Description of Research Expertise

Research Interests
Dr. Kaestner’s lab is employing modern genetic approaches (expression profiling, gene targeting, tissue-specific and inducible gene ablation) to understand the molecular mechanisms of organogenesis and physiology of the liver, pancreas and gastrointestinal tract.

Description of Research
Transcriptional control of pancreatic development and glucose homeostasis by Foxa2.
Recent evidence places the winged helix transcription factor Foxa2 on top of a transcription factor cascade that controls the development of the pancreas. Mutations in several of these transcription factor genes have been shown to cause non-insulin-dependent diabetes mellitus. The role of Foxa2 in pancreatic development and function has not yet been tested directly, as mice homozygous for a null mutation die at gastrulation, that is before the onset of pancreatic differentiation. We have employed conditional gene ablation to uncover a dramatic and unpredicted role for the winged-helix transcription factor Foxa2 in pancreatic beta cell differentiation and metabolism. Mice that lack Foxa2 specifically in beta cells (Foxa2loxP/loxP; Ins.Cre mice) are severely hypoglycemic and show dysregulated insulin secretion in response to both glucose and amino acids. This inappropriate hypersecretion of insulin in the face of profound hypoglycemia mimics pathophysiological and molecular aspects of familial hyperinsulinism. We have identified the two subunits of the beta cell ATP-sensitive K+ channel (KATP), the most frequently mutated genes linked to familial hyperinsulinism, as novel Foxa2 targets in islets. The Foxa2loxP/loxP; Ins.Cre mice will as a unique model to investigate the regulation of insulin secretion by the beta-cell.

Control of hepatic transcription and glucose homeostasis by the Foxa proteins.
We are investigating the role of transcription factors in the organogenesis of the liver. The liver project focuses on the winged helix transcription factors Foxa1, 2 and 3, which have been shown to regulate many liver-specific genes in vitro. We have now generated null as well as loxP-flanked alleles for all three Foxa genes and are currently analyzing the phenotypic consequences of the mutations for liver development and physiology. Recent experiments have shown that simultaneous deletion of both Foxa1 and Foxa2 in the foregut prevents the development of the hepatic primordium, which is the first in vivo evidence for a role of the Foxa genes in liver formation.

Regulatory cascades in differentiation and proliferation of the gastrointestinal epithelium.
The mammalian gut epithelium is a highly organized and dynamic system which requires continuous controlled proliferation and differentiation throughout life. Proliferation, cell migration and cell adhesion all must be tightly controlled in order to prevent either inflammatory diseases or epithelial cancers. As with many other vertebrate organs, the digestive tract develops from heterogeneous embryonic origins. While the musculature and the connective tissue are derived from lateral plate mesoderm, the epithelium is derived from the endoderm. We have identified a novel member of the winged helix gene family termed Foxl1 which is expressed in the gut mesoderm and have begun its functional analysis in vivo through targeted mutagenesis in mice. Null mutations in the mesodermal transcription factor Foxl1 result in dramatic alterations in endoderm development, including epithelial hyperproliferation. We have now identified APC/Min and GKLF as downstream targets of Foxl1 and have begun the analysis of these genes in gastrointestinal differentiation by tissue-specific gene ablation.

Functional Genomics of the endocrine pancreas.
We are also pursuing a project related to genome-wide expression analysis of the pancreatic beta-cell in the context of our NIDDK grant “Functional Genomics of the beta cell”. For this purpose, we have generated a large collection of ESTs and cDNAS expressed in the endocrine pancreas and spotted them on glass-based microarrays. We are currently using a 13,000 gene mouse and a 14,000 spot human cDNA microarray for screening of multiple disease paradigms. We are also providing functional annotation to all of these clones through our database “EPConDB” Finally, we are developing the first promoter chip for large-scale chromatin immunoprecipitation experiments in the mammalian pancreas and liver.


Rotation Projects for 2006-2007
(subject to change at a moment’s notice):

1. Analysis of gene expression in Foxa deficient mice by real time PCR and microarray analysis

2. Chromatin immunoprecipitation using various transcription factor antibodies. Ligation-mediated PCR, labeling and hybridization to our promoter chip.

3. Construction of expression plasmids or gene targeting vectors.

4. Modification of bacterial artificial chromosomes (BACs) by “recombineering”.

Lab Personnel:
Lindsay McKenna, Student
Sebastian Rieck, Student
Irina Bochkis, Student
Sara Dutton Sackett, Student
Geetu Tuteja, Student
Maria Golson, Student
Dr. Peter White, Technical Director, Functional Genomics Core
Dr. Diana Zi, Postoc
Dr. Nan Gao, Postdoc
Dr. Milena Gould, Postdoc
Dr. Blair Madison, Postdoc
Dr. Zhaoyu Li, Postdoc
Dr. John Lelay, Postdoc

Alan Fox, Research Specialist
Elizabeth Helmbrecht, Research Specialist
Olga Smirnova, Research Specialist