Steroids Regulate SLC2A1 and SLC2A3 to Deliver Glucose Into Trophectoderm for Metabolism via Glycolysis.

The conceptuses (embryo/fetus and placental membranes) of pigs require energy to support elongation and implantation, and amounts of glucose and fructose increase in the uterine lumen during the peri-implantation period. Conceptuses from day 16 of pregnancy were incubated with either 14C-glucose or 14C-fructose and amounts of radiolabeled CO2 released from the conceptuses measured to determine rates of oxidation of glucose and fructose. Glucose and fructose both transport into conceptuses, and glucose is preferentially metabolized in the presence of fructose, whereas fructose is actively metabolized in the absence of glucose and to a lesser extent in the presence of glucose. Endometrial and placental expression of glucose transporters SLC2A1, SLC2A2, SCL2A3, and SLC2A4 were determined. SLC2A1 messenger RNA (mRNA) and protein, and SLC2A4 mRNA were abundant in the uterine luminal epithelium of pregnant compared to cycling gilts, and increased in response to progesterone and conceptus-secreted estrogen. SLC2A2 mRNA was expressed weakly by conceptus trophectoderm on day 15 of pregnancy, whereas SLC2A3 mRNA was abundant in trophectoderm/chorion throughout pregnancy. Therefore, glucose can be transported into the uterine lumen by SLC2A1, and then into conceptuses by SLC2A3. On day 60 of gestation, the cell-specific expression of these transporters was more complex, suggesting that glucose and fructose transporters are precisely regulated in a spatial-temporal pattern along the uterine-placental interface of pigs to maximize hexose sugar transport to the pig conceptus/placenta.

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping.

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation.

Significant embryo loss remains a serious problem in pig production. Reactive oxygen species (ROS) play a critical role in embryonic implantation and placentation. However, the potential mechanism of ROS on porcine trophectoderm (pTr) cell fate during the peri-implantation period has not been investigated. This study aimed to elucidate the effects of ROS on pTr cell phenotypes and the regulatory role in cell attachment and differentiation. Herein, results showed that exogenous H2O2 inhibited pTr cell viability, arrested the cell cycle at S and G2/M phases, and increased cell apoptosis and autophagy protein light chain 3B and Beclin-1, whereas these effects were reversed by different concentrations of N-acetyl-l-cysteine (NAC) posttreatment. In addition, NAC abolished H2O2-induced autophagic flux, inhibited intracellular and mitochondrial ROS, and restored expression of genes important for mitochondrial DNA and biogenesis, cell attachment, and differentiation. NAC reversed H2O2-activated MAPK and Akt/mammalian target of rapamycin pathways in dose-dependent manners. Furthermore, analyses with pharmacological and RNA interference approaches suggested that autophagy regulated cell apoptosis and gene expression of caudal-related homeobox 2 and IL-1ß. Collectively, these results provide new insights into the role of the ROS-induced autophagy in pTr cell apoptosis, attachment, and differentiation, indicating a promising target for decreasing porcine conceptus loss during the peri-implantation period.

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3/4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRß, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

In vitro cultures of ectodermal monolayers from the model sea anemone Nematostella vectensis.

We report here a novel approach for the extraction, isolation and culturing of intact ectodermal tissue layers from a model marine invertebrate, the sea anemone Nematostella vectensis. A methodology is described in which a brief exposure of the animal to the mucolytic agent N-acetyl-L-cysteine (NAC) solution triggers the dislodging of the ectodermis from its underlying basement membrane and mesoglea. These extracted fragments of cell sheets adherent to culture-dish substrates, initially form 2D monolayers that are transformed within 24 h post-isolation into 3D structures. These ectodermal tissues were sustained in vitro for several months, retaining their 3D structure while continuously releasing cells into the surrounding media. Cultures were then used for cell type characterizations and, additionally, the underlying organization of actin filaments in the 3D structures are demonstrated. Incorporation of BrdU and immunohistochemical labeling using p-histone H3 primary antibody were performed to compare mitotic activities of ectodermal cells originating from intact and from in vivo regenerating animals. Results revealed no change in mitotic activities at 2 h after bisection and a 1.67-, 1.71- and 3.74-fold increase over 24, 48 and 72 h of regeneration, respectively, depicting a significant correlation coefficient (p < 0.05; R 2 = 0.74). A significant difference was found only between the control and 3-day regenerations (p = 0.016). Cell proliferation was demonstrated in the 3D ectodermis after 6 culturing days. Moreover, monolayers that were subjected to Ca++/Mg++ free medium for the first 2 h after isolation and then replaced by standard medium, showed, at 6 days of culturing, profuse appearance of positive p-histone H3-labeled nuclei in the 3D tissues. Cytochalasin administered throughout the culturing period abolished all p-histone H3 labeling. This study thus depicts novel in vitro tissue culturing of ectodermal layers from a model marine invertebrate, demonstrating the ease with which experiments can be performed and cellular and molecular pathways can be revealed, thus opening studies on 2D tissue organizations and morphogenesis as well as the roles of cellular components in the formation of tissues in this organism.

Correlation between IL-6 and invasiveness of ectoderm cells of embryo in early pregnancy.

This study aimed to explore the correlation between Interleukin-6 (IL-6) and invasiveness of ectoderm cells of embryo in early pregnancy, in order to further discuss whether IL-6 can enhance invasiveness of ectoderm cells. The study lays the foundation for determination of pathogenesis of some gestation period-related diseases. Differences in mRNA and protein expression of trophoblastic cell line JEG-3 cells in IL-6, matrix metalloproteinase-2 (MMP-2) and MMP-9 were analyzed; the regulating effect of different concentrations of IL-6 on invasive ability of trophoblast cells was studied by Transwell assay; the effect of IL-6 on proliferation of ectodermal cell line JEG-3 of embryo was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The invasive number of JEG-3 cells incubated by IL-6 (10 ng/ml) was higher than that of the control group, and the difference had statistical significance (p < 0.05). Results of using MMT assay to detect the effect of IL-6 on proliferation of trophoblastic cell line JEG-3 showed that JEG-3 cells before and after processing had no significant difference from the control group (p >0.05). Therefore, IL-6 can enhance invasiveness of ectoderm cells of embryo through activation of MMP-2.

RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins.

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.

Proliferation-stimulating effect of colony stimulating factor 2 on porcine trophectoderm cells is mediated by activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase.

Colony-stimulating factor 2 (CSF2), also known as granulocyte macrophage colony-stimulating factor, facilitates mammalian embryonic development and implantation. However, biological functions and regulatory mechanisms of action of porcine endometrial CSF2 in peri-implantation events have not been elucidated. The aim of present study was to determine changes in cellular activities induced by CSFs and to access CSF2-induced intracellular signaling in porcine primary trophectoderm (pTr) cells. Differences in expression of CSF2 mRNA in endometrium from cyclic and pregnant gilts were evaluated. Endometrial CSF2 mRNA expression increases during the peri-implantation period, Days 10 to 14 of pregnancy, as compared to the estrous cycle. pTr cells obtained in Day 12 of pregnancy were cultured in the presence or absence of CSF2 (20 ng/ml) and LY294002 (20 µM), U0126 (20 µM), rapamycin (20 nM), and SB203580 (20 µM). CSF2 in pTr cell culture medium at 20 ng/ml significantly induced phosphorylation of AKT1, ERK1/2, MTOR, p70RSK and RPS6 protein, but not STAT3 protein. Also, the PI3K specific inhibitor (LY294002) abolished CSF2-induced increases in p-ERK1/2 and p-MTOR proteins, as well as CSF2-induced phosphorylation of AKT1. Changes in proliferation and migration of pTr cells in response to CSF2 were examined in dose- and time-response experiments. CSF2 significantly stimulated pTr cell proliferation and, U0126, rapamycin and LY294002 blocked this CSF2-induced proliferation of pTr cells. Collectively, during the peri-implantation phase of pregnancy in pigs, endometrial CSF2 stimulates proliferation of trophectoderm cells by activation of the PI3K-and ERK1/2 MAPK-dependent MTOR signal transduction cascades.

Insulin-like growth factor I induces proliferation and migration of porcine trophectoderm cells through multiple cell signaling pathways, including protooncogenic protein kinase 1 and mitogen-activated protein kinase.

During early pregnancy, the developing conceptus is dependent upon a wide range of growth factors and nutrients that are secreted by or transported by uterine epithelia into the uterus at the maternal-conceptus interface for successful implantation and placentation. Among these factors, insulin-like growth factor-I (IGF-I) is known to play an important role in development of the early embryo and uterine endometrium. However, few studies have been conducted with pigs to determine IGF-I-induced functional effects on peri-implantation embryos such as activation of cell signaling cascades responsible for growth, proliferation and differentiation of cells of the conceptus. Therefore, the aim of this study was to analyze mRNA expression of endometrial IGF-I and its receptor, to examine the functional role of IGF-I on primary porcine trophectoderm (pTr) cells and to assess potential signaling pathways responsible for biological activities of IGF-1. In the present study, expression of endometrial type I IGF receptor (IGF-IR) mRNA increased significantly from Day 10 to Day 12 of pregnancy and the increase was greater for pregnant than cyclic gilts. Both IGF-I and IGF-IR mRNAs were abundant in endometrial luminal-, glandular epithelia, and stratum compactum stroma on Day 12 of pregnancy. In addition, IGF-I significantly induced phosphorylation of AKT1, ERK1/2 and RPS6 in a time- and concentration-dependent manner in pTr cells. Immunofluorescence microscopy revealed that IGF-I treated pTr cells exhibited increased abundance of phosphorylated (p)-AKT1 and p-ERK1/2 MAPK proteins in the nucleus and cytoplasm, and p-RPS6 proteins in the cytosol as compared to non-treated pTr cells. In the presence of the ERK1/2 MAPK inhibitor (U0126), IGF-I-induced AKT1 phosphorylation was not affected, whereas the PI3K inhibitor (LY294002) decreased IGF-I-induced phosphorylation of ERK1/2 and AKT1 proteins, and both the PI3K-AKT1 and ERK1/2 MAPK pathways were blocked by LY294002. Furthermore, IGF-I significantly stimulated both proliferation and migration of pTr cells, but these effects were blocked by P38 inhibitor (SB203580), U0126, MTOR inhibitor (rapamycin) and LY294002. Taken together, these results indicate that IGF-I coordinately regulates multiple cell signaling pathways including PI3K-AKT1-RPS6 and ERK1/2 MAPK signaling pathways that are critical to proliferation, migration and survival of trophectoderm cells during early pregnancy in pigs.

Human trophectoderm apposition is regulated by interferon γ-induced protein 10 (IP-10) during early implantation.

INTRODUCTION: The first step in human implantation is the attraction of the blastocyst to the endometrium. We aimed to study attraction of the human blastocyst to the endometrium, and how this process is accomplished by chemokines secreted by the endometrium. MATERIALS AND METHODS: Blastocyst trophectoderm cells and other trophoblast lineage cells were subjected to attraction assays by IP-10 and other chemokines using transwell migration and chemotaxis assays. Chemokine expression and secretion were investigated using immunohistochemistry, ELISA, FACS analysis, and RT-PCR on material from flushing of the uterine cavity in endometrial biopsies. Chemokine receptor expression by blastocyst trophectoderm following PGD biopsy, trophectoderm derived from hES, placental villi, and other trophoblast lineage cells were characterized by the same methods. RESULTS: IP-10 dramatically attracted trophectoderm derived from hES cells and other lineages by interaction with CXCR3 chemokine receptors, as shown by both chemotaxis and transwell migration. High levels of IP-10 were detected throughout the menstrual cycle at flushing of the uterine cavity. Immunohistochemistry, FACS analysis, and RT-PCR of endometrial biopsy detected IP-10 in glandular and stromal cells of the endometrium. High levels of IP-10 were detected in condition medium of the endometrial stromal and glandular cells. Of all of the chemokine/chemokine receptor combinations examined, the IP-10/CXCR3 interaction was the only cytokine that was significantly elevated. DISCUSSION: While they await the wandering blastocyst, IP-10 is produced by many cells of the endometrium, but not by endometrial natural killer cells. CONCLUSION: Endometrial IP-10 may specifically attract human blastocyst trophectoderm cells early in implantation.

Depletion of Suds3 reveals an essential role in early lineage specification.

Preimplantation development culminates with the emergence of three distinct populations: the inner cell mass, primitive endoderm and trophectoderm. Here, we define the mechanisms underlying the requirement of Suds3 in pre/peri-implantation development. Suds3 knockdown blastocysts exhibit a failure of both trophectoderm proliferation as well as a conspicuous lack of primitive endoderm. Expression of essential lineage factors Nanog, Sox2, Cdx2, Eomes, Elf5 and Sox17 are severely reduced in the absence of Suds3. Importantly, we document deficient FGF4/ERK signaling and show that exogenous FGF4 rescues primitive endoderm formation and trophectoderm proliferation in Suds3 knockdown blastocysts. We also show that Hdac1 knockdown reduces Sox2/FGF4/ERK signaling in blastocysts. Collectively, these data define a role for Suds3 in activation of FGF4/ERK signaling and determine an essential molecular role of Suds3/Sin3/HDAC complexes in lineage specification in vivo.

Directing differentiation of human embryonic stem cells toward anterior neural ectoderm using small molecules.

Based on knowledge of early embryo development, where anterior neural ectoderm (ANE) development is regulated by native inhibitors of bone morphogenic protein (BMP) and Nodal/Activin signaling, most published protocols of human embryonic stem cell differentiation to ANE have demonstrated a crucial role for Smad signaling in neural induction. The drawbacks of such protocols include the use of an embryoid body culture step and use of polypeptide secreted factors that are both expensive and, when considering clinical applications, have significant challenges in terms of good manufacturing practices compliancy. The use of small molecules to direct differentiation of pluripotent stem cells toward a specified lineage represents a powerful approach to generate specific cell types for further understanding of biological function, for understanding disease processes, for use in drug discovery, and finally for use in regenerative medicine. We therefore aimed to find controlled and reproducible animal-component-free differentiation conditions that would use only small molecules. Here, we demonstrate that pluripotent stem cells can be reproducibly and efficiently differentiated to PAX6(+) (a marker of neuroectoderm) and OCT4(-) (a marker of pluripotent stem cells) cells with the use of potent small inhibitors of the BMP and Activin/Nodal pathways, and in animal-component-free conditions, replacing the frequently used Noggin and SB431542. We also show by transcript analysis, both at the population level and for the first time at the single-cell level, that differentiated cells express genes characteristic for the development of ANE, in particular for the development of the future forebrain.

L-Arginine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells.

Impairment of placental growth is a major factor contributing to intrauterine growth retardation (IUGR) in both human pregnancy and animal production. Results of recent studies indicate that administration of L-arginine (Arg) to gestating pigs or sheep with IUGR fetuses can enhance fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that Arg stimulates the mammalian target of rapamycin (mTOR) signaling pathway and protein synthesis in porcine conceptus trophectoderm (pTr2) cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's Ham medium containing 10, 50, 100, 200, 350 or 500 µM Arg. Cell numbers, protein synthesis and degradation, as well as total and phosphorylated levels of mTOR, ribosomal protein S6 kinase 1 (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. The pTr2 cells exhibited time (0-6 days)- and Arg concentration (10-350 µM)-dependent increases in proliferation. Addition of 100 and 350 µM Arg to culture medium dose-dependently increased (a) protein synthesis and decreased protein degradation and (b) the abundance of total and phosphorylated mTOR, p70S6K and 4EBP1 proteins. Effects of 350 µM Arg on intracellular protein turnover were only modestly affected when nitric oxide synthesis was inhibited. Collectively, these results indicate a novel and important role for Arg in promoting growth of porcine placental cells largely via a nitric-oxide-independent pathway. Additionally, these findings help to explain beneficial effects of Arg supplementation on improving survival and growth of embryos/fetuses in mammals.

The response of human ectomesenchymal dental pulp stem cells to cisplatin treatment.

AIM: To determine the response of dental pulp stem cells (DPSCs) to DNA-damaging cytostatic cisplatin and compare it with the response of normal human dermal fibroblasts (HDFs). METHODOLOGY: Dental pulp stem cells were exposed to 5, 10, 20 or 40 µmol L(-1) of cisplatin. The proliferation of affected cells was assessed by a Z2 Counter and viability was assessed by means of a Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis and induction of apoptosis were performed by flow cytometry. Induction of apoptosis was determined by monitoring the activities of caspases. The expression of proteins was detected by electrophoresis and Western blotting. The descriptive statistics of the results was analyzed by Student's t-test. RESULTS: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. All three main Mitogen-activated protein kinases (MAPK) families - extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinase (JNK) and p38 were activated after treatment of DPSCs with cisplatin. The activation of MAPK pathways was not observed in HDFs exposed to cisplatin. The exposure of DPSCs and HDFs to cisplatin provoked an increase in p53 and p21 expression and p53 phosphorylation of serine 15. Higher concentrations of cisplatin reduced the viability of DPSCs and HDFs and induced the activation of caspases 3/7 and 9. CONCLUSION: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. Cisplatin in higher concentrations triggered activation of MAPK and apoptosis in DPSCs but not in HDFs.

Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation.

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs. EXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points, day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C. KEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2, TUBB III, PAX6, TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1 and COL1A2. In addition, no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated. CONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments.

Sensitivity to cadmium-chloride-induced forelimb ectrodactyly is independent of the p53 gene-dosage in the C57BL/6J mouse.

BACKGROUND: The p53 pathway plays an important role in the regulation of apoptosis, osteoblast differentiation, skeletal development, and teratogenic sensitivity. The administration of cadmium chloride (CdCl(2)) on gestational day 9 in susceptible mouse strains causes postaxial forelimb ectrodactyly in a percentage of fetuses through unknown mechanisms. In this study, the hypothesis that the p53 gene dosage might affect the incidence or severity of CdCl(2)-induced forelimb ectrodactyly was examined. METHODS: Heterozygous p53-null female mice, on the C57BL/6J background known to be sensitive to CdCl(2)-induced forelimb ectrodactyly, were mated with heterozygous males and then treated with a single intraperitoneal (ip) dose of CdCl(2) (4 mg x kg(-1)) at embryonic day (ED) 9. Embryos and fetuses, genotyped using DNA isolated from the yolk sacs, were collected at ED10 and examined for the pattern of cell death in the limb buds or collected at ED18 and examined for limb malformations. RESULTS: In the wild type and heterozygous p53 embryonic limb buds, CdCl(2)-induced apoptosis involved mesenchymal cells as well as the apical ectodermal ridge (AER), whereas CdCl(2)-induced apoptosis was restricted mainly to the AER in the homozygous p53-null limb buds. No difference in the incidence or severity of forelimb ectrodactyly in the embryos of different p53 genotypes was observed. CONCLUSION: Despite the fact that CdCl(2) induced both p53-dependent (in the mesenchyme) and p53-independent (in the AER) cell death in the developing limb bud, CdCl(2)-induced ectrodactyly was independent of the p53 gene dosage at the studied time point.

Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-beta/activin/nodal signaling using SB-431542.

Directing differentiation of human embryonic stem cells (hESCs) into specific cell types using an easy and reproducible protocol is a prerequisite for the clinical use of hESCs in regenerative-medicine procedures. Here, we report a protocol for directing the differentiation of hESCs into mesenchymal progenitor cells. We demonstrate that inhibition of transforming growth factor beta (TGF-beta)/activin/nodal signaling during embryoid body (EB) formation using SB-431542 (SB) in serum-free medium markedly upregulated paraxial mesodermal markers (TBX6, TBX5) and several myogenic developmental markers, including early myogenic transcriptional factors (Myf5, Pax7), as well as myocyte-committed markers [NCAM, CD34, desmin, MHC (fast), alpha-smooth muscle actin, Nkx2.5, cTNT]. Continuous inhibition of TGF-beta signaling in EB outgrowth cultures (SB-OG) enriched for myocyte progenitor cells; markers were PAX7(+) (25%), MYOD1(+) (52%), and NCAM(+) (CD56) (73%). DNA microarray analysis revealed differential upregulation of 117 genes (>2-fold compared with control cells) annotated to myogenic development and function. Moreover, these cells showed the ability to contract (80% of the population) and formed myofibers when implanted intramuscularly in vivo. Interestingly, SB-OG cells cultured in 10% fetal bovine serum (FBS) developed into a homogeneous population of mesenchymal progenitors that expressed CD markers characteristic of mesenchymal stem cells (MSCs): CD44(+) (100%), CD73(+) (98%), CD146(+) (96%), and CD166(+) (88%) with the ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and in vivo. Furthermore, microarray analysis of these cells revealed downregulation of genes related to myogenesis: MYH3 (-167.9-fold), ACTA1 (-161-fold), MYBPH (-139-fold), ACTC (-100.3-fold), MYH8 (-45.5-fold), and MYOT (-41.8-fold) and marked upregulation of genes related to mesoderm-derived cell lineages. In conclusion, our data provides a simple and versatile protocol for directing the differentiation of hESCs into a myogenic lineage and then further into mesenchymal progenitors by blocking the TGF-beta signaling pathway.

High dose cotinine may induce neural tube defects in a chick embryo model.

AIM: Nicotine is a well-known agent among 4000 chemicals in cigarettes. About 70 to 80% of nicotine is converted to cotinine, a major metabolite. The aim of the present study is to investigate the effect of cotinine on neural tube development in a chick embryo model. MATERIAL AND METHODS: Sixty fertile, specific pathogen free eggs were divided into 6 groups for this study. In the first group, a fixed cotinine concentration for each egg was calculated just to simulate the concentration of a smoker's blood level. A second experimental group was designed at a higher cotinine concentration. Embryos that succeeded to reach Hamburger-Hamilton stage 12 from each group were then embedded into paraffin for permanent sections. These two groups were compared with eggs subjected to vehicle (standard alcohol and ten times more alcohol concentration) and control groups (saline and sham groups). RESULTS: Embryos of the cotinine (regular dose), vehicle and control groups were normal, but embryos subjected to higher cotinine concentrations were malformed at the cranial part of the thoracic neural tube. CONCLUSION: Association of cotinine with neural tube defects was demonstrated in the present study. Cigarette smoking may induce hazardous effects on neural tube development.

Respecification of ectoderm and altered Nodal expression in sea urchin embryos after cobalt and nickel treatment.

In the sea urchin embryo, Nodal is the earliest known signal to play a role in the specification of the oral ectodermal territory. Nodal, a TGF-beta ligand, is first expressed in the presumptive oral ectoderm at approximately 7 H of development. Nodal overexpression produces a distinctive bell-shaped phenotype with expanded oral ectoderm, which resembles the oralized phenotype obtained as a result of nickel (Ni) treatment. To date, a detailed analysis of gene expression in Ni-treated embryos has not been undertaken. Because treatment with cobalt (Co) produces similar results to those seen with Ni treatment in other systems, we were interested in determining how Co influences sea urchin embryonic development. Here we report that Co also induces oralization of the ectoderm, and the effects of Ni and Co depend on functional Nodal signaling. Although both metals upregulate nodal gene expression, they do not initiate nodal transcription precociously. Analysis of the perturbation of Nodal receptor function suggests that Ni and Co contribute to nodal upregulation in the absence of nodal autoregulation, but cannot fully oralize the ectoderm in the absence of Nodal signaling.

Microarray analysis of murine limb bud ectoderm and mesoderm after exposure to cadmium or acetazolamide.

BACKGROUND: A variety of drugs, environmental chemicals, and physical agents induce a common limb malformation in the offspring of pregnant mice exposed on day 9 of gestation. This malformation, postaxial, right-sided forelimb ectrodactyly, is thought to arise via an alteration of hedgehog signaling. METHODS: We have studied two of these teratogens, acetazolamide and cadmium, using the technique of microarray analysis of limb bud ectoderm and mesoderm to search for changes in gene expression that could indicate a common pathway to postaxial limb reduction. RESULTS: Results indicated a generalized up-regulation of gene expression after exposure to acetazolamide but a generalized down-regulation due to cadmium exposure. An intriguing observation was a cadmium-induced reduction of Mt1 and Mt2 expression in the limb bud mesoderm indicating a lowering of embryonic zinc. CONCLUSIONS: We propose that these two teratogens and others (valproic acid and ethanol) lower sonic hedgehog signaling by perturbation of zinc function in the sonic hedgehog protein.