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

Zhang P, Ha T, Larouche M, Swanson D, Goldowitz D - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC, V5Z 4H4, Canada.

ABSTRACT
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- 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.

No MeSH data available.


Related in: MedlinePlus

Quantification of Pax6 cell number and expression down-regulation in Klf4- cerebellum.3a) 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 (***). 3b) Real-time PCR showing the expression of Pax6 in the wild-type and Klf4- 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- 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-.
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pone.0134390.g003: Quantification of Pax6 cell number and expression down-regulation in Klf4- cerebellum.3a) 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 (***). 3b) Real-time PCR showing the expression of Pax6 in the wild-type and Klf4- 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- 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-.

Mentions: We observe a marked reduction in Pax6 immunocytochemistry in Klf4-/- EGL cells at E13.5 (Fig 2a and 2b); and an almost complete elimination of staining in the E15.5 Klf4- when compared to the wild type cerebellum (Fig 2c and 2d). These observations suggest that Klf4 positively regulates Pax6 expression. To examine if the reduction of Pax6 expression is due to a smaller number of cells expressing similar levels of Pax6 or the same number of cells expressing Pax6 at a lower level; we quantified the number of Pax6+ cells. Indeed, fewer Pax6-positive cells are found in the Klf4-/- cerebellum at E13.5 (Fig 3a, p<0.05). A similar reduction of Pax6+ cells is found in the rhombic lip (Fig 3a, p<0.001) but not the proliferative neuroepithelium above the 4th ventricle. The quantitative assessment of Pax6 expression showed the similar reduction as seen in the sectioned and stained material at E13.5 (Fig 3b, p<0.01 and 15.5 (Fig 3b, p<0.01). Interestingly, at E16.5, there is a return of Pax6-immunopositivity in the EGL and RL (Fig 2e and 2f). The return of Pax6-positive staining appears almost complete by E18.5 (Fig 2g and 2h). The return of Pax6 expression at E18.5 is confirmed with real-time PCR (Fig 3b). Our observations indicate that Klf4 normally, positively regulates Pax6 during early granule cell development, prior to E16.5.


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

Zhang P, Ha T, Larouche M, Swanson D, Goldowitz D - PLoS ONE (2015)

Quantification of Pax6 cell number and expression down-regulation in Klf4- cerebellum.3a) 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 (***). 3b) Real-time PCR showing the expression of Pax6 in the wild-type and Klf4- 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- 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-.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520560&req=5

pone.0134390.g003: Quantification of Pax6 cell number and expression down-regulation in Klf4- cerebellum.3a) 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 (***). 3b) Real-time PCR showing the expression of Pax6 in the wild-type and Klf4- 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- 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-.
Mentions: We observe a marked reduction in Pax6 immunocytochemistry in Klf4-/- EGL cells at E13.5 (Fig 2a and 2b); and an almost complete elimination of staining in the E15.5 Klf4- when compared to the wild type cerebellum (Fig 2c and 2d). These observations suggest that Klf4 positively regulates Pax6 expression. To examine if the reduction of Pax6 expression is due to a smaller number of cells expressing similar levels of Pax6 or the same number of cells expressing Pax6 at a lower level; we quantified the number of Pax6+ cells. Indeed, fewer Pax6-positive cells are found in the Klf4-/- cerebellum at E13.5 (Fig 3a, p<0.05). A similar reduction of Pax6+ cells is found in the rhombic lip (Fig 3a, p<0.001) but not the proliferative neuroepithelium above the 4th ventricle. The quantitative assessment of Pax6 expression showed the similar reduction as seen in the sectioned and stained material at E13.5 (Fig 3b, p<0.01 and 15.5 (Fig 3b, p<0.01). Interestingly, at E16.5, there is a return of Pax6-immunopositivity in the EGL and RL (Fig 2e and 2f). The return of Pax6-positive staining appears almost complete by E18.5 (Fig 2g and 2h). The return of Pax6 expression at E18.5 is confirmed with real-time PCR (Fig 3b). Our observations indicate that Klf4 normally, positively regulates Pax6 during early granule cell development, prior to E16.5.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC, V5Z 4H4, Canada.

ABSTRACT
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- 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.

No MeSH data available.


Related in: MedlinePlus