Molecular pathways controlling pancreas induction

Recent advances in generating pancreatic cell types from human pluripotent stem cells has depended on our knowledge of the developmental processes that regulate pancreas development in vivo. The developmental events between gastrulation and formation of the embryonic pancreatic primordia are both rapid and dynamic and studies in frog, fish, chick, and mouse have identified the molecular basis of how the pancreas develops from multipotent endoderm progenitors. Here, we review the current status of our understanding of molecular mechanisms that control endoderm formation, endoderm patterning, and pancreas specification and highlight how these discoveries have allowed for the development of robust methods to generate pancreatic cells from human pluripotent stem cells.     

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.     

Molecular insights into evolution of the vertebrate gut: focus on stomach and parietal cells in the marsupial, Macropus eugenii

Gastrulation in vertebrate embryos results in the formation of the primary germ layers: ectoderm, mesoderm and endoderm, which contain the progenitors of the tissues of the entire fetal body. Extensive studies undertaken in Xenopus, zebrafish and mouse have revealed a high degree of conservation in the genes and cellular mechanisms regulating endoderm formation. Nodal, Mix and Sox gene factor families have been implicated in the specification of the endoderm across taxa. Considerably less is known about endoderm development in marsupials. In this study we review what is known about the molecular aspects of endoderm development, focusing on evolution and development of the stomach and parietal cells and highlight recent studies on parietal cells in the stomach of Tammar Wallaby, Macropus eugenii. Although the regulation of parietal cells has been extensively studied, very little is known about the regulation of parietal cell differentiation. Intriguingly, during late-stage forestomach maturation in M. eugenii, there is a sudden and rapid loss of parietal cells, compared with the sharp increase in parietal cell numbers in the hindstomach region. This has provided a unique opportunity to study the development and regulation of parietal cell differentiation. A PCR-based subtractive hybridization strategy was used to identify candidate genes involved in this phenomenon. This will allow us to dissect the molecular mechanisms that underpin regulation of parietal cell development and differentiation, which have been a difficult process to study and provide markers that can be used to study the evolutionary origin of these cells in vertebrates.     

Vertebrate head induction by anterior primitive endoderm

In vertebrates the antero-posterior organization of the embryonic body axis is thought to result from the activity of two separate centers, the head organizer and the trunk organizer, as operationally defined by Spemann in the 1920s. Current molecular studies have supported the existence of a trunk organizer activity while the presence of a distinct head inducing center has remained elusive. Mainly based on analyses of headless mutants in mice, it has been proposed that the anterior axial mesoderm plays a determining role in head induction. Recent gain- and loss-of-function studies in various organisms, however, provide compelling evidence that a largely ignored region, the anterior primitive endoderm, specifies rostral identity. In this review we discuss the emerging concept that the anterior primitive endoderm, rather than the prechordal plate mesoderm, induces head development in the vertebrate embryo.     

Establishing three blastocyst lineages--then what?

Development of the mouse embryo to the blastocyst stage occurs over 3 to 4 days following fertilization of the oocyte. During this time, several molecular and morphological events take place that result in the formation of three distinct cell lineages: the trophectoderm, the epiblast, and the primitive endoderm. Many studies have investigated the processes that control lineage specification in the blastocyst including gene expression, cell signaling, cell-cell contact/positional relationships, and most recently, epigenetics. Here we review, at the molecular level, recent contributions to our understanding of the mechanisms that play a role in formation of these lineages. Additionally, we focus on the next steps in differentiation to highlight processes important in the development of those lineages that contribute to the extraembryonic tissues. In this context, we discuss the establishment of extraembryonic ectoderm and the contributions of parietal and visceral endoderm to yolk sac formation.     

The production of distinct forms of plasminogen activator by mouse embryonic cells

We have examined cultured parietal endoderm, visceral endoderm, and extraembryonic mesoderm cells from the mouse embryo for production of the protease plasminogen activator. All of these cell types synthesize and secrete the enzyme, but the molecular characteristics of the plasminogen activators differ as defined by the apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gels, the antigenic properties as defined by two antisera to distinct plasminogen activators, and the interaction with an inhibitor present in fetal bovine serum. The parietal endoderm plasminogen activator has a predominant molecular weight of 79,000, is immunoprecipitated and inhibited by an antiserum raised against a human melanoma plasminogen activator, but not by an antiserum against mouse urokinase, and is only partially inhibited by the serum inhibitor. The visceral endoderm and extraembryonic mesoderm plasminogen activators, which are identical by all criteria, have molecular weights of 48,000, are inactivated only by the anti-urokinase antibodies, and are inhibited by an inhibitor in fetal bovine serum. These results establish the presence of at least two different forms of plasminogen activator in the early mouse embryo. The distinctive nature of the enzyme produced by parietal endoderm can be used as a diagnostic marker for this cell type at this stage of development. When F9 teratocarcinoma stem cells are induced to differentiate by retinoic acid and dibutyryl cAMP, they secrete a plasminogen activator of the parietal endoderm type.     

Requirement for endoderm and FGF3 in ventral head skeleton formation

The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and we explore here the function of endoderm in this process. By genetic ablation and reintroduction of endoderm in zebrafish, we show that it is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, we further identify Fgf3 as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, our results reveal for the first time that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3.     

casanova plays an early and essential role in endoderm formation in zebrafish.

The cellular and molecular mechanisms that regulate endoderm development in vertebrates have only recently begun to be explored. Here we show that the zebrafish locus casanova plays an early and essential role in this process. casanova mutants lack a gut tube and do not express any molecular markers of endoderm differentiation. The early endodermal expression of genes such as axial, gata5, and fkd2 does not initiate in casanova mutants, indicating that the endoderm is defective from the onset of gastrulation. Mosaic analysis demonstrates that casanova functions cell autonomously within the endodermal progenitors. We also report the isolation of a zebrafish homologue of Mixer, a gene important for early endoderm formation in Xenopus. casanova does not encode zebrafish Mixer, and mixer expression is normal in casanova mutants, indicating that casanova acts downstream of, or parallel to, mixer to promote endoderm formation. We further find that the forerunner cells, a specialized group of noninvoluting dorsal mesendodermal cells, do not form in casanova mutants. Studies of casanova mutants do not support an important role for the forerunner cells in either dorsal axis or tail development, as has been previously proposed. In addition, although different populations of mesodermal precursors are generated normally in casanova mutants, morphogenetic defects in the heart, vasculature, blood, and kidney are apparent, suggesting a possible role for the endoderm in morphogenesis of these organs.     

Development of definitive endoderm from embryonic stem cells in culture

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.     

Extraembryonic Endoderm cells as a model of endoderm development

In recent years the multipotent extraembryonic endoderm (XEN) stem cells have been the center of much attention. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endoderm and later on, the yolk sac. Recent data have shown that the distinction between embryonic and extraembryonic endoderm is not as strict as previously thought due to the integration, and not the displacement, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembryonic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling the potential and ability of XEN cells to both support differentiation and/or differentiate into endoderm‐like tissues as an alternative to embryonic stem (ES) cells. Moreover, the conversion of ES to XEN cells, shown recently without genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonic endoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalian lineage that complements the established ES and trophoblast stem cell lines. Through the study of essential genes and signaling requirements for XEN cells in vitro, insights will be gained about the developmental program of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overview on the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endoderm as well as the potential of using these cells for therapeutic applications.     

The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio)

The development of the vertebrate head is a highly complex process involving tissues derived from all three germ layers. The endoderm forms pharyngeal pouches, the paraxial mesoderm gives rise to endothelia and muscles, and the neural crest cells, which originate from the embryonic midbrain and hindbrain, migrate ventrally to form cartilage, connective tissue, sensory neurons, and pigment cells. All three tissues form segmental structures: the hindbrain compartmentalizes into rhombomeres, the mesoderm into somitomeres, and the endoderm into serial gill slits. It is not known whether the different segmented tissues in the head develop by the same molecular mechanism or whether different pathways are employed. It is also possible that one tissue imposes segmentation on the others. Most recent studies have emphasized the importance of neural crest cells in patterning the head. Neural crest cells colonize the segmentally arranged arches according to their original position in the brain and convey positional information from the hindbrain into the periphery. During the screen for mutations that affect embryonic development of zebrafish, one mutant, called van gogh (vgo), in which segmentation of the pharyngeal region is absent, was isolated. In vgo, even though hindbrain segmentation is unaffected, the pharyngeal endoderm does not form reiterated pouches and surrounding mesoderm is not patterned correctly. Accordingly, migrating neural crest cells initially form distinct streams but fuse when they reach the arches. This failure to populate distinct pharyngeal arches is likely due to the lack of pharyngeal pouches. The results of our analysis suggest that the segmentation of the endoderm occurs without signaling from neural crest cells but that tissue interactions between the mesendoderm and the neural crest cells are required for the segmental appearance of the neural crest-derived cartilages in the pharyngeal arches. The lack of distinct patches of neural crest cells in the pharyngeal region is also seen in mutants of one-eyed pinhead and casanova, which are characterized by a lack of endoderm, as well as defects in mesodermal structures, providing evidence for the important role of the endoderm and mesoderm in governing head segmentation.     

Endoderm is required for vascular endothelial tube formation, but not for angioblast specification

Angioblasts, the precursor cells that comprise the endothelial layer of blood vessels, arise from a purely mesodermal population. Individual angioblasts coalesce to form the primary vascular plexus through a process called vasculogenesis. A number of reports in the literature suggest that signals from the adjacent endoderm are necessary to induce angioblast specification within the mesoderm. We present evidence, using both embryological and molecular techniques, indicating that endoderm is not necessary for the induction of angioblasts. Xenopus embryos that had endoderm physically removed at the onset of gastrulation still express vascular markers. Furthermore, animal caps stimulated with bFGF form angioblasts in the absence of any detectable endodermal markers. These results show that endoderm is not required for the initial formation of angioblasts. While Xenopus embryos lacking endoderm contain aggregates of angioblasts, these angioblasts fail to assemble into endothelial tubes. Endothelial tube formation can be rescued, however, by implantation of endodermal tissue from sibling embryos. Based on these studies in Xenopus, and corroborating experiments using the quail embryo, we conclude that endoderm is not required for angioblast specification, but does play an essential role in the formation of vascular tubes.     

eXtraembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation

Background

Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.

Methodology/Principal Findings

Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.

Conclusions/Significance

These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.