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. 2013 Mar 15;375(2):128-39.
doi: 10.1016/j.ydbio.2012.12.010. Epub 2012 Dec 22.

Kruppel-like factor 5 controls villus formation and initiation of cytodifferentiation in the embryonic intestinal epithelium

Sheila M Bell  1 , Liqian Zhang, Yan Xu, Valerie Besnard, Susan E Wert, Noah Shroyer, Jeffrey A Whitsett
Affiliations

Kruppel-like factor 5 controls villus formation and initiation of cytodifferentiation in the embryonic intestinal epithelium

Sheila M Bell et al. Dev Biol. .

Abstract

Kruppel-like factor 5 (Klf5) is a transcription factor expressed by embryonic endodermal progenitors that form the lining of the gastrointestinal tract. A Klf5 floxed allele was efficiently deleted from the intestinal epithelium by a Cre transgene under control of the Shh promoter resulting in the inhibition of villus morphogenesis and epithelial differentiation. Although proliferation of the intestinal epithelium was maintained, the expression of Elf3, Pparγ, Atoh1, Ascl2, Neurog3, Hnf4α, Cdx1, and other genes associated with epithelial cell differentiation was inhibited in the Klf5-deficient intestines. At E18.5, Klf5(Δ/Δ) fetuses lacked the apical brush border characteristic of enterocytes, and a loss of goblet and enteroendocrine cells was observed. The failure to form villi was not attributable to the absence of HH or PDGF signaling, known mediators of this developmental process. Klf5-deletion blocked the decrease in FoxA1 and Sox9 expression that accompanies normal villus morphogenesis. KLF5 directly inhibited activity of the FoxA1 promoter, and in turn FOXA1 inhibited Elf3 gene expression in vitro, linking the observed loss of Elf3 with the persistent expression of FoxA1 observed in Klf5-deficient mice. Genetic network analysis identified KLF5 as a key transcription factor regulating intestinal cell differentiation and cell adhesion. These studies indicate a novel requirement for KLF5 to initiate morphogenesis of the early endoderm into a compartmentalized intestinal epithelium comprised of villi and terminally differentiated cells.

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Figures

Figure 1
Figure 1. Efficient deletion of Klf5 from the intestinal epithelium by ShhEgfp/Cre
KLF5 immunohistochemistry: arrows indicate the normal expression pattern throughout the intestinal epithelium in (A, A′) and absence of KLF5 in most of the intestine of Klf5Δ/Δ embryos (B). Inefficient recombination occurs in the duodenum (arrowhead in B). Gross appearance of the E18.5 gastrointestinal tract (C–D). In the Klf5Δ/Δ embryos, the intestinal length was decreased and the gut tube appeared more translucent and dilated. St (stomach), Ce (cecum).
Figure 2
Figure 2. KLF5 is required for villus morphogenesis and epithelial differentiation
At E18.5, the goblet cell marker CLCA3 stained goblet cells on the villi of the small intestine (arrows in A) and within the colon of control fetuses (arrowhead). Villus-like structures were rarely observed in the Klf5Δ/Δ epithelium (B) and few goblet cells were present in either the intestine (B, arrows) or colon (arrowhead). Chromogranin A positive staining enteroendocrine cells were present in controls (C, arrows) but rare in the Klf5Δ/Δ tissue (D, arrows). The enterocyte brush border was abnormal in the Klf5Δ/Δ epithelium (E, vs. F, arrowheads) as assessed by transmission electron microscopy. The location of tight junctions was similar in both tissues (E, F arrows). E-cadherin staining indicates that like the control (G) the epithelium lining the Klf5Δ/Δ (H) intestine was of columnar morphology on the flattened regions and on the rudimentary villus-like structures. Lumen of intestine (lu), colon (c), nucleus (nu). Scale bars for each stain are present in the Klf5Δ/Δ image.
Figure 3
Figure 3. Proliferation continues in the absence of KLF5
Proliferating cells were identified by immunohistochemical staining for phospho-histone H3 (A–B), bromodeoxyuridine (Brdu)(C–D), cyclin D1 (E), or Ki67 (G–H) at the indicated ages, n≥3 embryos per age and genotype. Similar levels of proliferation were observed within the epithelium and mesenchyme of control and Klf5Δ/Δ embryos at each time point. Cyclin D1 was detected in the mesenchyme and intervillus regions of controls (C, arrowheads) but was absent in the Klf5Δ/Δ epithelium (C, blue arrows). Epithelial proliferation was restricted to the intervillus region in control epithelium (C, G, arrowheads) but was present throughout the Klf5Δ/Δ epithelium (D, H, arrows). Scale bar in A is for images A–F. Scale bar in G is for G–H.
Figure 4
Figure 4. KLF5 is required for villus emergence at E15.5
At E14.5 a pseudostratified intestinal epithelium was present in control and Klf5Δ/Δ embryos that uniformly expressed CDX2 (A, B). At E15.5, villi are forming in proximal regions of control fetuses accompanied by a down regulation of SOX9 at the villus tips (C, arrows inset). Uniform SOX9 expression was present in distal regions of the intestine (C, arrowhead). SOX9 was uniform throughout the intestine of Klf5Δ/Δ embryos (D, arrowheads). At E16.5, defined villi were present in controls where FOXA1 was localized to a subset of villus cells (G, arrows). Villi were absent within epithelium of Klf5Δ/Δ embryos and FOXA1 staining was present throughout the epithelium (H, arrowheads). At E18.5 SOX9 (E, arrows) and FOXA1 (I, arrows) expression were restricted in controls and both were expressed at high levels in the flattened epithelium of Klf5Δ/Δ fetuses (F, J, arrowheads) but absent in the rare villus-like structures (blue arrows, F and J). Scale bars for each pair of images represent 100 μm.
Figure 5
Figure 5. KLF5 regulates villus morphogenesis
In control and Klf5Δ/Δ E15.5 embryos, immunohistochemistry detected PDGFRα in the mesenchyme in a thin line of cells subjacent to the epithelium (arrowheads, A, B) and in mesenchymal cell clusters within proximal regions of the small intestine (arrows). By E16.5, PDGFRα staining was detected in the tips of formed villi throughout the small intestine in controls (C, arrows). Although some mesenchymal clusters expressing PDGFRα were observed in distal regions of the Klf5Δ/Δ intestine (D, arrows) elongated villi were absent and clusters of PDGFRα expressing cells were not associated with flattened regions of the epithelium (D, arrowheads). Bmp4 mRNA was present within the subepithelial mesenchyme in both the control and Klf5Δ/Δ intestine (E, F, arrowheads). Note absence of forming villi in Klf5Δ/Δ embryos compared to controls. SOX2 was detected within the mesenchyme of the intestine (Int) and colon in both genotypes (G, H, arrows). Morphogenesis of the colon epithelium was perturbed in Klf5Δ/Δ embryos (G vs. H, arrowheads). Significant differences in Pdgfa (P≤.03), Ihh (P <.02), Ptc1 (P<.03), and Gli2 (P≤.007) mRNAs were detected by QRT-PCR, n=5 per genotype (I). αSMA detected an outer (arrow) and inner (arrowhead) muscle layer in the mesenchyme of Klf5Δ/Δ and control embryos (J, K). Lu (lumen).
Figure 6
Figure 6. Transcriptome analysis in KLF5 deficient intestines
Heat map of all genes differentially expressed between Klf5Δ/Δ and control intestines in which mRNA expression changed > 1.5 fold with P ≥ .05. Green indicates the presence of a lower level of mRNA (A). QRT-PCR analysis of key transcription factors known to mediate terminal differentiation of epithelial cells at E14.5 and E16.5 (*, P≤.006), (**, P≤ .001)(***, P≤.0005)(B). QRT-PCR of E14.5 intestines also confirmed differential expression of signaling molecules and structural proteins associated with terminal maturation (*, P≤ .04)(**, P≤ .01)(***, P≤.005) (C). For QRT-PCR, the two-tailed student T-test was used to assess statistical differences, (n=5 at E16.5; n=4 at E14.5).
Figure 7
Figure 7. Identification and characterization of intestinal transcription networks regulated by KLF5
Ingenuity Pathway Analysis of the differentially expressed genes within the microarray identified gene networks functionally enriched in “transcriptional regulation of cell differentiation and “cell adhesion” (A). Solid lines (direct interactions), dashed lines (indirect interactions). B–C) Representative transient transfection assays of HEK293T cells. Luciferase activity was normalized to Renilla activity 24 hours post transfection. For all experiments, empty vector was pcDNA3.1. KLF5 over-expression inhibited all mFoxA1 promoter constructs containing the region +43/+355 (B). FOXA1 expression repressed both mElf3 promoter pGL3 luciferase constructs, whereas KLF5 had no effect(C). FOXA1 expression was confirmed by Western blot analysis (data not shown). The two-tailed student T-test was used to assess statistical differences (*, P≤.03), (***, P≤.005).
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