Endodermal Precursors Research Articles

Architecture of the cortical actomyosin network driving apical constriction in C. elegans.

Apical constriction is a cell shape change that drives key morphogenetic events during development, including gastrulation and neural tube formation. The forces driving apical constriction are primarily generated through the contraction of apicolateral and/or medioapical actomyosin networks. In the Drosophila ventral furrow, the medioapical actomyosin network has a sarcomere-like architecture, with radially polarized actin filaments and centrally enriched non-muscle myosin II and myosin activating kinase. To determine if this is a broadly conserved actin architecture driving apical constriction, we examined actomyosin architecture during C. elegans gastrulation, in which two endodermal precursor cells internalize from the surface of the embryo. Quantification of protein localization showed that neither the non-muscle myosin II NMY-2 nor the myosin-activating kinase MRCK-1 is enriched at the center of the apex. Further, visualization of barbed- and pointed-end capping proteins revealed that actin filaments do not exhibit radial polarization at the apex. Our results demonstrate that C. elegans endodermal precursor cells apically constrict using a mixed-polarity actin filament network and with myosin and a myosin activator distributed throughout the network. Taken together with observations made in other organisms, our results demonstrate that diverse actomyosin architectures are used in animal cells to accomplish apical constriction.

Canine induced pluripotent stem cells efficiently differentiate into definitive endoderm in 3D cell culture conditions using high-dose activin A.

Endoderm-derived organs support indispensable functions in the body. Pluripotent stem cells can generate endoderm-derived cells or tissues and have excellent therapeutic potential to replace the functions of endodermal tissues. However, there is no viable method to induce endodermal precursor cells, definitive endoderm (DE), from canine induced pluripotent stem cells (ciPSCs). A ciPSC line was used in this study. In order to induce DE, ciPSCs were cultured with high dose activin A and fetal bovine serum. We considered the optimal differentiation period and starting cell density. Next, to reduce the remaining undifferentiated cells and improve the DE induction efficiency, DE was induced from 3D cell aggregates with knockout serum replacement instead of fetal bovine serum. Finally, hepatic and pancreatic induction were performed to investigate whether DE could differentiate into downstream lineages. After differentiation, some cells expressed the DE markers FOXA2 and SOX17. DE induction period and starting cell density were found to be important for efficient DE induction. However, some cells remained undifferentiated even after optimization of cell density and culture period. Cell differentiation under 3D culture conditions reduced undifferentiated cells and the replacement of fetal bovine serum with knockout serum replacement improved the DE induction efficiency. After hepatic and pancreatic induction, cells expressed some early hepatic and pancreatic markers. A ciPSC line was successfully differentiated to DE efficiently using a high dose of activin A with knockout serum replacement under 3D cell culture conditions. We believe that this study will be fundamental to achieving the generation of canine endodermal tissues from ciPSCs.

Definitive Endoderm from EpiSC Aggregates in Matrigel.

This chapter describes the protocol to derive definitive endoderm cells from epiblast stem cells (EpiSCs) via a process analogous to gastrulation in embryos. The basis of this protocol mimicking the in vivo gastrulation process makes a contrast with those using sequential administration of pharmacological molecules and recombinant signaling proteins even at nonphysiological levels. In the experimental setup, EpiSCs are first freed from the dish-adherent condition to form free-floating aggregates, where endoderm precursor pools are produced. Embedding the EpiSC aggregates in the Matrigel allows the endoderm precursors to interact with the Matrigel mimicking the laminin-rich basement membrane underlying the egg cylinder epiblast in embryos, and let the precursors migrate into the Matrigel-filled external zone and develop into endodermal epithelial tissues.

Cell Lineage Infidelity in PDAC Progression and Therapy Resistance.

Infidelity to cell fate occurs when differentiated cells lose their original identity and either revert to a more multipotent state or transdifferentiate into a different cell type, either within the same embryonic lineage or in an entirely different one. Whilst in certain circumstances, such as in wound repair, this process is beneficial, it can be hijacked by cancer cells to drive disease initiation and progression. Cell phenotype switching has been shown to also serve as a mechanism of drug resistance in some epithelial cancers. In pancreatic ductal adenocarcinoma (PDAC), the role of lineage infidelity and phenotype switching is still unclear. Two consensus molecular subtypes of PDAC have been proposed that mainly reflect the existence of cell lineages with different degrees of fidelity to pancreatic endodermal precursors. Indeed, the classical subtype of PDAC is characterised by the expression of endodermal lineage specifying transcription factors, while the more aggressive basal-like/squamous subtype is defined by epigenetic downregulation of endodermal genes and alterations in chromatin modifiers. Here, we summarise the current knowledge of mechanisms (genetic and epigenetic) of cell fate switching in PDAC and discuss how pancreatic organoids might help increase our understanding of both cell-intrinsic and cell-extrinsic factors governing lineage infidelity during the distinct phases of PDAC evolution.

Wnt Signaling Induces Asymmetric Dynamics in the Actomyosin Cortex of the C. elegans Endomesodermal Precursor Cell.

During asymmetrical division of the endomesodermal precursor cell EMS, a cortical flow arises, and the daughter cells, endodermal precursor E and mesodermal precursor MS, have an enduring difference in the levels of F-actin and non-muscular myosin. Ablation of the cell cortex suggests that these observed differences lead to differences in cortical tension. The higher F-actin and myosin levels in the MS daughter coincide with cell shape changes and relatively lower tension, indicating a soft, actively moving cell, whereas the lower signal in the E daughter cell is associated with higher tension and a more rigid, spherical shape. The cortical flow is under control of the Wnt signaling pathway. Perturbing the pathway removes the asymmetry arising during EMS division and induces subtle defects in the cellular movements at the eight-cell stage. The perturbed cellular movement appears to be associated with an asymmetric distribution of E-cadherin across the EMS cytokinesis groove. ABpl forms a lamellipodium which preferentially adheres to MS by the E-cadherin HMR-1. The HMR-1 asymmetry across the groove is complete just at the moment cytokinesis completes. Perturbing Wnt signaling equalizes the HMR-1 distribution across the lamellipodium. We conclude that Wnt signaling induces a cortical flow during EMS division, which results in a transition in the cortical contractile network for the daughter cells, as well as an asymmetric distribution of E-cadherin.

A Nodal/Eph signalling relay drives the transition from apical constriction to apico-basal shortening in ascidian endoderm invagination.

Gastrulation is the first major morphogenetic event during animal embryogenesis. Ascidian gastrulation starts with the invagination of 10 endodermal precursor cells between the 64- and late 112-cell stages. This process occurs in the absence of endodermal cell division and in two steps, driven by myosin-dependent contractions of the acto-myosin network. First, endoderm precursors constrict their apex. Second, they shorten apico-basally, while retaining small apical surfaces, thereby causing invagination. The mechanisms that prevent endoderm cell division, trigger the transition between step 1 and step 2, and drive apico-basal shortening have remained elusive. Here, we demonstrate a conserved role for Nodal and Eph signalling during invagination in two distantly related ascidian species, Phallusia mammillata and Ciona intestinalis Specifically, we show that the transition to step 2 is triggered by Nodal relayed by Eph signalling. In addition, our results indicate that Eph signalling lengthens the endodermal cell cycle, independently of Nodal. Finally, we find that both Nodal and Eph signals are dispensable for endoderm fate specification. These results illustrate commonalities as well as differences in the action of Nodal during ascidian and vertebrate gastrulation.

Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices.

Endoderm precursors expressing FoxA2 and Sox17 develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm‐generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed with Foxa2. Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiate Foxa2 expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g., Eomes, Gsc, and Sox17. In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E‐cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin‐rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E‐cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post‐gastrulation stage embryos: (1) definitive endoderm co‐expressing high SOX17, GATA4, and E‐cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E‐cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E‐cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin‐rich matrix models the gastrulation‐dependent endoderm precursor development.

Apelin Receptor Signaling During Mesoderm Development.

The Apelin receptor (Aplnr) is a G-protein coupled receptor which has a wide body distribution and various physiological roles including homeostasis, angiogenesis, cardiovascular and neuroendocrine function. Apelin and Elabela are two peptide components of the Aplnr signaling and are cleaved to give different isoforms which are active in different tissues and organisms.Aplnr signaling is related to several pathologies including obesity, heart disases and cancer in the adult body. However, the developmental role in mammalian embryogenesis is crucial for migration of early cardiac progenitors and cardiac function. Aplnr and peptide components have a role in proliferation, differentiation and movement of endodermal precursors. Although expression of Aplnr signaling is observed in endodermal lineages, the main function is the control of mesoderm cell movement and cardiac development. Mutant of the Aplnr signaling components results in the malformations, defects and lethality mainly due to the deformed heart function. This developmental role share similarity with the cardiovascular functions in the adult body.Determination of Aplnr signaling and underlying mechanisms during mammalian development might enable understanding of regulatory molecular mechanisms which not only control embryonic development process but also control tissue function and disease pathology in the adult body.

Production of iPSCs from a small volume of cryopreserved human umbilical cord blood buffy coat under “gmp-compliant” conditions

Background & Aim An iPSC bank derived from cord blood selected on the basis of human leukocyte antigens (HLA) requires the development and validation of protocols to generate iPSC lines under good manufacturing practice (GMP) conditions. Working towards this goal, we aim to create clinically compliant GMP grade iPSCs from small volumes of banked cord blood (CB) buffy coat samples for potential clinical use. Methods, Results & Conclusion We have established a protocol to generate iPSC lines from a minimum of 50 µl of cryopreserved CB buffy coat using defined medium and reagents manufactured under GMP. This allows us to use only segments or pilot tube of the stored CB unit without compromising the whole CB unit. Thawed CB cells were washed and an erythroblast cell population expanded in Animal Component Free (ACF) erythroblast growth medium prior to reprograming. Up to 0.11% reprogramming efficiency was obtained. iPSC colonies formed on Vitronectin coated plates were manually picked and passaged on the same matrix in defined Xeno-free E8 Flex medium. Karyotype integrity of the generated iPSC lines was confirmed with SNP Illumina Infinium CoreExome-24 v1.1 array at 0.50Mb resolution. Flow cytometry was used to show that iPSCs expressed the known stemness markers SSEA-3, SSEA-4, TRA-1-81 and TRA-1-60R but lacked expression of SSEA-1. In vitro monolayer differentiation experiments indicated that CB derived iPSCs could differentiate into cells representing the 3 germline lineages: beating cardiac cells (mesoderm), neuronal cells (ectoderm) and endodermal precursor cells. All derived cell types demonstrated the appropriate lineage markers by FACS or immunochemistry. The established iPSC line was passaged for >45 times under current culture conditions to demonstrate the stability of karyotype integrity and pluripotency potential of the cells, consistent with the earlier passages. Differentiation into cardiomyocytes indicates that differentiation capacity is similar to the earlier passages. We have demonstrated that GMP-compliant iPSC lines can be reproducibly and stability generated from small volumes of cryopreserved umbilical cord blood. By using the protocols established through this work we will create clinically compliant cell lines that will facilitate clinical research and the creation of an Australian CB-derived iPSC “haplobank” for cellular therapies.

Abiotic stress modulates root patterning via ABA-regulated microRNA expression in the endodermis initials.

Patterning of the root xylem into protoxylem (PX) and metaxylem is regulated by auxin-cytokinin signaling and microRNA miR165a/166b-mediated suppression of genes encoding Class III HOMEODOMAIN LEU-ZIPPER (HD-ZIPIII) proteins. We found that, in Arabidopsis, osmotic stress via core abscisic acid (ABA) signaling in meristematic endodermal cells induces differentiation of PX in radial and longitudinal axes in association with increased VND7 expression. Similarly, in tomato, ABA enhanced PX differentiation longitudinally and radially, indicating an evolutionarily conserved mechanism. ABA increased expression of miR165a/166b and reduced expression of the miR165a/166b repressor ZWILLE (ZLL) (also known as ARGONAUTE10), resulting in reduced levels of all five HD-ZIPIII RNAs. ABA treatments failed to induce additional PX files in a miR165a/166b-resistant PHB mutant, phb1-d, and in scr and shr mutants, in which miR165a/166b expression is strongly reduced. Thus, ABA regulates xylem patterning and maturation via miR165a/166b-regulated expression of HD-ZIPIII mRNAs and associated VND7 levels. In lateral root initials, ABA induced an increase in miR165a levels in endodermal precursors and inhibited their reduction in the future quiescent center specifically at pre-emergence stage. Hence, ABA-induced inhibition of lateral root is associated with reduced HD-ZIPIII levels.

Ectomesoderm and epithelial-mesenchymal transition-related genes in spiralian development.

Spiralians (e.g., annelids, molluscs, and flatworms) possess two sources of mesoderm. One is from endodermal precursors (endomesoderm), which is considered to be the ancestral source in metazoans. The second is from ectoderm (ectomesoderm) and may represent a novel cell type in the Spiralia. In the mollusc Crepidula fornicata, ectomesoderm is derived from micromere daughters within the A and B cell quadrants. Their progeny lie along the anterolateral edges of the blastopore. There they undergo epithelial-mesenchymal transition (EMT), become rounded and undergo delamination/ingression. Subsequently, they assume the mesenchymal phenotype, and migrate beneath the surface ectoderm to differentiate various cell types, including muscles and pigment cells. We examined expression of several genes whose homologs are known to regulate Type 1 EMT in other metazoans. Most of these genes were expressed within spiralian ectomesoderm during EMT. We propose that spiralian ectomesoderm, which exhibits analogous cellular behaviors to other populations of mesenchymal cells, may be controlled by the same genes that drive EMT in other metazoans. Perhaps these genes comprise a conserved metazoan EMT gene regulatory network (GRN). This study represents the first step in elucidating the GRN controlling the development of a novel spiralian cell type (ectomesoderm). Developmental Dynamics 247:1097-1120, 2018. © 2018 Wiley Periodicals, Inc.

Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells

Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for invitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.

PDGFRα+ Cells in Embryonic Stem Cell Cultures Represent the InVitro Equivalent of the Pre-implantation Primitive Endoderm Precursors.

SummaryIn early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα+ subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα+ cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here we identify a myosin light-chain kinase MRCK-1 as a key regulator of C.elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endoderm precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, β-catenin, and cadherin become highly enriched at the apical junctions of apically constricting cells and that MRCK-1 and myosin activity are required invivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis.

Progenitor cell niches in the human pancreatic duct system and associated pancreatic duct glands: an anatomical and immunophenotyping study.

Pancreatic duct glands (PDGs) are tubule-alveolar glands associated with the pancreatic duct system and can be considered the anatomical counterpart of peribiliary glands (PBGs) found within the biliary tree. Recently, we demonstrated that endodermal precursor niches exist fetally and postnatally and are composed functionally of stem cells and progenitors within PBGs and of committed progenitors within PDGs. Here we have characterized more extensively the anatomy of human PDGs as novel niches containing cells with multiple phenotypes of committed progenitors. Human pancreata (n=15) were obtained from cadaveric adult donors. Specimens were processed for histology, immunohistochemistry and immunofluorescence. PDGs were found in the walls of larger pancreatic ducts (diameters >300μm) and constituted nearly 4% of the duct wall area. All of the cells identified were negative for nuclear expression of Oct4, a pluripotency gene, and so are presumably committed progenitors and not stem cells. In the main pancreatic duct and in large interlobular ducts, Sox9(+) cells represented 5-30% of the cells within PDGs and were located primarily at the bottom of PDGs, whereas rare and scattered Sox9(+) cells were present within the surface epithelium. The expression of PCNA, a marker of cell proliferation, paralleled the distribution of Sox9 expression. Sox9(+) PDG cells proved to be Pdx1(+) /Ngn3(+/-) /Oct4A(-) . Nearly 10% of PDG cells were positive for insulin or glucagon. Intercalated ducts contained Sox9(+) /Pdx1(+) /Ngn3(+) cells, a phenotype that is presumptive of committed endocrine progenitors. Some intercalated ducts appeared in continuity with clusters of insulin-positive cells organized in small pancreatic islet-like structures. In summary, PDGs represent niches of a population of Sox9(+) cells exhibiting a pattern of phenotypic traits implicating a radial axis of maturation from the bottoms of the PDGs to the surface of pancreatic ducts. Our results complete the anatomical background that links biliary and pancreatic tracts and could have important implications for the common patho-physiology of biliary tract and pancreas.

Murine Sca1(+)Lin(-) bone marrow contains an endodermal precursor population that differentiates into hepatocytes.

The direct differentiation of hepatocytes from bone marrow cells remains controversial. Several mechanisms, including transdifferentiation and cell fusion, have been proposed for this phenomenon, although direct visualization of the process and the underlying mechanisms have not been reported. In this study, we established an efficient in vitro culture method for differentiation of functioning hepatocytes from murine lineage-negative bone marrow cells. These cells reduced liver damage and incorporated into hepatic parenchyma in two independent hepatic injury models. Our simple and efficient in vitro protocol for endodermal precursor cell survival and expansion enabled us to identify these cells as existing in Sca1+ subpopulations of lineage-negative bone marrow cells. The endodermal precursor cells followed a sequential developmental pathway that included endodermal cells and hepatocyte precursor cells, which indicates that lineage-negative bone marrow cells contain more diverse multipotent stem cells than considered previously. The presence of equivalent endodermal precursor populations in human bone marrow would facilitate the development of these cells into an effective treatment modality for chronic liver diseases.

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis.

BackgroundThe C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis.ResultsWe found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.ConclusionsPTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.

The Maternal-to-Zygotic Transition in C. elegans.

In Caenorhabditis elegans, the first zygotic transcription can be detected in the 4-cell stage C. elegans embryo, a little over 2h after fertilization. However, early development until the onset of gastrulation at approximately the 28-cell stage takes place normally even in the absence of zygotic transcription. Therefore, posttranslational and posttranscriptional regulation of the maternal proteins and mRNAs, respectively, that are loaded into the developing oocytes is sufficient to direct development prior to gastrulation. Protein phosphorylation is extensively used throughout the C. elegans maternal-to-zygotic transition (MZT): (1) for maternal protein activation, (2) for coordination of the meiotic and mitotic cell cycle, (3) to mark specific proteins for degradation, and/or (4) to switch the biochemical activity of specific proteins. Maternally loaded mRNAs are regulated primarily by a set of maternal RNA-binding proteins (RBPs), each of which binds to sometimes overlapping target sequences within the mRNA 3'UTRs and either promotes or inhibits translation. Most maternal transcripts are uniformly distributed throughout the embryo but specific transcripts are translated only in certain blastomeres. This control is achieved by the asymmetric distribution of the maternal RBPs, such that the blastomere-specific constellation of RBPs present, and their relative levels, determines the translational readout for their target transcripts. In certain well-studied cases, such as the specification of the sole endodermal precursor in the 8-cell embryo, the maternal transcripts and proteins along with their directly targeted zygotic genes have been identified.

Molecular conservation of metazoan gut formation: evidence from expression of endomesoderm genes in Capitella teleta (Annelida).

BackgroundMetazoan digestive systems develop from derivatives of ectoderm, endoderm and mesoderm, and vary in the relative contribution of each germ layer across taxa and between gut regions. In a small number of well-studied model systems, gene regulatory networks specify endoderm and mesoderm of the gut within a bipotential germ layer precursor, the endomesoderm. Few studies have examined expression of endomesoderm genes outside of those models, and thus, it is unknown whether molecular specification of gut formation is broadly conserved. In this study, we utilize a sequenced genome and comprehensive fate map to correlate the expression patterns of six transcription factors with embryonic germ layers and gut subregions during early development in Capitella teleta.ResultsThe genome of C. teleta contains the five core genes of the sea urchin endomesoderm specification network. Here, we extend a previous study and characterize expression patterns of three network orthologs and three additional genes by in situ hybridization during cleavage and gastrulation stages and during formation of distinct gut subregions. In cleavage stage embryos, Ct-otx, Ct-blimp1, Ct-bra and Ct-nkx2.1a are expressed in all four macromeres, the endoderm precursors. Ct-otx, Ct-blimp1, and Ct-nkx2.1a are also expressed in presumptive endoderm of gastrulae and later during midgut development. Additional gut-specific expression patterns include Ct-otx, Ct-bra, Ct-foxAB and Ct-gsc in oral ectoderm; Ct-otx, Ct-blimp1, Ct-bra and Ct-nkx2.1a in the foregut; and both Ct- bra and Ct-nkx2.1a in the hindgut.ConclusionsIdentification of core sea urchin endomesoderm genes in C. teleta indicates they are present in all three bilaterian superclades. Expression of Ct-otx, Ct-blimp1 and Ct-bra, combined with previously published Ct-foxA and Ct-gataB1 patterns, provide the most comprehensive comparison of these five orthologs from a single species within Spiralia. Each ortholog is likely involved in endoderm specification and midgut development, and several may be essential for establishment of the oral ectoderm, foregut and hindgut, including specification of ectodermal and mesodermal contributions. When the five core genes are compared across the Metazoa, their conserved expression patterns suggest that ‘gut gene’ networks evolved to specify distinct digestive system subregions, regardless of species-specific differences in gut architecture or germ layer contributions within each subregion.Electronic supplementary materialThe online version of this article (doi:10.1186/2041-9139-5-39) contains supplementary material, which is available to authorized users.

Progenitor Cells in Liver Development, Regeneration and Repair

During organogenesis, the liver develops from the foregut endoderm and grows into the adjacent septum transversum resulting in the formation of the liver bud. Growth factors released from the septum transversum and the cardiac mesenchyme induce endodermal differentiation and proliferation, thus, forming the primordial liver and extrahepatic biliary structures. Endodermal precursor cells within the liver bud comprise bi-potential liver progenitor cells called hepatoblasts, which differentiates into hepatocytes and cholangiocytes. While the postnatal liver has remarkable capacity to regenerate via the proliferation of mature hepatocytes, this compensatory mechanism may be overwhelmed during states of chronic injury. Under these conditions, resident stem/progenitor cells proliferate to replace lost liver parenchyma. Significant progress has been made recently in elucidating the role of various signaling pathways and progenitor cells in liver development and regeneration. In this review, we summarize our recent understanding of progenitor cells in liver development, regeneration and repair.