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Am J Physiol Gastrointest Liver Physiol 292: G1757-G1769, 2007. First published March 22, 2007; doi:10.1152/ajpgi.00013.2007
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MUCOSAL BIOLOGY

Dysregulation of intestinal crypt cell proliferation and villus cell migration in mice lacking Krüppel-like factor 9

Frank A. Simmen,1,2 Rijin Xiao,1,2 Michael C. Velarde,1,2 Rachel D. Nicholson,2 Margaret T. Bowman,2 Yoshiaki Fujii-Kuriyama,3 S. Paul Oh,4 and Rosalia C. M. Simmen1,2

Department of 1Physiology and Biophysics and 2Arkansas Children's Nutrition Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas; 3TARA Center, University of Tsukuba, 1-1-1 Tennodai Tsukuba 305-8577, Japan; and 4Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida

Submitted 6 January 2007 ; accepted in final form 14 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Krüppel-like factor 9 (Klf9), a zinc-finger transcription factor, is implicated in the control of cell proliferation, cell differentiation, and cell fate. Using Klf9-null mutant mice, we have investigated the involvement of Klf9 in intestine crypt-villus cell renewal and lineage determination. We report the predominant expression of Klf9 gene in small and large intestine smooth muscle (muscularis externa). Jejunums null for Klf9 have shorter villi, reduced crypt stem/transit cell proliferation, and altered lineage determination as indicated by decreased and increased numbers of goblet and Paneth cells, respectively. A stimulatory role for Klf9 in villus cell migration was demonstrated by bromodeoxyuridine labeling. Results suggest that Klf9 controls the elaboration, from intestine smooth muscle, of molecular mediator(s) of crypt cell proliferation and lineage determination and of villus cell migration.

Krüppel-like; Igfbp4; Ptk6; stem cells; colon; smooth muscle

KRÜPPEL-LIKE FACTOR 9 (Klf9), previously designated basic transcription element binding protein (Bteb) 1, is a transcriptional regulator whose primary sequence is highly conserved among vertebrate organisms (11, 44). Klf9 belongs to a family of transcriptional mediators [specificity protein 1 (SP1)-like/Krüppel-like factors, SP/KLF family], defined by the presence of an 81-amino-acid DNA-binding region located in the carboxy terminus and comprised of three contiguous C2-H2 zinc fingers (2, 12, 38). There are 25 SP/KLF genes/proteins known at present. SP/KLF proteins bind to GC/GT boxes (consensus binding site: 5'-NGGGNGNGG-3') resident in promoter and enhancer/silencer regions of multiple chromosomal genes (38). Although each SP/KLF family member is homologous to all others by virtue of the Krüppel-like DNA-binding domain, each is unique with respect to the sequence amino terminal to this domain. Some KLFs are widely expressed in multiple tissues, whereas others are more restricted in their expression (38). KLFs function as transcriptional activators and/or repressors depending on target gene, cis element(s), and tissue context and by partnering with other nuclear proteins (12, 49). Data, albeit limited, suggest nonoverlapping functional roles for SP/KLF proteins in tissue growth, morphogenesis, and stem cell biology (38).

Klf9 was first isolated as a transcriptional inducer of the hepatic CYP1A1 gene (11). Later work identified stimulatory roles in vitro for Klf9 in neuronal cell differentiation (7) and in endometrial cell proliferation (35, 48). In support of the latter, subsequent studies described a role for Klf9, in concert with progesterone receptor, in determining the program of endometrial secretory protein gene expression (34, 36, 42, 47). Studies of the Klf9-null mutant (Klf9–/–) mouse have borne out findings from in vitro studies, namely that this transcription factor has a functional role in the developing brain (mutant mice have deficits in cerebellum function as gauged by certain behavioral tests) (22) and in female reproductive function, the latter at the level of the maternal uterus (37, 41). Female Klf9–/– mice exhibit reduced numbers of implantation sites, smaller uteri, and developmental asynchrony of embryos and uterus and as a consequence are subfertile compared with wild-type (WT) dams (37, 41).

While elucidating the reproductive phenotype of Klf9 mutant mice, we observed a significant degree of mortality of Klf9–/– newborn mice as well as mild growth retardation of preweaning Klf9–/– mice relative to WT counterparts (37). This, coupled with the well-characterized roles for two other Klf proteins (Klf4, Klf5) in intestinal crypt-villus growth and differentiation (5, 30, 31), prompted us to characterize the expression and in vivo function of Klf9 in mouse intestine during postnatal ontogeny. In this report, we document the expression of the Klf9 gene in small and large intestine smooth muscle and describe a subtle, albeit significant, intestine mucosal phenotype in mice lacking this gene. In addition, we identify potential downstream (direct and indirect) gene targets and pathways of Klf9 by using microarrays. Our results suggest that Klf9 controls elaboration, from intestine smooth muscle, of molecular mediator(s) of crypt cell proliferation, villus cell migration, and Paneth and goblet cell differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Animal use protocols were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences. Klf9 mutant mice (Klf9lacZ) in the C57BL/6J background were previously generated by insertion of the bacterial beta-galactosidase (LacZ) gene in frame within exon 1 of the mouse Klf9 gene (22). Mice were maintained on a 12-h light/12-h dark schedule with ad libitum access to food and water. Animals were genotyped as previously described (37).

Tissue collection. The small intestine was divided into three equal segments that were operationally defined as duodenum, jejunum, and ileum. Tissue samples from the midpoint of jejunum and ileum were placed in formalin for later histochemistry or immunohistochemistry analyses or were cryopreserved (see below) for 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining. The colon was divided into two halves, proximal and distal, and the midpoint of each half-segment was fixed in 10% neutral-buffered formalin or was cryopreserved. Fixed tissues were subsequently embedded in paraffin.

Histochemistry. Fixed tissues were embedded in paraffin. Sections (5 µm) were deparaffinized in xylene, rehydrated through a series of alcohols, stained with hematoxylin and eosin (H&E), and coverslipped. Differentiated cell types within intestinal crypts and villi were identified by histochemistry. Grimelius stain (18) was used to identify enteroendocrine cells; the Lendrum's phloxine-tartrazine stain/procedure (1) was used to identify Paneth cells; and Alcian Blue (pH 2.5) (3), mucicarmine (http://www.ihcworld.com/_protocols/special_stains/southgate_mucicarmine_ellis.htm), or H&E stains were used for goblet cell identification. For purposes of X-gal staining, freshly isolated gastrointestinal tissues were soaked in PBS containing 20% sucrose, embedded in optimum cutting temperature medium (Fisher Scientific), and frozen in liquid nitrogen. Sections (7 µm) were cut by using a cryostat, postfixed in PBS containing 2% paraformaldehyde and 0.2% glutaradehyde for 5 min, and then stained with X-gal (1 mg/ml) at 37°C overnight, followed by counterstaining with neutral red (1%) in sodium acetate (50 mM, pH 3.3). Lengths of villi, depths of crypts, and muscularis thicknesses were measured by using MCID software (Interfocus, Linton, UK).

Immunohistochemistry. Thirty-day-old male mice were injected intraperitoneally with 150 µl of bromodeoxyuridine (BrdU) labeling reagent (Zymed Laboratories, South San Francisco, CA) at 2 and 48 h before tissue collection. Tissues were fixed in 10% neutral-buffered formalin and were embedded in paraffin. Sections (4 µm) were cut, applied to ProbeOn Plus slides, dewaxed in xylene, and rehydrated through a series of graded alcohols. Antigen retrieval was done in 1x Citra-plus (Biogenics, Napa, CA). After being cooled, slides were washed in 1x PBS and H2O, quenched in 3% H2O2, and blocked with 10% donkey serum-1x PBS. Anti-BrdU mouse IgG1 monoclonal antibody (Roche Diagnostics, Indianapolis, IN) was diluted in blocking buffer (1:500) and was incubated on slides overnight at 4°C. After a wash with PBS, biotin-SP-conjugated affinity-purified F(ab')2 fragment donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was diluted (1:100) in blocking buffer and was applied to the slides for 30 min at 37°C. Fluorescein (DTAF)-conjugated streptavidin-RITC (Jackson ImmunoResearch) was diluted in blocking buffer (1:100) and was applied to slides for 30 min at 37°C. After being washed, slides were counterstained with 0.01% Evans blue and were viewed on an Axiovert 200M microscope. Images were captured by AxioCam HRc (Zeiss Oberkochen) and were processed by Axiovision software release 4.5 SPI (03-2006) (Zeiss Oberkochen). BrdU-labeled cells in crypts were counted manually in a blinded fashion. Immunohistochemistry of PCNA followed standard methodologies described previously for other antigens (41, 45). Mouse monoclonal antibody to PCNA was from Dako (Carpinteria, CA).

Microarrays. Total RNA was extracted in parallel from the jejunums of five WT and five Klf9–/– male mice at postnatal day (PND) 30 by using Trizol reagent (Invitrogen, Carlsbad, CA). Conversion of each RNA preparation to the corresponding fragmented cRNA probe was as previously described (45). Fifteen micrograms of each cRNA were hybridized for 16 h to an Affymetrix mouse 430A GeneChip. Ten GeneChips (each corresponding to a single animal) were hybridized, washed, and scanned in parallel. Following the wash, signal-amplification, and signal-detection steps, GeneChips were scanned (Agilent GeneArray laser scanner) and the resultant images were quantified by using Affymetrix MAS 5.0 software. The average of the fluorescent intensities of all probe sets on a given array was scaled to a constant target intensity of 500, and the data from each GeneChip were log2 transformed and normalized by using median values within each treatment group (genotype) (13). Differentially expressed transcripts were identified by the t-test function in Spotfire DecisionSite (Somerville, MA) using P < 0.05. Differentially expressed transcripts were further filtered by using a fold-change cutoff of 1.3, a value approaching the practical limit for detectable differences with Affymetrix microarrays (28). An additional filter required that all transcripts expressed at higher levels in WT jejunum had to be called "present" on all five corresponding GeneChips, whereas transcripts expressed at higher levels in Klf9–/– jejunums had to be called "present" on all five corresponding GeneChips. Unsupervised nearest-neighbor hierarchical clustering was performed by using Spotfire DecisionSite. Gene lists were annotated by using NETAFFX (http://www.affymetrix.com/analysis/index.affx), Gene Ontology (http://www.geneontology.org/), and NCBI (http://www.ncbi.nlm.nih.gov/). The complete microarray dataset was deposited in Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE6443.

Real time RT-PCR. Primer sequences designed by using Primer Express (Applied Biosystems, Foster City, CA) were as follows: Klf9, forward 5'-CGTTGCCCACTGTGTGAGAA-3', reverse 5'-TTGATCATGCTGGGATGGAA-3'; Klf5, forward 5'-TCCGTCCTATGCCGCTACAA-3', reverse 5'-CCAGATCCGGGTTACTCCTTCT-3'; Klf13, forward 5'-ACACAGGTGAGAGGCCTTTCG-3', reverse 5'-AGCATGCCTGGGTGGAAG-3'; Klf16, forward 5'-CCTTACTCCCACTGGGTTAGGG-3', reverse 5'-AGCACATGACGGCAG ACCA-3'; Klf4, forward 5'-AGAGGAGCCCAAGCCAAAGA-3', reverse 5'-AGTTCGCAGGTGTGCCTTGA-3'; IGF binding protein gene Igfbp4, forward 5'-CCAAACTGTGACCGCAACG-3', reverse 5'-CCAAACCCCCAGGAAGCTT-3'; protein tyrosine kinase gene Ptk6, forward 5'-TCCCAAGTGCTGGGATCAAA-3', reverse 5'-TCCACAAGGCCTGTTGCCTA-3'; pituitary homeobox 2 (Pitx2), forward 5'-AACCTTACGGAAGCCCGAGTC-3', reverse 5'-CCCAAAGCCATTCTTGCACA-3'; cyclin D2 gene Ccnd2, forward 5'-CTTTGTGGTAGGACGGTGGGT-3', reverse 5'-TGTGCAGTGCGTGAGCTCTG-3'; myotubularin 1 (Mtm1), forward 5'-TGTCTCAAGATGGAGTCAGT-3', reverse 5'-GACCATAGGAATTTTCTCCTC-3'; CEA-related cell adhesion molecule gene Ceacam1, forward 5'-TTGTTGTCTTCAGCAACCTGG-3', reverse 5'-AGGACTACTGCTCACAGCCTC-3'; Igfbp5, forward 5'-GCTCGCCGTAGCTCTTTTC-3', reverse 5'-GGTTCTTTCGTGCACTGTGA-3'; cyclophilin A gene Ppia, forward 5'-TGTGCCAGGGTGGTGACTTTA-3', reverse 5'-AGATGCCAGGACCTGTATGCTT-3'. One microgram of RNA from each jejunum was reverse transcribed by using random hexamers and MultiScribe Reverse Transcriptase (Applied Biosystems). Real-time PCR was performed with an ABI Prism 7000 Sequence Detector as described previously (45).

Statistical analysis. Statistical analysis was performed by using SigmaStat for Windows (SPSS, Chicago, IL). Values are presented as means ± SE or as box plots. Differences between treatment means were considered significant at P < 0.05, whereas 0.05 < P < 0.1 indicated a tendency for a difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Klf9–/– on gastrointestinal tissue weight at postweaning. We previously observed reduced body weights for young Klf9–/– mice (37). To elucidate the physiological basis for this observation, the gastrointestinal tracts of Klf9–/– and heterozygous mutant Klf9+/– male mice and their corresponding WT counterparts were evaluated at early postweaning. At 28 days of age, Klf9–/– mice had reductions in body weight compared with heterozygous mutant and WT counterparts (Table 1), in agreement with our previous report (37). Gastrointestinal tissue weights (normalized to individual body weight) of Klf9–/– mice were reduced compared with heterozygous mutant and WT counterparts (Table 1). For the stomach and colon, numerical differences in wet weight were statistically significant; by contrast, numerical reductions in small intestine weight of Klf9–/– mice were not. Klf9 protein abundance was reduced and absent in heterozygous and homozygous null jejunums, respectively (data not shown).


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  		Table 1. Body weights and gastrointestinal tissue weights of postweaning male mice

 
Effects of Klf9 gene ablation on intestinal crypts and villi. We measured small intestine villi and crypts as indices of mucosal tissue growth. In jejunums of male mice at 4 and 6 wk of age, absence of Klf9 resulted in significant reductions (by up to 40%) in villus length, without any alteration in crypt depth (Fig. 1, A–D). Interestingly, differences in villus length were not apparent at 6 mo of age, because knockout jejunum villus lengths had reached that of WT (Fig. 1C). At 6 mo of age, however, knockout jejunum crypt depth tended (P = 0.06) to be reduced compared with WT (Fig. 1D). Quantification of number of cells staining positive for PCNA (a marker primarily of the crypt transit cell population) at postweaning revealed decreases (~40% for jejunum, ~29% for ileum) with absence of Klf9 (Fig. 1, E and F). The measured width (in cross-section) of the muscularis externa in jejunum was unaffected by Klf9 gene deletion (data not shown). The data indicate reductions in villus length that can be predicted to result in lower capacity for nutrient absorption and hence lower body weight.


Figure 1
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  		Fig. 1. Shorter villi in Krüppel-like factor (Klf) 9 knockout (Klf9–/–) mice at 4 wk and 6 wk but not 6 mo of age. Representative sections of jejunum from wild-type (WT) [A, postnatal day (PND) 29] and Klf9–/– (B, PND 31) males were hematoxylin and eosin-stained; me, muscularis externa; vi, villus. C and D: villus length and crypt depth were measured for a minimum of 20 crypt-villus units per slide, with 2–3 slides analyzed per animal. Data were analyzed for effects of genotype within each age group (3–8 animals per age/genotype) with the Student's t-test. Crypt depths did not differ between genotypes (P > 0.1) at 4 and 6 wk of age, but a reduction in length of Klf9–/– crypts, which approached statistical significance (P = 0.06), was found for animals of 6 mo of age. E: 3 or 4 tissue sections from each WT (n = 4) and Klf9–/– (n = 3) male mouse jejunum, respectively (ages ranged from PND 25 to PND 33) underwent immunohistochemistry for PCNA. F: 3 or 4 sections from each WT (n = 2) and Klf9–/– (n = 4) male mouse ileum, respectively (PND 25–33) stained for PCNA. PCNA-positive cells (exhibiting nuclear staining) in crypt-villus epithelium were counted, and data (means ± SE per crypt-villus unit) were analyzed by Student's t-test.

 
Mitosis of stem cells deep within crypts of the small intestine yields transit cells that further divide (over several generations) to provide villus enterocyte, goblet, and endocrine cell lineages (cell differentiation commences with migration out of the crypts) or Paneth cells (migrate to the crypt base). Because Klf9 knockout resulted in a reduced number of PCNA-positive cells in small intestine crypt epithelium (Fig. 1E), we examined if cell lineage determination was consequently perturbed. Quantification of enteroendocrine cells in tissue sections subjected to Grimelius argyrophil stain revealed no differences in numbers of this cell type for WT vs. Klf9–/– jejunum (data not shown). Paneth cells were identified histochemically by using Lendrum's procedure. Interestingly, a small but significant increase in number of Paneth cells was found for Klf9–/– jejunum crypts compared with WT (Fig. 2, A–C). Klf9–/– jejunum manifested fewer numbers of goblet cells in crypt but not in villus epithelium (Fig. 2, D–E). Thus absence of Klf9 led to reduced crypt transit cell proliferation (reduced number of PCNA-positive cells) and subtle changes in goblet and Paneth cell specification processes/pathways within the small intestine.


Figure 2
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  		Fig. 2. Lineage determination is perturbed in jejunum villus epithelium of Klf9–/– mice. A and B: representative sections of jejunum stained with Lendrum's reagent to reveal Paneth cells (arrows). Note the typical granular appearance of Paneth cell cytoplasm. C: quantification of Paneth cells in jejunum crypts. Paneth cells were counted in a minimum of 20 well-oriented crypts per animal (n = 3 animals/genotype), and results were analyzed by Student's t-test. D and E: goblet cell numbers per crypt and villus, respectively, for WT (n = 3) and Klf9–/– (n = 3) jejunums at PND 30. Goblet cell numbers within crypt, but not villus, epithelium were reduced with Klf9 knockout.

 
Effects of Klf9 gene deletion on colon histomorphology. Tissue sections from midpoints of proximal and distal colons of ~ 4-wk-old mice were subjected to H&E staining, followed by determination of crypt depth, goblet cell number per crypt, and cross-sectional thickness of the muscularis externa (Fig. 3, A–C). Results demonstrated a tendency (P = 0.054) for reduced (by ~12%) colon crypt depths in knockout animals with no interaction of genotype and colon location. Goblet cell number per crypt was increased (by 2–3 cells/crypt, P < 0.05) in proximal colons of Klf9 KO animals (Fig. 3D), with no such differences observed for distal colons. There was no difference in thickness of the muscularis with genotype (P = 0.928; Fig. 3E).


Figure 3
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  		Fig. 3. A and B: representative hematoxylin and eosin-stained sections of proximal colon from WT (PND 27) and Klf9–/– (PND 33) male mice. Crypt depths and muscularis externa thicknesses were measured for proximal and distal colons (PND 30 ± 3 days; minimum of 40 crypts and 5 muscle measurements per slide, 1 slide per region/animal). C: data corresponding to proximal colon (n = 10 WT and n = 9 Klf9–/– animals) and distal colon (n = 6 WT and n = 6 Klf9–/– animals) were analyzed by using 2-way ANOVA. Crypt depths did not differ between colon regions (P = 0.139), but a tendency for a difference between genotypes (P = 0.054, both regions combined) was found. D: in proximal colon only, Klf9–/– crypts had more goblet cells than did WT crypts (P = 0.015). E: smooth muscle thickness did not differ by genotype; however, smooth muscle of the proximal colon was thicker than that of the distal colon (P = 0.018).

 
Tissue-expression domains of Klf9. In view of the above results, it was important to establish the cellular location of Klf9 expression. To address this, we took advantage of the targeted insertion of the Escherichia coli LacZ gene within Klf9 exon 1, which allows for in situ staining of Klf9 gene expression with X-gal (22). WT tissues served as negative controls because they lack the inserted LacZ gene. As expected, WT tissue sections had no X-gal-stained (blue) cells (Fig. 4A). In Klf9+/– and Klf9–/– jejunums, X-gal staining was observed throughout the muscularis externa, with rare stained cells observed in the lamina propria (Fig. 4, B and C). In colon, the muscularis externa was strongly stained, as were cells comprising the surface epithelium (Fig. 4D). Sporadic X-gal-stained lamina propria cells were apparent also (Fig. 4D). The identities of the X-gal stained lamina propria cells in small and large intestines are unknown.


Figure 4
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  		Fig. 4. Klf9 gene promoter activity in jejunum and colon. A: WT jejunum, PND 22; B: Klf9 heterozygous (Klf9+/–) jejunum, PND 27; C: Klf9–/– jejunum, PND 25. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal)-stained muscularis externa was apparent in mutant but not WT jejunum (B; large arrow, inner circular layer; small arrows, outer longitudinal layer). Rare X-gal-stained cells were observed within the villus lamina propria (C, arrows) of mutant animals. D: proximal colon of Klf9–/– animals at PND 27, stained with X-gal. Stained cells were prevalent in the luminal surface epithelium (left, large arrow; image corresponds to upper red-boxed area of middle) and muscularis externa (right, arrows; image corresponds to lower red-boxed area of middle) with lesser staining observed in the upper crypts and some lamina propria cells. No X-gal-stained cells were observed in negative control (WT) colon (not shown).

 
Gene transcripts that are differentially expressed between WT and Klf9–/– small intestines. We next performed gene expression profiling to identify underlying mechanism(s) of Klf9 action. Initial efforts used quantitative real-time RT-PCR to examine relative abundance of key Klf9-related genes in small and large intestine. We chose Klf5 (previously referred to as intestinal Klf, Bteb2), Klf13(Bteb3, most similar in sequence to Klf9), Klf16 (Bteb4), and Klf4 (gut Klf, Gklf) for analysis. In WT mice, colon Klf9 and Klf4 transcript expression exceeded that for jejunum (Fig. 5A and E). No effects of Klf9 gene ablation (heterozygous or homozygous) on Klf gene expression in jejunum were observed, thus eliminating the possibility of compensatory expression of one or more of these Klf-related genes in the tissue (Fig. 5, B–E). In colon, there was a tendency (P = 0.073) for a small increase in Klf16 transcript abundance with loss of Klf9 allele(s) (Fig. 5D). There was no detectable expression of Klf14 (Bteb5) mRNA in tissue of either WT or knockout animals (data not shown).


Figure 5
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  		Fig. 5. Klf mRNA abundance in WT and Klf9 heterozygous (Klf9+/–) and Klf9–/– mutant mouse jejunums and proximal colons (PC). A: relative abundance of Klf9 transcript in WT and Klf9+/– jejunum and proximal colon. B–E: relative abundance of Klf5, Klf13, Klf16, and Klf4 mRNAs in PND 30 male mouse jejunum (n = 3, 3, and 4 for WT, heterozygous, and null-mutant animals, respectively) and PC (n = 3, 4, and 4 for WT, heterozygous, and null-mutant animals, respectively) was evaluated by real-time quantitative RT-PCR. All data were normalized to cyclophilin A (Ppia) mRNA. There was no detectable amplification of Klf14 mRNA in any samples.

 
We also employed Affymetrix microarray technology to monitor global gene expression in postweaning (PND 30) WT and knockout mouse jejunums. Unsupervised hierarchical clustering of the resultant data demonstrated a major effect of Klf9 on jejunum gene-expression profile (data not shown), in agreement with the phenotypic changes observed above. Loss of Klf9 affected expression of multiple genes, with inductions (185 transcripts) and repressions (204 transcripts) in abundance of mRNAs observed in knockout jejunums relative to WT (Supplementary Tables S1 and S2; see the online version of this article for supplemental data). Knockout of Klf9 led to downregulation of genes encoding cell-signaling proteins, transcription factors, angiogenic/vasculogenic factors, defense/pathogen-related molecules, cell adhesion and matrix proteins, cell cycle and migration proteins, Wnt pathway members, and transporters (partial list in Table 2). Transcripts upregulated with Klf9 knockout encode signaling molecules and transcription factors, muscle and mitochondrial proteins, adhesion and proliferation proteins, Wnt pathway members, and transporters (partial list in Table 3). The increased expression of muscle and mitochondrial transcripts in knockout jejunum is consistent with less mucosal mass (i.e., shorter villi) and more smooth muscle transcript representation in the tissue. The complete gene lists with fold changes and functional annotation for differentially expressed RNA transcripts (1.3-fold cutoff, P < 0.05) are in Supplementary Tables S1 and S2.


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  		Table 2. Partial list of transcripts identified by microarray analysis to be downregulated in the jejunum of Klf9–/– mice

 

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  		Table 3. Partial list of transcripts identified by microarray analysis to be upregulated in jejunum of Klf9–/– mice

 
Molecular basis for Klf9 knockout small intestine phenotype. The PCNA immunohistochemistry data suggested that crypt stem/transit cell proliferation is reduced with Klf9 knockout. We confirmed this by analyzing BrdU-labeled jejunum of WT and Klf9–/– mice at 2 h after BrdU administration. There were fewer BrdU-positive cells in knockout than in WT crypts at 2 h after BrdU treatment (Fig. 6, A–C, G–I, and M), confirming reduced crypt cell proliferation. Interestingly, however, knockout villi exhibited more BrdU-positive cells than WT villi at 48 h after BrdU (Fig. 6, D–F, J–L, and N). Closely migrating cohorts of BrdU-labeled cells in villus epithelium were apparent near the termini of WT villi at 48 h (Fig. 6, D–F). Corresponding cells in knockout villi were not as advanced in relative position (Fig. 6, J–L and P), demonstrating a reduced rate of villus epithelial cell migration toward the villus tips. Staining of lamina propria cells was observed also; however, this was also apparent for animals that did not receive BrdU and hence was judged to be nonspecific (Fig. 6O). Terminal deoxynucleotidyl transferase nick-end labeling staining revealed no differences in numbers of apoptotic cells in villus epithelium of WT and knockout animals (data not shown).


Figure 6
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  		Fig. 6. Crypt cell proliferation and villus epithelial cell migration are inhibited in Klf9–/– mouse jejunum. Animals were injected with bromodeoxyuridine (BrdU) at 2 and 48 h before tissue collection. BrdU-positive cells were identified by immunohistochemistry (n = 4 and 3 of WT and null-mutant animals, respectively, with 2 sections used per animal). A, B, C, G, H, and I represent animals injected with BrdU 2 h before tissue collection to identify proliferating cells in S-phase (C is a magnification of an area of A). D, E, F, J, K, and L represent animals injected with BrdU 48 h before tissue collection to monitor cell migration over that period. Crypt cell proliferation was reduced in knockout jejunum (M). At 48 h, there was a significant increase (P = 0.001) in BrdU-labeled cells in villi of Klf9–/– mice (N). The crypt-villus unit was measured, and the midpoint was established (arrows); BrdU-labeled cells in the bottom half were counted (P). A representative negative control (jejunum from a mouse that did not receive BrdU; O) was subjected to BrdU immunohistochemistry, and nonspecific binding was observed in the lamina propria (boxed areas). Nonspecific binding did not interfere with quantitation of BrdU-labeled cells in crypt and villus epithelium.

 
Six transcripts identified as differentially expressed by microarray were examined by real-time RT-PCR of an expanded number of PND 30 jejunums (Fig. 7A). Ptk6 and Ceacam1 were confirmed to be more highly expressed in WT than knockout jejunum, and Ccnd2 (cyclin D2) was confirmed to be more highly expressed in knockout than WT jejunum, whereas Pitx2 and Mtm1 did not differ by genotype and are thus false positives by microarray. Igfbp4 was elevated in knockout jejunum, in agreement with the microarray results, although the difference by RT-PCR was not statistically significant. Igfbp5 was not differentially expressed by microarray, and this was borne out by RT-PCR. Ptk6 gene is expressed in transit cells as they exit the crypt (40); therefore, reduction in this mRNA's abundance in knockouts may be reflective of fewer transit cells per crypt or fewer numbers of cells exiting crypts. Augmentation of cyclin D2 expression may be a compensatory response to reduced Wnt signaling in Klf9 knockout crypts, as this gene product functions in Wnt/Disheveled signaling pathways in other proliferative tissues (16). Expression of mushashi-1 mRNA (a presumptive marker of intestinal stem cells; Ref. 26) in jejunum was unaffected by altered genotype as analyzed by microarray and RT-PCR (data not shown). The differential expression (knockout > WT) of Igfbp4 mRNA was confirmed in jejunum smooth muscle tissue obtained by dissection (Fig. 7B).


Figure 7
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  		Fig. 7. Differentially expressed genes in WT and Klf9–/– jejunums. A: intact jejunum was obtained from WT (n = 12) and Klf9–/– (n = 11) male mice (PND 29–31). RNA was isolated from each animal's tissue and was subjected to real-time RT-PCR for various mRNAs (each transcript was normalized to Ppia mRNA). Ptk, protein tyrosine kinase; Ceacam, CEA-related cell adhesion molecule; Igfbp, IGF binding protein; Pitx, pituitary homeobox; Ccnd2, cyclin D2 gene; Mtm, myotubularin. B: Klf9 gene knockout leads to increased Igfbp4 mRNA abundance in intestinal smooth muscle. Jejunum smooth muscle was obtained by dissection from WT (n = 7) and Klf9–/– (n = 8) male mice (PND 30). RNA was isolated from each tissue and was subjected to real-time RT-PCR for Igfbp4 mRNA (normalized to Ppia mRNA). Normalized values for each mouse sample are shown along with corresponding box plot. Expression data for each gene in A or B were compared for effects of genotype by Student's t-test or Mann-Whitney rank sum test, with resultant P values indicated.

 

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Our results show that postnatal intestine development is perturbed in the Klf9–/– mouse. The original report of these mice (22) described a neurobehavioral phenotype for the knockout animals. Subsequent work demonstrated subfertility for Klf9-deficient dams, characterized by perturbations in embryo-maternal signaling during peri-implantation (37, 41). The present study further implicates Klf9 in control of cell growth, differentiation, and migration and in long-range signaling within the small intestine and colon. The emerging complex-yet-subtle phenotype of the Klf9–/– mouse is consistent with the postnatal induction of expression of Klf9 gene in multiple tissues (22).

Klf9 mRNA is expressed in epithelial and mesenchymal layers of the embryonic mouse gut (20). During embryogenesis, Klf4 and Klf5 genes are coexpressed in this tissue (20, 24). Our results for postweaning and adult mice did not indicate significant epithelial expression of Klf9 gene in small intestine; however, epithelial and smooth muscle expression of this gene were retained in the postnatal and adult colon. The tissue differences in postnatal Klf9 expression may be functionally significant, although further characterization of the colon of the Klf9 knockout is required to examine this possibility. An important question raised by the current body of work is whether loss of epithelial Klf9 expression in small intestine is coincident with crypt-villus morphogenesis during preweaning. Our results, taken in combination with those for embryo development (20), suggest that this is possible, and in one scenario, crypt formation and villus growth during development are accompanied by restrictions in cellular expression domains for Klf9 and other Klf genes. This process might serve to establish nonoverlapping expression but functionally interactive actions of these transcription factors, consequently driving small intestine morphogenesis as well as cell renewal in the maturing gut.

In mature mice, Klf4 mRNA abundance is maximal in the colon, with the transcript localized to surface and middle/upper crypt epithelium as well as scattered lamina propria cells (30, 31); this pattern of expression resembles that of Klf9 gene in colon mucosa (present study). In vivo, Klf4 knockout is lethal at PND 1 of mouse development because of loss of skin barrier function (14, 29). Moreover, Klf4knockout mice had complete loss of the goblet cell lineage in colon, without differences in cell proliferation or apoptosis (14), whereas the Klf9 knockout proximal colon had more goblet cells compared with WT (this study). The phenotypic differences for Klf4 and Klf9 knockout clearly indicate a lack of functional redundancy for these transcription factors in the colon. Interestingly, however, Klf4 gene ablation resulted in lineage perturbations in the pit-gland units of the gastric mucosa (15), and we observed reduced stomach weights for Klf9–/– mice. Studies to examine possible functional interactions or overlaps of gastric Klf4 and Klf9 will be interesting in this regard.

The Klf5 (Bteb2, Iklf) gene encodes another highly expressed Klf of the mouse gastrointestinal tract (5). This transcript is localized to the lower-third region of crypts in small intestine and colon (5), thus spatially distinguishing it from Klf9 and Klf4. Klf5–/– embryos die in utero before embryonic day 8.5 (32). Heterozygous Klf5+/– animals survive until adulthood but exhibit misshapen villi and a sparse submucosal mesenchyme (32). Although extensive studies of Klf5 action in small intestine crypt-villus epithelium have not been reported, it seems reasonable to speculate that this Klf promotes crypt stem and/or transit cell proliferation in concert with Klf9, the latter acting at a distance.

Evidence for the above is provided by the Ptk6 and Ceacam1 genes. Ptk6 (Brk/Sik) gene encodes an intracellular src-related tyrosine kinase highly expressed in the gastrointestinal tract (19, 40). In the murine small intestine, this gene is expressed in villus epithelial cells as they migrate out of crypts to begin differentiation (40). Genetic disruption of Ptk6 resulted in longer intestinal villi and an expanded crypt proliferative zone, indicating an inhibitory role in cell-cycle arrest and enterocyte differentiation (10). The Klf9 knockout manifested reduced Ptk6 expression and shorter intestinal villi, results that appear inconsistent with that for the Ptk6 knockout. However, Cdkn1b (p27 or Kip1) mRNA also was repressed in Klf9-null jejunum. This gene, like Ptk6, is expressed in the upper crypt region, where it stimulates enterocyte differentiation, perhaps independent of its actions as a cell-cycle inhibitor (27). Because we did not observe Klf9 promoter activity (measured by X-gal staining) in this cellular location, we infer that Ptk6 and Cdkn1b genes are indirect targets of Klf9 action. Reductions in mRNA abundance of both genes may reflect, in part, fewer numbers of transit cells initiating (or in the process of) differentiation within the upper crypt and lower villus regions, and that would correlate with reduced numbers of BrdU-labeled cells at 2 h after BrdU administration. Furthermore, Ptk6 promotes EGF-induced cell migration (4), and its downregulation may underlie reductions in villus cell motility observed with loss of Klf9. Similar arguments can be made for the Ceacam1 transcript and encoded protein. Ceacam1 is a multifunctional cell-adhesion protein that is upregulated in the early postproliferative, nondifferentiated phase of Caco-2 cell differentiation in vitro (http://www.ncbi.nlm.nih.gov/projects/geo/gds/gds_browse.cgi?gds=709). Thus its reduced expression in the Klf9 KO may again reflect a deficiency/block in onset of crypt cell differentiation in transit cells concomitant with their reduced migration. Crypt and villus cell migration is likely driven by stem/transit cell division (21); hence, diminished proliferation as reported here would contribute to slower migration rates. In colon, Ptk6 mRNA is manifest in epithelial cells of the upper crypt, the postmitotic cells undergoing terminal differentiation (10, 19). In the present study, these same cells exhibited Klf9 promoter activity. Thus it will be interesting to examine the relationships between Klf9 and Ptk6 in colon cell migration and goblet cell specification.

The present study implicates Klf9 and one or more of its target genes as long-range effector(s) of cell growth and migration in crypt-villus units. Signaling of epithelium by adjacent smooth muscle cells as well as the reciprocal interaction are not unprecedented (6). Wnts represent possible candidates for this signaling, since this signal-transduction pathway is well implicated in the maintenance of stem/transit cell proliferation as well as in differentiation of the intestinal cell lineages (25, 39). Absence of Klf9 may lead to dampening of Wnt signals and consequent reductions in crypt stem/progenitor cell proliferation and differentiation. Consistent with this, microarray results identified changes in mRNA abundance for several Wnt pathway-related proteins (Dab2, Ccnd2) that might underlie the Klf9–/– phenotype, although there were no apparent changes in expression of Wnt ligands, members of the Frizzled and LDL receptor-related proteins, or the Wnt antagonists. We speculate that upregulation of Ccnd2 transcripts in knockout jejunum may be indicative of a compensatory response to attenuated Wnt signaling, because this protein can function downstream of the Wnt-Disheveled-beta-catenin pathway (16, 46).

The observed increase in Igfbp4 mRNA abundance in the jejunum smooth muscle of Klf9–/– mice is of interest from the standpoint of hypothesized mesenchymal-derived signaling mediators. The Igfbp4 gene is expressed in the muscularis externa and lamina propria of rodent and human intestines (17, 33, 43). In vitro, this IGF binding protein is antiproliferative via sequestration of IGF and/or IGF-independent mechanism(s) and can inhibit IGF-dependent cell invasion (motility) in vitro (8). IGF-I is synthesized by differentiating enterocytes and goblet cells within intestinal crypts before their movement onto villi (9). Thus it is tempting to speculate that the induction of Igfbp4 in the muscularis externa with Klf9 knockout elicits a partial block in IGF-dependent proliferation and/or motility of cells within the confines of the crypts by sequestering IGF-I ligand. Nevertheless, this requires experimental verification. Transgenic overexpression of Igfbp4 in mouse intestinal smooth muscle caused hypoplasia of this tissue compartment (43). However, our morphological analysis of intestinal smooth muscle did not indicate reductions in tissue cross-sectional thickness with Klf9 knockout, perhaps reflecting functional antagonism by another Igfbp (23). However, the latter point remains speculative, as we have not documented mRNA expression for Igfbp family members other than Igfbp4 and Igfbp5 in WT and knockout intestines.

In summary, results demonstrate a regulatory role for Klf9 in crypt cell proliferation and villus cell migration in small intestine, which may be mediated by long-range signaling from the muscularis externa. Absence of Klf9 resulted in smaller villi, likely a consequence of reductions in proliferation of crypt cells and in villus cell migration. We speculate that blocks in cell proliferation/motility in crypts elicit compensatory increases in Wnt signaling that, although not overriding these blocks, result in augmented Paneth cell numbers and diminished goblet cell numbers in crypts. Knockout of Klf9 resulted in subtle changes in colon mucosal phenotype, which may indicate effects of this transcription factor on crypt cell proliferation and goblet cell differentiation in this tissue. Further study of intestinal Klf9 and its downstream targets may reveal novel pathways that can facilitate pharmacological approaches for treatment of short-bowel syndrome, mucositis, colitis, and other intestinal pathologies of compromised crypt-villus cell renewal.


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This work was supported by grants from the National Institute of Child Health and Human Development (2-RO1-HD-21961), Arkansas Children's Hospital Research Institute Dean's Research Development Fund, and Arkansas Biosciences Institute.


   ACKNOWLEDGMENTS
 
We thank Dr. Yan Geng, Amanda Linz, Renea Eason, Reneé Till, Julie Frank, and Charles Skinner for technical assistance; Dr. Bhuvanesh Dave for suggestions regarding BrdU histochemistry; Dr. Rick Helm for manuscript critique; and Bryan Hewlett for suggestions regarding staining of Paneth cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. A. Simmen, Arkansas Children's Nutrition Center, 1120 Marshall St., Little Rock, AR 72202 (e-mail: simmenfranka{at}uams.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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