Using stem cells to study and possibly treat type 1 diabetes.

Stem cells with the potential to form many different cell types are actively studied for their possible use in cell replacement therapies for several diseases. In addition, the differentiated derivatives of stem cells are being used as reagents to test for drugs that slow or correct disease phenotypes found in several degenerative diseases. This paper explores these approaches in the context of type 1 or juvenile diabetes, pointing to recent successes as well as the technical and theoretical challenges that lie ahead in the path to new treatments and cures.

Nuclear reprogramming in cells.

Nuclear reprogramming describes a switch in gene expression of one kind of cell to that of another unrelated cell type. Early studies in frog cloning provided some of the first experimental evidence for reprogramming. Subsequent procedures included mammalian somatic cell nuclear transfer, cell fusion, induction of pluripotency by ectopic gene expression, and direct reprogramming. Through these methods it becomes possible to derive one kind of specialized cell (such as a brain cell) from another, more accessible, tissue (such as skin) in the same individual. This has potential applications for cell replacement without the immunosuppression treatments that are required when cells are transferred between genetically different individuals. This article provides some background to this field, a discussion of mechanisms and efficiency, and comments on prospects for future nuclear reprogramming research.

Pathways to new beta cells.

Diabetes is a leading health problem of the world and its prevalence continues to rise. With Type I diabetes, and in some patients with Type II, the lack of insulin can be counterbalanced by providing new beta (insulin-producing) cells. For Type I diabetes, treating the autoimmune attack remains a serious challenge. Several strategies to produce new beta cells have been proposed. These include differentiation from embryonic stem cells, proliferation of existing adult beta cells, derivation from putative adult progenitors/stem cells, and reprogramming of non-beta cells to beta cells. Each of these strategies has distinct merits and risks, and they are at different stages of understanding and development. In particular, the approach based on differentiation from embryonic stem cells has had strong support and in recent years has made notable progress. Nevertheless, significant hurdles remain to transform the current research into future therapies. To expedite this transformation, we believe particular emphasis should be placed on overcoming key knowledge gaps in beta-cell biology, developing strategies that produce patient-specific beta cells, and carefully addressing potential treatment-related complications or limitations.

Analysis of pancreatic development in living transgenic zebrafish embryos.

Using DNA constructs containing regulatory sequences of the zebrafish Pdx-1 and insulin genes, germline transgenic zebrafish expressing the green fluorescent protein (GFP) reporter gene in the pancreas were generated. For both constructs, the GFP expression patterns in transgenic embryos were consistent with the mRNA expression patterns detected by RNA in situ hybridization. A deletion promoter analysis revealed that positive and negative cis-acting elements were involved in regulation of insulin gene expression. Three-dimensional reconstructions imaged from living embryos using two-photon laser-scanning microscopy (TPLSM) demonstrated that the zebrafish pancreas is formed from a single dorsal pancreatic cell mass. This is in contrast to mammals where the pancreas derives from both dorsal and ventral anlage. These transgenic fish should be useful for in vivo studies of factors involved in specifying and regulating pancreatic development and function.

Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes.

The mechanisms by which the epithelium of the digestive tract and its associated glands are specified are largely unknown. One clue is that several transcription factors are expressed in specific regions of the endoderm prior to and during organogenesis. Pdx-1, for example, is expressed in the duodenum and pancreas and Pdx-1 inactivation results in an arrest of pancreatic development after buds formation. Similarly, ngn3 is transiently expressed in the developing pancreas and a knockout results in the absence of endocrine cells. This paper focuses on the question of whether these and other transcription factors, known to be necessary for pancreatic development, are also sufficient to drive a program of pancreatic organogenesis. Using in ovo electroporation of chick embryos, we show that ectopic expression of Pdx-1 or ngn3 causes cells to bud out of the epithelium like pancreatic progenitors. The Pdx-1-expressing cells extinguish markers for other nonpancreatic regions of the endoderm and initiate, but do not complete, pancreatic cytodifferentiation. Ectopic expression of ngn3 is sufficient to turn endodermal cells of any region into endocrine cells that form islets expressing glucagon and somatostatin in the mesenchyme. The results suggest that simple gene combinations could be used in stem cells to achieve specific endodermal tissue differentiation.

Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of in vitro fertilized human blastocysts. We examined the potential of eight growth factors [basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-beta1), activin-A, bone morphogenic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), beta nerve growth factor (betaNGF), and retinoic acid] to direct the differentiation of human ES-derived cells in vitro. We show that human ES cells that have initiated development as aggregates (embryoid bodies) express a receptor for each of these factors, and that their effects are evident by differentiation into cells with different epithelial or mesenchymal morphologies. Differentiation of the cells was assayed by expression of 24 cell-specific molecular markers that cover all embryonic germ layers and 11 different tissues. Each growth factor has a unique effect that may result from directed differentiation and/or cell selection, and we can divide the overall effects of the factors into three categories: growth factors (Activin-A and TGFbeta1) that mainly induce mesodermal cells; factors (retinoic acid, EGF, BMP-4, and bFGF) that activate ectodermal and mesodermal markers; and factors (NGF and HGF) that allow differentiation into the three embryonic germ layers, including endoderm. None of the growth factors directs differentiation exclusively to one cell type. This analysis sets the stage for directing differentiation of human ES cells in culture and indicates that multiple human cell types may be enriched in vitro by specific factors.

Regulation of pancreas development by hedgehog signaling.

Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGF(beta+) signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Here we show that Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh(-)(/)(-) and Shh(-)(/)(-) Ihh(+/)(-) mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance.

Activin receptor patterning of foregut organogenesis.

Foregut development produces a characteristic sequence of gastrointestinal and respiratory organs, but the signaling pathways that ensure this developmental order remain largely unknown. Here, mutations of activin receptors ActRIIA and ActRIIB are shown to disrupt the development of posterior foregut-derived organs, including the stomach, pancreas, and spleen. Foregut expression of genes including Shh and Isl1 is shifted in mutant mice. The endocrine pancreas is particularly sensitive to the type and extent of receptor inactivation. ActRIIA(+/-)B(+/-) animals lack axial defects, but have hypoplastic pancreatic islets, hypoinsulinemia, and impaired glucose tolerance. Thus, activin receptor-mediated signaling regulates axial patterning, cell differentiation, and function of foregut-derived organs.

Development of the pancreas in Xenopus laevis.

Xenopus embryos have several experimental advantages for studying development. Although these advantages have traditionally been used to elucidate mechanisms of early development, they can also be exploited to investigate issues later in development such as organogenesis. We have begun to study pancreatic organogenesis in Xenopus. Using histological and molecular marker analysis, we characterized the anatomy of the developing pancreas in Xenopus embryos from the time of initial pancreatic rudiment formation to the time when the tadpole starts to feed. We examined the expression of various endocrine hormones, exocrine gene products, and pancreatic transcription factors. Interestingly, the endocrine hormone insulin has restricted expression in the dorsal pancreas. Investigation of pancreatic specification during gastrulation demonstrates that insulin expression is regionalized along the dorsoventral axis early in development.

Notch gene expression during pancreatic organogenesis.

Notch receptors are involved in regulating the balance between cell differentiation and stem cell proliferation during the development of numerous tissues (Artavanis-Tsakonas, S., Matsuno, K., Fortini, M. E., 1995. Notch signaling. Science 268, 225-232). Here the expression of all four vertebrate Notch genes, their ligands, and some down-stream targets is analyzed during mouse pancreatic organogenesis. Notch 1 is the first Notch gene expressed in the pancreatic epithelium, and coexpression with HES 1 suggests that the Notch 1 pathway is activated. Notch 2 expression follows later when pancreatic buds branch and is restricted to embryonic ducts, believed to be the source for endocrine and exocrine stem cells. Notch 3 and Notch 4 are expressed in pancreatic mesenchyme and later in endothelial cells. Together these descriptive data comprise a framework for understanding the cellular basis for Notch function during pancreatic development.

Hedgehog signals regulate multiple aspects of gastrointestinal development.

The gastrointestinal tract develops from the embryonic gut, which is composed of an endodermally derived epithelium surrounded by cells of mesodermal origin. Cell signaling between these two tissue layers appears to play a critical role in coordinating patterning and organogenesis of the gut and its derivatives. We have assessed the function of Sonic hedgehog and Indian hedgehog genes, which encode members of the Hedgehog family of cell signals. Both are expressed in gut endoderm, whereas target genes are expressed in discrete layers in the mesenchyme. It was unclear whether functional redundancy between the two genes would preclude a genetic analysis of the roles of Hedgehog signaling in the mouse gut. We show here that the mouse gut has both common and separate requirements for Sonic hedgehog and Indian hedgehog. Both Sonic hedgehog and Indian hedgehog mutant mice show reduced smooth muscle, gut malrotation and annular pancreas. Sonic hedgehog mutants display intestinal transformation of the stomach, duodenal stenosis (obstruction), abnormal innervation of the gut and imperforate anus. Indian hedgehog mutants show reduced epithelial stem cell proliferation and differentiation, together with features typical of Hirschsprung's disease (aganglionic colon). These results show that Hedgehog signals are essential for organogenesis of the mammalian gastrointestinal tract and suggest that mutations in members of this signaling pathway may be involved in human gastrointestinal malformations.

Early mouse endoderm is patterned by soluble factors from adjacent germ layers.

Endoderm that forms the respiratory and digestive tracts is a sheet of approximately 500-1000 cells around the distal cup of an E7.5 mouse embryo. Within 2 days, endoderm folds into a primitive gut tube from which numerous organs will bud. To characterize the signals involved in the developmental specification of this early endoderm, we have employed an in vitro assay using germ layer explants and show that adjacent germ layers provide soluble, temporally specific signals that induce organ-specific gene expression in endoderm. Furthermore, we show that FGF4 expressed in primitive streak-mesoderm can induce the differentiation of endoderm in a concentration-dependent manner. We conclude that the differentiation of gastrulation-stage endoderm is directed by adjacent mesoderm and ectoderm, one of the earliest reported patterning events in formation of the vertebrate gut tube.

Endoderm development: from patterning to organogenesis.

Although the ectoderm and mesoderm have been the focus of intensive work in the recent era of studies on the molecular control of vertebrate development, the endoderm has received less attention. Because signaling must occur between germ layers in order to achieve a properly organized body, our understanding of the coordinated development of all organs requires a more thorough consideration of the endoderm and its derivatives. This review focuses on present knowledge and perspectives concerning endoderm patterning and organogenesis. Some of the classical embryology of the endoderm is discussed and the progress and deficiencies in cellular and molecular studies are noted.

Vertebrate endoderm development.

Endoderm, one of the three principal germ layers, contributes to all organs of the alimentary tract. For simplicity, this review divides formation of endodermal organs into four fundamental steps: (a) formation of endoderm during gastrulation, (b) morphogenesis of a gut tube from a sheet of cells, (c) budding of organ domains from the tube, and (d) differentiation of organ-specific cell types within the growing buds. We discuss possible mechanisms that regulate how undifferentiated endoderm becomes specified into a myriad of cell types that populate the respiratory and gastrointestinal tracts.

Screening for novel pancreatic genes expressed during embryogenesis.

We have combined suppressive subtractive hybridization with in situ hybridization to identify genes expressed at early stages of pancreas development. By using polymerase chain reaction amplification and subtractive hybridization, this protocol for screening can be applied when the amount of RNA is limited. Seven genes expressed in or adjacent to the pancreas anlage were isolated, three of which show similarity to known genes. The expression pattern and sequence information indicate that some of the genes could govern pancreas development.

Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor.

Exposure to cyclopamine, a steroid alkaloid that blocks Sonic hedgehog (Shh) signaling, promotes pancreatic expansion in embryonic chicks. Heterotopic development of pancreatic endocrine and exocrine structures occurs in regions adjacent to the pancreas including stomach and duodenum, and insulin-producing islets in the pancreas are enlarged. The homeodomain transcription factor PDX1, required for pancreas development, is expressed broadly in the posterior foregut but pancreas development normally initiates only in a restricted region of PDX1-expressing posterior foregut where endodermal Shh expression is repressed. The results suggests that cyclopamine expands the endodermal region where Shh signaling does not occur, resulting in pancreatic differentiation in a larger region of PDX1-expressing foregut endoderm. Cyclopamine reveals the capacity of a broad region of the posterior embryonic foregut to form pancreatic cells and provides a means for expanding embryonic pancreas development.

Mixer, a homeobox gene required for endoderm development.

An expression cloning strategy in Xenopus laevis was used to isolate a homeobox-containing gene, Mixer, that can cause embryonic cells to form endoderm. Mixer transcripts are found specifically in the prospective endoderm of gastrula, which coincides with the time and place that endodermal cells become histologically distinct and irreversibly determined. Loss-of-function studies with a dominant inhibitory mutant demonstrate that Mixer activity is required for endoderm development. In particular, the expression of Sox17alpha and Sox17beta, two previously identified endodermal determinants, require Mixer function. Together, these data suggest that Mixer is an embryonic transcription factor involved in specifying the endodermal germ layer.

Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos.

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.

Notochord repression of endodermal Sonic hedgehog permits pancreas development.

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-betaB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.

Notochord to endoderm signaling is required for pancreas development.

The role of the notochord in inducing and patterning adjacent neural and mesodermal tissues is well established. We provide evidence that the notochord is also required for one of the earliest known steps in the development of the pancreas, an endodermally derived organ. At a developmental stage in chick embryos when the notochord touches the endoderm, removal of notochord eliminates subsequent expression of several markers of dorsal pancreas bud development, including insulin, glucagon and carboxypeptidase A. Pancreatic gene expression can be initiated and maintained in prepancreatic chick endoderm grown in vitro with notochord. Non-pancreatic endoderm, however, does not express pancreatic genes when recombined with the same notochord. The results suggest that the notochord provides a permissive signal to endoderm to specify pancreatic fate in a stepwise manner.