Characterization of expression in mice of a transgene containing 3.3 kb of the human lactase-phlorizin hydrolase (LPH) 5' flanking sequence.

BACKGROUND AND AIM: The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator. METHODS: Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22. RESULTS: In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28 days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7 days and declines significantly by 28 days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells. CONCLUSIONS: Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.

Developmental expression of Eph and ephrin family genes in mammalian small intestine.

BACKGROUND: Eph receptor tyrosine kinases EphB2 and EphB3, and ephrin-B1 ligand play a critical role in regulating small intestinal epithelial cell migration. Although well studied in developing brain, the expression pattern of Ephs/ephrins has not been delineated in the developing small intestine. AIMS: To examine the gene expression of all known members of Ephs/ephrins during development of mouse small intestine. METHODS: We examined the expression of 21 A- and B-Ephs/ephrins in mouse small intestine or the Caco-2 cell line using reverse-transcription polymerase chain reaction (RT-PCR), quantitative (q)RT-PCR, and immunohistochemical analyses. EphB2-expressing cells from isolated crypts were detected by immunofluorescence and fluorescence-activated cell sorting (FACS) analyses. RESULTS: With the exception of EphA5, all family members were expressed throughout the intestine at all ages examined. Most were uniformly expressed. In contrast, levels of EphA4, EphA8, EphB4, and ephrin-B2 messenger RNA (mRNA) were highest during early fetal development and declined with age. At E15, EphB2 and EphB4 proteins were diffusely expressed in proliferating stratified intestinal epithelial cells. By E18, the proteins had become localized to cell membranes of columnar epithelial cells within intervillus regions, and later were expressed on epithelial cell membranes in adult crypts. EphB2-expressing cells can be specifically isolated from crypt cell fractions. CONCLUSIONS: The current study represents the first analysis of Ephs/ephrins during intestinal development. The elevated expression of EphA4, EphA8, EphB4, and ephrin-B2 during the fetal period of intestinal morphogenesis suggests an important role in development. Continued intestinal expression of other family members implicates a role in differentiation.

Gata4 and Hnf1alpha are partially required for the expression of specific intestinal genes during development.

The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1alpha (Hnf1alpha) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1alpha regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1alpha resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1alpha had a partial effect ( approximately 50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1alpha protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1alpha mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1alpha that are dependent on the developmental time frame in which they are expressed.

Gata4 is essential for the maintenance of jejunal-ileal identities in the adult mouse small intestine.

Gata4, a member of the zinc finger family of GATA transcription factors, is highly expressed in duodenum and jejunum but is nearly undetectable in distal ileum of adult mice. We show here that the caudal reduction of Gata4 is conserved in humans. To test the hypothesis that the regional expression of Gata4 is critical for the maintenance of jejunal-ileal homeostasis in the adult small intestine in vivo, we established an inducible, intestine-specific model that results in the synthesis of a transcriptionally inactive Gata4 mutant. Synthesis of mutant Gata4 in jejuna of 6- to 8-week-old mice resulted in an attenuation of absorptive enterocyte genes normally expressed in jejunum but not in ileum, including those for the anticipated targets liver fatty acid binding protein (Fabp1) and lactase-phlorizin hydrolase (LPH), and a surprising induction of genes normally silent in jejunum but highly expressed in ileum, specifically those involved in bile acid transport. Inactivation of Gata4 resulted in an increase in the goblet cell population and a redistribution of the enteroendocrine subpopulations, all toward an ileal phenotype. The gene encoding Math1, a known activator of the secretory cell fate, was induced approximately 75% (P < 0.05). Gata4 is thus an important positional signal required for the maintenance of jejunal-ileal identities in the adult mouse small intestine.

Hepatocyte nuclear factor-1alpha is required for expression but dispensable for histone acetylation of the lactase-phlorizin hydrolase gene in vivo.

Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1alpha for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1alpha targets lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) in hnf1alpha null mice. We found that in adult jejunum, LPH mRNA in hnf1alpha(-/-) mice was reduced 95% compared with wild-type controls (P < 0.01, n = 4), whereas SI mRNA was virtually identical to that in wild-type mice. Furthermore, SI mRNA abundance was unchanged in the absence of HNF-1alpha along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1alpha occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas, where HNF-1alpha regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1alpha. Finally, we showed that a subset of hypothesized intestinal target genes is regulated by HNF-1alpha in vivo and that this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1alpha is an activator of a subset of intestinal genes and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling.

Developmental and regional expression and localization of mRNAs encoding proteins involved in RNA translocation.

RNA localization is a regulated component of gene expression of fundamental importance in development and differentiation. Several RNA binding proteins involved in RNA localization during development in Drosophila have been identified, of which Y14, Mago, Pumilio, and IMP-1 are known to be expressed in adult mammalian intestine. The present study was undertaken to define the developmental and regional expression of these proteins, as well as Staufen-1, in mouse intestinal cells and in other tissues and cell lines using RT-PCR, and localization using in situ hybridization and immunohistochemistry. Staufen-1, Y14, Mago-m, and Pumilio-1 were expressed in intestinal epithelial cells of both villus and crypt and in Caco-2 and IEC-6 cells. In contrast, expression of IMP-1 was age- and region-specific, showing clear expression in distal fetal and newborn intestine, but very low or no expression in adult. The mRNAs were cytosolic, with more apical than basal expression in enterocytes. Staufen protein showed a similar localization pattern to that of its cognate mRNA. Overall, the data suggest an essential role for these proteins in intestinal cells. Age and regional expression of IMP-1 may indicate a role in regulation of site-specific translation of intestinal genes or in RNA localization.