June 10, 1999 - Developmental and Molecular Biology of the Liver and Gastrointestinal (GI) Tract : NIDDK

June 10, 1999 - Developmental and Molecular Biology of the Liver and Gastrointestinal (GI) Tract

Member attendees

Jay Hoofnagle (Chair)
Frank Hamilton
Leslie Curtis

Mushtaq Khan

Nancy Ernst

Thomas Kresina (Executive Secretary)
Vishnudutt Purohit

Kenneth Gruber

James Butler


Guests from NIH

Michael Weinstein
Chuxia Deng

Other Guests

Lopa Mishra--Veterans Administration (VA) Medical Center, Washington, D.C.; Fels Cancer Institute, Temple University, Philadelphia, Pennsylvania
Andrew Mulberg--Children's Hospital of Philadelphia
Yi Tang--Temple University

2:10 p.m.

Dr. Jay Hoofnagle, Director, Division of Digestive Diseases and Nutrition (DDDN) of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH) welcomed attendees to this Digestive Diseases Interagency Coordinating Committee (DDICC) meeting on developmental and molecular biology of the liver and gastrointestinal (GI) tract.

Dr. Hoofnagle explained the purpose of DDICC and its mandate to meet four times per year. Each meeting consists of a topic of interest to several Institutes to assess progress in that area and whether new initiatives are needed.

After introductions, Dr. Hoofnagle reported that NIDDK held a 3.5-day meeting on hepatitis C (6th International Symposium on Hepatitis C and Related Viruses) in early June, which was supported by several other Institutes as well as the Department of Defense, the Centers for Disease Control and Prevention (CDC), and the Food and Drug Administration (FDA). He also announced the following upcoming meetings:

  • "Hepatitis C and African Americans" 1-day workshop
    • December 2, 1999
    • Lister Hill Auditorium, National Library of Medicine, Bethesda, Maryland
    • Chairman: Charles Howell, Chief of Hepatology at the University of Maryland
    • Issues to be addressed: (1) the higher incidence and prevalence of hepatitis C among African Americans; (2) the emerging data showing that hepatitis C among African Americans when compared with whites is worse, more likely to result in cancer, and less responsive to therapies and the poorer outcomes of liver transplantation among African Americans; and (3) the underrepresentation of African Americans in clinical trials.
  • (10th) Triennial Symposium on Viral Hepatitis and Liver Disease
    • April 2000
    • Atlanta, Georgia
    • Hosted by CDC, who will be looking to the Institutes for support
    • Expected attendance of about 3,000
  • "Complementary and Alternative Medicine in Liver Disease" 3-day workshop/scientific meeting
    • August 22-24, 1999
    • Natcher Conference Center, NIH, Bethesda, Maryland
    • Sponsored by NIDDK and co-sponsored by the National Center for Complementary and Alternative Medicine, the Office of Dietary Supplements, and the American Association of Naturopathic Physicians
    • Includes a patient forum, where lay individuals can meet and question experts in the field of alternative medicine
    • Abstracts being accepted through July 2
    • Proposed scientific sessions include:
      1. Complementary and Alternative Medicine and Chronic Liver Diseases: Introduction and Definitions
      2. Design of Outcome Research
      3. Liver Disease and Complementary and Alternative Medicine
      4. Spectrum of Botanicals for Use in Liver Diseases
      5. International Perspective of Botanicals in Liver Disease
      6. Adverse Effects, Hepatotoxicity, and Quality Control

    2:23 p.m.

    Dr. Hoofnagle introduced the first speaker, Lopa Mishra, M.D., Associate Professor at the Fels Cancer Institute at Temple University and Chief of GI Developmental Biology at the VA Medical Center in Washington, D.C., who presented new data in the developmental biology of the liver to DDICC committee members and guests.

    Dr. Mishra began by explaining that the goal of research in her lab is twofold: (1) to look at genes involved in liver development and (2) to translate this information into the disease states and put it to clinical use in diseases such as primary biliary cirrhosis (PBC). She then explained how she and her research team isolated more than 200 clones through subtractive hybridization of stage-specific embryonic liver cDNA libraries (the early liver libraries). Next, Dr. Mishra focused on a set of genes called the spectrins (specifically, b-spectrin embryonic liver fodrin [elf]); three isoforms of the elf gene (elf1, elf2, and elf3) were isolated through full-length sequencing of one particular clone (sc32).

    Dr. Mishra's research shows that elf1, found in the cytosol, may play a role in the emergence of hepatocyte polarity during liver development and that elf3, which is membrane-bound, plays a vital role in hepatocyte differentiation and intrahepatic bile duct formation. elf3 modulates interactions between various components of the cytoskeleton proteins controlling liver and bile duct formation. Inhibition of elf3, specifically the ankyrin-binding domain, causes a loss of intrahepatic bile ducts with an increase in lymphocytes, which can lead to PBC. Decreased membrane labeling of elf and a marked increase in cytoplasmic labeling characterize PBC.

    Dr. Mishra continued by explaining the mechanism by which elf disruption results in PBC. Through the work of Drs. Chuxia Deng and Michael Weinstein of NIDDK--the knockout of two transforming growth factor-beta (TGF-b) signaling molecules (smad2 and smad3)--Dr. Mishra and her colleagues were able to characterize this smad phenotype, rescue it, and tie elf and these smad proteins together. She defined TGF-b as a major cytokine involved in liver fibrosis and stated that it inhibits growth of hepatocytes and some hepatocellular carcinomas. Dr. Mishra also said that smad proteins serve as intracellular mediators of TGF-b and activins. Next, she explained that on TGF-b receptor activation, phosphorylation of smad2 and smad3 occurs; these signaling molecules form heteromeric complexes with smad4; and these complexes translocate to the nucleus to control expression of target genes. Disruption of the TGF-b pathway will result in a disease such as PBC.

    Animals lacking smad2 die before 8.5 days of development (smad2 is required for gastrulation and mesoderm induction), and animals lacking smad3 are viable but suffer mucosal immunodeficiency. Dr. Mishra's group intercrossed animal strains lacking smad2 and smad3, and these resulting mutant mice, which are doubly heterozygous for disruptions of the smad2 and smad3 genes, displayed novel phenotypes not present in either single heterozygote. The researchers found that mutants die at day 14 of development with marked hypoplasia, characterized by nodule formation and an absence of bile ducts. Dr. Mishra and her colleagues found a small reduction in differentiation of stem cells and a marked reduction in cell proliferation of hepatocytes.

    Furthermore, the researchers found that hepatocyte growth factor (HGF) rescues the smad phenotype. When they looked further into inducing smad expression, they found that HGF completely suppresses smad3. Also, smad2 and smad3 bind to elf proteins (smad2 more so than smad3), and when they are disrupted, spectrin binding is lost, resulting in a phenotype that is suggestive of PBC.

    Dr. Mishra summarized this final segment of her presentation by stating that

    • Smad3 is suppressed in PBC.
    • Smad2 nuclear localization is absent in PBC.
    • Smad2 and smad3 bind to elf spectrins in PBC tissue.
    From these findings, she and her colleagues concluded that smad2 and smad3 mutants have a severe PBC phenotype and that elf interactions with smad2 and smad3 play a pivotal role in the pathogenesis of PBC.

    3:15 p.m.

    Dr. Hoofnagle introduced the next speaker, Dr. Andrew Mulberg, Assistant Professor of Pediatrics and Director of the Gastroenterology Fellowship Program at Children's Hospital in Philadelphia, who discussed the developmental and molecular biology of the liver and GI tract.

    Dr. Mulberg began his presentation with an overview of GI development. In the general model of development, morphogenesis and cell proliferation are the first stage, and this leads to cell differentiation. In specific organs, the stages are cytodifferentiation, organogenesis, nutrient absorption, and exocrine and endocrine secretion (as it relates to pancreas and intestine). Dr. Mulberg outlined first expression of various human developmental milestones:

    Milestone Expression
    Gastrulation week 3
    Gut tube largely closed week 4
    Liver and pancreas buds form week 7
    Intestines start forming week 7
    Intestinal villus starts forming week 8
    Multiple organ systems completely formed week 12
    Bile secretion in fetus has been identified week 12

    Dr. Mulberg then discussed the drosophila model system of GI development. The homeobox/homeotic genes of drosophila encode for transcription factors, which are important for various developmental paradigms in segmentation and pattern formation. Dr. Mulberg said that the same regulation develops similarly in humans. In vertebrates, homologous genes called hox are very critical to the same proximal-distal organ-specific development.

    Dr. Mulberg said that various investigators have demonstrated that with targeted knockout of specific homeobox genes, GI development can be altered. Specific examples include

    • The hox C4 gene transcription factor, when knocked out, will lead to esophageal obstruction due to abnormal epithelial cell proliferation. Muscle development also is abnormal.
    • hox 3.1, now referred to as hox C8, when knocked out, will lead to abnormal gastric epithelial development.
    • hox A has been shown to be correlated with a model of congenital aganglionosis (Hirschsprung's disease).
    • hox D12 and hox D13 have been shown to affect the development of anal musculature.

    Dr. Mulberg discussed the developmental biology of specific organs, starting with the esophagus. Important work in this area includes research into the expression of neuropeptides. The function of neuropeptides in fetal life and how they interact with specific physiological dysfunction that characterizes neonates (for instance, gastroesophageal reflux) is not clearly understood. It also is not clearly understood how GI hormones expressed in multiple fetal tissues correlate with specific function. Glucagon and somatostatin are expressed very early and are expressed initially in the pancreas and stomach. The antrum and colon start expressing vasoactive intestinal peptide by week 12. Pancreatic polypeptide is expressed by week 10. Motilin, secretin, and gastric inhibitory polypeptide are expressed by week 16 of fetal development. The exact developmental expression and developmental maturation function of these hormones are not well understood.

    Dr. Mulberg then moved on to a discussion of the stomach and said that this is an area that needs more research. One of the important factors that characterize the neonate from the adult is the classical achlorhydria that is demonstrated for the first week of life. There is no molecular or cellular understanding of this process. No studies have been done on the function of the gastric proton pump in this period of time. Some studies have clarified that preduodenal lipolysis in neonates is important to understanding fat absorption. Gastric lipase is important in neonatal fat absorption as is/are bile salts _______ lipase that is/are present in breastmilk. [Dr. Mulberg, please fill in this blank; this was somewhat inaudible on the audiotape.] Intrinsic factor is expressed at week 11, but there is no clear understanding of what regulates motility in fetal life.

    With regard to the pancreas, Dr. Mulberg said much is known about the developmental biology of the pancreas as a result of recent research, including some exciting work being done at Children's Hospital in Boston with zebra fish that he believes will be instrumental in understanding the genesis of the pancreas. There is a positive inductive effect of mesenchyme upon the development of the pancreas from the pancreatic endoderm. Dr. Mulberg said that it is known that various transcription factors are important in the regulation of endocrine function, specifically insulin gene expression and the morphogenesis of beta cells.

    There is no clear understanding of the development of the enteric nervous system. It is known that another transcription factor--hlx1--plays a role in the development of the enteric nervous system, specifically the pacemaker cells, and this is being researched at the University of Texas Southwestern Medical Center at Dallas. Targeted disruption of this gene has shown that the induction of intestinal mesenchyme is very important for the differentiation of the enteric neurons.

    The small intestine and colon develop very similarly. Specifically, there is similarity in the development of stratified epithelium, with the villus architecture developing into cryptlike epithelium in the third trimester of gestation. Dr. Mulberg also discussed the expression of various enzymes, including sucrase-isomaltase, alkaline phosphatase, amino peptidase, and lactase. A chronology of development includes initial remodeling of the mucosa, formation of villi, and formation of cords of epithelial cells in crypts by 9-10 weeks; by 13 weeks, the small intestine is completed; by 22 weeks, the muscularis begins to form at the base of the crypts; and by 22 weeks, there is complete functional maturation of the small intestine (equal to that of the adult). With regard to the small intestine, Dr. Mulberg added that there is no clear understanding of the origin of the stem cell.

    Dr. Mulberg then focused on cdx1 and cdx2 as specific examples of intrinsic regulators of gastrointestinal development. The esophagus and the stomach show no specific cdx1 expression, but cdx1 is expressed very highly in the colon. Although cdx2 does seem to be expressed in all (proximal to distal) aspects of the intestine, it is very highly expressed in the jejunum. Cdx1 is highly pronounced in the proliferative cells. Cdx1-null mice have normal development, although adult cdx1-null mice show some evidence of thinned mucosa and changes in proliferation; cdx2-null mice do not survive.

    Dr. Mulberg next presented his current research on bile ducts. He and colleagues at Children's Hospital are using Alagille syndrome as a model system for studying bile duct development. Alagille syndrome is characterized by an absence of bile ducts in the portal tract. Dr. Mulberg is also interested in and is in the process of being published in the area of pancreatic insufficiency and jagged1 gene expression. jagged1 is the gene encoded by JAG1. It interacts with the intercellular signaling pathway encoded by the notch1 proteins. Exactly how this pathway relates to bile duct development is an active focus of research at Children's Hospital in Philadelphia.

    In closing, Dr. Mulberg provided suggestions for future initiatives, including further exploration of the development of the esophagus, stomach, and biliary system. He also said that transport, as it relates to bile duct flow and nutrient absorption, is underexplored. He thinks re-identification of the stem cell in the small intestine, liver, and other organs is important, and some investigators are actively working on this. Dr. Mulberg also suggested bridging the gap between the cellular and molecular mechanisms discussed by Dr. Mishra and the current understanding of liver disease. Finally, Dr. Mulberg suggested that the best way for developmental biologists/physician scientists to foster progress is to develop a more effective means of collaboration.

    3:58 p.m.

    In closing, Dr. Hoofnagle asked attendees to make their agency announcements.

    Dr. Kresina reported on two draft requests for applications (RFAs) from NIAAA. The first, "Hepatitis C and Alcoholic Liver Disease," solicits both basic and clinical studies on cellular and molecular mechanisms that initiate and promote the progression to end-stage liver disease in individuals with the hepatitis C virus. The second, "Peptide Regulation of Alcohol Intake," solicits research that will advance understanding of the peptidergic regulation of alcohol consumption in humans and animals, with the ultimate goal that novel pharmacotherapeutics for treating alcohol abuse and alcoholism will be developed through the information and knowledge gained from these studies.

    4:03 p.m.

    Dr. Hoofnagle reminded committee members that the next meeting is September 14, 1999, and then adjourned the meeting.

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