Localized expression of genes related to carbohydrate and lipid absorption along the crypt–villus axis of rat jejunum

Biochimica et Biophysica Acta 1790 (2009) 1624–1635

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Localized expression of genes related to carbohydrate and lipid absorption along the crypt–villus axis of rat jejunum Takuji Suzuki, Kazuki Mochizuki, Toshinao Goda ⁎ Laboratory of Nutritional Physiology, The University of Shizuoka Graduate School of Nutritional and Environmental Sciences and Global COE, Shizuoka, Japan

a r t i c l e

i n f o

Article history: Received 24 December 2008 Received in revised form 13 August 2009 Accepted 18 August 2009 Available online 26 August 2009 Keywords: Cryostat sectioning DNA microarray analysis Digestion/absorption of nutrient Gene expression Villus

a b s t r a c t Background: Enterocytes of the jejunum express several genes related to digestion/absorption of nutrients and ions when these cells rapidly differentiate from crypt to villus cells. However, it is unknown whether the distribution of extensive gene expression along the villus–crypt axis of the jejunum is altered during differentiation. Methods: We investigated the changes in jejunal gene expression during differentiation from crypt to villus cells in rats using DNA microarray analysis on cryostat sections of the villus–crypt columns. Results: During differentiation, the expression of many genes related to cell growth rapidly decreased, while expression of genes related to digestion and absorption of nutrients and ions increased. Expression of a subset of genes related to the digestion and absorption of starch and sucrose was highest at the middle of the villi, whereas expression of genes related to dietary fat absorption was highest at the top of the villi. Several transcriptional factors such as Pdx1, Foxa2 and Thra were expressed in the crypt, whereas Klf15 was highly expressed during the crypt–villus transition. Expression of Klf4 and Pparg was highest at the top of the villi. Conclusions: Subsets of genes related to the digestion and absorption of starch/sucrose and dietary fat as well as their transcriptional factors/co-factors are expressed in the specific locations along the crypt–villus axis. General Significance: The jejunum may absorb nutrients effectively by simultaneously expressing subsets of genes along the villus-crypt axis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Enterocytes, which are responsible for the digestion and absorption of nutrients and ions in the jejunum of the small intestine, go through a process of differentiation from crypt to villus cells. DNA synthesis rapidly decreases during this transition, indicating that the differentiation from crypt cells to villus cells is associated with cell cycle arrest [1–3]. Indeed, a recent study has shown by microarray analysis that the expression of genes related to DNA replication is markedly reduced during the time of transition from crypt cells to villus cells [4]. Other studies have demonstrated that expression of genes related to the digestion and absorption of nutrients, such as sucrase-isomaltase (Si), lactase-phlorizin hydrolase (Lct) [5], glucose transporters [Slc5a1 (SGLT1) and Slc2a5 (GLUT5)] [6], intestinal- and liver-type fatty acidbinding protein [Fabp2 (I-FABP) and Fabp1 (L-FABP)] [7,8] and cellular retinol-binding protein, type II [Rbp2 (CRBPII)] [9], is higher in the villi than the crypt. These results indicate that differentiation of enterocytes from crypt to villus cells is associated with a remarkable decrease in the expression of genes related to cell cycle, and an increase in the expression of genes related to digestion and the ab⁎ Corresponding author. Laboratory of Nutritional Physiology, School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5533; fax: +81 54 264 5565. E-mail address: [email protected] (T. Goda). 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.08.004

sorption of nutrients. These findings are supported by many cell studies using Caco-2 cells, which differentiate into villus-like cells after suppression of the cell cycle by contact inhibition. This is followed by the expression of many genes related to the digestion and absorption of nutrients such as Rbp2, Si and Slc2a5 [10–12]. However, the complete pattern of changes in gene expression during the crypt–villus transition has not yet been determined. One study has shown that the expression of cell cycle-related genes decreased during the transition [4]. Another study examined the distribution of the expression of genes of the solute carrier (Slc) family, which are related to membrane nutrient and ion transporter proteins, along the villus–crypt axis of the jejunum. This study showed that many genes in the Slc family had higher expression in the villi than in the crypt cells [13]. In addition, a recent study has shown differences in expression between crypt and villus cells using microarray analysis in mouse ileum [14]. The overall expressional pattern along the villuscrypt axis of the jejunum needs to be further investigated to improve our understanding of the digestion and absorption of nutrients and ions, since it is the jejunum rather than the ileum that is responsible for digestion and absorption of most nutrients and ions. In addition, these previous studies roughly divided the intestinal mucosa into two fractions (crypt and villus) using laser dissection microscopy or collagenase treatment. We have previously used cryostat sectioning to cut frozen jejunum into several fractions from the top of the villi to the crypt, and found that the pattern of gene expression differed between

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cells of the lower villi (close to the crypt) and those of the upper villi (top of the villus). For example, expression of Si, Rbp2 and Slc2a5 genes peaked at the middle of the villi [5,9,15], whereas Lct showed maximal expression at the upper villi [5]. The differential expression patterns along the villus–crypt axis of genes related to the digestion and absorption of nutrients and ions suggest that the various parts of the intestinal mucosa may make different contributions to these processes. To fully understand the changes in gene expression along the villus–crypt columns, we considered it pertinent to perform microarray analysis of well-fractionated villus of the jejunum. Therefore, microarray analysis on cryostat sections of the villus– crypt axis of the jejunum in adult rats was performed in this study. 2. Materials and methods 2.1. Animals Six-week-old male Sprague Dawley rats (Japan SLC Hamamatsu, Japan) had free access to a standard diet (MF; Oriental Yeast, Tokyo, Japan; Supplemental Table 1) and water until the experiment. Rats were killed by decapitation between 10:00 and 11:00 a.m. at the age of 8 weeks. The experimental procedures used in the present study conformed to the guidelines of the Animal Usage Committee of the University of Shizuoka. 2.2. Tissue preparation The entire small intestine was removed, and the region extending from the pylorus to the ligament of Treitz was discarded. The jejunoileum was divided into three segments of equal length. The proximal one-third (jejunum) of the jejunoileum was flushed with ice cold diethylpyrocarbonate (DEPC) in 0.154 M NaCl solution. A segment was excised from the middle region of the jejunal segment for total RNA extraction. This segment was opened longitudinally and flattened on a glass slide, serosal-side down, and immediately frozen in liquid nitrogen. For RNA extraction, frozen tissue blocks of approximately 7 × 7 mm2 were covered with a supporting medium (OCT Compound, Miles Laboratories, IN) and then transferred serosal-side down to a preflattened supporting surface of 1 g/L of agar inside a cryostat at −18 °C. Transverse sections of 10 μm thickness were taken through the submucosa into the muscular layer, as described previously [5]. At various depths on the villus–crypt unit, one section was attached to a microscope slide for inspection of the presence of the villus and the crypt architecture after staining with 3 g/L of methylene blue. Frozen tissue sections (10 μm sections × 10) were collected (100 μm as one fractionation). Typically, the villus–crypt column was divided into seven fractions, three of which were used for further analysis; the first fraction from the top of the villi was labeled “top of the villi,” the fourth fraction from the top of the villi was labeled “middle of the villi” and the sixth fraction from the top of the villi was labeled “crypt.” 2.3. Microarray analysis and real-time RT-PCR Total RNA was extracted from the cryostat-sectioned tissues using RNeasy® Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer's directions. For the DNA microarray experiments, 1 μg of the total RNA of each of the three fractions (crypt, middle villi and the top of the villi) of a typical rat was labeled with Cy3 and 1 μg was labeled with Cy5 using the Agilent Linear Amplification/Labeling Kit (Agilent Technologies, Kanagawa, Japan) according to the instructions given by the manufacturer. After assessing the efficiency of the Cy3 or Cy5 labeling of the cRNA, 1.5 μg aliquots of the Cy3- or Cy5-labeled cRNA of three fractions were separately hybridized to Agilent Rat Oligo Microarrays (22.5 K) according to the manufacturer's hybridization protocol. After the washing steps, the microarray slides were analyzed

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with ScanArray Lite (Perkin-Elmer, MA). Data analysis was performed using ScanArray Express (Perkin-Elmer) and Excel (Microsoft). Variability spots were removed by algorism in ScanArray express, and ≤2 signal/noise ratio in the spots in base 2 logarithms was removed. After removing variability spots, signals between Cy3- and Cy5-labeling did not differ (r2 = 0.97), indicating high reproducibility of microarray analyses. Normalization of Cy3 or Cy5 signal was carried out by global median normalization. Data were averaged for Cy3 and Cy5 signal and represented by base 2 logarithms. To confirm the reliability of the results from the DNA microarray analysis, we randomly selected several genes that showed marked changes in their expression patterns and performed real-time RT-PCR analysis. Expressional patterns of the transcript levels along the villuscrypt axis of the jejunum of three animals were correlated with those shown by microarray data (Supplemental Fig. 1). Microarray data were categorized into functional groups for cell growth, signal transduction and the digestion and absorption of nutrients and ions and transcriptional factors/co-factors, and then subdivided into groups with maximum expression in the top of the villi, middle of the villi or the crypt. Functional groups were determined by gene ontology (GO). The group of genes related to cell growth included categories for the cell cycle (GO:0007049), translation (GO:0006412), translation initiation factor activity (GO:0003743) and protein import into the nucleus (GO:0006606). A group of genes related to signal transduction was also analyzed (GO:0007165). Genes related to the digestion/absorption of nutrients and ions included categories for transporter activity (GO:0005215), specifically lipid transporter activity (GO:0005319), protein transporter activity (GO:0008565), amino acid transmembrane transporter activity (GO:0015171) and glucose transmembrane transporter activity (GO:0005355). Glucose transport (GO:0015758), lactase activity (GO:0000016), fructose transport (GO:0015755) and brush border genes were also investigated (GO:0005903). In addition, we added the Abc transporter and Slc family in the group of genes related to digestion and absorption of nutrients and ions because several genes in the Abc transporter and Slc family are not categorized in the GO clusters we selected. The group of genes related to transcriptional factors/co-factors included categories for transcription factor activity (GO:0003700). We carefully looked into the list of the genes, and genes unrelated to the functional groups were manually removed. The microarray data (Supplemental Table 2) was submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih. gov/geo/), accession number GSE15421. For real-time RT-PCR, total RNA samples (1 μg) were converted to cDNA by reverse transcription, using SuperScript™ III RT (Invitrogen, Tokyo, Japan) according to the manufacturer's instructions. To quantitatively estimate the mRNA levels of selected genes, PCR amplification was performed on a LightCycler® 480 instrument (Roche Molecular Biochemicals, Tokyo, Japan), as previously described [16]. The PCR primer sequences are listed in Supplemental Table 3. The cycle threshold (CT) values of each gene detected by real-time RT-PCR were converted into signal intensities by the delta-delta method [17], which calculates the difference of one CT value as twice the difference between the signal for each gene and the signal for a gene for normalization (HPRT). The formula used was [2(CT of test gene − CT of β-actin)]. 3. Results and discussions 3.1. DNA microarray analysis using total RNA samples fractionated from the villus–crypt axis of rat jejunum In this study, we performed microarray analysis using RNA samples of adult rat jejunum that was separated into several fractions along the villus-crypt axis by cryostat sectioning. Out of 20,500 genes printed to microarray, 6059 were expressed in the villus–crypt axis (Table 1). The coefficient of determination between the crypt and the

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Table 1 Number of genes with maximal expression at each locus along the villus–crypt columns. Pattern of expression

Number of genes

Crypt Middle of the villi Top of the villi Other Total

1416 (23%) 1434 (24%) 1312 (22%) 1897 (31%) 6059

top of the villi (r2 = 0.27) was lower than that of the crypt and the middle of the villi (r2 = 0.51), indicating that the difference in overall gene expression was markedly greater between the crypt and the top of the villi than between the crypt and the middle of the villi. These results suggest that gene expression sequentially changes during the differentiation process from the crypt to the top of the villi. To examine whether gene expression was different for the different locations along the villus–crypt axis, we divided the detected genes into groups related to cell growth, signal transduction, digestion and absorption of nutrients and ions and transcriptional factors/cofactors, according to categories of gene ontology (GO). In addition, genes were divided into three categories: those with a maximal expression at the top of the villi, at the middle of the villi or at the crypt. As shown in Table 2, most genes related to cell growth were highly expressed in the crypt (50 in 66 genes). Most genes related to the digestion and absorption of nutrients and ions were expressed highly at the villus (Table 2). The number of genes related to signal transduction that had maximum expression at the middle and top of the villi was higher than those that had maximum expression at the crypt (1.5- and 1.6-fold, respectively; Table 2). The number of genes related to transcriptional factors/co-factors that had maximal expression at the middle and top of the villi was lower than those that had maximal expression at the crypt (0.56- and 0.56-fold, respectively; Table 2). These results suggest that subsets of genes related to the cell cycle, digestion and absorption, signal transduction and transcriptional factors/co-factors are expressed in specific locations along the villus-crypt axis. 3.2. Cell growth-related genes, including eukaryotic translation initiation factors, ribosomal proteins and nucleocytoplasmic transporters, are expressed highly at the crypt Microarray data revealed that genes related to cell growth, such as many eukaryotic translation initiation factors (eIF), ribosomal proteins (Rp) and aminoacyl-tRNA synthetases such as Vars2, Kars, Farsb and Sars1, were highly expressed in the crypt. In fact, six out of nine genes for eIF, ribosomal proteins and aminoacyl-tRNA synthetases were exclusively detected in the crypt (Supplemental Table 4). We confirmed by real-time RT-PCR that some eukaryotic translation initiation factors (eIF) genes (Eif2s1 and Eif4a1) and ribosomal proteins (Rps3a and Rpl32) were expressed in crypt cells (Fig. 1A and B). A previous study has shown by microarray analysis that cell growth-related genes including eIF and ribosomal proteins Table 2 Functional classification of genes according to maximal expression along the villuscrypt axis. Functional group

Number of genes Pattern of expression

Cell growth Digestion/absorption of nutrients and ions Signal transduction Transcription factors/co-factors

Crypt

Villi Middle

Top

50 9 28 39

9 32 41 22

7 26 44 22

Total (% of all)

66 67 113 83

(1.1%) (1.1%) (1.9%) (1.4%)

Fig. 1. The distribution along the villus–crypt axis of cell growth-related genes. Graphical representations of relative mRNA levels of each gene normalized for corresponding β-actin mRNA abundance. (A) The distribution of eukaryotic translation initiation factors genes (Eif2s1 and Eif4a1). (B) The distribution of ribosomal protein genes (Rps3a and Rpl32). (C) The distribution of nucleocytoplasmic transporter genes (Kpna2 and Kpnb1). Values are means ± SEM for three animals. Values not sharing common superscripts (a and b) are significantly different from each other at p b 0.05 by Scheffe's test based on Kruskal-Wallis multiple range test.

decreased rapidly during the villus-crypt transition [4]. In addition, a study has demonstrated by in situ hybridization that a ribosomal protein, ribosomal protein L18a, had higher expression in the crypt than the villi [18]. Our results in this study are consistent with the findings of these previous studies. Additionally, several nucleocytoplasmic transporters known to transport many proteins from the cytoplasm to the nucleus or vice versa [19], such as the karyopherins Kpna2 and Kpnb1, were highly expressed in the crypt (real-time PCR; Fig. 1C). Recent studies have indicated that genes related to protein synthesis and nuclear transport are expressed during the cell cycle, particularly during the progression from the G1 to the S phase and from the S to the G2 phase [19–21]. Along with our current data, this indicates that the high expression of genes related to eukaryotic translation initiation factors and nucleocytoplasmic protein synthesis in crypt cells is related to cell growth. 3.3. Genes related to starch/sucrose digestion/absorption are expressed highly at the middle of the villi Many genes related to the digestion and absorption of nutrients and ions were categorized in the microarray data as genes with higher

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expression in the villi (Table 3). Previous studies have demonstrated that genes related to digestion and absorption of nutrients and ions, such as Rbp2, Si and many genes belonging to the Slc family, are expressed in the villi [5,9,13]. Several studies, including the present work, indicate by cryostat sectioning and in situ hybridization that the

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mRNA of Si [5], Rbp2 [9], Slc5a1 [22] and Slca5 [23] genes is expressed in the villi. In addition, the mRNA expression of Si, Lct and Rbp2 corresponds with protein levels [5,9]. In this study, we focused on several categorized genes related to digestion and absorption of nutrients and ions detected by microarray, and performed real-time

Table 3 DNA microarray results for genes related to the digestion and absorption of nutrients and ions. Pattern

Accession no.

Symbol

Gene name

Crypt

Middle

Top

Crypt

NM_144743 NM_130405 NM_017278 XM_341549 NM_013111 XM_220219 XM_243524 NM_080581 NM_144758 NM_012738 NM_013068 NM_012640 NM_057102 NM_053442 NM_012804 NM_012879 NM_053929 NM_012653 NM_031741 NM_031746 XM_236560 XM_238479 NM_013061 NM_031589 NM_130740 NM_031541 NM_022398 NM_019283 NM_053965 NM_013033 NM_031152 NM_138831 NM_053754 NM_133401 NM_001013427 NM_199081 NM_017315 XM_220013 NM_053582 NM_031013 NM_031760 NM_012737 NM_012556 NM_024149 NM_057121 NM_012501 NM_031703 XM_214047 NM_013170 NM_053332 XM_227765 NM_012697 XM_222899 XM_574504 NM_012833 NM_012654 NM_053411 NM_031704 NM_181381 NM_053502 NM_053924 XM_341115 XM_236186 NM_001009713 NM_130414 NM_001025649 NM_199118

Ces6 Khdrbs1 Psma1 Akr1c12 Slc7a1 Abca3 Lrp1 Abcc3 Slc15a4 Apoa1 Fabp2 Rbp2 Slc25a5 Slc7a8 Abcd3 Slc2a2 Slc7a9 Slc9a2 Slc2a5 Slc13a2 Pls1 Myo1a Si Slc37a4 Pacsin2 Scarb1 Slc25a11 Slc3a2 Slc25a20 Slc5a1 Rab11a Slc16a10 Abcg5 Abcb1a Rarres2 Slc35b1 Slc23a1 Tm9sf3 Lcn7 Abcc6 Abcb11 Apoa4 Fabp1 Arf5 Slc15a1 Apoc3 Aqp3 Pgm1 Gucy2c Cubn Mttp Slc22a1 Copa Nup98 Abcc2 Slc9a3 Snx1 Stx5a Abcg2 Abcg1 Abcc5 Lct Abcg4 Slc17a5 Abcg8 Tm9sf4 Gaa

carboxylesterase 6 src associated in mitosis, 68 kDa proteasome (prosome, macropain) subunit, alpha type 1 aldo-keto reductase family 1, member C12 solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 ATP-binding cassette, subfamily A (ABC1), member 3 low density lipoprotein receptor-related protein 1 ATP-binding cassette, subfamily C (CFTR/MRP), member 3 peptide/histidine transporter apolipoprotein A-I fatty acid-binding protein 2, intestinal retinol-binding protein 2, cellular solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 ATP-binding cassette, subfamily D (ALD), member 3 solute carrier family 2 (facilitated glucose transporter), member 2 solute carrier family 7 (cationic amino acid transporter, y+ system), member 9 solute carrier family 9, member 2 solute carrier family 2, member 5 solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 2 plastin 1 (I isoform) myosin IA sucrase-isomaltase solute carrier family 37 (glycerol-6-phosphate transporter), member 4 protein kinase C and casein kinase substrate in neurons 2 scavenger receptor class B, member 1 solute carrier family 25 (mitochondrial carrier oxoglutarate carrier), member 11 solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 solute carrier family 5 (sodium/glucose co-transporter), member 1 RAB11a, member RAS oncogene family solute carrier family 16 (monocarboxylic acid transporters), member 10 ATP-binding cassette, subfamily G (WHITE), member 5 ATP-binding cassette, subfamily B (MDR/TAP), member 1A retinoic acid receptor responder (tazarotene induced) 2 solute carrier family 35, member B1 solute carrier family 23 (nucleobase transporters), member 1 transmembrane 9 superfamily member 3 lipocalin 7 ATP-binding cassette, subfamily C (CFTR/MRP), member 6 ATP-binding cassette, subfamily B (MDR/TAP), member 11 apolipoprotein A-IV fatty acid-binding protein 1, liver ADP-ribosylation factor 5 solute carrier family 15 (oligopeptide transporter), member 1 apolipoprotein C-III aquaporin 3 phosphoglucomutase 1 guanylate cyclase 2C cubilin (intrinsic factor-cobalamin receptor) microsomal triglyceride transfer protein solute carrier family 22 (organic cation transporter), member 1 coatomer protein complex subunit alpha nucleoporin 98 ATP-binding cassette, subfamily C (CFTR/MRP), member 2 solute carrier family 9, member 3 sorting nexin 1 syntaxin 5a Rattus norvegicus ATP-binding cassette, subfamily G (WHITE), member 2 ATP-binding cassette, subfamily G (WHITE), member 1 ATP-binding cassette, subfamily C (CFTR/MRP), member 5 lactase ATP-binding cassette, subfamily G (WHITE), member 4 solute carrier family 17 (anion/sugar transporter), member 5 ATP-binding cassette, subfamily G (WHITE), member 8 transmembrane 9 superfamily protein member 4 glucosidase, alpha, acid

12.1 10.9 10.7 10.0 8.5 7.6 7.5 7.4 5.6 6.2 9.1 11.9 12.8 11.4 10.1 9.3 10.9 9.7 9.1 10.7 10.5 11.0 8.1 10.4 8.7 7.6 9.7 8.7 8.9 4.9 6.8 4.3 6.8 3.6 8.0 7.1 3.8 6.7 4.2 5.3 1.1 7.8 10.4 11.2 9.5 8.2 6.8 10.4 8.5 3.9 8.8 9.7 8.5 9.4 2.6 7.3 8.8 8.4 2.5 5.5 2.6 1.6 4.4 2.4 1.5 6.5 4.4

11.9 9.5 10.0 9.7 7.3 7.4 5.3 6.5 1.7 15.5 15.3 14.2 14.0 13.5 12.6 12.3 12.1 11.9 11.9 11.8 11.5 11.0 10.9 10.5 10.2 10.1 9.9 9.8 9.8 8.8 8.7 8.5 8.5 8.5 8.4 8.2 8.2 8.1 7.3 7.3 5.0 11.3 14.9 13.9 12.1 11.3 10.6 11.4 10.5 8.9 10.8 9.8 9.3 9.6 8.8 9.4 9.2 8.6 7.7 5.6 7.3 1.9 5.1 5.7 5.5 7.0 4.9

10.9 8.7 8.5 5.9 6.5 6.1 5.3 5.6 0.7 15.2 15.1 12.9 13.0 10.3 8.9 5.6 10.9 11.5 5.9 5.0 11.5 9.7 4.8 7.7 9.8 9.4 7.8 7.3 9.7 1.9 6.8 7.2 8.3 6.7 7.7 6.4 8.1 4.6 5.2 5.9 3.2 15.2 15.1 15.0 13.4 13.4 12.7 12.4 11.3 11.1 11.0 10.7 10.7 10.1 10.0 9.7 9.7 9.7 9.6 8.9 8.3 8.2 8.0 7.8 7.4 7.3 5.7

Middle of the villi

Top of the villi

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Fig. 2. The distribution of genes related to starch/sucrose digestion and absorption, and other transporters of soluble nutrients/ions along the villus–crypt axis. Graphical representations of relative mRNA levels of each gene normalized for corresponding β-actin mRNA abundance. (A) The distribution of genes related to digestion and absorption of carbohydrates. (B) The distribution of genes related to amino acid transporters. (C) The distribution of genes related to transport of various substances. Values are means ± SEM for three animals. Values not sharing common superscripts (a and b) are significantly different from each other at p b 0.05 by Scheffe's test based on Kruskal-Wallis multiple range test.

RT-PCR. As shown in Fig. 2A, Slc2a5, Slc5a1 and Si expression was highest at the middle of the villi and decreased toward the top of the villi (p b 0.05). SI digests sucrose to glucose and fructose, and hydrolyses malt oligosaccharides to glucose. Considering that Slc5a1 is a sodium–glucose co-transporter, whereas Slc2a5 is a fructose transporter [24–26], our results indicate that genes related to the digestion and absorption of sucrose and starch are expressed simultaneously along the villus–crypt axis for effective absorption. Regarding amino acid transporters, real-time RT-PCR shows that expression of Slc15a1, which is an oligopeptide transporter known as PEPT1 (intestinal-type peptide transporter), increased gradually from the crypt to the top of the villi, whereas Slc7a1 decreased gradually from the crypt to the top of villi (p b 0.05; Fig. 2). Slc3a2, co-transporter for dibasic and neutral amino acid, and Slc7a9, cationic amino acid transporters, were also verified to be expressed highly at the middle of the villi by real-time RT-PCR, but this expression level was not significant. Regarding other transporters, Slc16a3, a monocarboxylic acid transporter (p b 0.05;

Fig. 2B), Slc28a1, sodium-coupled nucleoside transporter (p b 0.05), and Slc31a1, a copper transporter, tended to increase gradually from the crypt to the top of the villi. Slc35a1, CMP-sialic acid transporter, was expressed highly in the crypt, and decreased rapidly toward the villi (p b 0.05; Fig. 2C). Taken together, these results show that during the transition from crypt to villus cells the expression of many genes related to the digestion and absorption of nutrients increases. In addition, many genes related to dietary fat absorption were highly expressed at the top and middle of the villi (Table 3). Real-time RT-PCR confirmed that expression of Apoc3, Apoa4 and Fabp1 increased gradually from the crypt toward the top of villi (p b 0.05; Fig. 3A). Expression of Abcg5 and Abcg8 as well as Abcc2 and Abcb1a tended to show a gradual increase from the crypt to the middle of the villi and remained high at the top of the villi. Abca3 and Abcc3 had a peak at the crypt, whereas Abcd3, Abcc6 and Abcb11 tended to peak at the middle villi (however this observation was not significant; Fig. 3B). Previous studies showed that the mRNA and proteins of

Fig. 3. The distribution along the villus–crypt axis of genes related to dietary fat absorption. Graphical representations of relative mRNA levels of each gene normalized for corresponding β-actin mRNA abundance. (A) The distribution of genes related to absorption of lipids. (B) The distribution of genes related to ATP-binding cassette transporters. Values are means ± SEM for three animals. Values not sharing common superscripts (a, b and c) are significantly different from each other at p b 0.05 by Scheffe's test based on Kruskal-Wallis multiple range test.

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Table 4 DNA microarray results for transcriptional factors/co-factors. Pattern

Crypt

Middle of villus

Top of villus

Accession no.

NM_019201 NM_031121 NM_022598 NM_031563 NM_012992 NM_031789 NM_031979 NM_031816 XM_341863 XM_217279 NM_053948 NM_172336 NM_031129 NM_031627 NM_022593 XM_234508 NM_001004206 NM_001011914 NM_053920 NM_001012356 XM_216759 NM_001025409 XM_214996 NM_031824 XM_345861 NM_001015027 NM_139113 NM_001008309 NM_001024236 NM_022852 NM_012743 NM_030989 XM_213849 NM_001025735 XM_234299 NM_181081 XM_577103 NM_001017960 NM_024388 NM_031642 NM_130430 NM_057133 NM_001001504 NM_054004 XM_225368 NM_031626 NM_012543 NM_022186 NM_144730 NM_052980 NM_031041 NM_001024252 XM_341596 NM_021745 NM_053698 NM_012576 NM_053536 XM_227586 XM_341072 XM_218630 NM_019334 NM_053328 NM_031642 NM_138875 NM_053713 NM_181628 NM_024134 NM_183329 NM_032616 NM_178091 NM_017325 NM_001024252 NM_013131 NM_133381

Symbol

Ctbp1 Ssrp1 Cnbp1 Nsep1 Npm1 Nfe2l2 Csda Rbbp7 Gtf2h1 Scap Polr2g Atf5 Tceb2 Nr1h3 Tceb1 Papola Pa2g4 Dr1 Trip10 Nono Med6 Hdac1 Eed Ctcf Tcf20 Crebl2 Nr2f6 Btf3 Gtf2h2 Pdx1 Foxa2 Tp53 Nfix Tcea1 Snapc1 Myst2 Ncor1 Thra Nr4a1 Copeb Psmd9 Nr0b2 Gtf2ird1 Tip120A Abt1 Nr1h2 Dbp Nrbf2 Gata4 Nr1i2 Gtf2b Pcaf Pfdn1 Nr1h4 Cited2 Nr3c1 Klf15 Taf13 Ncor2 Tead2 Pitx2 Bhlhb2 Copeb Jund Klf4 Tsg101 Ddit3 Sra1 Lisch7 Insig2 Runx1 Pcaf Nr3c2 Crebbp

Gene name

C-terminal-binding protein 1 structure specific recognition protein 1 cellular nucleic acid-binding protein 1 nuclease sensitive element-binding protein 1 nucleophosmin 1 nuclear factor, erythroid derived 2, like 2 cold shock domain protein A retinoblastoma-binding protein 7 general transcription factor II H, polypeptide 1 SREBP cleavage activating protein polymerase (RNA) II (DNA directed) polypeptide G activating transcription factor 5 transcription elongation factor B (SIII), polypeptide 2 nuclear receptor subfamily 1, group H, member 3 transcription elongation factor B (SIII), polypeptide 1 poly (A) polymerase alpha proliferation-associated 2G4, 38kDa down-regulator of transcription 1 thyroid hormone receptor interactor 10 non-POU domain-containing, octamer-binding mediator of RNA polymerase II transcription, subunit 6 homolog histone deacetylase 1 embryonic ectoderm development CCCTC-binding factor transcription factor 20 cAMP responsive element-binding protein-like 2 nuclear receptor subfamily 2, group F, member 6 (EAR2, orphan nuclear receptor ) basic transcription factor 3 general transcription factor IIH, polypeptide 3 pancreatic and duodenal homeobox gene 1 forkhead box A2 tumor protein p53 nuclear factor I/X transcription elongation factor A (SII) 1 small nuclear RNA activating complex, polypeptide 1 MYST histone acetyltransferase 2 nuclear receptor co-repressor 1 thyroid hormone receptor alpha nuclear receptor subfamily 4, group A, member 1 (Nerve growth factor-induced protein I-B) core promoter element-binding protein proteasome (prosome, macropain) 26S subunit, non-ATPase, 9 nuclear receptor subfamily 0, group B, member 2 general transcription factor II I repeat domain-containing 1 TBP-interacting protein 120A activator of basal transcription 1 nuclear receptor subfamily 1, group H, member 2 (LXR-beta) D site albumin promoter-binding protein nuclear receptor-binding factor 2 GATA-binding protein 4 nuclear receptor subfamily 1, group I, member 2 (Pregnane X receptor, PXR) general transcription factor IIB p300/CBP-associated factor prefoldin 1 nuclear receptor subfamily 1, group H, member 4 (Farnesoid X-activated receptor, FXR) Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 nuclear receptor subfamily 3, group C, member 1 (Glucocorticoid receptor, GR) Kruppel-like factor 15 TAF13 RNA polymerase II, TATA box-binding protein (TBP)-associated factor nuclear receptor co-repressor 2 TEA domain family member 2 paired-like homeodomain transcription factor 2 basic helix-loop-helix domain-containing, class B2 core promoter element-binding protein Jun D proto-oncogene Kruppel-like factor 4 (gut) tumor susceptibility gene 101 DNA-damage inducible transcript 3 steroid receptor RNA activator 1 liver-specific bHLH-Zip transcription factor 7 insulin induced gene 2 runt related transcription factor 1 p300/CBP-associated factor nuclear receptor subfamily 3, group C, member 2 CREB-binding protein

Log2 ratio Crypt

Middle

Top

13.8 12.1 11.6 11.5 11.3 11.3 11.0 10.9 10.7 10.7 10.6 10.4 10.4 10.1 10.1 10.0 9.9 9.7 9.7 9.5 9.1 9.1 9.0 8.8 8.4 8.3 8.1 8.0 7.9 7.7 7.7 7.5 7.2 7.0 6.8 6.0 5.8 5.8 5.0 6.0 9.8 5.4 9.4 8.9 8.5 7.2 4.1 7.9 7.9 7.8 8.0 7.6 7.7 7.7 7.7 8.2 2.5 7.4 7.2 7.0 6.9 2.7 9.8 10.4 9.5 10.3 8.6 9.1 8.5 8.2 7.0 9.0 6.1 8.1

12.8 10.4 10.2 9.9 9.5 10.4 7.9 9.3 10.1 10.0 9.7 9.7 9.8 10.0 7.2 9.8 7.4 8.9 8.6 9.3 8.6 9.0 8.5 7.9 7.8 7.9 7.5 7.1 4.7 7.4 4.9 6.4 7.1 3.9 6.5 5.0 5.3 0.0 4.2 10.3 10.3 9.9 9.4 9.4 9.1 8.9 8.8 8.7 8.7 8.6 8.6 8.4 8.4 8.4 8.3 8.2 8.2 8.1 7.8 7.8 7.7 2.7 11.7 11.6 10.7 10.9 8.9 9.5 9.8 9.1 8.4 9.4 7.6 8.1

11.7 9.9 9.1 9.1 7.0 8.2 6.7 8.4 9.6 9.2 9.6 9.2 9.3 9.8 7.0 9.7 5.8 8.8 7.6 6.5 8.6 8.7 7.2 7.1 6.2 7.8 5.3 4.8 4.6 7.0 3.2 3.8 1.3 3.8 2.2 3.9 3.6 0.0 3.6 10.2 8.7 8.5 8.5 9.2 6.9 6.5 7.0 8.5 7.4 6.8 8.4 5.0 7.4 3.3 7.3 7.7 0.1 3.9 6.3 7.7 2.7 1.1 14.5 13.4 12.7 12.6 11.9 10.5 10.2 9.9 9.8 9.8 9.5 9.2

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Table 4 (continued) Pattern

Top of villus

Other

Accession no.

NM_013221 NM_017058 NM_031528 XM_232064 NM_012555 XM_342552 NM_212495 NM_013124 XM_576312 XM_217232 NM_022858 XM_234281 NM_024403 NM_013154 XM_343002 XM_232354 XM_342379 XM_226624 NM_053345 NM_031042 XM_238362 XM_220698 NM_031553 NM_012866 NM_053611 XM_220644 XM_343548 NM_138888 NM_053624 XM_344659 NM_031595 NM_031094 NM_019384 NM_147177 NM_022394 XM_226380 XM_219965 NM_001012463 NM_001004198 XM_225983 NM_012669 XM_345861 NM_053369 XM_216917 NM_031139 NM_133423

Symbol

Hbp1 Vdr Rara Tcf3 Ets1 Ncoa6 Brd2 Pparg p300 Tfdp2 Foxq1 Arid4a Atf4 Cebpd Cebpz Chd4 Ehmt1 Ell2 Gtf2a2 Gtf2f2 Hdac11 Mnt Nfyb Nfyc Nupr1 Phf12 Phf3 Phf5a Pitx1 Polr2d Psmc3 Rbl2 Scaf1 Ruvbl1 Safb Smarca5 Taf5 Taf9_ps Tbp Tcerg1 Tcf1 Tcf20 Tcf4 Thrap6 Usf2 Yt521

Gene name

Log2 ratio

high mobility group box transcription factor 1 vitamin D receptor retinoic acid receptor, alpha transcription factor 3 v-ets erythroblastosis virus E26 oncogene homolog 1 nuclear receptor coactivator 6 bromodomain-containing 2 peroxisome proliferator activated receptor, gamma E1A-binding protein p300 transcription factor Dp-2 (E2F dimerization partner 2) HNF-3/forkhead homolog-1 AT rich interactive domain 4A activating transcription factor 4 CCAAT/enhancer-binding protein (C/EBP), delta CCAAT/enhancer-binding protein zeta chromodomain helicase DNA-binding protein 4 euchromatic histone methyltransferase 1 elongation factor RNA polymerase II 2 general transcription factor IIa 2 general transcription factor IIF, polypeptide 2 histone deacetylase 11 max-binding protein nuclear transcription factor-Y beta nuclear transcription factor-Y gamma nuclear protein 1 PHD finger protein 12 PHD finger protein 3 transcription factor INI paired-like homeodomain transcription factor 1 polymerase (RNA) II (DNA directed) polypeptide D proteasome (prosome, macropain) 26S subunit, ATPase 3 retinoblastoma-like 2 CTD-binding SR-like rA1 RuvB-like protein 1 scaffold attachment factor B SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 5 TAF5 RNA polymerase II, TATA box-binding protein (TBP)-associated factor TAF9 RNA polymerase II, TATA box-binding protein (TBP)-associated factor TATA box-binding protein transcription elongation regulator 1 transcription factor 1 transcription factor 20 transcription factor 4 thyroid hormone receptor associated protein 6 upstream transcription factor 2 splicing factor YT521-B

Fabp1 and Fabp2, which trap fatty acids in the cytoplasm of enterocytes and play a role in the transport of fatty acids to chylomicrons, were located at the top of the villi [8,27]. The results for Apoa4 are in agreement with results previously reported for in situ hybridization [18]. It is also reported that apolipoproteins (Apoa1, Apoa4 and Apoc3) are related protein components of chylomicrons and very low density lipoproteins (VLDL) [28–32]. Mttp is a microsomal triglyceride transfer protein which plays a central role in chylomicron assembly [33]. In addition, the superfamily of ATP-binding cassette transporters, Abcg5 and Abcg8, which are known to promote the efflux of cholesterol and plant sterols from enterocytes back into the intestinal lumen for excretion [34], and Scarb1, facilitates the uptake of dietary cholesterols into the small intestinal brush border [35]. Considering these insights and our results, genes related to the absorption of dietary fat from the lumen into enterocytes, and the release of chylomicrons or VLDL from the enterocytes into the lymph vessels, are expressed simultaneously in the middle and top of the villi. This presumably aids in improving the efficiency of dietary fat absorption. It should be noted that microarray data showed that Abcg5 and Abcg8 had a peak at the middle of the villi and top of the villi, respectively, although real-time RT-PCR showed that these genes were simultaneously expressed. This difference between the microarray data and the realtime RT-PCR data may be due to the number of the rats that were

Crypt

Middle

Top

5.6 4.8 4.3 4.1 6.7 6.1 8.0 1.1 6.8 3.0 3.5 6.1 11.8 9.8 8.0 9.5 7.9 5.0 6.8 8.0 9.4 7.8 7.4 9.1 7.2 1.6 1.4 11.2 7.7 7.7 7.4 9.4 8.7 9.5 7.7 8.9 7.9 9.2 4.4 7.8 7.1 6.8 2.1 5.5 7.0 11.1

7.5 6.8 7.5 8.0 7.4 7.3 8.0 6.0 7.2 5.0 5.2 4.4 11.6 8.9 2.3 9.4 3.5 4.3 3.3 4.5 5.2 7.4 5.1 8.2 4.1 1.5 1.0 10.3 1.3 3.6 6.0 8.8 6.4 6.6 4.5 6.4 1.0 6.5 2.8 4.5 2.8 4.7 0.1 3.2 5.1 9.8

8.7 8.6 8.6 8.4 8.3 8.1 8.1 7.8 7.5 7.1 6.5 6.9 11.8 9.0 6.6 9.7 6.1 7.7 6.6 5.6 6.6 8.6 7.2 8.5 5.3 4.7 4.4 11.0 7.0 7.0 6.1 9.6 8.4 7.1 6.3 7.7 3.2 7.8 3.8 4.6 6.3 7.7 7.1 5.1 7.6 10.3

used. Microarray analysis was performed on one animal, whereas real-time RT-PCR was performed on three animals. 3.4. The distribution of genes related to signal transduction and transcriptional factors/co-factors To further explore the mechanism behind the expressional changes during the crypt–villus transition, we focused on genes related to signal transduction and transcriptional factors/co-factors. Membrane receptors, such as G protein coupled receptors and tyrosine kinases, induce the expression of many genes through activating kinase cascades. Transcriptional factors/co-factors directly regulate transcription by forming transcriptional complexes on the genes. Interestingly, many genes related to transcriptional factors/cofactors and signal transduction were found in groups of microarray data with maximal expression at all three locations (Supplemental Table 5, Table 4). These results indicate that expressional changes of genes related to transcriptional factors/co-factors and signal transduction during the crypt–villus transition may regulate intestinal genes related to cell growth and to digestion and absorption of nutrients and ions. Real-time RT-PCR confirmed that Crebbp (CREBbinding protein), p300 (E1A-binding protein p300) and Ncoa6 (nuclear receptor coactivator 6), which are known as co-activators,

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Fig. 4. The distribution along the villus–crypt axis of genes related to signal transduction. Graphical representations of relative mRNA levels of each gene normalized for corresponding β-actin mRNA abundance. (A) Crypt pattern. (B) Middle of villus pattern. (C) Top of villus pattern. Values are means ± SEM for three animals. Values not sharing common superscripts (a and b) are significantly different from each other at p b 0.05 by Scheffe's test based on Kruskal-Wallis multiple range test.

were found at the top of the villus pattern in the microarray. Their expression gradually decreased from the crypt to the middle of the villi, although expression increased again at the middle of the villi toward the top of villi (Fig. 5). In addition, Rps6kb1 (ribosomal protein S6 kinase, polypeptide 1), which is known as a regulator for translation and an inducer of cell growth phase G1 [36], was highly expressed in the crypt (Fig. 4). Cell growth arresting and differentiation-inducing genes such as Gps1 (G protein pathway suppressor 1) [37] were also highly expressed in the crypt (Fig. 4). In addition, Ppp5c (protein phosphatase 5, catalytic subunit), which is known to inhibit cell growth by activating glucocorticoid hormone receptor signaling [38], was also highly expressed in this region, as were important nuclear transcriptional factors for intestinal differentiation and inducing intestinal genes such as Pdx1 (pancreatic and duodenal homeobox gene 1) [39], Foxa2 (forkhead box A2) [40] and Thra (thyroid hormone receptor α) [41] (Figs. 3 and 4). Indeed, target genes for Pdx1 and Thra such as Si (Fig. 2) and Lct (Supplemental Fig. 1) [39,41,42] were up-regulated during the crypt–villus transition. In addition, real-time PCR showed that histone deacetylases such as

Hdac1 and histone acetyltransferase such as Myst2, Crebbp and p300 rapidly decreased during the crypt–villus transition (Fig. 5). Recent studies have shown that abrupt changes in gene expression, which occurs frequently in differentiating cells, are accompanied by major chromatin structural changes that are triggered by modifications, such as acetylation, methylation and phosphorylation, of the histone tail [43,44]. Indeed, we recently revealed that histone acetylation on the Si gene is associated with the induction of Si gene expression during the crypt–villus transition [16]. The results in this study suggest that expressional changes of these transcriptional factors/co-factors may regulate genes related to digestion and absorption of nutrients and ions during the transition from the crypt to the middle of the villi. Regarding genes with peak expression at the villi, we also confirmed microarray data by real-time PCR (Fig. 5) such that Cideb in signal transduction, and Pparg and Klf4, which are transcriptional factors, gradually increased from the crypt to the top of the villi. Many recent studies have demonstrated that one of the subtypes of PPAR, PPARA, regulates the expression of many genes, including fatty acid absorption related genes such as Fabp1[45], Fabp2 [46] and Apoa1

Fig. 5. The distribution along the villus–crypt axis of genes related to transcriptional factors/co-factors. Graphical representations of relative mRNA levels of each gene normalized for corresponding β-actin mRNA abundance. (A) Crypt pattern. (B) Middle of villus pattern. (C) Top of villus pattern. Values are means ± SEM for three animals. Values not sharing common superscripts (a and b) are significantly different from each other at p b 0.05 by Scheffe's test based on Kruskal-Wallis multiple range test.

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[47], whose expressions increased gradually from the crypt to the top of the villi (Fig. 3A). Indeed, Pparg expression along the villus-crypt axis was closely associated with the expression of these genes (Fig. 5C). It should be investigated whether the expression of these genes along the villus-crypt axis is regulated by Pparg. Regarding Klf4, a recent study reports that this gene regulates zinc transporter 4 in mouse small intestine, although other target genes have not yet been identified [48]. Taken together, these results suggest that expressional changes of the genes related to transcriptional factors/co-factors and signal transduction during the crypt–villus transition may regulate the genes related to cell growth and the digestion and absorption of nutrients and ions. Whether these transcriptional factors/co-factors directly regulate genes related to cell growth and the digestion and absorption of nutrients and ions altered during the crypt–villus transition needs to be further investigated. 3.5. Comparison between the present microarray data in rat jejunum and the database for mouse ileum Differences in gene expression patterns between the crypt and villus examined by microarray analysis have previously been investigated in mouse ileum (MouseCVDB: http://gastro.imbg.ku.dk/ mousecv/)[14]. The major difference between the jejunum and ileum is a function of digestion and absorption of nutrients or ions. It is known that major nutrients including fat, carbohydrate, protein, vitamin A and vitamin D are predominantly absorbed in the jejunum, whereas some nutrients such as vitamin B2, B12 and bile acid are absorbed in the ileum. In addition, the ileum contains larger numbers of Peyer's patches than the other parts of the small intestine. We found that, similar to the ileum, several genes related to digestion and absorption of nutrients and ions, such as Apoa4, Abcc2, Cubn, Fabp2, Slc15a1 and Slca2, had higher expression in the villi than the crypt of the jejunum. Our study demonstrated that 58 genes related to digestion and absorption were highly expressed in the villi of the jejunum, whereas past studies revealed only 10 genes were expressed in the villi of the murine ileum, although the quality and criteria of microarray data were different between the two experiments. These differences indicate that the villi of the jejunum expressed more genes related to digestion and absorption of nutrients and ions than those of the ileum, supporting the notion that many nutrients are absorbed from the jejunum rather than the ileum. Whereas some genes such as Gaa (glucosidase, alpha, acid) are expressed highly in the villi, Nb1l (neuroblastoma, suppression of tumorigenicity 1) and Hbs1l (HBS1like) had different expression patterns between rat jejunum and mouse ileum. Because we did not perform real-time RT-PCR for Gaa, Nb1l and Hbs1l, further real-time RT-PCR studies are needed to investigate whether the expression patterns along the villus-crypt axis of these genes are different between the jejunum and the ileum. 4. Conclusion In conclusion, the jejunum may absorb nutrients effectively by simultaneously expressing subsets of genes related to the digestion and absorption of nutrients and their transcriptional factors/co-factors at specific locations along the villus-crypt axis. Further investigation is required to determine whether a subset of genes related to the digestion and absorption of carbohydrates and/or those related to the absorption of dietary fat is regulated by common transcriptional mechanisms. In addition, protein levels along the villus-crypt axis of the genes highlighted in this study need to be determined using immunostaining and ELISA of cryostat fractions. Acknowledgments This work was supported by a Grant-in-Aid for JSPS Researcher Fellows for Young Scientists (20-11635), a Grant-in-Aid for Scientific

Research from the Ministry of Education, Science, Sports and Culture of Japan (18590220, 18790171), and a grant from Uehara Memorial Foundation and the Global COE program for the Center of Excellence for Innovation in Human Health Sciences, from the Ministry of Education, Science, Sports and Culture of Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbagen.2009.08.004. References [1] A. Morrison, J.W. Porteous, Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick, Biochem. J. 188 (1980) 609–618. [2] Y. Maheshwari, M. Rao, D.E. Sykes, A.L. Tyner, M.M. Weiser, Changes in ribosomal protein and ribosomal RNA synthesis during rat intestinal differentiation, Cell Growth Differ. 4 (1993) 745–752. [3] Z. Uni, R. Platin, D. 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[38]

[39]

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[41]

[42]

[43]

[44]

[45]

[46]

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