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.     

Novel regulation of yolk utilization by thyroid hormone in embryos of the direct developing frog Eleutherodactylus coqui

Thyroid hormone (TH) is required for metamorphosis of the long, coiled tadpole gut into the short frog gut. Eleutherodactylus coqui, a direct developing frog, lacks a tadpole. Its embryonic gut is a miniature adult form with a mass of yolky cells, called nutritional endoderm, attached to the small intestine. We tested the TH requirement for gut development in E. coqui. Inhibition of TH synthesis with methimazole arrested gut development in its embryonic form. Embryos treated with methimazole failed to utilize the yolk in their nutritional endoderm, and survived for weeks without further development. Conversely, methimazole and 3,3',5-tri-iodo-l-thyronine, the active form of TH, stimulated gut development and utilization and disappearance of the nutritional endoderm. In Xenopus laevis, the receptor for TH, TRβ, is upregulated in response to TH. Similarly, EcTRβ, the E. coqui ortholog, was upregulated by TH in the gut. EcTRβ expression was high in the nutritional endoderm, suggesting a direct role for TH in yolk utilization by these cells. An initial step in the breakdown of yolk in X. laevis is acidification of the yolk platelet. E. coqui embryos in methimazole failed to acidify their yolk platelets, but acidification was stimulated by TH indicating its role in an early step of yolk utilization. In addition to a conserved TH role in gut development, a novel regulatory role for TH in yolk utilization has evolved in these direct developers.     

Phorbol esters alter cell fate during development of sea urchin embryos

Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.     

Specification of pharyngeal endoderm is dependent on early signals from axial mesoderm.

The development of taste buds is an autonomous property of the pharyngeal endoderm, and this inherent capacity is acquired by the time gastrulation is complete. These results are surprising, given the general view that taste bud development is nerve dependent, and occurs at the end of embryogenesis. The pharyngeal endoderm sits at the dorsal lip of the blastopore at the onset of gastrulation, and because this taste bud-bearing endoderm is specified to make taste buds by the end of gastrulation, signals that this tissue encounters during gastrulation might be responsible for its specification. To test this idea, tissue contacts during gastrulation were manipulated systematically in axolotl embryos, and the subsequent ability of the pharyngeal endoderm to generate taste buds was assessed. Disruption of both putative planar and vertical signals from neurectoderm failed to prevent the differentiation of taste buds in endoderm. However, manipulations of contact between presumptive pharyngeal endoderm and axial mesoderm during gastrulation indicate that signals from axial mesoderm (the notochord and prechordal mesoderm) specify the pharyngeal endoderm, conferring upon the endoderm the ability to autonomously differentiate taste buds. These findings further emphasize that despite the late differentiation of taste buds, the tissue-intrinsic mechanisms that generate these chemoreceptive organs are set in motion very early in embryonic development.     

Developmental potential for tissue differentiation of fully dissociated cells of the ascidian embryo

Summary Initially, each tissue-progenitor blastomere of embryos of the ascidian Halocynthia was identified and isolated manually at the 110-cell (late-blastula) stage, the time at which most of the blastomeres have assumed a particular fate, such that each gives rise to a single type of tissue. The isolates were allowed to develop as partial embryos, then tissue differentiation was examined by monitoring the expression of specific molecular markers for differentiation of epidermis, endoderm, muscle and notochord. Essentially, all of the precursor blastomeres of these four kinds of tissue expressed the appropriate features of tissue differentiation in isolation, indicating that determination is already complete in most of the blastomeres by the 110-cell stage. Next, in order to evaluate the absolute capacity of cells for autonomous development, embryos were maintained continuously in a dissociated state from the first cleavage to the 110-cell stage, then the cells were allowed to develop into partial embryos. Tissue differentiation in the partial embryos was examined. The results showed the striking autonomy of the processes of segregation of developmental potential, as well as the autonomy of the processes of expression of differentiated phenotypes, namely those of epidermis and endoderm. Autonomous muscle differentiation was also observed; however, excess formation of muscle partial embryos occurred. The hypothesis that fate determination is mediated by localized maternal information in the egg cytoplasm is supported by the evidence of development of these tissues. By contrast, no evidence of notochord differentiation was observed in the partial embryos.     

Generating human intestinal tissue from pluripotent stem cells in vitro

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.     

Beta-catenin is required for the establishment of vegetal embryonic fates in the nemertean, Cerebratulus lacteus

Downstream components of the canonical Wnt signaling pathway that result in the nuclear localization of beta-catenin are involved in diverse developmental processes including the formation of the mesendoderm, the regulation of axial properties and asymmetric cell divisions in a wide array of metazoans. The nemertean worm, Cerebratulus lacteus, represents a member of the understudied lophotrochozoan clade that exhibits a highly stereotyped spiral cleavage program in which ectodermal, endodermal, and mesodermal origins are known from intracellular fate mapping studies. Here, the embryonic distribution of beta-catenin protein was studied using injection of synthetic mRNA, encoding GFP-tagged beta-catenin, into fertilized eggs. During the early cleavage stages beta-catenin was destabilized/degraded in animal hemisphere blastomeres and became localized to the nuclei of the four vegetal-most cells at the 64-cell stage, which give rise to definitive larval and adult endoderm. Functional assays indicate that beta-catenin plays a key role in the development of the endoderm. Morpholino knockdown of endogenous beta-catenin, as confirmed by Western analysis, resulted in the failure to gastrulate, absence of the gut and an animalized phenotype in the resulting larvae, including the formation of ectopic (anterior) apical organ tissue with elongated apical tuft cilia and no indications of dorsoventral polarity. Similarly, over-expression of the cytoplasmic domain of cadherin or a beta-catenin-engrailed repressor fusion construct prevented endoderm formation and generated the same animalized phenotype. Injections of mRNA encoding either a stabilized, constitutively activated form of beta-catenin or a dominant negative form of GSK3-beta converted all or nearly all cells into endodermal fates expressing gut-specific esterase. Thus, beta-catenin appears to be both necessary and sufficient to promote endoderm formation in C. lacteus, consistent with its role in endoderm and endomesoderm formation in anthozoan cnidarians, ascidians, and echinoderms. Consistent with the results of other studies, beta-catenin may be viewed as playing a role in the development of posterior/vegetal larval fates (i.e., endoderm) in C. lacteus. However, unlike the case found in polychaete annelid and soil nematode embryos, there is no evidence for a role of beta-catenin in regulating cell fates and asymmetric cell divisions along the entire anterior-posterior axis.     

Acquisition of developmental autonomy in the equatorial region of the Xenopus embryo

This paper describes a continuing effort to define the location and mode of action of morphogenetic determinants which direct the development of dorsal body axis structures in embryos of the frog Xenopus laevis. Earlier results demonstrated that presumptive endodermal cells in one vegetal quadrant of the 64-cell embryo can, under certain experimental conditions, induce partial or complete body axis formation by progeny of adjacent equatorial cells. (R.L. Gimlich and J.C. Gerhart, 1984, Dev. Biol. 104, 117-130). I have now assessed the importance of other blastomeres for embryonic axis formation in a series of transplantation experiments using cells from the equatorial level of the 32-cell embryo. The transplant recipients were embryos which had been irradiated with ultraviolet light before first cleavage. Without transplantation, embryos failed to develop the dorsal structures of the embryonic body axis. However, cells of these recipients were competent to respond to inductive signals from transplanted tissue and to participate in normal embryogenesis. Dorsal equatorial cells, but not their lateral or ventral counterparts, often caused partial or complete body axis development in irradiated recipients, and themselves formed much of the notochord and some prechordal and somitic mesoderm. These are the same structures that they would have formed in the normal donor. Thus, the dorsal equatorial blastomeres were often at least partially autonomous in developing according to their prospective fates. In addition, they induced progeny of neighboring host cells to contribute to the axial mesoderm and to form most of the central nervous system. The frequency with which such transplants caused complete axis formation in irradiated hosts increased when they were made at later and later cleavage stages. In contrast, the inductive activity of vegetal cells remained the same or declined during the cleavage period. These and other results suggest that the egg cytoplasmic region containing "axial determinants" is distributed to both endodermal and mesodermal precursors in the dorsal-most quadrant of the early blastula.     

Sizes and rates of elongation of replicons in the frog embryo

Summary DNA fiber autoradiography has shown an increase in size of replicons during early development of the frog embryo. Replicons of endoderm cells were considerably larger than those of dorsal ectoderm and mesoderm cells in tailbud embryos. Late replicating DNA in partially synchronized tailbuds has a more rapid rate of replicon elongation than does early replicating DNA.     

Cleavage and gastrulation in the shrimp Penaeus (Litopenaeus) vannamei (Malacostraca, Decapoda, Dendrobranchiata)

While most malacostracan crustaceans develop through superficial cleavage, in the Amphipoda, Euphausiacea, and Dendrobranchiata (Decapoda) cleavage is complete. Euphausiaceans and dendrobranchiate shrimp share a similar early cleavage pattern, early cleavage arrest and ingression of mesendoderm progenitor cells, a ring of crown cells (prospective naupliar mesoderm) around the blastopore, and hatching as a nauplius larva. Yet recent phylogenies do not support a close relationship between Euphausiacea and Decapoda. In addition, some variation is reported in the timing of mesendoderm cell arrest and number of crown cells for a number of dendrobranchiates. To determine the representative pattern of development in the Dendrobranchiata, embryos of the Pacific white shrimp Penaeus (Litopenaeus) vannamei were stained with Sytox Green to label chromosomes and nuclei and examined with confocal microscopy. The early cleavage pattern, mesendoblast arrest and subsequent ingression at the 32-cell stage, presence of 8 initial crown cells, and fates of the mesendoblasts are the same for P. vannamei (family Peneaeidae) and Sicyonia ingentis (family Sicyoniidae). The lineage of the primordial endoderm cells differs from that reported for P. kerathurus. These characters were discussed in the context of the evolution of development in the Dendrobranchiata and in comparison to the Euphausiacea.     

Formation of a Polarised Primitive Endoderm Layer in Embryoid Bodies Requires Fgfr/Erk Signalling

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.     

Wnt6 induces the specification and epithelialization of F9 embryonal carcinoma cells to primitive endoderm

Epithelial-to-mesenchymal transitions (EMTs) play key roles in the normal development of an organism as well as its demise following the metastasis of a malignant tumour. An EMT during early mouse development results in the differentiation of primitive endoderm into the parietal endoderm that forms part of the parietal yolk sac. In the embryo, primitive endoderm develops from cells in the inner cell mass, but the signals that instruct these cells to become specified and adopt an epithelial fate are poorly understood. The mouse F9 teratocarcinoma cell line, a model that can recapitulate the in vivo primitive to parietal endoderm EMT, has been used extensively to elucidate the signalling cascades involved in extraembryonic endoderm differentiation. Here, we identified Wnt6 as a gene up-regulated in F9 cells in response to RA and show that Wnt6 expressing cells or cells exposed to Wnt6 conditioned media form primitive endoderm. Wnt6 induction of primitive endoderm is accompanied by beta-catenin and Snail1 translocation to the nucleus and the appearance of cytokeratin intermediate filaments. Attenuating glycogen synthase kinase 3 activity using LiCl gave similar results, but the fact that cells de-differentiate when LiCl is removed reveals that other signalling pathways are required to maintain cells as primitive endoderm. Finally, Wnt6-induced primitive endodermal cells were tested to determine their competency to complete the EMT and differentiate into parietal endoderm. Towards that end, results show that up-regulating protein kinase A activity is sufficient to induce markers of parietal endoderm. Together, these findings indicate that undifferentiated F9 cells are responsive to canonical Wnt signalling, which negatively regulates glycogen synthase kinase 3 activity leading to the epithelialization and specification of primitive endoderm competent to receive additional signals required for EMT. Considering the ability of F9 cells to mimic an in vivo EMT, the identification of this Wnt6-beta-catenin-Snail signalling cascade has broad implications for understanding EMT mechanisms in embryogenesis and metastasis.     

Frizzled1/2/7 signaling directs β-catenin nuclearisation and initiates endoderm specification in macromeres during sea urchin embryogenesis

In sea urchins, the nuclear accumulation of β-catenin in micromeres and macromeres at 4th and 5th cleavage activates the developmental gene regulatory circuits that specify all of the vegetal tissues (i.e. skeletogenic mesoderm, endoderm and non-skeletogenic mesoderm). Here, through the analysis of maternal Frizzled receptors as potential contributors to these processes, we found that, in Paracentrotus lividus, the receptor Frizzled1/2/7 is required by 5th cleavage for β-catenin nuclearisation selectively in macromere daughter cells. Perturbation analyses established further that Frizzled1/2/7 signaling is required subsequently for the specification of the endomesoderm and then the endoderm but not for that of the non-skeletogenic mesoderm, even though this cell type also originates from the endomesoderm lineage. Complementary analyses on Wnt6 showed that this maternal ligand is similarly required at 5th cleavage for the nuclear accumulation of β-catenin exclusively in the macromeres and for endoderm but not for non-skeletogenic mesoderm specification. In addition, Wnt6 misexpression reverses Frizzled1/2/7 downregulation-induced phenotypes. Thus, the results indicate that Wnt6 and Frizzled1/2/7 are likely to behave as the ligand-receptor pair responsible for initiating β-catenin nuclearisation in macromeres at 5th cleavage and that event is necessary for endoderm specification. They show also that β-catenin nuclearisation in micromeres and macromeres takes place through a different mechanism, and that non-skeletogenic mesoderm specification occurs independently of the nuclear accumulation of β-catenin in macromeres at the 5th cleavage. Evolutionarily, this analysis outlines further the conserved involvement of the Frizzled1/2/7 subfamily, but not of specific Wnts, in the activation of canonical Wnt signaling during early animal development.     

Intercellular Interactions,Position, and Polarity in Establishing Blastocyst Cell Lineages and Embryonic Axes

The formation of the three lineages of the mouse blastocyst provides a powerful model system to study interactions among cell behavior, cell signaling, and lineage development. Hippo signaling differences between the inner and outer cells of the early cleavage stages, combined with establishment of a stably polarized outer epithelium, lead to the establishment of the inner cell mass and the trophectoderm, whereas FGF signaling differences among the individual cells of the ICM lead to gradual separation and segregation of the epiblast and primitive endoderm lineages. Events in the late blastocyst lead to the formation of a special subset of cells from the primitive endoderm that are key sources for the signals that establish the subsequent body axis. The slow pace of mouse early development, the ability to culture embryos over this time period, the increasing availability of live cell imaging tools, and the ability to modify gene expression at will are providing increasing insights into the cell biology of early cell fate decisions.     

Germ layer patterning in bichir and lamprey; an insight into its evolution in vertebrates

Amphibian holoblastic cleavage in which all blastomeres contribute to any one of the three primary germ layers has been widely thought to be a developmental pattern in the stem lineage of vertebrates, and meroblastic cleavage to have evolved independently in each vertebrate lineage. In extant primitive vertebrates, agnathan lamprey and basal bony fishes also undergo holoblastic cleavage, and their vegetal blastomeres have been generally thought to contribute to embryonic endoderm. However, the present marker analyses in basal ray-finned fish bichir and agnathan lamprey embryos indicated that their mesoderm and endoderm develop in the equatorial marginal zone, and their vegetal cell mass is extraembryonic nutritive yolk cells, having non-cell autonomous meso-endoderm inducing activity. Eomesodermin (eomes), but not VegT, orthologs are expressed maternally in these animals, suggesting that VegT is a maternal factor for endoderm differentiation only in amphibian. The study raises the viewpoint that the lamprey/bichir type holoblastic development would have been ancestral to extant vertebrates and retained in their stem lineage; amphibian-type holoblastic development would have been acquired secondarily, accompanied by the exploitation of new molecular machinery such as maternal VegT.     

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.