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ABSTRACT: The endoderm gives rise to the lining of the esophagus, stomach and intestines, as well as associated organs. To generate a functional intestine, a series of highly orchestrated developmental processes must occur. In this review, we attempt to cover major events during intestinal development from gastrulation to birth, including endoderm formation, gut tube growth and patterning, intestinal morphogenesis, epithelial reorganization, villus emergence, as well as proliferation and cytodifferentiation. Our discussion includes morphological and anatomical changes during intestinal development as well as molecular mechanisms regulating these processes.
Developmental Dynamics 03/2011; 240(3):501-20. · 2.54 Impact Factor
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Jason R Spence,
Christopher N Mayhew,
Scott A Rankin,
Matthew F Kuhar,
Jefferson E Vallance,
Kathryn Tolle,
Elizabeth E Hoskins,
Vladimir V Kalinichenko,
Susanne I Wells,
Aaron M Zorn,
Noah F Shroyer,
James M Wells
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ABSTRACT: Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal 'organoids' consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.
Nature 02/2011; 470(7332):105-9. · 36.28 Impact Factor
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ABSTRACT: 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.
Nature Protocol 01/2011; 6(12):1920-8. · 8.36 Impact Factor
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ABSTRACT: The ventral pancreas, biliary system, and liver arise from the posterior ventral foregut, but the cell-intrinsic pathway by which these organ lineages are separated is not known. Here we show that the extrahepatobiliary system shares a common origin with the ventral pancreas and not the liver, as previously thought. These pancreatobiliary progenitor cells coexpress the transcription factors PDX1 and SOX17 at E8.5 and their segregation into a PDX1+ ventral pancreas and a SOX17+ biliary primordium is Sox17-dependent. Deletion of Sox17 at E8.5 results in the loss of biliary structures and ectopic pancreatic tissue in the liver bud and common duct, while Sox17 overexpression suppresses pancreas development and promotes ectopic biliary-like tissue throughout the PDX1+ domain. Restricting SOX17+ biliary progenitor cells to the ventral region of the gut requires the notch effector Hes1. Our results highlight the role of Sox17 and Hes1 in patterning and morphogenetic segregation of ventral foregut lineages.
Developmental cell 08/2009; 17(1):62-74. · 13.36 Impact Factor
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ABSTRACT: Diseases that affect endodermally derived organs such as the lungs, liver, and pancreas include cystic fibrosis, chronic hepatitis, and diabetes, respectively. Despite the prevalence of these diseases, cures remain elusive. While several promising transplantation-based therapies exist for some diseases such as Type 1 diabetes, they are currently limited by the availability of donor-derived tissues. Embryonic stem cells are a promising and renewable source of tissue for transplantation; however, directing their differentiation into specific, adult cell lineages remains a significant challenge. In this review, we will focus on one endodermally derived organ, the pancreas, and discuss how studies of embryonic pancreas development have been used as the basis for the directed, step-wise differentiation of mouse and human embryonic stem cells into pancreatic endocrine cells that are capable of rescuing Type 1 diabetes in animal models.
Developmental Dynamics 01/2008; 236(12):3218-27. · 2.54 Impact Factor
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ABSTRACT: The canonical Wnt pathway is necessary for gut epithelial cell proliferation, and aberrant activation of this pathway causes intestinal neoplasia. We report a novel mechanism by which the Sox family of transcription factors regulate the canonical Wnt signaling pathway. We found that some Sox proteins antagonize while others enhance beta-catenin/T-cell factor (TCF) activity. Sox17, which is expressed in the normal gut epithelium but exhibits reduced expression in intestinal neoplasia, is antagonistic to Wnt signaling. When overexpressed in SW480 colon carcinoma cells, Sox17 represses beta-catenin/TCF activity in a dose-dependent manner and inhibits proliferation. Sox17 and Sox4 are expressed in mutually exclusive domains in normal and neoplastic gut tissues, and gain- and loss-of-function studies demonstrate that Sox4 enhances beta-catenin/TCF activity and the proliferation of SW480 cells. In addition to binding beta-catenin, both Sox17 and Sox4 physically interact with TCF/lymphoid enhancer factor (LEF) family members via their respective high-mobility-group box domains. Results from gain- and loss-of-function experiments suggest that the interaction of Sox proteins with beta-catenin and TCF/LEF proteins regulates the stability of beta-catenin and TCF/LEF. In particular, Sox17 promotes the degradation of both beta-catenin and TCF proteins via a noncanonical, glycogen synthase kinase 3beta-independent mechanism that can be blocked by proteasome inhibitors. In contrast, Sox4 may function to stabilize beta-catenin protein. These findings indicate that Sox proteins can act as both antagonists and agonists of beta-catenin/TCF activity, and this mechanism may regulate Wnt signaling responses in many developmental and disease contexts.
Molecular and cellular biology 12/2007; 27(22):7802-15. · 6.06 Impact Factor
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ABSTRACT: The embryonic chick is able to regenerate the retina after it has been removed. We have previously shown that proliferating stem/progenitor cells present in the ciliary body/ciliary marginal zone (CB/CMZ) of the chick eye are responsible for regeneration, which can be induced by ectopic fibroblast growth factor-2 (FGF2) or Sonic hedgehog (Shh). Here, we reveal the mechanisms showing how FGF2 and Shh signaling are interdependent during retina regeneration. If the FGF pathway is inhibited, regeneration stimulated by Shh is inhibited. Likewise, if the Hedgehog pathway is inhibited, regeneration stimulated by FGF2 is inhibited. We examined early signaling events in the CB/CMZ and found that FGF2 or Shh induced a robust Erk phosphorylation during the early stages of retina regeneration. Shh also up-regulated the expression of several members of the FGF signaling pathway. We show that ectopic FGF2 or Shh overexpression increased the number of phosphohistone 3 (PH3)-positive cells in the CB/CMZ and inhibition of either pathway decreased the number of PH3-positive cells. Additionally, both FGF and Hh signaling are required for cell survival in the CB/CMZ, whereas Hh and not FGF signaling plays a role in maintaining the identity of the retinal progenitor population in this region. Combined, our results support a model where the FGF and Hedgehog pathways work together to stimulate retina regeneration.
Developmental Dynamics 06/2007; 236(5):1161-74. · 2.54 Impact Factor
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ABSTRACT: To elucidate the early cellular events that take place during induction of retina regeneration in the embryonic chick, focusing on the relationship between fibroblast growth factor (FGF) signaling and the regulation of Pax6 and Mitf.
The retina of embryonic day 4 (E4) chicks was removed and a heparin coated bead soaked in fibroblast growth factor 2 (FGF2) was placed into the optic cup. The pharmacological inhibitor PD173074 was used to inhibit FGF receptors, PD98059 was used to inhibit MAP kinase-kinase/extracellular signal-regulated kinase (MEK/Erk) signaling. Retroviral constructs for paired box 6 (Pax6), MEK, and microphthalmia (Mitf) were also used in overexpression studies. Immunohistochemistry was used to examine pErk, Pax6, Mitf, and melanosomal matrix protein 115 (MMP115) immunoreactivity and bromodeoxyuridine (BrdU) incorporation at different time points after removing the retina.
The embryonic chick has the ability to regenerate a new retina by the process of transdifferentiation of the retinal pigment epithelium (RPE). We observed that during the induction of transdifferentiation, downregulation of Mitf was not sufficient to induce transdifferentiation at E4 and that FGF2 was required to drive Pax6 protein expression and cell proliferation, both of which are necessary for transdifferentiation. Furthermore, we show that FGF2 works through the FGFR/MEK/Erk signaling cascade to increase Pax6 expression and proliferation. Ectopic Mitf expression was able to inhibit transdifferentiation by acting downstream of FGFR/MEK/Erk signaling, likely by inhibiting the increase in Pax6 protein in the RPE.
FGF2 stimulates Pax6 expression during induction of transdifferentiation of the RPE through FGFR/MEK/Erk signaling cascade. This Pax6 expression is accompanied by an increase in BrdU incorporation. In addition, we show that Mitf is spontaneously downregulated after removal of the retina even in the absence of FGF2. This Mitf downregulation is not accompanied by Pax6 upregulation, demonstrating that FGF2 stimulated Pax6 upregulation is required for transdifferentiation of the RPE. Furthermore, we show that ectopic Mitf expression is able to protect the RPE from FGF2 induced transdifferentiation by inhibiting Pax6 upregulation.
Molecular vision 02/2007; 13:57-65. · 2.20 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are capable of controlling gene expression by targeting complimentary sequences in many mRNAs. Thus, a small number of miRNAs are capable of regulating expression of many different genes. miRNAs have been found in all animals from Drosophila to human and they are highly conserved. This work was undertaken in order to identify such RNAs in the newt eye.
Cloning of these RNAs was attempted after isolating and fractionating total RNA from the adult newt eye. A gel slice ranging from about 15 to 30 nucleotides in length was cut and the extracted RNA was cloned after several processes involving reverse transcription and linker addition. For expression analysis and verification during the process of lens regeneration we used as a probe mir-124a.
Several microRNAs, piRNAs and other small RNAS were identified. Some of them have eye specific gene targets in other species, but for many a function in the eye remains to be attributed. Expression of miR-124a showed an interesting regulation in the lens regeneration-competent dorsal iris.
The cloned miRNAs and other small RNAs are the first to be reported for this animal and might bear significance in regulating processes that are unique to the newt eye, i.e., regeneration of the lens and retina.
Molecular vision 02/2006; 12:1386-91. · 2.20 Impact Factor
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ABSTRACT: This study identifies and characterizes retinal stem cells (RSCs) in early postnatal to seventh-decade human eyes. Different subregions of human eyes were dissociated and cultured by using a clonal sphere-forming assay. The stem cells were derived only from the pars plicata and pars plana of the retinal ciliary margin, at a frequency of approximately 1:500. To test for long-term self-renewal, both the sphere assay and monolayer passaging were used. By using the single sphere passaging assay, primary spheres were dissociated and replated, and individual spheres demonstrated 100% self-renewal, with single spheres giving rise to one or more new spheres in each subsequent passage. The clonal retinal spheres were plated under differentiation conditions to assay the differentiation potential of their progeny. The spheres were produced all of the different retinal cell types, demonstrating multipotentiality. Therefore, the human eye contains a small population of cells (approximately equal to 10,000 cells per eye) that have retinal stem-cell characteristics (proliferation, self-renewal, and multipotentiality). To test the in vivo potential of the stem cells and their progeny, we transplanted dissociated human retinal sphere cells, containing both stem cells and progenitors, into the eyes of postnatal day 1 NOD/SCID mice and embryonic chick eyes. The progeny of the RSCs were able to survive, migrate, integrate, and differentiate into the neural retina, especially as photoreceptors. Their facile isolation, integration, and differentiation suggest that human RSCs eventually may be valuable in treating human retinal diseases.
Proceedings of the National Academy of Sciences 12/2004; 101(44):15772-7. · 9.68 Impact Factor
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ABSTRACT: The embryonic chick has the ability to regenerate its retina after it has been completely removed. Here, we provide a detailed characterization of retina regeneration in the embryonic chick at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). We show that regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2, but we show that it can be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, we show that the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE), as ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, our results suggest that the hedgehog pathway is an important modulator of retina regeneration.
Development 10/2004; 131(18):4607-21. · 6.60 Impact Factor
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ABSTRACT: Lens regeneration in the adult newt is a classic example of replacing a lost organ by the process of transdifferentiation. After lens removal, the pigmented epithelial cells of the dorsal iris proliferate and dedifferentiate to form a lens vesicle, which subsequently differentiates to form a new lens. In searching for factors that control this remarkable process, we investigated the expression and role of hedgehog pathway members. These molecules are known to affect retina and pigment epithelium morphogenesis and have been recently shown to be involved in repair processes. Here we show that Shh, Ihh, ptc-1, and ptc-2 are expressed during lens regeneration. The expression of Shh and Ihh is quite unique since these genes have never been detected in lens. Interestingly, both Shh and Ihh are only expressed in the regenerating and developing lens, but not in the intact lens. Interfering with the hedgehog pathway results in considerable inhibition of the process of lens regeneration, including decreased cell proliferation as well as interference with lens fiber differentiation in the regenerating lens vesicle. Down-regulation of ptc-1 was also observed when inhibiting the pathway. These results provide the first evidence of a novel role for the hedgehog pathway in specific regulation of the regenerating lens.
Developmental Biology 04/2004; 267(2):450-61. · 4.07 Impact Factor
