Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 26 (36), 9184-95

Unipolar Brush Cells of the Cerebellum Are Produced in the Rhombic Lip and Migrate Through Developing White Matter

Affiliations

Unipolar Brush Cells of the Cerebellum Are Produced in the Rhombic Lip and Migrate Through Developing White Matter

Chris Englund et al. J Neurosci.

Abstract

Unipolar brush cells (UBCs) are glutamatergic interneurons in the cerebellar cortex and dorsal cochlear nucleus. We studied the development of UBCs, using transcription factor Tbr2/Eomes as a marker for UBCs and their progenitors in embryonic and postnatal mouse cerebellum. Tbr2+ UBCs appeared to migrate out of the upper rhombic lip via two cellular streams: a dorsal pathway into developing cerebellar white matter, where the migrating cells dispersed widely before entering the internal granular layer, and a rostral pathway along the cerebellar ventricular zone toward the brainstem. Ablation of the rhombic lip in organotypic slice cultures substantially reduced the production of Tbr2+ UBCs. In coculture experiments, Tbr2+ UBCs migrated from rhombic lip explants directly into the developing white matter of adjacent cerebellar slices. The origin of Tbr2+ UBCs was confirmed by colocalization with beta-galactosidase expressed from the Math1 locus, a molecular marker of rhombic lip lineages. Moreover, the production of Tbr2+ UBCs was Math1 dependent, as Tbr2+ UBCs were severely reduced in Math1-null cerebellum. In reeler mutant mice, Tbr2+ UBCs accumulated near the rhombic lip, consistent with impaired migration through developing white matter. Our results suggest that UBCs arise from the rhombic lip and migrate via novel pathways to their final destinations in the cerebellum and dorsal cochlear nucleus. Our findings support a model of cerebellar neurogenesis, in which glutamatergic and GABAergic neurons are produced from separate progenitor pools located mainly in the rhombic lip and the cerebellar ventricular zone, respectively.

Figures

Figure 1.
Figure 1.
Distribution of Tbr2+ cells in the adult mouse cerebellum (P201). The locations of Tbr2+ cell nuclei (dots) were mapped by tracing from digital images of sagittal sections. Thick lines indicate the cerebellar surface; thin lines indicate the boundary between cerebellar cortex and white matter. Tbr2+ cells were located in the internal granular layer, mainly in lobule X (nodulus) and the ventral part of lobule IX (uvula), with a secondary concentration in lobules VI-VII. The abundance of Tbr2+ cells was highest near the midline and decreased sharply in the hemispheres. The distance from the midline is shown above each section. Roman numerals indicate lobules of the vermis. The asterisk indicates a tissue fold. Orientation: C, caudal; D, dorsal; R, rostral; V, ventral. Scale bar, 1 mm.
Figure 2.
Figure 2.
Tbr2 is specifically expressed by UBCs in adult mouse cerebellum. A, Tbr2 (red) and class III β-tubulin (green). All Tbr2+ cells expressed class III β-tubulin and thus were identified as neurons. B, C, Tbr2 (red) and Pax6 (green). Tbr2+ cells (UBCs) expressed low levels of Pax6. Granule neurons expressed high levels of Pax6 and were slightly smaller than UBCs. The bracketed area in B is shown at a higher magnification in C. D, Tbr2 (red) and calretinin (green). The calretinin+ subtype of UBCs (Nunzi et al., 2002) expressed Tbr2. Note calretinin in the cell body with Tbr2+ nucleus (arrow) and in the dendritic brush (arrowhead). E, Tbr2 (red) and mGluR1α (green). The mGluR1α protein is expressed by another subtype of UBCs (Nunzi et al., 2002). Tbr2+ nuclei (arrow) were often adjacent to mGluR1α+ brushes (arrowhead), consistent with coexpression in the same cell. F, Tbr2 (red) and GluR2 (green). GluR2 is a pan-UBC marker (Sekerková et al., 2004). Scale bar (in A): A, C, E, F, 20 μm; B, 60 μm; D, 13 μm.
Figure 3.
Figure 3.
Tbr2+ cells in the developing cerebellum. A, Tbr2 (red) and DAPI (blue), E13.5. Tbr2+ cells were present in the nuclear transitory zone (ntz), external granular layer (egl), and rhombic lip (rl). Asterisks indicate edge artifact. cp, Choroid plexus; ctz, cortical transitory zone; cvz, cerebellar ventricular zone. B, Tbr2 (red) and DAPI (blue), E15.5. Tbr2+ cells began streaming from the rhombic lip into the developing white matter (wm). Tbr2 was also expressed in the deep cerebellar nuclei (dcn), which develop from the nuclear transitory zone, and in the external granular layer. C, D, Tbr2 (red) and Pax6 (green), E16.5. Tbr2 and Pax6 were coexpressed by some cells in the core of the rhombic lip. Cells at the ventricular and subpial surfaces of the rhombic lip expressed only Pax6 and not Tbr2. Tbr2+ cells and Pax6+ cells avoided the developing Purkinje cell layer (pcl). Neither Pax6 nor Tbr2 was expressed in the cerebellar ventricular zone. However, Pax6 expression did extend a short distance rostral from the rhombic lip (C, arrowhead) and may have defined the boundary between progenitor compartments more accurately than morphological criteria. The bracketed area in C is shown at a higher magnification in D. E, F, Tbr2 (red), calretinin (green), and DAPI (blue), E17.5. Some Tbr2+ cells expressed calretinin in the developing white matter (E, arrowheads) and rhombic lip (F, arrowheads), suggesting that some UBCs differentiated as the calretinin+ subtype before completing migration. Calretinin was also expressed by some projection neurons in the deep cerebellar nuclei (Fink et al., 2006). The bracketed area in E is shown at a higher magnification in F. G, Tbr2 (red) and DAPI (blue), P0.5. Tbr2+ cells streamed out of the rhombic lip and into developing white matter, avoiding the Purkinje cell layer and the deep cerebellar nuclei. One pathway appeared to lead rostrally from the rhombic lip toward the brainstem (bottom arrow), whereas another pathway appeared to lead dorsally toward the cerebellar cortex (top arrows). H, Tbr2 (red) and Pax6 (green), P0.5 (same section as G). Migrating Tbr2+ cells avoided the developing internal granular layer (igl), marked by abundant Pax6+ cells. I–K, Tbr2 (red) and Pax6 (green), P3–P10. On P3 (I), most Tbr2+ UBCs were in the developing white matter. Many Tbr2+ UBCs reached the internal granular layer by P5 (J), and most entered the internal granular layer by P10 (K). Roman numerals indicate cerebellar lobules rich in Tbr2+ cells. Sagittal sections: rostral, left; dorsal, top. Scale bar (in A): A–C, E, 100 μm; D, F, 40 μm; G, H, 150 μm; I–K, 200 μm.
Figure 4.
Figure 4.
Tbr2+ progenitor cells in the rhombic lip. A, Tbr2 (red) and acute BrdU (green), E14.5 rhombic lip (rl). Some Tbr2+ cells were in S-phase (BrdU+), and some were in M-phase, as indicated by chromatin condensation (arrow). B, A Tbr2+ cell in metaphase (arrow), E14.5 rhombic lip. C, Tbr2 (red) and PCNA (green), E14.5 nuclear transitory zone (ntz). Tbr2+ cells in the nuclear transitory zone did not express PCNA or other mitotic markers. In contrast, many cells in the meninges (mn) expressed PCNA. D, E, Tbr2 (red) and PCNA (green), rhombic lip. A subset of Tbr2+ cells continued to express PCNA (arrowheads) on E16.5 (D) and E18.5 (E). F, Tbr2 (red) and acute BrdU (green), E19.5 rhombic lip. Very few Tbr2+ cells expressed mitotic markers at this age. Sagittal sections: rostral, left; dorsal, top. Scale bar (in A): A, C, 30 μm; B, 9 μm; D–F, 45 μm.
Figure 5.
Figure 5.
Migrations of Tbr2+ UBCs labeled with BrdU. A–D, Stages in the migration of Tbr2+ UBCs born on E16.5. The locations of double-labeled Tbr2+/BrdU+ cells (dots) were plotted after survival to E18.5 (A), P0.5 (B), P5 (C), and P10 (D) by tracing from digital images of sagittal sections (thick and thin lines as in Fig. 1). Many E16.5-born Tbr2+ cells remained in the rhombic lip until E18.5–P0.5 (A, B, arrowheads). Most Tbr2+ UBCs entered the internal granular layer by P10. The number of E16.5-born Tbr2+ cells in the vermis appeared to increase postnatally, suggesting that some UBCs may have migrated from lateral to medial (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Steps in the migration of UBCs born on E15.5 and E17.5 (data not shown) were similar to those of E16.5-born UBCs. Roman numerals indicate lobules of the vermis with high density of Tbr2+ cells. Orientation: rostral, left; dorsal, top. E–G, Final distributions of Tbr2+ UBCs born on E15.5 (E), E16.5 (F), and E17.5 (G). Pups were killed after survival to P19 (E) or P21 (F, G). The locations of double-labeled, Tbr2+/BrdU+ cells (dots) were plotted as in A–D. Tbr2+ UBCs born on E15.5, E16.5, and E17.5 showed similar overall distributions, with high abundance in lobule X and ventral lobule IX, intermediate abundance in lobules VI-VII, and low abundance in other lobules. Orientation: rostral, left; dorsal, top. Scale bar: A–D, 0.5 mm; E–G, 1 mm.
Figure 6.
Figure 6.
Rhombic lip ablation reduces Tbr2+ cell production in organotypic slice cultures. A, Diagram of E14.5 sagittal slice, illustrating the rhombic lip (rl), cerebellar ventricular zone (cvz), external granular layer (egl), nuclear transitory zone (ntz), and cortical transitory zone (ctz). Orientation: rostral, left; dorsal, top. B, Tbr2 (red) and Pax6 (green) immunofluorescence in control cerebellar slice, E14.5 plus 3 DIV, cryostat section. The developing white matter contained numerous Tbr2+ cells (arrowheads), which appeared to migrate from the rhombic lip. Pax6+ cells formed a well-defined external granular layer. In this experiment, some Tbr2+ cells and Pax6+ cells migrated aberrantly into the brainstem (asterisk) across an artifactual contact in vitro. C, Plot traced from section in B, showing the positions of Tbr2+ cells (red dots) outside the rhombic lip (yellow) and the external granular layer (green). D, Diagram of E14.5 cerebellar slice after rhombic lip ablation (x). E, F, Tbr2 (red) and Pax6 (green) in rhombic lip-ablated cerebellar slice, E14.5 plus 3 DIV, cryostat section (E) and plot (F), prepared as in B and C. The number of Tbr2+ cells in developing white matter was markedly reduced, compared with controls (B, C). G, Diagram of E16.5 cerebellar slice, illustrating regions as in A. dcn, Deep cerebellar nuclei; pcl, Purkinje cell layer. Orientation: rostral, left; dorsal, top. H, I, Tbr2 (red) and Pax6 (green) in control cerebellar slice, E16.5 plus 3 DIV, cryostat section (E) and plot (F). The inset in H shows Tbr2 (red) and calretinin (green) immunofluorescence in an adjacent cryosection, revealing typical UBC morphology. J, Diagram of E16.5 cerebellar slice after rhombic lip ablation (x). K, L, Tbr2 (red) and Pax6 (green) in rhombic lip-ablated cerebellar slice, E16.5 plus 3 DIV, cryostat section (K), and plot (L). The number of Tbr2+ cells in developing white matter was markedly reduced, compared with controls (H, I). M, N, Areal density of Tbr2+ cells (M) and Pax2+ cells (N) in control (blue bars) and rhombic lip-ablated (red bars) slices after 3 DIV. The density of Tbr2+ cells (UBCs) decreased after rhombic lip ablation, but Pax2+ cells (GABAergic interneurons) were not affected. Data are mean ± SD of n = 9 (E14.5) or n = 5 (E16.5) pairs of cultures. *p < 0.05; **p < 0.01; n.s., not significant. Scale bar: B, E, H, K, 100 μm; H, inset, 17 μm.
Figure 7.
Figure 7.
Cells migrate from rhombic lip explants into cerebellar white matter and express Tbr2 in organotypic slice coculture. A, Diagram showing placement of a tau-GFP transgenic rhombic lip (rl) explant (green) adjacent to a slice of E16.5 cerebellum from which the endogenous rhombic lip was removed. Abbreviations as in Figure 6G. B, Tbr2 (red) and GFP (green) immunofluorescence in cryostat section through an E16.5 cerebellar slice and rhombic lip explant, cocultured for 4 DIV. Numerous tau-GFP+ rhombic lip cells migrated into the developing white matter of the nontransgenic cerebellum. Most of the migrating cells expressed Tbr2 (arrowheads). The tau-GFP+/Tbr2+ cells avoided the deep cerebellar nuclei, as did endogenous, nontransgenic tau-GFP/Tbr2+ cells (arrows). The dashed line shows the boundary between tau-GFP transgenic rhombic lip and nontransgenic cerebellum. Choroid plexus (cp) was included with the rhombic lip explant and expressed tau-GFP. C, D, High-magnification views of bracketed regions in B, counterstained with DAPI (blue). E, Plot traced from section in B, showing the positions of tau-GFP+/Tbr2+ cells (yellow dots), tau-GFP/Tbr2+ cells (red dots), and tau-GFP+/Tbr2 cells (green dots). Only cells with visible nuclei (assessed from DAPI counterstain) were plotted. F, Tbr2 (red) and GFP (green) immunofluorescence in another E16.5 cerebellar slice and rhombic lip explant, cocultured for 4 DIV. The migrating, tau-GFP+/Tbr2+ cells displayed long processes, some of them branched. G, H, Pax6 (red) and GFP (green) immunofluorescence in cryosections of E16.5 cerebellar slice and explant cultured for 3 DIV (G) or 4 DIV (H). Pax6+ cells were located mainly in the external granular layer. Cells migrating through the developing white matter (tau-GFP+) expressed low levels of Pax6 (H, arrowheads). I, J, Pax2 (red) and GFP (green) immunofluorescence in E16.5 cerebellar slice and rhombic lip explant cultured for 4 DIV. Most tau-GFP+ cells did not express Pax2 (I, arrowheads). The blue counterstain in I is DAPI. Scale bar (in B): B, E, F, G, I, 100 μm; C, D, H, J, 50 μm.
Figure 8.
Figure 8.
Math1 is required for the production of Tbr2+ UBCs. A–C, Math1 gene expression, indicated by β-galactosidase (green) immunoreactivity, was detected in Tbr2+ cells (red) on E14.5 (A), E16.5 (B), and E19.5 (C). Many Tbr2+ progenitors exhibited diffuse cytoplasmic β-galactosidase immunoreactivity, suggestive of active Math1 gene expression. In contrast, Tbr2+ cells migrating out of the rhombic lip contained a cytoplasmic dot of β-galactosidase immunoreactivity (B, C, arrowheads), consistent with perdurance after active gene expression. D–K, Rhombic lip (rl)-derived cell types were selectively reduced in Math1-null (LacZ/-) cerebellum (E, G, I, K) compared with the Math1 heterozygous (LacZ/+) controls (D, F, H, J) on E16.5 (D–G) and E19.5 (H–K). Double immunofluorescence for Tbr2 (red) and Pax6 (green) (D, E, H, I) revealed a severe reduction of Tbr2+ UBCs and Pax6+ granule cell progenitors in Math1-null cerebellum. The arrows in E indicate small clusters of Pax6+ cells in the rudimentary external granular layer (egl). Pax2+ cells (red) (F, G, J, K), which are GABAergic interneurons derived from the cerebellar ventricular zone (cvz), were relatively spared. See text for cell counting results. Other abbreviations as in previous figures. The blue fluorescence (D–K) is DAPI. Scale bars: (in A) A–C, 20 μm; (in D) D–G, 100 μm; H–K, 200 μm.
Figure 9.
Figure 9.
Tbr2+ UBCs, reelin expression, and abnormal migrations in reeler. A, Tbr2 (red) and reelin (green) immunofluorescence, E16.5 cerebellum. Many Tbr2+ UBCs expressed reelin (arrowheads) after exiting the rhombic lip (rl). cvz, Cerebellar ventricular zone. B, Tbr2 (red) immunofluorescence and DAPI counterstain (blue), reeler cerebellum, P0.5. Tbr2+ UBCs were abundant near the rhombic lip (rl), suggesting their migration was impaired. Few Tbr2+ UBCs migrated dorsally through developing white matter compared with controls (see Fig. 3G). The rostral pathway along the cerebellar ventricular zone (cvz) appeared relatively preserved. C, Tbr2 (red) immunofluorescence and DAPI (blue), reeler cerebellum, P22. The number of Tbr2+ UBCs was overall reduced, compared with age-matched controls (data not shown) (for comparison with P10, see Fig. 5D) (Ilijic et al., 2005). Nevertheless, Tbr2+ UBCs were most abundant in the caudoventral sector (arrowheads), corresponding to lobules IX and X. D, Tbr2 (red) and Pax6 (green) immunofluorescence, reeler cerebellum, P22 (same section as C). Many Tbr2+ UBCs were abnormally isolated from Pax6+ granule neurons (arrows), suggesting they did not integrate into cerebellar circuitry (sagittal sections). Orientation: dorsal, top; rostral, left. Scale bar (in A): A, 30 μm; B–D, 200 μm.

Similar articles

See all similar articles

Cited by 78 articles

  • Maturation of Human Pluripotent Stem Cell-Derived Cerebellar Neurons in the Absence of Co-culture.
    Silva TP, Bekman EP, Fernandes TG, Vaz SH, Rodrigues CAV, Diogo MM, Cabral JMS, Carmo-Fonseca M. Silva TP, et al. Front Bioeng Biotechnol. 2020 Feb 14;8:70. doi: 10.3389/fbioe.2020.00070. eCollection 2020. Front Bioeng Biotechnol. 2020. PMID: 32117945 Free PMC article.
  • Functional Outcomes of Cerebellar Malformations.
    Gill JS, Sillitoe RV. Gill JS, et al. Front Cell Neurosci. 2019 Oct 4;13:441. doi: 10.3389/fncel.2019.00441. eCollection 2019. Front Cell Neurosci. 2019. PMID: 31636540 Free PMC article. Review.
  • Identification of novel cerebellar developmental transcriptional regulators with motif activity analysis.
    Ha TJ, Zhang PGY, Robert R, Yeung J, Swanson DJ, Mathelier A, Wasserman WW, Im S, Itoh M, Kawaji H, Lassmann T, Daub CO, Arner E; FANTOM Consortium, Carninci P, Hayashizaki Y, Forrest ARR, Goldowitz D. Ha TJ, et al. BMC Genomics. 2019 Sep 18;20(1):718. doi: 10.1186/s12864-019-6063-9. BMC Genomics. 2019. PMID: 31533632 Free PMC article.
  • Resolving medulloblastoma cellular architecture by single-cell genomics.
    Hovestadt V, Smith KS, Bihannic L, Filbin MG, Shaw ML, Baumgartner A, DeWitt JC, Groves A, Mayr L, Weisman HR, Richman AR, Shore ME, Goumnerova L, Rosencrance C, Carter RA, Phoenix TN, Hadley JL, Tong Y, Houston J, Ashmun RA, DeCuypere M, Sharma T, Flasch D, Silkov A, Ligon KL, Pomeroy SL, Rivera MN, Rozenblatt-Rosen O, Rusert JM, Wechsler-Reya RJ, Li XN, Peyrl A, Gojo J, Kirchhofer D, Lötsch D, Czech T, Dorfer C, Haberler C, Geyeregger R, Halfmann A, Gawad C, Easton J, Pfister SM, Regev A, Gajjar A, Orr BA, Slavc I, Robinson GW, Bernstein BE, Suvà ML, Northcott PA. Hovestadt V, et al. Nature. 2019 Aug;572(7767):74-79. doi: 10.1038/s41586-019-1434-6. Epub 2019 Jul 24. Nature. 2019. PMID: 31341285 Free PMC article.
  • Dynamic Expression and New Functions of Early B Cell Factor 2 in Cerebellar Development.
    Badaloni A, Casoni F, Croci L, Chiara F, Bizzoca A, Gennarini G, Cremona O, Hawkes R, Consalez GG. Badaloni A, et al. Cerebellum. 2019 Dec;18(6):999-1010. doi: 10.1007/s12311-019-01051-3. Cerebellum. 2019. PMID: 31273610
See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback