Martin F. Offield

PDX-1 is Required for Posterior Poregut Patterning and Differentiation of the Pancreas and Duodenum

Martin F. Offield — Mon, 23 Nov 2009 09:16:34 PST

The Xenopus homeobox gene, XlHbox8, has been proposed to be involved in endodermal differentiation, specifically in pancreatic and duodenal development (Wright et al., 1988. Development 105, 787-794). To test this hypothesis directly, the mouse homolog, pdx-1, was cloned and gene targeting was used to produce two separate null alleles. In one, the second pdx-1 exon, including homeobox sequences, was replaced by a neomycin resistance cassette. In the second, a lacZ reporter was fused in-frame with the N-terminus of PDX-1, replacing most of the homeodomain. Neonatal $pdx{-}1\ {-}/{-}$ mice for both mutations are apancreatic, in confirmation of the report by Jonsson et al. (Jonsson. J., Carlsson, L., Edlund, T. and Edlund, H. 1994. Nature 371, 606-609.). However, the data presented in this dissertation show that the pancreatic buds form in homozygous mutants, with the dorsal bud undergoing limited proliferation and outgrowth to form a small, irregularly branched, ductular tree. No insulin or amylase-positive cells are found in these outgrowths, but glucagon-expressing GLUT2-positive cells are found. The rostral duodenum suffers a local absence of the normal columnar epithelial lining, villi, and Brunner's glands, which are replaced by a GLUT2-positive cuboidal epithelium resembling the bile duct lining. The abundance of enteroendocrine cells in the rostral duodenal villi is greatly reduced in pdx-$1\ {-}/{-}$ embryos. The PDX-1/$\beta$-galactosidase fusion allele is expressed in the pancreatic and duodenal cells in the absence of functional PDX-1, and the majority of these cells express PDX-1/$\beta$-galactosidase fusion protein into perinatal stages without changes in the boundaries or levels of expression. These results are discussed in terms of a role for pdx-1 in posterior foregut patterning, specifically in the differentiation of the pancreas and rostral duodenum.

The Development of Xenopus tropicalis Transgenic Lines and their Use in Studying Lens Developmental Timing in Living Embryos

Martin F. Offield et al. — Tue, 23 Jun 2009 07:51:04 PDT

The generation of reporter lines for observing lens differentiation in vivo demonstrates a new strategy for embryological manipulation and allows us to address a long-standing question concerning the timing of the onset of differentiation. Xenopus tropicalis was used to make GFP reporter lines with &#;1-crystallin promoter elements directing GFP expression within the early lens. X. tropicalis is a close relative of X. laevis that shares the same ease of tissue manipulation with the added benefits of a diploid genome and faster life cycle. The efficiency of the Xenopus transgenic technique was improved in order to generate greater numbers of normal, adult transgenic animals and to facilitate in vivo analysis of the crystallin promoter. This transgene is transmitted through the germline, providing an accurate and consistent way to monitor lens differentiation. This line permitted us to distinguish models for how the onset of differentiation is controlled: by a process intrinsic to differentiating tissue or one dependent on external cues. This experiment would not have been feasible without the sensitivity and accuracy provided by the in vivo reporter. We find that, in specified lens ectoderm transplanted from neural tube stage donors to younger neural-plate-stage hosts, the onset of differentiation, as measured by expression of the crystallin/GFP transgene, is delayed by an average of 4.4 hours. When specified lens ectoderm is explanted into culture, the delay was an average of 16.3 hours relative to control embryos. These data suggest that the onset of differentiation in specified ectoderm can be altered by the environment and imply that this onset is normally controlled by external cues rather than by an intrinsic mechanism.

Hepatocyte Nuclear Factor 3beta is Involved in Pancreatic Beta-Cell-Specific Transcription of the PDX-1 Gene

Kou-Liang Wu et al. — Tue, 23 Jun 2009 07:51:04 PDT

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdx-1 is expressed principally within insulin-secreting pancreatic islet b cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb 24.5 to 18.2 was used to drive a b-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp 22560 and 21880 (site 1), bp 21330 and 2800 (site 2), and bp 2260 and 1180 (site 3), were identified within the 5*-flanking region of the endogenous pdx-1 gene. Pancreatic b-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdx-1–herpes simplex virus thymidine kinase promoter expression constructs in transfected b-cell and non-b-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven b-galactosidase reporter construct was directed to islet b-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in b-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides 22007 and 21996. Gel shift analysis indicated that HNF3b present in islet b cells binds to this element. Immunohistochemical studies revealed that HNF3b was present within the nuclei of almost all islet b cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3b, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.

PDX-1 is required for pancreatic out-growth and differentiation of the rostral duodenum

Martin F. Offield et al. — Tue, 23 Jun 2009 07:51:03 PDT

It has been proposed that the Xenopus homeobox gene, XlHbox8, is involved in endodermal differentiation during pancreatic and duodenal development (Wright, C. V. E., Schnegelsberg, P. and De Robertis, E. M. (1988). Development 105, 787-794). To test this hypothesis directly, gene targeting was used to make two different null mutations in the mouse XlHbox8 homolog, pdx-1. In the first, the second pdx-1 exon, including the homeobox, was replaced by a neomycin resistance cassette. In the second, a lacZ reporter was fused in-frame with the N terminus of PDX-1, replacing most of the homeodomain. Neonatal pdx-1-/- mice are apancreatic, in confirmation of previous reports (Jonsson, J., Carlsson, L., Edlund, T. and Edlund, H. (1994). Nature 371, 606-609). However, the pancreatic buds do form in homozygous mutants, and the dorsal bud undergoes limited proliferation and outgrowth to form a small, irregularly branched, ductular tree. This outgrowth does not contain insulin or amylase-positive cells, but glucagon-expressing cells are found. The rostral duodenum shows a local absence of the normal columnar epithelial lining, villi, and Brunner’s glands, which are replaced by a GLUT2-positive cuboidal epithelium resembling the bile duct lining. Just distal of the abnormal epithelium, the numbers of enteroendocrine cells in the villi are greatly reduced. The PDX-1/b-galactosidase fusion allele is expressed in pancreatic and duodenal cells in the absence of functional PDX-1, with expression continuing into perinatal stages with similar boundaries and expression levels. These results offer additional insight into the role of pdx-1 in the determination and differentiation of the posterior foregut, particularly regarding the proliferation and differentiation of the pancreatic progenitors.