Huwe1 ubiquitin ligase is essential to synchronize neuronal and glial differentiation in the developing cerebellum

Abstract

We have generated a knockout mouse strain in which the gene coding for the ubiquitin ligase Huwe1 has been inactivated in cerebellar granule neuron precursors (CGNPs) and radial glia. These mice have a high rate of postnatal lethality and profound cerebellar abnormalities. The external granule layer of the cerebellum, which contains CGNPs, is expanded and displays aberrant proliferation and impaired differentiation of the progenitor cell population. The uncontrolled proliferation of the CGNPs is associated with accumulation of the N-Myc oncoprotein, a substrate of Huwe1, and con-sequent activation of the signaling events downstream to N-Myc. Furthermore, loss of Huwe1 in Bergmann glia leads to extensive disorganization of this cell population with layering aberrations, severe granule neuron migration defects, and persistence of ectopic clusters of granule neurons in the external granule layer. Our findings uncover an unexpected role for Huwe1 in regulating Berg-mann glia differentiation and indicate that this ubiquitin ligase orchestrates the programming of the neural progenitors that give rise to neurons and glia in the cerebellum.

Fig. 1.

Ataxic phenotype and postnatal lethality in Huwe1^F/YGFAP mice. (A) The upper three panels are results from tail DNA genotyping for Huwe1 loxP allele, GFAP-Cre, and Sex-determining Region Y, SRY in representative P5 mice from heterozygous floxed Huwe1 females bred onto GFAP-Cre heterozygous males (lanes 4 and 5). Bottom panel is genomic PCR for Huwe1 using DNA from the cerebella of mice in the Upper panels. Lane 1 is PCR control for the Huwe1 WT allele. Lane 2 is PCR control for the recombined Huwe1 allele. Lane 3 is PCR control for Huwe1 loxP plus Huwe1 recombined alleles. (B) Western blot analysis of cerebellum lysates from mice in lanes 4 and 5 of A using an antibody against the HECT domain of the Huwe1 protein (HECT) or the N-terminal region (N-Ter). α-Tubulin is shown as control for loading. (C) Expression of Huwe1 protein in the cerebellum of Huwe1^F/YGFAP and control mice at P8. Sagittal sections were immunostained using Huwe1 N-Ter antibody and antibodies against GLAST (Upper), or GFAP (Lower). Besides granule neurons, Bergmann glia, GLAST/GFAP-positive cells (white arrowhead), and GFAP-postive astrocytes in the IGL (arrows) were also positive for Huwe1 in control but not mutant cerebellum. Purkinjie cells (yellow arrowheads) expressed high levels of Huwe1 in wild-type and mutant cerebella. (D) Immunofluorescence using the Huwe1-HECT antibody confirmed that the HECT domain of Huwe1 was efficiently deleted in the Huwe1^F/YGFAP granule neurons, Bergmann glia, and astrocytes but not Purkinjie cells (P15). (E) Kaplan-Meier curves of wild-type and Huwe1^F/YGFAP mice. (F) Growth retardation and ataxia in Huwe1^F/YGFAP mice. EGL, external granule layer; ML, molecular layer; IGL, internal granule layer.

Fig. 2.

Histological analysis of Huwe1^F/YGFAP cerebellum. (A) Hematoxylin and eosin staining of sagittal sections from P0 Huwe1^F/Y and Huwe1^F/YGFAP cerebella. (B) Hematoxylin and eosin staining from P8 Huwe1^F/Y and Huwe1^F/YGFAP cerebella. Higher magnificaton view (Middle and Lower) of framed area in the Upper panels shows an enlarged EGL, decreased thickness of the ML, and reduced cell density in the IGL in Huwe1^F/YGFAP compared to Huwe1^F/Y. (C) Immunofluorescence for the Purkinje cell marker calbindin in Huwe1^F/Y and Huwe1^F/YGFAP P8 mice. (D) Hematoxylin and eosin staining from P15 Huwe1^F/Y and Huwe1^F/YGFAP cerebella. (Lower) Higher magnificaton view of framed area in Upper. P15 Huwe1^F/YGFAP mice exhibit a residual EGL and migrating granule cells are visible in the ML (arrows).

Fig. 3.

Huwe1^F/YGFAP mice display increased proliferation and accumulation of N-Myc and cyclin D2 in the EGL of postnatal cerebella. (A) Sagittal sections of the cerebellum of wild-type and Huwe1^F/YGFAP mice at P8 were immunostained with anti-Ki67 antibody. (B) Histograms are quantification of Ki67⁺ cells normalized by area (150 μm²) in P8 (Left) and P15 (Right) cerebella. Bars indicate mean ± SD n = 3 for Huwe1^F/Y and n = 3 for Huwe1^F/YGFAP; P = 1.85293E-07 (P8) and P = 0.000975 (P15). (C) Sagittal sections of cerebella from P8 Huwe1^F/Y and Huwe1^F/YGFAP mice were immunostained for the mitotic marker pHH3. (D) Histograms are quantification of pHH3⁺ cells normalized by area (150 μm²) in P8 (Left) and P15 (Right) cerebella. Bars indicate mean ± SD n = 3 for Huwe1^F/Y and n = 3 for Huwe1^F/YGFAP; P = 7.59553E-05 (P8) and P = 0.001447 (P15). Sagittal sections of cerebella from P8 Huwe1^F/Y and Huwe1^F/YGFAP mice were immunostained for N-Myc (E) and cyclin D2 (F).

Fig. 4.

Defective differentiation of granule neurons in Huwe1^F/YGFAP mice. (A) Sagittal sections of cerebella from P8 Huwe1^F/Y and Huwe1^F/YGFAP mice were immunostained for p18^Ink4C. (B Left) Sagittal sections of cerebella from P8 Huwe1^F/Y and Huwe1^F/YGFAP mice were immunostained for p27^Kip1 (red). Nuclei were counterstained with DAPI (blue). (B Right) Histogram shows the percentage of p27^Kip1-positive cells in the EGL. Bars indicate mean ± SD n = 3 for Huwe1^F/Y and n = 3 for Huwe1^F/YGFAP; P = 4.85935E-05. (C) Immunofluorescence using DCX, a marker of migrating neuroblasts in P8 wild-type and Huwe1^F/YGFAP cerebella. Spindle-shaped cells (arrows in Upper Left) are present in the premigratory area of EGL presumably migrating along parallel fibers (yellow arrowheads). Huwe1 mutant EGL shows accumulation of round cells (arrows in Upper Right). Migratory leading processes perpendicular to the pia (white arrowheads in Upper Left) are identified in controls but are rudimentary or absent in the Huwe1-null granule cells. DCX-positive cells are present in the IGL of control mice but are markedly reduced in Huwe1^F/YGFAP mice (Lower). (D) Immunofluorescence using NeuN (red), a marker of terminally differentiated neurons, in P8 wild-type and Huwe1^F/YGFAP cerebella. Nuclei were counterstained with DAPI (blue). In mutant cerebella NeuN immunostaining exhibits an irregular pattern in the premigratory region of the EGL (IEGL, Upper) and IGL, in which cell density is markedly reduced (Lower).

Fig. 5.

Defects in Bergman glia scaffold in Huwe1^F/YGFAP mice. (A) Immunofluorescence for BLBP on sagittal sections from Huwe1^F/Y and Huwe1^F/YGFAP P8 mice. (B) Immunofluorescence for GLAST on sagittal sections from Huwe1^F/Y and Huwe1^F/YGFAP P8 mice. (C) Immunofluorescence for GFAP on sagittal sections from Huwe1^F/Y and Huwe1^F/YGFAP P8 mice. In control littermates, Bergmann glia are aligned with Purkinje cell bodies and extend processes to the pial surface. Huwe1 mutant Bergmann glia are misplaced, Bergmann glia fibers are enlarged, and most of them lack their contacts to the pial surface (arrows in C). An increased number of BLBP-positive cells occupy the mutant IGL (arrowheads in A Right).

Fig. 6.

Huwe1^F/YGFAP mice display profound defects of granule neuron migration. (A) Granule cell migration was analyzed by BrdU pulse labeling. Mice at P6 were injected intraperitoneally with BrdU and distribution of BrdU-labeled neurons (red) was determined at 20 h (Upper) and 110 h (Lower) after injection. Calbindin immunostaining (green) was used to mark the border between ML and IGL. Compared with littermate controls, Huwe1 mutant mice exhibited increased number of BrdU-positive granule cells in the ML and correspondingly reduced number of BrdU-positive granule cells in the IGL. (B) Bar graph shows quantification of BrdU-labeled neurons expressed as the ratio between BrdU-labeled neurons in ML and IGL. (20 h, n = 3; P = 0.007055; 110 h, P = 0.000699). (C) Double immunofluorescence for DCX (neurons) and GFAP (glia) shows that most of the interactions among granule neurons and radial glia are lost in mutant cerebella (white arrows, migrating granule cells; white arrowheads, radial trailing fibers; yellow arrowheads, parallel fibers).