon after they exit the cell cycle in the cerebellum (27). To begin to address the role of LIM-homeodomain proteins in cerebellar development, we examined the expression of Lhx1, Lhx5, and Ldb1 mRNAs in the developing cerebellum.…”

Differentiation of ES cells into cerebellar neurons

“…To differentiate the ES cells into cerebellar granule cells, we cultured them in BME supplemented with N2 Supplement-B (ϫ100) (StemCell Technologies), B27 (StemCell Technologies), and a BMP [BMP7 (100 ng/ml), GDF7 (100 ng/ml), and BMP6 (20 ng/ml)] (13) for 4 days (16 DIV). To promote proliferation of granule neuron precursors, we cultured the cells in the medium above containing SHH (100 ng/ml), JAG1 (20 ng/ml), NT3 (100 ng/ml), and BDNF (100 ng/ml) for 7-8 days (16)(17)(18)(19)(20)(21)(22)(23)(24). Throughout the period of cell culture (22-23 DIV), we replaced the indicated medium with fresh medium (replacing 50% of the medium) every 2 days.…”

“…Recent studies reveal that the anterior rhombic lip, once thought to exclusively generate granule neurons of the cerebellar cortex (17), generates precursors of the cerebellar and precerebellar nuclei, which project afferent fibers to the cerebellar cortex or receive efferent fibers from the cerebellum. Progenitors of the Purkinje neuron arise from the ventricular zone of the cerebellar territory and express specific transcription factors (20).…”

“…The GENSAT Project has generated hundreds of Egfp-BAC transgenic lines and identified markers for cerebellar granule cells, including Pde1c for granule cells. Pde1c is expressed by GCPs very early in the program of development, commencing at E10, when the earliest GCP markers, including Math1 (18), Zic1,2 (19), and Meis1 (20), are expressed. Expression continues throughout the lifetime of the animal, making Pde1c a granule neuron marker that is expressed at all stages of life.…”