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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

The expression levels of genes involved in complementary cell proliferative pathways in the Klf4- with real-time PCR.a) RT-PCR and 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-. c) RT-PCR and d) Immunohistochemistry showing Klf5 a Kruppel-like factor belonging to the same gene family as Klf4, do not show expression changes in the Klf4-. 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-.
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pone.0134390.g006: The expression levels of genes involved in complementary cell proliferative pathways in the Klf4- with real-time PCR.a) RT-PCR and 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-. c) RT-PCR and d) Immunohistochemistry showing Klf5 a Kruppel-like factor belonging to the same gene family as Klf4, do not show expression changes in the Klf4-. 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-.

Mentions: While the findings on Pax6 expression and granule cell proliferation were robust in the Klf4- at E13.5, the effects of Klf4 knockout were diminished at E18.5. One explanation for this result could be that the dynamic expression of Klf4 in the cerebellum—it is expressed highest in the cerebellum at E13.5 and lowest at E18.5. Thus, the Klf4- phenotypes may be due to expression level differences over developmental time. Another possibility for the observed temporal differences in the Klf4- is that the proliferative roles of Klf4 in the cerebellum could be replaced by other genes and pathways by E18.5. Other members of the Kruppel-like factor family are candidates for complete or partial functional redundancy since they are structurally similar. Therefore, we investigated potential functional redundant or functional complementary genes to Klf4. Expression level of Klf2 and Klf5, two other Klf transcription factors that have overlapping functions with Klf4 in the iPS cells, were not altered in the Klf4-/- at E13.5, E15.5 and P0 (Fig 6a and 6c). Immunohistochemistry staining at E13.5 and E15.5 also showed similar expression pattern for Klf2 (Fig 6b) and Klf5 (Fig 6d). The summarized expression data of all 17 Klf family members are shown in Table 1. Previous studies have shown that granule cell proliferation is regulated by at least two other molecular pathways: Zic and Wnt[21]. To examine these alternative granule cell proliferation pathways, the expression of the transcription factor Zic1 and β-catenin were measured with RT-PCR. We find that the expression level of Zic1 was normal in the Klf4-/- at E13.5 and E15.5 (Fig 7a). Gene expression of β-catenin at E13.5, determined by RT-PCR, showed a suggestive increase in the Klf4-/- when compared with wild-type, however, this increase was not significant (Fig 7b). Further validation with Anti-β-catenin immunohistochemistry showed that β-catenin is enhanced at E13.5 at the rhombic lip and EGL in the mutant compared to the wildtype (Fig 7c). At E15.5, there is a significant increase in β-catenin expression in the Klf4- cerebellum (Fig 7b, p<0.05) determined by RT-PCR; however, this increased expression is not obvious with immunohistochemical staining of β-catenin at E15.5. At this time, the immunostaining of β-catenin in both Klf4-/- and wild-type is much weaker compared with staining at E13.5 (Fig 7c).


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)

The expression levels of genes involved in complementary cell proliferative pathways in the Klf4- with real-time PCR.a) RT-PCR and 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-. c) RT-PCR and d) Immunohistochemistry showing Klf5 a Kruppel-like factor belonging to the same gene family as Klf4, do not show expression changes in the Klf4-. 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-.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134390.g006: The expression levels of genes involved in complementary cell proliferative pathways in the Klf4- with real-time PCR.a) RT-PCR and 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-. c) RT-PCR and d) Immunohistochemistry showing Klf5 a Kruppel-like factor belonging to the same gene family as Klf4, do not show expression changes in the Klf4-. 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-.
Mentions: While the findings on Pax6 expression and granule cell proliferation were robust in the Klf4- at E13.5, the effects of Klf4 knockout were diminished at E18.5. One explanation for this result could be that the dynamic expression of Klf4 in the cerebellum—it is expressed highest in the cerebellum at E13.5 and lowest at E18.5. Thus, the Klf4- phenotypes may be due to expression level differences over developmental time. Another possibility for the observed temporal differences in the Klf4- is that the proliferative roles of Klf4 in the cerebellum could be replaced by other genes and pathways by E18.5. Other members of the Kruppel-like factor family are candidates for complete or partial functional redundancy since they are structurally similar. Therefore, we investigated potential functional redundant or functional complementary genes to Klf4. Expression level of Klf2 and Klf5, two other Klf transcription factors that have overlapping functions with Klf4 in the iPS cells, were not altered in the Klf4-/- at E13.5, E15.5 and P0 (Fig 6a and 6c). Immunohistochemistry staining at E13.5 and E15.5 also showed similar expression pattern for Klf2 (Fig 6b) and Klf5 (Fig 6d). The summarized expression data of all 17 Klf family members are shown in Table 1. Previous studies have shown that granule cell proliferation is regulated by at least two other molecular pathways: Zic and Wnt[21]. To examine these alternative granule cell proliferation pathways, the expression of the transcription factor Zic1 and β-catenin were measured with RT-PCR. We find that the expression level of Zic1 was normal in the Klf4-/- at E13.5 and E15.5 (Fig 7a). Gene expression of β-catenin at E13.5, determined by RT-PCR, showed a suggestive increase in the Klf4-/- when compared with wild-type, however, this increase was not significant (Fig 7b). Further validation with Anti-β-catenin immunohistochemistry showed that β-catenin is enhanced at E13.5 at the rhombic lip and EGL in the mutant compared to the wildtype (Fig 7c). At E15.5, there is a significant increase in β-catenin expression in the Klf4- cerebellum (Fig 7b, p<0.05) determined by RT-PCR; however, this increased expression is not obvious with immunohistochemical staining of β-catenin at E15.5. At this time, the immunostaining of β-catenin in both Klf4-/- and wild-type is much weaker compared with staining at E13.5 (Fig 7c).

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