Date

11-17-2009

UMMS Affiliation

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Document Type

Dissertation, Doctoral

Subjects

Zebrafish; Zebrafish Proteins; Pancreas; Endoderm; SOX Transcription Factors

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

The pancreas is located below the liver and adjacent to the small intestine where it connects to the duodenum. It consists of exocrine and endocrine components. The exocrine portion makes enzymes which are deposited in the duodenum to digest fats, proteins, and carbohydrates. Exocrine tissue also makes bicarbonates that neutralize stomach acids. The endocrine portion produces hormones such as insulin and glucagon which are released into the blood stream. These hormones regulate glucose transport into the body's cells and are crucial for energy production. The pancreas is associated with diseases such as cancer, diabetes, Annular pancreas and Nesidioblastosis. Annular pancreas and Nesidioblastosis are congenital malformations associated with excess endocrine tissue of the pancreas and its structures. Understanding the development of the pancreas might lead to insight of these diseases.

The pancreas arises from the endoderm. In zebrafish, Nodal signaling activates mix-type and gata genes that then function together to regulate sox32 expression which is necessary and sufficient to induce endoderm formation. Interestingly, sox32 is exclusive to zebrafish and works synergistically with pou5f1 to regulate its own expression and turn on sox17 expression. sox17 is evolutionarily conserved from zebrafish to mouse and is necessary for endoderm formation.

Signals from within the endoderm and the surrounding mesoderm specify regions in the endoderm to develop into the pancreas and other endodermal organs. Sonic hedgehog (shh) expression in the foregut establishes the anterior boundary of the pancreas primordium while cdx4 expression establishes the posterior boundary, but what regulates these factors is unclear. We determined that two Three Amino Acid Loop Extension (TALE) homeodomain transcription cofactors, Meis3 and Pbx4, regulate shh expression in the anterior endoderm. Disrupting either meis3 or pbx4 reduces shh expression in the anterior endoderm. As a result, anterior ectopic insulin expression occurs outside the normal pancreatic domain. Therefore, we discovered upstream regulatory factors of shh expression in the anterior endoderm, which is necessary for patterning the endoderm and pancreas primordium.

We performed an ENU (N-ethyl-N-nitrosurea) haploid screen to look for endocrine pancreas mutants and to find other factors involved in pancreas development and patterning. From the screen, we characterized two mutants. We identified an aldh1a2 mutant, aldh1a2um22, which blocks the production of Retinoic Acid (RA) from vitamin A. While RA is known to be necessary for differentiation of the pancreas and liver, we also found it to be necessary for intestine differentiation. Two other aldh family genes exist in the zebrafish genome, but our data suggests that aldh1a2 is the only Aldh that functions in endoderm differentiation and it is maternally deposited.

From the screen, we discovered a second mutant, 835.4, that spontaneously arose within the background. pou5f1 expression is normal in mutant embryos, but sox32 expression is reduced and sox17 expression is lost. Downstream endoderm genes of sox17 are also lost and as a result no endodermal organs develop. Rescue experiments indicate that the mutation is located between sox32 and sox17 in the endoderm pathway. We currently have not been successful at mapping this mutation and therefore are unable to rule out the possibility that it lies in the sox17 gene. However, our data suggest that the mutation occurs in a new gene that is necessary for sox17 expression, potentially working with sox32 and/or pou5f1.