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

Effects of Klf4-knockout on cell death and/or cell proliferation in the developing cerebellum.

<p><b>A) Cresyl-violet staining of wild-type and Klf4-null cerebellum</b>. The appearance of heterochromatic cells is a hallmark of the Klf4-null EGL compared to the wildtype at E15.5 (black arrows). The proliferating cells were identified as having condensed heterochromatin in one of the phases of mitosis. <b>b) and c) BrdU-staining demonstrates a reduced proliferation of EGL and RL cells in the Klf4-null at E13.5</b>. b) Immunolabeling of BrdU in the cerebellum and c) counting of BrdU+ cells at E13.5. Proliferative cells incorporate BrdU into newly synthesized DNA and become BrdU+. There is a decreased number of BrdU+ cells in the EGL (p<0.01) and RL (p<0.05) of the Klf4-null cerebellum. BrdU+ cells were identified as a dark brown staining after histochemical reaction with DAB. Number of BrdU+ cells were compared with one-tail students’ T-test and results were represented with p<0.05(*), p<0.01 (**) and p<0.001 (***). X-axis: EGL—external granular layer, RL-Rhombic lip, NE- neuroepithelium.</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

Klf4 expression in the cerebellum and its co-expression with Pax6.

<p><b>a-c) Klf4 expression at: a) E13.5, b)E15.5, and c)E18.5</b>. Immunohistochemistry of Klf4 in the developing cerebellum. Klf4 is expressed in the EGL of the cerebellum at E13.5 and E15.5 but virtually no expression is seen at E18.5 (black arrows). <b>d-f) Co-expression of Klf4 and Pax6 in the EGL at E15.5</b>. Immunofluorescence staining of Klf4 (green, d), Pax6 (red, e) and merged picture (f) in the developing cerebellum. Klf4 and Pax6 are co-expressed in the EGL (white arrows) of the cerebellum at E15.5. In (f), green arrow indicates EGL cells that express only Klf4, red arrow indicates cells in the cerebellar core that express only Pax6, and yellow arrow indicates EGL cells that co-express Klf4 and Pax6. EGL- external granular layer, NE—neuroepithelium, RL- Rhombic lip.</p

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

<p><b>a)</b> Zic1, an early granule cell proliferation gene at E13.5, does not show expression changes in the Klf4-null. <b>b)</b> β-catenin, a member of Wnt pathway, shows an activated expression in the Klf4-null (p<0.05) at E15.5. E13.5 and E15.5 immunohistochemistry against β-catenin, there is no difference observed at E15.5 as the stain is generally weaker than E13.5. 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

Pax6 is down-regulated in Klf4-null cerebellum at E13.5 and E15.5.

<p>Immunohistochemical demonstration of Pax6 expression during development in Klf4-wildtype (a,c,e,g) and -null (b,d,f,h) cerebellum. Pax6 immunocytochemistry is similar in the developing EGL of the wildtype cerebellum from E13.5 to E18.5. Pax6’s expression is greatly reduced at E13.5 and E15.5 in the Klf4-null cerebellum but rebounds at E16.5 and E18.5.</p

Quantification of Pax6 cell number and expression down-regulation in Klf4-null cerebellum.

<p><b>3a)</b> E13.5 Pax6 positive granule cell count in Klf4-/- compared with wild-type in the EGL (p<0.05), RL (p<0.001), and NE. One-tail students’ T-test was used and results were represented with p<0.05(*), p<0.01 (**) and p<0.001 (***). <b>3b)</b> Real-time PCR showing the expression of Pax6 in the wild-type and Klf4-null at E13.5, E15.5 and E18.5 in the whole cerebellum. The expression of Pax6 is ~23% of the wild-type expression level in the Klf4-null in the E13.5 (p<0.01) and 15.5 (p<0.01). One-tail students’ T-test was used 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: EGL- external granular layer, NE—neuroepithelium, RL- Rhombic lip, WT- wild-type, Mut—Klf4-null.</p