Endodermal Signaling Research Articles

Foxa1 and Foxa2 orchestrate development of the urethral tube and division of the embryonic cloaca through an autoregulatory loop with Shh

Congenital anomalies of external genitalia affect approximately 1 in 125 live male births. Development of the genital tubercle, the precursor of the penis and clitoris, is regulated by the urethral plate epithelium, an endodermal signaling center. Signaling activity of the urethral plate is mediated by Sonic hedgehog (SHH), which coordinates outgrowth and patterning of the genital tubercle by controlling cell cycle kinetics and expression of downstream genes. The mechanisms that govern Shh transcription in urethral plate cells are largely unknown. Here we show that deletion of Foxa1 and Foxa2 results in persistent cloaca, an incomplete separation of urinary, genital, and anorectal tracts, and severe hypospadias, a failure of urethral tubulogenesis. Loss of Foxa2 and only one copy of Foxa1 results in urethral fistula, an additional opening of the penile urethra. Foxa1/a2 participate in an autoregulatory feedback loop with Shh, in which FOXA1 and FOXA2 positively regulate transcription of Shh in the urethra, and SHH feeds back to negatively regulate Foxa1 and Foxa2 expression. These findings reveal novel roles for Foxa genes in development of the urethral tube and in division of the embryonic cloaca.

Region-specific endodermal signals direct neural crest cells to form the three middle ear ossicles.

Defects in the middle ear ossicles - malleus, incus and stapes - can lead to conductive hearing loss. During development, neural crest cells (NCCs) migrate from the dorsal hindbrain to specific locations in pharyngeal arch (PA) 1 and 2, to form the malleus-incus and stapes, respectively. It is unclear how migratory NCCs reach their proper destination in the PA and initiate mesenchymal condensation to form specific ossicles. We show that secreted molecules sonic hedgehog (SHH) and bone morphogenetic protein 4 (BMP4) emanating from the pharyngeal endoderm are important in instructing region-specific NCC condensation to form malleus-incus and stapes, respectively, in mouse. Tissue-specific knockout of Shh in the pharyngeal endoderm or Smo (a transducer of SHH signaling) in NCCs causes the loss of malleus-incus condensation in PA1 but only affects the maintenance of stapes condensation in PA2. By contrast, knockout of Bmp4 in the pharyngeal endoderm or Smad4 (a transducer of TGFβ/BMP signaling) in the NCCs disrupts NCC migration into the stapes region in PA2, affecting stapes formation. These results indicate that region-specific endodermal signals direct formation of specific middle ear ossicles.

Symplastic signaling instructs cell division, cell expansion, and cell polarity in the ground tissue of Arabidopsis thaliana roots

Cell-to-cell communication is essential for the development and patterning of multicellular organisms. In plants, plasmodesmata (PD) provide direct routes for intercellular signaling. However, the role that PD-mediated signaling plays in plant development has not been fully investigated. To gain a comprehensive view of the role that symplastic signaling plays in Arabidopsis thaliana, we have taken advantage of a synthetic allele of CALLOSE SYNTHASE3 (icals3m) that inducibly disrupts cell-to-cell communication specifically at PD. Our results show that loss of symplastic signaling to and from the endodermis has very significant effects on the root, including an increase in the number of cell layers in the root and a misspecification of stele cells, as well as ground tissue. Surprisingly, loss of endodermal signaling also results in a loss of anisotropic elongation in all cells within the root, similar to what is seen in radially swollen mutants. Our results suggest that symplastic signals to and from the endodermis are critical in the coordinated growth and development of the root.

Molecular Characterization of the Genital Organizer: Gene Expression Profile of the Mouse Urethral Plate Epithelium

Lower urinary tract malformations are among the most common congenital anomalies in humans. Molecular genetic studies of mouse external genital development have begun to identify mechanisms that pattern the genital tubercle and orchestrate urethral tubulogenesis. The urethral plate epithelium is an endodermal signaling region that has an essential role in external genital development. However, little is known about the molecular identity of this cell population or the genes that regulate its activity. We used microarray analysis to characterize differences in gene expression between urethral plate epithelium and surrounding tissue in mouse genital tubercles. In situ hybridizations were performed to map gene expression patterns and ToppCluster (https://toppcluster.cchmc.org/) was used to analyze gene associations. A total of 84 genes were enriched at least 20-fold in urethral plate epithelium relative to surrounding tissue. The majority of these genes were expressed throughout the urethral plate in males and females at embryonic day 12.5 when the urethral plate is known to signal. Functional analysis using ToppCluster revealed genetic pathways with known functions in other organ systems but unknown roles in external genital development. Additionally, a 3-dimensional molecular atlas of genes enriched in urethral plate epithelium was generated and deposited at the GUDMAP (GenitoUrinary Development Molecular Anatomy Project) website (http://gudmap.org/). We identified dozens of genes previously unknown to be expressed in urethral plate epithelium at a crucial developmental period. It provides a novel panel of genes for analysis in animal models and in humans with external genital anomalies.

Network Analysis Identifies Crosstalk Interactions Governing TGF-β Signaling Dynamics during Endoderm Differentiation of Human Embryonic Stem Cells

The fate choice of human embryonic stem cells (hESCs) is controlled by complex signaling milieu synthesized by diverse chemical factors in the growth media. Prevalence of crosstalks and interactions between parallel pathways renders any analysis probing the process of fate transition of hESCs elusive. This work presents an important step in the evaluation of network level interactions between signaling molecules controlling endoderm lineage specification from hESCs using a statistical network identification algorithm. Network analysis was performed on detailed signaling dynamics of key molecules from TGF-β/SMAD, PI3K/AKT and MAPK/ERK pathways under two common endoderm induction conditions. The results show the existence of significant crosstalk interactions during endoderm signaling and they identify differences in network connectivity between the induction conditions in the early and late phases of signaling dynamics. Predicted networks elucidate the significant effect of modulation of AKT mediated crosstalk leading to the success of PI3K inhibition in inducing efficient endoderm from hESCs in combination with TGF-β/SMAD signaling.

Eph-Pak2a signaling regulates branching of the pharyngeal endoderm by inhibiting late-stage epithelial dynamics

Branching morphogenesis depends on the precise temporal and spatial control of epithelial dynamics. In the vertebrate head, endodermal branches, called pharyngeal pouches, form through the transient stratification, collective migration and reorganization of epithelial cells into bilayers. Here, we report novel requirements for the EphrinB ligands B2a and B3b, the Ephb4a receptor and the Pak2a kinase in the development of pouches and the posterior facial skeleton that depends on pouches for its segmentation. Time-lapse imaging in zebrafish shows that EphB-Pak2a signaling is required to stabilize pouch epithelial cells at the end of branching morphogenesis. Transgenic rescue experiments further demonstrate that endodermal Eph-ephrin signaling promotes pouch integrity by targeting Pak2a to the plasma membrane, where subsequent activation by Wnt4a-Cdc42 signaling increases junctional E-cadherin in maturing pouches. Integration of Eph-ephrin and Wnt4a signaling through Pak2a thus signals the end of branching morphogenesis by increasing intercellular adhesion that blocks further epithelial rearrangements.

CstF-64 is necessary for endoderm differentiation resulting in cardiomyocyte defects

Although adult cardiomyocytes have the capacity for cellular regeneration, they are unable to fully repair severely injured hearts. The use of embryonic stem cell (ESC)-derived cardiomyocytes as transplantable heart muscle cells has been proposed as a solution, but is limited by the lack of understanding of the developmental pathways leading to specification of cardiac progenitors. Identification of these pathways will enhance the ability to differentiate cardiomyocytes into a clinical source of transplantable cells. Here, we show that the mRNA 3′ end processing protein, CstF-64, is essential for cardiomyocyte differentiation in mouse ESCs. Loss of CstF-64 in mouse ESCs results in loss of differentiation potential toward the endodermal lineage. However, CstF-64 knockout (Cstf2E6) cells were able to differentiate into neuronal progenitors, demonstrating that some differentiation pathways were still intact. Markers for mesodermal differentiation were also present, although Cstf2E6 cells were defective in forming beating cardiomyocytes and expressing cardiac specific markers. Since the extraembryonic endoderm is needed for cardiomyocyte differentiation and endodermal markers were decreased, we hypothesized that endodermal factors were required for efficient cardiomyocyte formation in the Cstf2E6 cells. Using conditioned medium from the extraembryonic endodermal (XEN) stem cell line we were able to restore cardiomyocyte differentiation in Cstf2E6 cells, suggesting that CstF-64 has a role in regulating endoderm differentiation that is necessary for cardiac specification and that extraembryonic endoderm signaling is essential for cardiomyocyte development.

Pulmonary endoderm, second heart field and the morphogenesis of distal outflow tract in mouse embryonic heart

The second heart field (SHF), foregut endoderm and sonic hedgehog (SHH) signaling pathway are all reported to associate with normal morphogenesis and septation of outflow tract (OFT). However, the morphological relationships of the development of foregut endoderm and expression of SHH signaling pathway members with the development of surrounding SHF and OFT are seldom described. In this study, serial sections of mouse embryos from ED9 to ED13 (midgestation) were stained with a series of marker antibodies for specifically highlighting SHF (Isl-1), endoderm (Foxa2), basement membrane (Laminin), myocardium (MHC) and smooth muscle (α-SMA) respectively, or SHH receptors antibodies including patched1 (Ptc1), patched2 (Ptc2) and smoothened, to observe the spatiotemporal relationship between them and their contributions to OFT morphogenesis. Our results demonstrated that the development of an Isl-1 positive field in the splanchnic mesoderm ventral to foregut, a subset of SHF, is closely coupled with pulmonary endoderm or tracheal groove, the Isl-1 positive cells surrounding pulmonary endoderm are distributed in a special cone-shaped pattern and take part in the formation of the lateral walls of the intrapericardial aorta and pulmonary trunk and the transient aortic-pulmonary septum, and Ptc1 and Ptc2 are exclusively expressed in pulmonary endoderm during this Isl-l positive field development, suggesting special roles played in inducing the Isl-l positive field formation by pulmonary endoderm. It is indicated that pulmonary endoderm plays a role in the development and specification of SHF in midgestation, and that pulmonary endoderm-associated Isl-l positive field is involved in patterning the morphogenesis and septation of the intrapericardial arterial trunks.

The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

Control of Cell Identity in Pancreas Development and Regeneration

The endocrine and exocrine cells in the adult pancreas are not static, but can change their differentiation state in response to injury or stress. This concept of cells in flux means that there may be ways to generate certain types of cells (such as insulin-producing β-cells) and prevent formation of others (such as transformed neoplastic cells). We review different aspects of cell identity in the pancreas, discussing how cells achieve their identity during embryonic development and maturation, and how this identity remains plastic, even in the adult pancreas.

Pharyngeal endoderm and FGF signaling in induction and patterning of the chick middle ear columella condensation

Pharyngeal endoderm and FGF signaling in induction and patterning of the chick middle ear columella condensation

Dlx5 and Dlx6 expression in the anterior neural fold is essential for patterning the dorsal nasal capsule

Morphogenesis of the vertebrate facial skeleton depends upon inductive interactions between cephalic neural crest cells (CNCCs) and cephalic epithelia. The nasal capsule is a CNCC-derived cartilaginous structure comprising a ventral midline bar (mesethmoid) overlaid by a dorsal capsule (ectethmoid). Although Shh signalling from the anterior-most region of the endoderm (EZ-I) patterns the mesethmoid, the cues involved in ectethmoid induction are still undefined. Here, we show that ectethmoid formation depends upon Dlx5 and Dlx6 expression in a restricted ectodermal territory of the anterior neural folds, which we name NF-ZA. In both chick and mouse neurulas, Dlx5 and Dlx6 expression is mostly restricted to NF-ZA. Simultaneous Dlx5 and Dlx6 inactivation in the mouse precludes ectethmoid formation, while the mesethmoid is still present. Consistently, siRNA-mediated downregulation of Dlx5 and Dlx6 in the cephalic region of the early avian neurula specifically prevents ectethmoid formation, whereas other CNCC-derived structures, including the mesethmoid, are not affected. Similarly, NF-ZA surgical removal in chick neurulas averts ectethmoid development, whereas grafting a supernumerary NF-ZA results in an ectopic ectethmoid. Simultaneous ablation or grafting of both NF-ZA and EZ-I result, respectively, in the absence or duplication of both dorsal and ventral nasal capsule components. The present work shows that early ectodermal and endodermal signals instruct different contingents of CNCCs to form the ectethmoid and the mesethmoid, which then assemble to form a complete nasal capsule.

Sonic Hedgehog Mediator Gli2 Regulates Bladder Mesenchymal Patterning

Congenital bladder anomalies are a major challenge to pediatric urologists. Understanding the mechanism of bladder development is crucial for advancing patient treatment. Current evidence suggests that Shh (R&D Systems) is an epithelial signal regulating bladder development, although the mechanism of the regulation is still unclear. We examined the regulation of bladder mesenchymal development. Mutation analysis, immunohistochemistry, immunoblot, in situ hybridization, and primary cell culture and transfection were performed. The mesenchyma proximal to the epithelium was defined as the inner zone and that distal to the epithelium was defined as the outer zone. We found that the Shh transcriptional factor Gli2 and the Shh target gene Bmp4 (R&D Systems) were expressed in the inner mesenchymal zone of the bladder, where active cell proliferation was observed. In Gli2(-/-) bladder primary mesenchymal cell cultures transfection with adenoviruses expressing DeltaNGli2, a constitutionally active form of Gli2, up-regulated Bmp4 expression and promoted cell proliferation. In the outer mesenchymal zone, where Gli2 and Bmp4 expression was not detectable, smooth muscle alpha-actin was expressed. In Gli2(-/-) embryo bladders Bmp4 expression in the inner zone was lost and ectopic smooth muscle was detected in the inner mesenchymal zone. Exogenous Bmp4 (10 ng/ml) in primary smooth muscle cell culture repressed smooth muscle differentiation and repression was partially rescued by the Bmp4 antagonist Noggin (R&D Systems) (300 ng/ml). Our data suggests that the Shh transcriptional factor Gli2 regulates radial patterning of the bladder mesenchyma.

Hedgehog induction of murine vasculogenesis is mediated by Foxf1 and Bmp4

The first vasculature of the developing vertebrate embryo forms by assembly of endothelial cells into simple tubes from clusters of mesodermal angioblasts. Maturation of this vasculature involves remodeling, pruning and investment with mural cells. Hedgehog proteins are part of the instructive endodermal signal that triggers the assembly of the first primitive vessels in the mesoderm. We used a combination of genetic and in vitro culture methods to investigate the role of hedgehogs and their targets in murine extraembryonic vasculogenesis. We show that Bmps, in particular Bmp4, are crucial for vascular tube formation, that Bmp4 expression in extraembryonic tissues requires the forkhead transcription factor Foxf1 and that the role of hedgehog proteins in this process is to activate Foxf1 expression in the mesoderm. We show in the allantois that genetic disruption of hedgehog signaling (Smo(-/-)) has no effect on Foxf1 expression, and neither Bmp4 expression nor vasculogenesis are disturbed. By contrast, targeted inactivation of Foxf1 leads to loss of allantoic Bmp4 and vasculature. In vitro, the avascular Foxf1(-/-) phenotype can be rescued by exogenous Bmp4, and vasculogenesis in wild-type tissue can be blocked by the Bmp antagonist noggin. Hedgehogs are required for activation of Foxf1, Bmp4 expression and vasculogenesis in the yolk sac. However, vasculogenesis in Smo(-/-) yolk sacs can be rescued by exogenous Bmp4, consistent with the notion that the role of hedgehog signaling in primary vascular tube formation is as an activator of Bmp4, via Foxf1.

Hedgehog Signaling in Development and Homeostasis of the Gastrointestinal Tract

The Hedgehog family of secreted morphogenetic proteins acts through a complex evolutionary conserved signaling pathway to regulate patterning events during development and in the adult organism. In this review I discuss the role of Hedgehog signaling in the development, postnatal maintenance, and carcinogenesis of the gastrointestinal tract. Three mammalian hedgehog genes, sonic hedgehog (Shh), indian hedgehog (Ihh), and desert hedgehog (Dhh) have been identified. Shh and Ihh are important endodermal signals in the endodermal-mesodermal cross-talk that patterns the developing gut tube along different axes. Mutations in Shh, Ihh, and downstream signaling molecules lead to a variety of gross malformations of the murine gastrointestinal tract including esophageal atresia, tracheoesophageal fistula, annular pancreas, midgut malrotation, and duodenal and anal atresia. These congenital malformations are also found in varying constellations in humans, suggesting a possible role for defective Hedgehog signaling in these patients. In the adult, Hedgehog signaling regulates homeostasis in several endoderm-derived epithelia, for example, the stomach, intestine, and pancreas. Finally, growth of carcinomas of the proximal gastrointestinal tract such as esophageal, gastric, biliary duct, and pancreatic cancers may depend on Hedgehog signaling offering a potential avenue for novel therapy for these aggressive cancers.

Cardiopoiesis: Cardiac myocyte creation by adult and embryonic cells

The capacity of mammalian myocardium for self-repair is meager, and a cell-based approach to cardiac regeneration is highly cogent. But barriers to successful translation are many, highlighted by the small effect of stem cells from bone marrow even in the most encouraging phase II human trials. While effects alternative to myocyte creation might be beneficial, the principal focus here is on two cell types for which cardiogenic differentiation is incontrovertible (embryonic stem cells and adult heart-derived progenitor cells). For ES cells, work will be presented concerning a Sox17-Hex circuit for endodermal signals, which patterns primitive mesoderm into heart-forming mesoderm. For adult cardiac progenitor cells, work will be presented on the prospective identification of a clonogenic population of mouse cardiac Sca-1+ cells, and recent discoveries involving their potential human equivalent.

SOX7 and SOX18 are essential for cardiogenesis in Xenopus

Early in vertebrate development, endodermal signals act on mesoderm to induce cardiogenesis. The F-type SOXs SOX7 and SOX18beta are expressed in the cardiogenic region of the early Xenopus embryo. Injection of RNAs encoding SOX7 or SOX18beta, but not the related F-type SOX, SOX17, leads to the nodal-dependent expression of markers of cardiogenesis in animal cap explants. Injection of morpholinos directed against either SOX7 or SOX18mRNAs lead to a partial inhibition of cardiogenesis in vivo, while co-injection of SOX7 and SOX18 morpholinos strongly inhibited cardiogenesis. SOX7 RNA rescued the effects of the SOX18 morpholino and visa versa, indicating that the proteins have redundant functions. In animal cap explants, it appears that SOX7 and SOX18 act indirectly through Xnr2 to induce mesodermal (Eomesodermin, Snail, Wnt11), organizer (Cerberus) and endodermal (endodermin, Hex) tissues, which then interact to initiate cardiogenesis. Versions of SOX7 and SOX18 with their C-terminal, beta-catenin interaction domains replaced by a transcriptional activator domain failed to antagonize beta-catenin activation of Siamois, but still induced cardiogenesis. These observations identify SOX7 and SOX18 as important, and previously unsuspected, regulators of cardiogenesis in Xenopus.

Requirements for endoderm and BMP signaling in sensory neurogenesis in zebrafish

Cranial sensory neurons largely derive from neurogenic placodes (epibranchial and dorsolateral), which are ectodermal thickenings that form the sensory ganglia associated with cranial nerves, but the molecular mechanisms of placodal development are unclear. Here, we show that the pharyngeal endoderm induces epibranchial neurogenesis in zebrafish, and that BMP signaling plays a crucial role in this process. Using a her5:egfp transgenic line to follow endodermal movements in living embryos, we show that contact between pharyngeal pouches and the surface ectoderm coincides with the onset of neurogenesis in epibranchial placodes. By genetic ablation and reintroduction of endoderm by cell transplantation, we show that these contacts promote neurogenesis. Using a genetic interference approach we further identify bmp2b and bmp5 as crucial components of the endodermal signals that induce epibranchial neurogenesis. Dorsolateral placodes (trigeminal, auditory, vestibular, lateral line) develop independently of the endoderm and BMP signaling, suggesting that these two sets of placodes are under separate genetic control. Our results show that the endoderm regulates the differentiation of cranial sensory ganglia, which coordinates the cranial nerves with the segments that they innervate.

Disturbing endoderm signaling to anterior neural plate of vertebrates by the teratogen cadmium

Cadmium accumulation in the mouse gut endoderm occurs until the closure of the vitelline duct (day 9 post-coitus; p.c.), producing anterior neural tube defects (NTD). The anterior part of the primitive endoderm, designated as the primary signaling center for anterior patterning, expresses several transcription factors of importance for head formation. Here, we studied the expression levels of some of these transcription factors ( Hesx1, HNF3β, Cerl, Otx2 and Sox2), and cell death induced after single cadmium administration to dams on days 7, 8 and 9 p.c. Stage specific down-regulation of Hesx1, Cerl, and Sox2, and an up-regulation of HNF3β were observed. No effect was seen in Otx2 expression levels. Cell death was increased in the neuroepithelium of the cranial neural folds, and in areas where neural crest cells migrate, but not in the gut endoderm. It is proposed that cadmium-induced NTD is due to interference with head-inductive signals from the endoderm to the adjacent layers.

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.