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Published on: October 19, 2013
Zebrafish krüppel-like factor 4a represses intestinal cell proliferation and promotes differentiation of intestinal
I-Chen Li1, Chein-Tso Chan, Yu-Fen Lu
1Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan.
Background:
Mouse krüppel-like factor 4 (Klf4) is a zinc finger-containing transcription factor required for terminal differentiation of goblet cells in the colon. However, studies using either Klf4(-/-) mice or mice with conditionally deleted Klf4 in their gastric epithelia showed different results in the role of Klf4 in epithelial cell proliferation. We used zebrafish as a model organism to gain further understanding of the role of Klf4 in the intestinal cell proliferation and differentiation.
Methodology/Principal Findings:
We characterized the function of klf4a, a mammalian klf4 homologue by antisense morpholino oligomer knockdown. Zebrafish Klf4a shared high amino acid similarities with human and mouse Klf4. Phylogenetic analysis grouped zebrafish Klf4a together with both human and mouse Klf4 in a branch with high bootstrap value. In zebrafish, we demonstrate that Klf4a represses intestinal cell proliferation based on results of BrdU incorporation, p-Histone 3 immunostaining, and transmission electron microscopy analyses. Decreased PepT1 expression was detected in intestinal bulbs of 80- and 102-hours post fertilization (hpf) klf4a morphants. Significant reduction of alcian blue-stained goblet cell number was identified in intestines of 102- and 120-hpf klf4a morphants. Embryos treated with γ-secretase inhibitor showed increased klf4a expression in the intestine, while decreased klf4a expression and reduction in goblet cell number were observed in embryos injected with Notch intracellular domain (NICD) mRNA. We were able to detect recovery of goblet cell number in 102-hpf embryos that had been co-injected with both klf4a and Notch 1a NICD mRNA.
Conclusions/Significance:
This study provides in vivo evidence showing that zebrafih Klf4a is essential for the repression of intestinal cell proliferation. Zebrafish Klf4a is required for the differentiation of goblet cells and the terminal differentiation of enterocytes. Moreover, the regulation of differentiation of goblet cells in zebrafish intestine by Notch signaling at least partially mediated through Klf4a.
Insights
Zebrafish Klf4a represses intestinal cell proliferation and is crucial for goblet cell differentiation. Notch signaling regulates goblet cell differentiation, partly through Klf4a.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- Krüppel-like factor 4 (Klf4) is a transcription factor involved in goblet cell differentiation in mice.
- Conflicting results exist regarding Klf4's role in epithelial cell proliferation in mammalian models.
- Zebrafish offer a model to investigate Klf4's function in intestinal cell proliferation and differentiation.
Purpose of the Study:
- To characterize the function of zebrafish klf4a, a homolog of mammalian Klf4, in intestinal development.
- To elucidate the role of Klf4a in intestinal cell proliferation and goblet cell differentiation in vivo.
- To investigate the interplay between Notch signaling and Klf4a in regulating goblet cell differentiation.
Main Methods:
- Antisense morpholino oligomer knockdown was used to inhibit klf4a expression in zebrafish embryos.
- Cell proliferation was assessed using BrdU incorporation and p-Histone 3 immunostaining.
- Goblet cell differentiation was evaluated by alcian blue staining and PepT1 expression analysis.
Main Results:
- Zebrafish Klf4a was found to repress intestinal cell proliferation.
- Klf4a knockdown led to decreased PepT1 expression and a significant reduction in goblet cell numbers.
- Notch signaling pathway activation (via γ-secretase inhibitor) increased klf4a expression, while inhibition (via NICD mRNA) decreased it and goblet cell numbers.
Conclusions:
- Zebrafish Klf4a is essential for repressing intestinal cell proliferation and promoting goblet cell and enterocyte differentiation.
- Notch signaling pathway regulates goblet cell differentiation in the zebrafish intestine, at least partially through Klf4a.
- This study provides in vivo evidence for Klf4a's critical role in intestinal homeostasis and differentiation.
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