Barhl1 regulates migration and survival of cerebellar granule cells by controlling expression of the neurotrophin-3 gene

Abstract

The neurons generated at the germinal rhombic lip undergo long distance migration along divergent pathways to settle in widely dispersed locations within the hindbrain, giving rise to cerebellar granule cells and precerebellar nuclei. Neurotrophin-3 (NT-3) signaling has been shown to be required for proper migration and survival of cerebellar granule cells. The molecular bases that govern NT-3 expression within the cerebellum, however, remain unknown at present. Here we report that, during early mouse neurogenesis, the Barhl1 homeobox gene is highly expressed by the rhombic lip and rhombic lip-derived migratory neurons. Its expression is later restricted to cerebellar granule cells and precerebellar neurons extending mossy fibers, two groups of neurons that synaptically connect in the adult cerebellar system. Loss of Barhl1 function causes cerebellar phenotypes with a striking similarity to those of NT-3 conditional null mice, which include attenuated cerebellar foliation as well as defective radial migration and increased apoptotic death of granule cells. Correlating with these defects, we find that NT-3 expression is dramatically downregulated in granule cells of the posterior lobe of Barhl1(-)/- cerebella. Moreover, in the precerebellar system of Barhl1(-/-) mice, all five nuclei that project mossy fibers fail to form correctly because of aberrant neuronal migration and elevated apoptosis. These results suggest that Barhl1 plays an essential role in the migration and survival of cerebellar granule cells and precerebellar neurons and functionally link Barhl1 to the NT-3 signaling pathway during cerebellar development.

Figure 1.

Expression of Barhl1 in developing cerebellar and precerebellar systems. A, The Barhl1 transcript was detected by in situ hybridization in a whole-mount embryo at E11.5. B, C, Localization of Barhl1 transcripts in sagittal (B) and coronal (C) sections through brains of E14.5 embryos. D,E, Neurons within the reticulotegmental and lateral reticular nuclei in coronal sections through brains of P0 mice were immunolabeled with an anti-Barhl1 antibody. F–H, Localization of Barhl1 transcripts in coronal sections through brains of P2 (F) and P8 (G, H) animals. Barhl1 expression is seen in restricted areas within the diencephalon, mesencephalon, cerebellum, brainstem, and neural tube. Note the strong expression of Barhl1 in the rhombic lip, external granule layer, anterior and posterior extramural migratory streams, and precerebellar nuclei extending mossy fibers (A–H), but not in the inferior olivary neurons (C). aes, Anterior extramural migratory stream; ARL, anterior rhombic lip; Cb, cerebellum; ECN, external cuneate nucleus; EGL, external granule layer; IC, inferior colliculus; IGL, internal granule layer; ION, inferior olivary nucleus; LRN, lateral reticular nucleus; M, mesencephalon; Me; medulla; MM, mammillary region; NT, neural tube; pes, posterior extramural migratory stream; PGN, pontine gray nucleus; PRL, posterior rhombic lip; R, rhombencephalon; RTN, reticulotegmental nucleus. Scale bar: E, 25 μm; D, G, 50 μm; F, H, 100 μm; B, C, 250 μm; A, 385 μm.

Figure 2.

Expression of the lacZ reporter during CNS development in Barhl1^+/– mice. A–I, β-galactosidase activity was visualized by X-gal staining in whole-mount embryos (A–C) and in brain sections from various stages counterstained with Fast Red (D–I). The embryo in C was cleared in benzyl alcohol–benzyl benzoate after X-gal staining. lacZ was expressed in two small stripes (indicated by arrows in A) within the diencephalon at E10.5. Its expression in the neural tube was seen in the dorsal tip at E11.5 (B), but localized in two symmetric lateral columns in the middle of the spinal cord (its thickness is indicated in C) at E14.5. Within the cerebellar and precerebellar systems, strong β-galactosidase activity was found in the rhombic lip, the external granule cells of the cerebellum, anterior and posterior extramural migratory streams, and all precerebellar neurons extending mossy fibers (D–I). J, A medullary section from a P6 Barhl1^+/– pup was double-stained with X-gal and an anti-Brn3a antibody. lacZ-expressing cells (blue) were restricted in lateral reticular and vestibular nuclei, which did not overlap with those that expressed Brn3a (brown) in the inferior olivary nucleus. The inset indicates that all Brn3a⁺ cells are free from blue stain. K, Schematic diagram illustrating the expression pattern of Barhl1 in the cerebellar and precerebellar systems. The inferior olivary nucleus (dashed oval) lacks any Barhl1 expression. CP, choroid plexus; D, diencephalon; SpC, spinal cord; VN, vestibular nucleus. Scale bar: D, F, 50 μm; E, I, 100 μm; C, 125 μm; J, 192 μm; G, H, 250 μm; A, 400 μm; B, 500 μm.

Figure 3.

Abnormalities in the cerebella of Barhl1^–/– mice. A–D, Attenuated foliation and size reduction in the cerebella of P23 Barhl1^–/– mice. Macroscopic views of ink-stained wild-type (A) and mutant (B) cerebella showed a diminution in foliation and size of the Barhl1^–/– cerebellum. Cresyl violet labeling of sagittal cerebellar sections further revealed the loss of vermis lobule VII in the Barhl1^–/– cerebellum (C, D). The vermis lobules are indicated by numerals. E–P, Defective migration of granule cells in postnatal Barhl1^–/– cerebella. E–J, Cerebellar sections from the indicated stages and genotypes were stained with X-gal (E–H) or cresyl violet (I, J). Compared with the wild-type and heterozygote, many clusters of granule cells (indicated by black arrows) get stuck on the surface of the mutant cerebellum at P19 and P100 (G–J). The arrowheads point to migrating granule cells in the molecular layer (E, F). K–P, Dividing granule cells in wild-type and Barhl1^–/– littermates were pulse-labeled at P9 by BrdU and then visualized by BrdU immunohistochemistry (red) along with a weak DAPI counterstain at the indicated times after injection. All images were taken from lobules VI and VII. In the mutant, there are fewer BrdU⁺ cells within the IGL at 2 d after BrdU labeling and more BrdU⁺ cells within the EGL at 3 d post-BrdU labeling (K–N). Some BrdU⁺ cells (indicated by white arrows) persist in granule cell ectopias even 8 d after BrdU labeling in the mutant (O, P). CI, crus I; CII, crus II; EGL, external granule layer; IGL, internal granule layer; ML, molecular layer; P, paramedian lobule; PCL, Purkinje cell layer; S, simplex. Scale bar: E-J, 25 μm; K-P, 16.7 μm; C, D, 400 μm; A, B, 927 μm.

Figure 4.

Downregulation of NT-3 expression within the cerebella of Barhl1^–/– mice. A, Northern blot comparing NT-3 mRNA levels in P6 cerebella of Barhl1^+/+, Barhl1^+/–, and Barhl1^–/– animals. The blot was stripped and rehybridized with a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe as shown below. B, Real-time RT-PCR analysis of NT-3, BDBF, Barhl1, and GAPDH mRNA levels in P6 cerebella of three genotypes. Each histogram represents the mean ± SD for five cerebella. C–N, P6 (C, D), P3 (E,F), and P1 (G–N) cerebellar sections from wild-type and mutant mice were hybridized with the indicated riboprobes. The inset in C or D is a higher magnification of the boxed area in each panel. Note the strong NT-3 signal within the IGL of the posterior lobe (PL) in the wild-type cerebellum (C) but the near absence of NT-3 signal in both anterior (AL) and posterior lobes of the mutant (D). In the EGL, comparable expression levels of EphB2 (E, F), netrin-1 (G, H), Math1 (I, J), NeuroD (K, L), and Pax6 (M, N) were seen between the wild-type and mutant cerebella. Scale bar: E, F, 50 μm; C, D, G, H, 100 μm; I-N, 250 μm.

Figure 5.

Abnormalities in all precerebellar nuclei that extend mossy fibers in Barhl1^–/– mice. A, B, X-gal staining of P5 and P19 Barhl1^+/– and Barhl1^–/– whole-mount brains. C–H, X-gal staining of P6 coronal brain sections from Barhl1^+/– (C, E, G) and Barhl1^–/– (D, F, H) mice. The pontine gray (PGN), reticulotegmental (RTN), external cuneate (ECN), and lateral reticular (LRN) nuclei are all reduced in size in Barhl1^–/– mice (A–H). Instead of two in the control (E), a single fused RTN is present in the mutant (F). The arrowheads point to midlines of brain sections (E, F). Scale bar: C, D, 50 μm; G, H, 100 μm; E, F, 250 μm; A, 1000 μm; B, 1429 μm.

Figure 6.

Abnormalities in the pontine gray (PGN) and reticulotegmental (RTN) nuclei of Barhl1^–/– mice. A, B, P19 whole-mount brains were stained with X-gal. Compared with Barhl1^+/– mice (A), the pontine gray nuclei in Barhl1^–/– mice (B) were smaller, elongated, and incompletely separated at the midline (indicated by arrows). C–H, Coronal brain sections from postnatal animals at the indicated developmental stages were stained with X-gal (C–F) or cresyl violet (G, H). In the pontine gray nuclei of Barhl1^–/– mice, lacZ-expressing cells failed to form tight clusters at P0 (C, D), and many of them became lost by P6 (E, F). At P100, the mutant PGN became much smaller than the control (G, H). I, Quantitation of neuron numbers in pontine gray and reticulotegmental nuclei of P100 Barhl1^+/– and Barhl1^–/– mice. Each histogram represents the mean ± SD for four nuclei. Scale bar: C, D, 156 μm; G, H, 200 μm; E, F, 250 μm; A, B, 400 μm.

Figure 7.

Loss of neurons by apoptotic cell death in Barhl1^–/– pontine gray nuclei. A–D, Cells undergoing apoptosis were immunostained with an anti-active caspase-3 antibody in E16.5 (A, B) and P2 (C, D) Barhl1^+/– (A, C) and Barhl1^–/– (B, D) pontine gray nuclei. A significant increase of apoptotic neurons was observed in the Barhl1^–/– pontine gray nucleus at P2 (C, D). E, Quantitation of apoptotic cell death in Barhl1^–/– and controlBarhl1^+/– pontine gray nuclei during development. Each histogram represents the mean ± SD for four nuclei. Scale bar: A, B, 25 μm; C, D, 50 μm.

Figure 8.

Retrograde labeling of precerebellar nuclei in control and mutant newborn mice after unilateral DiI injections in the cerebellum. In both control and null mice, the vestibular (VN) and lateral reticular nuclei (LRN) were ipsilaterally labeled, whereas the inferior olivary nuclei (ION) were contralaterally labeled (A–D). Unilateral retrograde tracing also similarly labeled pontocerebellar fibers derived from the contralateral pontine gray nuclei (PGN) in control and null mice (E, F). Scale bar: C, D, 100 μm; A, B, E, F, 250 μm.