Jonathan M.W. Slack

Molecular basis of transdifferentiation of pancreas to liver

The appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology. Here we show that pancreatic cells can be converted into hepatocytes by treatment with a synthetic glucocorticoid, dexamethasone. This occurs both in a pancreatic cell line, AR42J-B13, and in organ cultures of pancreatic buds from mouse embryos. We have established several features of the mechanism behind this transdifferentiation. We show that a proportion of the hepatocytes arises directly from differentiated exocrine-like cells, with no intervening cell division. This conversion is associated with induction of the transcription factor C/EBPβ and the activation of differentiated hepatic products. Transfection of C/EBPβ into the cells can provoke transdifferentiation; conversely, a dominant-negative form of C/EBPβ can inhibit the process. These results indicate that C/EBPβ is a key component that distinguishes the liver and pancreatic programmes of differentiation.

How cells change their phenotype

Recent attention has focused on the remarkable ability of adult stem cells to produce differentiated cells from embryologically unrelated tissues. This phenomenon is an example of metaplasia and shows that embryological commitments can be reversed or erased under certain circumstances. In some cases, even fully differentiated cells can change their phenotype (transdifferentiation). This review examines recently discovered cases of metaplasia, and speculates on the potential molecular and cellular mechanisms that underlie the switches, and their significance to developmental biology and medicine.

eFGF regulates Xbra expression during Xenopus gastrulation.

We show that, in addition to a role in mesoderm induction during blastula stages, FGF signalling plays an important role in maintaining the properties of the mesoderm in the gastrula of Xenopus laevis. eFGF is a maternally expressed secreted Xenopus FGF with potent mesoderm‐inducing activity. However, it is most highly expressed in the mesoderm during gastrulation, suggesting a role after the period of mesoderm induction. eFGF is inhibited by the dominant negative FGF receptor. Embryos overexpressing the dominant negative receptor show a change of behaviour of the dorsal mesoderm such that it moves around the blastopore lip instead of elongating in an antero‐posterior direction. In such embryos there is a reduction in Xbra expression during gastrulation. We show that during blastula stages eFGF and Xbra are able to activate the expression of each other, suggesting that they are components of an autocatalytic regulatory loop. Moreover, we show that Xbra expression in isolated gastrula mesoderm cells is maintained by eFGF, suggesting that eFGF continues to regulate the expression of Xbra in the blastopore region. In addition, overexpression of eFGF after the mid‐blastula transition results in the up‐regulation of Xbra expression during gastrula stages and causes suppression of the head and enlargement of the proctodeum, which is the converse of the posterior reductions of the FGF dominant negative receptor phenotype. These data suggest an important role for eFGF in regulating the expression of Xbra and for the eFGF‐Xbra regulatory pathway in the control of mesodermal cell behaviour during gastrula stages.

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.

Regulation of Hox gene expression and posterior development by the Xenopus caudal homologue Xcad3

The caudal gene codes for a homeodomain transcription factor that is required for normal posterior development in Drosophila. In this study the biological activities of the Xenopus caudal (Cdx) family member Xcad3 are examined. A series of domain‐swapping experiments demonstrate that the N‐terminus of Xcad3 is necessary for it to activate Hox gene expression and that this function can be replaced by the activation domain from the viral protein VP16. In addition, experiments using an Xcad3 repressor mutant (XcadEn‐R), which potently blocks the activity of wild‐type Xcad3, are reported. Overexpression of XcadEn‐R in embryos inhibits the activation of the same subset of Hox genes that are activated by wild‐type Xcad3 and leads to a dramatic disruption of posterior development. We show that Xcad3 is an immediate early target of the FGF signalling pathway and that Xcad3 posteriorizes anterior neural tissue in a similar way to FGF. Furthermore, Xcad3 is required for the activation of Hox genes by FGFs. These data provide strong evidence that Xcad3 is required for normal posterior development and that it regulates the expression of the Hox genes downstream of FGF signalling.

Metaplasia and transdifferentiation: from pure biology to the clinic

Transformations from one tissue type to another make up a well established set of phenomena that can be explained by the principles of developmental biology. Although these phenomena might be rare in nature, we can now imagine the possibility of deliberately reprogramming cells from one tissue type to another by manipulating the expression of transcription factors. This approach could generate new therapies for many human diseases.

Transdifferentiation and metaplasia — switching cell types

States of developmental commitment are encoded as combinations of transcription factors and changes in their expression can bring about transdifferentiation or metaplasia. For example, ectopic expressions of Vestigial can convert Drosophila leg to wing; of C/EBPbeta can convert pancreatic exocrine cells to hepatocytes; and expression of C/EBPalpha and PPARgamma can convert myoblasts to adipocytes.

Cell lineage tracing during Xenopus tail regeneration

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures – the spinal cord, the notochord and the segmented myotomes – are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.

Origin of Stem Cells in Organogenesis

The development of individual organs in animal embryos involves the formation of tissue-specific stem cells that sustain cell renewal of their own tissue for the lifetime of the organism. Although details of their origin are not always known, tissue-specific stem cells usually share the expression of key transcription factors with cells of the embryonic rudiment from which they arise, and are probably in a similar developmental state. On the other hand, the isolation of pluripotent stem cells from the postnatal organism has encouraged the formulation of models of embryonic and postnatal development that are at variance with the conventional ones. Possible explanations for the existence of such cells, and the issue of whether they also exist in vivo, are discussed.

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.