Cerebella from mice heterozygous for the <i>Apoer2</i> and <i>Vldlr</i> deletions have Purkinje ectopia restricted to a small subset of zebrin II-immunonegative Purkinje cells.

<p>Serial sagittal sections from adult cerebella immunoreacted with anti-calbindin (CaBP -A, E), anti-zebrin II (ZII-B, F), or anti-phospholipase C ß4 (PLCß4-C, G) antibodies reveal the presence of a small cluster of Purkinje cells (e.g. dotted circle–D) that fail to express zebrin II but do express PLCß4 (F). Roman numerals denote putative lobule assignments. Boxes in A–D indicate magnified areas. Scale bar in F = 1mm for A–C and 250 µm for D–F.</p

Purkinje cell ectopia in the <i>Vldlr</i> null cerebellum is parasagittally organized.

<p>Serial transverse cryosections through adult <i>Vldlr</i> null cerebellum immunostained with calbindin to reveal the location of all Purkinje cells (CaBP-A, D, G, J, M), or with zebrin II (ZII-B, E, H, K, N) phospholipase C ß4 (PLCß4-C, F, I, L, O) antibodies to reveal the location of parasagittal subsets of Purkinje cells. Boxes in A–F mark areas of higher magnification presented in the photomicrographs beneath as indicated by the letter on the corner of the box. Cells immunopositive for any of these three markers (CaBP, ZII, or PLCß4) are observed properly positioned within the Purkinje cell monolayer at the cerebellar cortex however numerous ectopic cells are also distributed throughout the cerebellar intralobular white matter. Most ectopic Purkinje cells in the anterior cerebellum are ZII-/PLCß4+ (5G–I). Some ZII-expressing Purkinje cells were observed in the granular layer (i.e. arrowheads–4E, 5H) and these ectopic Purkinje cells align in rough parasagittal stripes consistent with the overlying Purkinje cell topography in the cerebellar cortex (dotted lines-5H). M–O: high power views of ventral lobule IX (NZ) reveals that misaligned Purkinje cells are arranged into parasagittally-restricted groups, that are all zebrin II-positive (N). P1+, and P3+ mark zebrin II-immunopositive/PLCß4-immunonegative stripes and P1- and P2- mark zebrin II- immunonegative/PLCß4-immunopositive stripes. Arrowheads in G mark the Purkinje cell layer, the vertical dotted line denotes the midline, and numbers denote the location of ectopic clusters of Purkinje cells within the lobular white matter. Roman numerals denote cerebellar lobules. Scale bar in O = 1 mm for A–F and 250 µm for G–O.</p

Cresyl violet staining reveals that the cerebellar cortex of <i>Apoer2</i> and <i>Vldlr</i> mutants is abnormal.

<p>Sagittal sections through the medial cerebellum of adult wild type (A, D), <i>Apoer2</i> (B, E) or <i>Vldlr⁻</i> (C, F) null animals indicate that mutant cerebella are smaller and have fewer lobules when compared to wild type mice. Higher-power views reveal that a trilaminar structure is present in both mutants and wild type (D–F) consisting of an outer molecular layer (ML), Purkinje cell layer (PCL) and inner granule cell layer (GL). White matter tracts (WM) can also be observed in each animal. High magnification views of the <i>Vldlr</i> null cerebellum reveal the presence of Purkinje cell-sized somata in the granular layer and white matter (e.g. black arrowheads–F) as well as gaps in the Purkinje cell layer (white arrowheads–F). Measurements of the length of lobules in <i>Apoer2</i> null (G) or <i>Vldlr</i> null (H) cerebella are expressed as a percentage of the length in wild-type littermates. Length measurements reveal a reduction in several areas of each mutant cerebellum. These reductions are most prominent in the anterior cerebellum of both mutants. Error bars on the graph depict SEM. Dotted line indicates the length of the equivalent lobule in wild type animals. Scale bar = 1 mm for A–C and 125 µm for D–F. * indicates p<0.05 as determined by one way ANOVA.</p

KLC3 immunolabeling reveals that ectopic Purkinje cells do not intermingle with the deep cerebellar nuclei.

<p>Sagittal cryosections from adult cerebella immunofluorescence labeled by using antibodies against KLC3 (magenta) to identify cerebellar nuclear neurons and calbindin (green) to identify Purkinje cells in wild type (A–C), <i>Vldlr</i> (D–F), or <i>Apoer2</i> (G–I) null cerebella. Merged images reveal that ectopic Purkinje clusters lie near to, but outside of the deep cerebellar nuclei in both <i>Apoer2</i> (A-/-) and <i>Vldlr</i> (V-/-) null mice. KLC-immunopositive cerebellar nuclear neurons are surrounded by the calbindin-immunoreactive axons of the Purkinje cells and these observations are consistent with previous studies of deep cerebellar nuclear neuron labeling (Chung et al., 2006). Wild type littermates (WT) have few ectopic Purkinje cells. Scale bar = 250 µm.</p

Adult <i>Apoer2</i> null cerebella have Purkinje cell ectopia that is largely restricted to zebrin II-immunonegative cells.

<p>Sagittal sections are taken from either adult wild type (A–C) or <i>Apoer2</i> null (D–L) cerebella. Cerebella have been immunostained with antibodies against calbindin (CaBP-A, D, G, J), zebrin II (ZII-B, E, H), phospholipase Cß4 (PLCß4-C, F, I, L) or heat shock protein 25 (HSP25-K) to reveal immunopositive Purkinje cell bodies in the Purkinje cell layer (P) as well as their dendrites located within the molecular layer (M). Sections from the <i>Apoer2</i> null cerebellum are serial sections (zebrin II-calbindin-PLCß4) while wild type sections are not. Boxes in D–F indicate areas where higher-magnification pictures are presented below. High-magnification panels (G, H, I, J, L) illustrate the presence of discrete groups of ectopic Purkinje cells in the white matter of the <i>Apoer2</i> null cerebellum, as identified with CaBP-immunostaining (G, J).The absence of zebrin II immunoreactivity in these cells (H) indicates that the predominant phenotype of Purkinje cells in the white matter of these mutants is ZII-/PLC ß4+ (I, L). Black arrows in E point to areas in lobules IX and X where Purkinje cells are misaligned within the Purkinje cell monolayer. Arrowheads in H point to the rare occurrence of zebrin II immunopositive Purkinje cells in the ectopic clusters. K–HSP25 immunoreactivity is revealed in Purkinje cells throughout the NZ (dotted line). Roman numerals indicate lobules. Scale bar in L = 1 mm for A–F and 250 µm for E–L.</p

Immunostaining of sagittal sections from adult <i>Vldlr</i> null cerebella reveals that Purkinje cell ectopia includes cells from both zebrin II-immunonegative and -immunopositive subsets.

<p>A series of sagittal cryosections is illustrated from the vermis of adult <i>Vldlr</i> null cerebella immunostained for calbindin to reveal the location of all Purkinje cells (CaBP-A, D, G, J, M), as well as zebrin II (ZII-B, E, H, K, N), phospholipase C ß4 (PLCß4-C, F), or heat shock protein 25 (HSP25-I, L, O) to reveal the location of select subsets of Purkinje neurons. All four markers reveal that some Purkinje cells are correctly located within the Purkinje cell monolayer at the cerebellar cortex (e.g. between arrows–4D) as well as ectopically within the cerebellar white matter (e.g. 4D, G–dotted circles). The transition from posterior zone ( = PZ-lobule VIII and dorsal IX-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001653#pone-0001653-g004" target="_blank">Fig. 4J</a>) into nodular zone ( = NZ-ventral IX and X–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001653#pone-0001653-g004" target="_blank">Fig. 4J</a>) is revealed in the form of Purkinje cell ectopia (J, K). In the dorsal aspect of lobule IX Purkinje cells are restricted to a monolayer, with some ectopic cells located in the lobule white matter (J, K). In the ventral aspect of IX, the area of transition between the PZ->NZ, is highlighted by Purkinje cells misalignment and this misalignment extends the length of the NZ to include lobule X (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001653#pone-0001653-g004" target="_blank">Fig. 4M, N</a>). Roman numerals denote putative lobule assignments. Scale bar = 1 mm for A–C and 250 µm for D–O.</p

Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development

<div><p>Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene’s function in neuronal development.</p></div

The expression levels of genes involved in complementary cell proliferative pathways in the Klf4-null with real-time PCR.

<p>a) RT-PCR and <b>b)</b> Immunohistochemistry showing Klf2 expression, a Kruppel-like factor belonging to the same gene family as Klf4, do not show expression changes in the Klf4-null. c) RT-PCR and <b>d)</b> Immunohistochemistry showing Klf5 a Kruppel-like factor belonging to the same gene family as Klf4, do not show expression changes in the Klf4-null. One-tail students’ T-test was used for analysis and results were represented with p<0.05(*), p<0.01 (**) and p<0.001 (***). Y-axis: Relative Quantity vs H2O –target gene expression of the sample compared against with a negative control where H2O were used as template. X-axis: WT- wild-type, Mut—Klf4-null.</p

The expression of Klf family members in mouse cerebellum.

<p>The expression of Klf family members in mouse cerebellum.</p

Klf4 has dual effects on the proliferation of epithelial cells in the cerebellum at E15.5.

<p>a) Immunolabeling of BrdU in the cerebellum and b) counting of BrdU+ cells at E15.5. There is an increased number of proliferating cells in the EGL (p<0.01) and RL (p<0.01), but a decreased number of proliferating cells in the NE (p<0.01) in the Klf4-null cerebellum, indicated by a one-tailed Students’ T-test p<0.05(*), p<0.01 (**) and p<0.001 (***). X-axis: EGL—external granular layer, RL-Rhombic lip, NE- neuroepithelium.</p