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Research Agenda for Pediatric Gastroenterology, Hepatology and Nutrition: Developmental Physiology and Pathophysiology: Report of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition for the Children's Digestive Health and Nutrition Foundation

Montgomery, Robert; Ghishan, Fayez K.; Moyer, Mary Susan; Polk, D. Brent; Thompson, John F.

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Boston

Tucson

New Haven

Nashville

Miami

Address requests for reprints to: Executive Director, Children's Digestive Health and Nutrition Foundation, PO Box 6, Flourtown, PA, 19031, U.S.A. (e-mail: NASPGHAN@naspghan.org).

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RATIONALE

Considerable progress has been made in recent years in elucidating the mechanisms regulating development of the gastrointestinal (GI) tract. Important insights have been gained through the use of model systems, including Caenorhabditis elegans, Drosophila melanogaster, Xenopus and transgenic mice. Recent findings have emphasized the common mechanisms and similar genes involved in regulating GI development in diverse organisms. Of necessity, much of this work has focused on individual genes and specific regulatory pathways.

It is becoming clear that an understanding of physiology and pathophysiology requires an integrated approach to the simultaneous interaction of regulatory factors. Such an analysis is now becoming feasible through the use of accumulated genetic data provided by the genome projects currently under way or recently completed. The combination of these data with the rapidly developing technology for synthesizing and screening deoxyribonucleic acid (DNA) arrays should make possible the comprehensive analysis of patterns of gene expression in the developing GI tract as well as of aberrant developments leading to pathophysiologic states.

The availability of data from the human genome project will facilitate the identification of human genes involved in pathophysiology. Support for a major new emphasis on the comprehensive analysis of development should lead to advances in child health. In addition, many promising research directions have been identified recently, for which additional support should be generated. Involvement of clinically trained investigators in these emerging research areas for the study of GI development should be strongly encouraged.

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AREAS OF EMPHASIS

Carry Out A Comprehensive Analysis of Gene Expression Patterns in Development and Pathophysiology of the GI Tract

The hox genes regulate development in specific regions of the GI tract and likely regulate the overall pattern of development (1,2). The hox genes encode transcription factors, but few of the target genes are currently known. Cdx2, a divergent homeobox gene that mediates intestine-specific expression of a number of genes, interacts with a hox protein, and its binding site in lactase has been reported to also bind a hox protein. Although the hox genes are expressed in both developing and adult GI tissues, how they carry out their functions is not understood.

For a number of individual transcription factors and growth factors, specific roles have been established in the development of the GI tract. For example, the transcription factors cdx2, GATA and HNFI regulate a number of genes critical for GI function. Few data are currently available, however, on how they function together either in maturing cells or in the developing GI tract. Similarly, epidermal growth factor (EGF) and transforming growth factor–beta (TGF-β) both regulate enterocyte proliferation, but their integrated function in the organ is not well understood.

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Research Goals

With both the technology and an extensive database available, it is now feasible to carry out a comprehensive analysis of gene expression in the GI tract to address these questions. It should be possible to perform such analyses in great detail both spatially and temporally in the GI tract. These data would then serve as a benchmark for analyzing the changes in pathologic conditions. For example, delineation of the changes in expression of cytokines and related immune factors and resulting changes in cellular gene expression during the inflammatory response would enhance understanding of these processes.

A similar approach to analyzing adaptation following resection would also provide important information. Such an analysis of age-related changes and the impact of caloric restriction on these changes in mouse muscle was recently reported (3). Ultimately, such analyses should enable the identification of critical changes during the onset of pathology, and will facilitate the development of therapeutic agents to target critical factors. Such a possibility is supported by the successful administration of an antisense oligonucleotide to inhibit NF-kappa B and relieve symptoms of inflammatory bowel disease in mice (4).

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Research Strategies

This would be an appropriate use of DNA microchip technology. The increasing availability of genetic sequence information and the capability to examine changes in multiple genes allow an evaluation of the integrated physiology of development as well as changes in disease states. This could lead to the development of specific arrays for diagnostic use (e.g., screening for genetic disease) or for the evaluation of disease processes and effectiveness of treatment interventions.

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Projected Timetable and Funding Requirements

This research area could be approached both through individual investigator-initiated grants (R01 grants) and through center grants to support the establishment and maintenance of core facilities for computer analysis of microarray data. Some of the necessary reagents are already commercially available. Development of arrays specific for analysis of GI development should be encouraged. The possibility of partnerships with companies interested in commercializing standardized arrays as diagnostic tools should be explored.

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Elucidate the Role of Growth Factors in GI Development
Research Goals

In addition to some of the better-studied growth factors, such as EGF and TGF-β, evidence is now emerging for a role in gut development by several less well-studied factors, including trefoil factor and fibroblast growth factor (FGF). One growth factor in particular—bone morphogenetic protein 4 (Bmp4), a member of the TGF-β superfamily—has been shown to be important in early intestinal development. Recent studies demonstrated that several members of the FGF family and their receptors are critical for the process of liver induction (5). The signal transduction pathways and cross-talk among these growth factors in the GI tract should be investigated. More basic work on expression patterns and their effects needs to be done for these less well-understood growth factors. Trophic effects have been suggested, but not definitively established, for many gut hormones, whose involvement has been postulated in the maturation of the GI tract in human newborns.

Elucidation of cellular mechanisms regulating the diverse effects of these growth factors on the GI epithelium, including specific roles and cross-talk, is an important research goal. Another priority area for study is the role of growth factors during epithelial-mesenchymal interactions in the GI tract. Epithelial-mesenchymal interactions have long been known to be critical in GI tract development, but few data are available on the mechanism of these interactions. Evidence is now emerging that growth factors such as Bmp4 mediate these interactions (6). Other soluble mediators, such as sonic hedgehog and several genes of currently unknown function (e.g., Nkx2.3 and Hlx), have recently been shown to be critical for epithelial-mesenchymal interactions during development.

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Research Strategies

The use of knockout models and transgenic technology is appropriate. Peptide hormones have been postulated to have developmental effects, especially after the first postnatal feeding. Careful analysis of emerging knockout models for peptide hormones is required. For example, analysis of knockout mice identified a role for gastrin in differentiation of the stomach and proliferation of colonic cells (7). Transgenic technology is a key tool for investigating the trophic effects of these factors.

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Projected Timetable and Funding Requirements

Investigations in these areas primarily lend themselves to individual investigator-initiated grants. As our understanding increases, comprehensive analysis as described above would become a priority.

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Elucidate the Role of Non-Epithelial Cell Types and Their Interactions With the Epithelium in Development and Pathophysiology of the GI Tract
Research Goals

There is emerging evidence for involvement of dendritic cells, smooth-muscle cells, pericryptal fibroblasts, immune cells and neural cells in pathophysiology of the GI tract. Development of the enteric nervous system clearly is an important pathophysiologic factor, as evidenced by the genetic defects now identified as the cause of Hirschsprung's disease (1). Another example is a study indicating a role for the enteric nervous system in rotavirus-related diarrhea (8).

Similarly, cells of the immune system within the GI tract are emerging as important factors in pathophysiology. Although the gut is the largest immune organ of the body, interaction of this immune system with the luminal environment is not well understood, especially in the human neonate. Much work needs to be done in this area, which has important implications for clinical care.

Although pericryptal fibroblasts were well described some time ago (9), evidence has only recently begun to be presented on their function. Mice heterozygous for a knockout of the phosphatase PTEN show increased numbers of fibroblasts surrounding hyperplastic crypts, suggesting that part of the abnormality may be due to disordered interactions between these fibroblasts and epithelial cells (10). Recent knockouts of the fkh6, Hlx and Nkx2.3 genes have produced abnormal epithelial development, most likely the result of abnormal epithelial-mesenchymal interaction (1). These findings point the way toward dissecting the mechanisms of GI epithelial-mesenchymal interaction. This is a critical area that until recently has been largely descriptive, but key genes are now being identified.

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Research Strategies

These areas are appropriate for investigator-initiated studies, which would benefit from greater support.

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Delineate the Relation Between Nutrient Intake and Intestinal Development
Research Goals

Although many of the relevant transporters have been cloned, there are few data on developmental patterns and their regulation in the human GI tract. For example, inherited glucose-galactose malabsorption is due to a defect in the SGLT1 gene, suggesting the potential clinical relevance of additional data in this area (11). Mobilization of transporters to the luminal surface is regulated by nutrient intake. There is also some evidence for the regulation of specific digestive enzyme gene expression by nutrient levels. Some studies suggest a direct effect of nutrients on the expression of transporters and digestive enzyme genes, such as that for sucrase-isomaltase. This is a little-explored field of potential importance to infant nutrition.

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Develop Animal Models and Cell Lines to Address Specific Questions in GI Physiology and Pathophysiology
Research Goals

Gene knockout technology is a powerful tool for identifying developmental effects of a specific gene. However, if such knockouts are embryonic lethals, it is impossible to study the role of the gene at later stages, such as in the immediate postnatal period. Recent studies report the development of an inducible gene knockout in cells of the small intestine and colon (12). With this approach, it is now possible to eliminate the gene of choice at any stage of postnatal life. This technology would have obvious applications for a number of questions in developmental physiology.

Another approach that is being developed is the use of immortalized cells from the commercially available “Immortomouse” (sold by Charles River), both to generate cell lines directly and, by crossing to mice, to generate mouse cell lines carrying the desired gene knockout (13). This approach can be used to dissect complex regulatory pathways. Intestinal cell lines lacking the receptor for EGF, for example, can be generated for study.

Another research goal should be to identify stem cells and to develop culture methods to maintain and differentiate such stem cells from the GI tract. A successful effort would not only provide a model for investigation of basic questions in development, but also raise the possibility of using such cells to treat damaged or genetically defective GI tissues. It is known that stem cells exist, for example, in the crypts of the small intestine; their rough location and number within a single crypt are also known (14). Currently, identification and culture of stem cells originating from the gut have not been accomplished. Recent work demonstrated that the transcription factor Tcf-4 is required for the maintenance of stem cells in the murine small intestine (15). Possibly Tcf-4 will provide a marker to successfully identify and culture human small-intestinal stem cells. Stem cell culture is a long term goal, but one of great importance.

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Research Strategies

Additional experience with techniques of cell immortalization should make possible the application of these methods to the development of nontransformed human cell lines of intestinal origin, which would provide an invaluable research model. Conditionally immortalized human intestinal cell lines have been reported, but have not so far been widely used. Furthermore, it should be possible to develop and use such cell lines for therapeutic purposes, as was recently suggested for reversibly immortalized human hepatocytes (16).

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Projected Timetable and Funding Requirements

The described models obviously would be of use to individual investigators, but might be developed by a core center as part of a center grant, so that funding could support a group of investigators. The model systems might be generated by a core facility and individuals supported to carry out specific projects by pilot project grants, preliminary to obtaining R01 grants.

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REFERENCES

1. Montgomery RK, Muhlberg AE, Grand RJ. Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology 1999; 116:702–31.

2. Pitera JE, Smith VV, Thorogood P, Milla PJ. Coordinated expression of 3'hox genes during murine embryonal gut development: an enteric Hox code. Gastroenterology 1999; 117:1339–51.

3. Lee CK, Klopp RG, Weindruch R, Prolla TA. Gene expression profile of aging and its retardation by caloric restriction. Science 1999; 285:1390–3.

4. Neurath MF, Pettersson S, Meyer zum Buschenfelde KH, Strober W. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice. Nat Med 1996; 2:998–1004.

5. Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 1999; 284:1998–2003.

6. Kaestner KH, Silberg DG, Traber PG, Schutz G. The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation. Genes Dev 1997; 11:1583–95.

7. Koh TJ, Goldenring JR, Ito S, Mashimo H, Kopin AS, Varro A, Dockray GJ, Wang TC. Gastrin deficiency results in altered gastric differentiation and decreased colonic proliferation in mice. Gastroenterology 1997; 113:1015–25.

8. Lundgren O, Peregrin AT, Persson K, Kordasti S, Uhnoo I, Svensson L. Role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea. Science 2000; 287:491–5.

9. Marsh MN, Trier JS. Morphology and cell proliferation of subepithelial fibroblasts in adult mouse jejunum. Gastroenterology 1974; 67:622–45.

10. Di Cristofano A, Pesce B, Cordon-Cardo C, Pandolfi PP. Pten is essential for embryonic development and tumour suppression. Nat Genet 1998; 19:348–55.

11. Martin MG, Turk E, Lostao MP, Kerner C, Wright EM. Defects in Na+/glucose cotransporter (SGLT1) trafficking and function cause glucose-galactose malabsorption. Nat Genet 1996; 12:216–20.

12. Saam JR, Gordon JI. Inducible gene knockouts in the small intestinal and colonic epithelium. J Biol Chem 1999; 274:38071–82.

13. Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 1991; 88:5096–100.

14. Potten CS. Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos Trans R Soc Lond B Biol Sci 1998; 353:821–30.

15. Korinek V, Barker N, Moerer P, van Donselaar E, Huls G, Peters PJ, Clevers H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet 1998; 19:379–83.

16. Kobayashi N, Fujiwara T, Westerman KA, Inoue Y, Sakaguchi M, Noguchi H, Miyazaki M, Cai J, Tanaka N, Fox IJ, Leboulch P. Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes. Science 2000; 287:1258–62.

© 2002 Lippincott Williams & Wilkins, Inc.

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