Marko E. Horb

Experimental Conversion of Liver to Pancreas

BACKGROUND The liver and the pancreas arise from adjacent regions of endoderm in embryonic development. Pdx1 is a key transcription factor that is essential for the development of the pancreas and is not expressed in the liver. The aim of this study was to determine whether a gene overexpression protocol based on Pdx1 would be able to cause conversion of liver to pancreas. RESULTS We show that a modified form of Pdx1, carrying the VP16 transcriptional activation domain, can cause conversion of liver to pancreas, both in vivo and in vitro. Transgenic Xenopus tadpoles carrying the construct TTR-Xlhbox8-VP16:Elas-GFP were prepared. Xlhbox8 is the Xenopus homolog of Pdx1, the TTR (transthyretin) promoter directs expression to the liver, and the GFP is under the control of an elastase promoter and provides a real-time visible marker of pancreatic differentiation. In the transgenic tadpoles, part or all of the liver is converted to pancreas, containing both exocrine and endocrine cells, while liver differentiation products are lost from the regions converted to pancreas. The timing of events is such that the liver is differentiating by the time Xlhbox8-VP16 is expressed, so we consider this a transdifferentiation event rather than a reprogramming of embryonic development. Furthermore, this same construct will bring about transdifferentiation of human hepatocytes in culture, with formation of both exocrine and endocrine cells. CONCLUSIONS We consider that the conversion of liver to pancreas could be the basis of a new type of therapy for insulin-dependent diabetes. Although expression of the transgene is transient, once the ectopic pancreas is established, it persists thereafter.

Transdifferentiation of pancreas to liver

Transdifferentiation is the name used to describe the direct conversion of one differentiated cell type into another. Cells which have the potential to interconvert by transdifferentiation generally arise from adjacent regions in the developing embryo. For example, the liver and pancreas arise from the same region of the endoderm. The transdifferentiation of pancreas to liver (and vice versa) has been observed in animal experiments and in certain human pathologies. Understanding transdifferentiation is important to developmental biologists because it will help elucidate the cellular and molecular differences that distinguish neighbouring regions of the embryo. While the in vivo models for the transdifferentiation of liver to pancreas have been valuable, it is more difficult to extrapolate from these studies to individual changes at the cellular or molecular levels. The recent development of two in vitro systems (AR42J cells and embryonic pancreatic cultures) for the transdifferentiation of pancreas to liver has shown that an environmental change in the form of an exogenous glucocorticoid can cause the conversion of pancreatic exocrine cells into hepatocytes. The AR42J cell system has been used to elucidate the cell lineage and the molecular basis of transdifferentiation of pancreas to liver.

In vitro transdifferentiation of hepatoma cells into functional pancreatic cells

We have characterised the transdifferentiation of human HepG2 (hepatoma) cells to pancreatic cells following introduction of an activated version of the pancreatic transcription factor Pdx1 (XlHbox8-VP16). The following questions are addressed: (1) are all types of pancreatic cells produced? (2) is the requirement for expression of the transgene temporary or permanent? (3) are the transdifferentiated beta-cells responsive to physiological stimuli? The results showed that both pancreatic exocrine cells (by detection of amylase protein), and endocrine cells (by detecting insulin, glucagon and somatostatin proteins) are induced after XlHbox8VP16 transfection. Moreover, the hepatic phenotype becomes suppressed during transdifferentiation of hepatocytes to pancreatic cells. Requirement for the transgene is only temporary and it is no longer required once the pancreatic differentiation program is activated. Finally, we provided results to suggest that the transdifferentiated cells are functional by detecting: (1) functional markers for pancreatic beta-cells including prohormone convertase 1/3 (PC1/3), insulin C-peptide and glucagon-like peptide 1 receptor (GLP-1R), (2) increased insulin mRNA expression after treatment of cells with GLP-1 and betacellulin, physiological stimuli that regulate pancreatic function and (3) elevated insulin secretion after glucose challenge. The transdifferentiation of hepatic to pancreatic cells represents one possible source of beta-cells for human islet transplantation and this study shows that such a transdifferentiation can be achieved in vitro.

On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development

A biochemical explanation of development from the fertilized egg to the adult requires an understanding of the proteins and RNAs expressed over time during embryogenesis. We present a comprehensive characterization of protein and mRNA dynamics across early development in Xenopus. Surprisingly, we find that most protein levels change little and duplicated genes are expressed similarly. While the correlation between protein and mRNA levels is poor, a mass action kinetics model parameterized using protein synthesis and degradation rates regresses protein dynamics to RNA dynamics, corrected for initial protein concentration. This study provides detailed data for absolute levels of ∼10,000 proteins and ∼28,000 transcripts via a convenient web portal, a rich resource for developmental biologists. It underscores the lasting impact of maternal dowry, finds surprisingly few cases where degradation alone drives a change in protein level, and highlights the importance of transcription in shaping the dynamics of the embryonic proteome.

The tetraspanin Tm4sf3 is localized to the ventral pancreas and regulates fusion of the dorsal and ventral pancreatic buds

During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each region to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild-type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas-derived cells. To identify ventral pancreas-specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from NF38/39 Xenopus laevis tadpoles and compared their gene expression profiles (NF refers to the specific stage of Xenopus development). As a result of this screen, we have identified several new ventral pancreas-specific genes, all of which are expressed in the same location within the ventral pancreas at the junction where the two ventral pancreatic buds fuse. Morpholino-mediated knockdown of one of these ventral-specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion, as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.

Expression of amylase and other pancreatic genes in Xenopus

To better understand the relationship between the endocrine and exocrine cell types in the Xenopus pancreas, we have cloned the Xenopus amylase cDNA and compared its expression profile with that of four other pancreatic markers: insulin, glucagon, elastase and trypsinogen. Our results demonstrate that the first pancreatic marker to be expressed is insulin, exclusively in the dorsal pancreas. These insulin-expressing cells form small groups which resemble islets, but no insulin is detected in the ventral pancreas until stage 47. In contrast, the exocrine markers, amylase, elastase and trypsinogen are first expressed only in the ventral pancreas beginning at stage 41; by stage 45 their expression extends into the dorsal pancreas. Glucagon, on the other hand, is not expressed in the pancreas until stage 45. In the endocrine cell clusters we do not find glucagon-expressing cells surrounding insulin-expressing cells, either in the tadpole or in the mature frog pancreas.

Xenopus pancreas development

Understanding how the pancreas develops is vital to finding new treatments for a range of pancreatic diseases, including diabetes and pancreatic cancer. Xenopus is a relatively new model organism for the elucidation of pancreas development, and has already made contributions to the field. Recent studies have shown benefits of using Xenopus for understanding both early patterning and lineage specification aspects of pancreas organogenesis. This review focuses specifically on Xenopus pancreas development, and covers events from the end of gastrulation, when regional specification of the endoderm is occurring, right through metamorphosis, when the mature pancreas is fully formed. We have attempted to cover pancreas development in Xenopus comprehensively enough to assist newcomers to the field and also to enable those studying pancreas development in other model organisms to better place the results from Xenopus research into the context of the field in general and their studies specifically. Developmental Dynamics 238:1271–1286, 2009.

Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling

The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations.

Functional analysis of Rfx6 and mutant variants associated with neonatal diabetes

Mutations in rfx6 were recently associated with Mitchell-Riley syndrome, which involves neonatal diabetes, and other digestive system defects. To better define the function of Rfx6 in early endoderm development we cloned the Xenopus homologue. Expression of rfx6 begins early, showing broad expression throughout the anterior endoderm; at later stages rfx6 expression becomes restricted to the endocrine cells of the gut and pancreas. Morpholino knockdown of rfx6 caused a loss of pancreas marker expression, as well as other abnormalities. Co-injection of exogenous wild-type rfx6 rescued the morpholino phenotype in Xenopus tadpoles, whereas attempts to rescue the loss-of-function phenotype using mutant rfx6 based on Mitchell-Riley patients were unsuccessful. To better define the pleiotropic effects, we performed microarray analyses of gene expression in knockdown foregut tissue. In addition to pancreatic defects, the microarray analyses revealed downregulation of lung, stomach and heart markers and an upregulation of kidney markers. We verified these results using RT-PCR and in situ hybridization. Based on the different rfx6 expression patterns and our functional analyses, we propose that rfx6 has both early and late functions. In early development Rfx6 plays a broad role, being essential for development of most anterior endodermal organs. At later stages however, Rfx6 function is restricted to endocrine cells.

Patterning the endoderm: the importance of neighbours

The endodermal germ layer gives rise to the inner epithelial lining of the gastrointestinal tract, while that of the mesoderm gives rise to the outer smooth muscle layer. Much of the work in chick shows that the mesoderm plays an important role in endodermal differentiation, and recent results in Xenopus have begun to elucidate the factors involved in establishing endodermal cell fate. However, little is know about the signals responsible for the initial specification and pattern of the endoderm. In a recent paper, Wells and Melton have investigated the importance of early mesectodermal-endodermal interactions in the initial specification of the early mouse endoderm.(1) They demonstrate that the initial specification and differentiation of the endoderm does not occur cell-autonomously, but requires signals released from the mesectoderm.