Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay

Development ◽  
1992 ◽  
Vol 116 (1) ◽  
pp. 55-66 ◽  
Author(s):  
M. Wagner ◽  
B. Han ◽  
T.M. Jessell
Keyword(s):  
Spinal Cord ◽  
Retinoic Acid ◽  
Neural Tube ◽  
Neural Tissue ◽  
Floor Plate ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Embryonic Tissues ◽  
Low Concentrations

Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.

Control of dorsoventral pattern in vertebrate neural development: induction and polarizing properties of the floor plate

Development ◽  
1991 ◽  
Vol 113 (Supplement_2) ◽  
pp. 105-122 ◽  
Author(s):  
Marysia Placzek ◽  
Toshiya Yamada ◽  
Marc Tessier-Lavigne ◽  
Thomas Jessell ◽  
Jane Dodd
Keyword(s):  
Neural Tube ◽  
Neural Development ◽  
Cell Types ◽  
Floor Plate ◽  
Neural Cells ◽  
Dorsoventral Patterning ◽  
Cellular Mechanisms ◽  
Cell Position

Distinct classes of neural cells differentiate at specific locations within the embryonic vertebrate nervous system. To define the cellular mechanisms that control the identity and pattern of neural cells we have used a combination of functional assays and antigenic markers to examine the differentiation of cells in the developing spinal cord and hindbrain in vivo and in vitro. Our results suggest that a critical step in the dorsoventral patterning of the embryonic CNS is the differentiation of a specialized group of midline neural cells, termed the floor plate, in response to local inductive signals from the underlying notochord. The floor plate and notochord appear to control the pattern of cell types that appear along the dorsoventral axis of the neural tube. The fate of neuroepithelial cells in the ventral neural tube may be defined by cell position with respect to the ventral midline and controlled by polarizing signals that originate from the floor plate and notochord.

scholarly journals Transcriptional response of Hoxb genes to retinoid signalling is regionally restricted along the neural tube rostrocaudal axis

2017 ◽  
Vol 4 (4) ◽  
pp. 160913 ◽  
Author(s):  
Nicoletta Carucci ◽  
Emanuele Cacci ◽  
Paola S. Nisi ◽  
Valerio Licursi ◽  
Yu-Lee Paul ◽  
...  
Keyword(s):  
Neural Tube ◽  
Neural Development ◽  
Transcriptional Response ◽  
Neural Tissue ◽  
Positional Information ◽  
Multiple Roles ◽  
Chromatin Activation ◽  
Neural Stem Progenitor Cells ◽  
Rostrocaudal Axis

During vertebrate neural development, positional information is largely specified by extracellular morphogens. Their distribution, however, is very dynamic due to the multiple roles played by the same signals in the developing and adult neural tissue. This suggests that neural progenitors are able to modify their competence to respond to morphogen signalling and autonomously maintain positional identities after their initial specification. In this work, we take advantage of in vitro culture systems of mouse neural stem/progenitor cells (NSPCs) to show that NSPCs isolated from rostral or caudal regions of the mouse neural tube are differentially responsive to retinoic acid (RA), a pivotal morphogen for the specification of posterior neural fates. Hoxb genes are among the best known RA direct targets in the neural tissue, yet we found that RA could promote their transcription only in caudal but not in rostral NSPCs. Correlating with these effects, key RA-responsive regulatory regions in the Hoxb cluster displayed opposite enrichment of activating or repressing histone marks in rostral and caudal NSPCs. Finally, RA was able to strengthen Hoxb chromatin activation in caudal NSPCs, but was ineffective on the repressed Hoxb chromatin of rostral NSPCs. These results suggest that the response of NSPCs to morphogen signalling across the rostrocaudal axis of the neural tube may be gated by the epigenetic configuration of target patterning genes, allowing long-term maintenance of intrinsic positional values in spite of continuously changing extrinsic signals.

scholarly journals Knockdown of Long Noncoding RNA HOXA-AS2 Suppresses Chemoresistance of Acute Myeloid Leukemia via the miR-520c-3p/S100A4 Axis

2018 ◽  
Vol 51 (2) ◽  
pp. 886-896 ◽  
Author(s):  
Xiaoya Dong ◽  
Zhigang Fang ◽  
Mingxue Yu ◽  
Ling Zhang ◽  
Ruozhi Xiao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Online Programs ◽  
Acute Myeloid

Background/Aims: Among different molecular candidates, there is growing data to support that long noncoding RNAs (lncRNAs) play a significant role in acute myeloid leukemia (AML). HOXA-AS2 is significantly overexpressed in a variety of tumors and associated with anti-cancer drug resistance, however, little is known regarding the expression and function of HOXA-AS2 in the chemoresistance of AML. In this study, we aimed to determine the role and molecular mechanism of HOXA-AS2 in adriamycin-based chemotherapy resistance in AML cells. Methods: Quantitative real-time PCR was used to detect HOXA-AS2 expression in the BM samples and ADR cell lines, U/A and T/A cells. Furthermore, the effects of HOXA-AS2 silencing on cell proliferation and apoptosis were assessed in vitro by CCK8 and flow cytometry, and on tumor growth in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in AML. Results: In this study, we showed that HOXA-AS2 is significantly upregulated in BM samples from AML patients after treatment with adriamycin-based chemotherapy and in U/A and T/A cells. Knockdown of HOXA-AS2 inhibited ADR cell proliferation in vitro and in vivo and promoted apoptosis. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3’-UTR with complementary binding sites, which was validated using luciferase reporter assay and anti-Ago2 RIP assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in ADR cells. S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay. Conclusion: Our results suggest that HOXA-AS2 plays an important role in the resistance of AML cells to adriamycin. Thus, HOXA-AS2 may represent a therapeutic target for overcoming resistance to adriamycin-based chemotherapy in AML.

Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2271-2282 ◽  
Author(s):  
Felix A. Mic ◽  
Robert J. Haselbeck ◽  
Arnold E. Cuenca ◽  
Gregg Duester
Keyword(s):  
Spinal Cord ◽  
Retinoic Acid ◽  
Neural Tube ◽  
The Novel ◽  
Vertebrate Development ◽  
Targeted Disruption ◽  
Surface Ectoderm ◽  
Eye Field ◽  
Null Mutant Mice ◽  
Dorsal Retina

Retinoid control of vertebrate development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). The RA biosynthetic enzyme RALDH2 catalyzes much, but not all, RA production in mouse embryos, as revealed here with Raldh2 null mutants carrying an RA-responsive transgene. Targeted disruption of Raldh2 arrests development at midgestation and eliminates all RA synthesis except that associated with Raldh3 expression in the surface ectoderm of the eye field. Conditional rescue of Raldh2–/– embryos by limited maternal RA administration allows development to proceed and results in the establishment of additional sites of RA synthesis linked to Raldh1 expression in the dorsal retina and to Raldh3 expression in the ventral retina, olfactory pit and urinary tract. Unexpectedly, conditionally rescued Raldh2–/– embryos also possess novel sites of RA synthesis in the neural tube and heart that do not correspond to expression of Raldh1-3. RA synthesis in the mutant neural tube was localized in the spinal cord, posterior hindbrain and portions of the midbrain and forebrain, whereas activity in the mutant heart was localized in the conotruncus and sinus venosa. In the posterior hindbrain, this novel RA-generating activity was expressed during establishment of rhombomeric boundaries. In the spinal cord, the novel activity was localized in the floorplate plus in the intermediate region where retinoid-dependent interneurons develop. These novel RA-generating activities in the neural tube and heart fill gaps in our knowledge of how RA is generated spatiotemporally and may, along with Raldh1 and Raldh3, contribute to rescue of Raldh2–/– embryos by producing RA locally.

Retinoic acid modulation of the early development of the inner ear is associated with the control of c-fos expression

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1081-1090 ◽  
Author(s):  
J. Represa ◽  
A. Sanchez ◽  
C. Miner ◽  
J. Lewis ◽  
F. Giraldez
Keyword(s):  
Cell Proliferation ◽  
Retinoic Acid ◽  
Inner Ear ◽  
Early Development ◽  
Otic Vesicle ◽  
Low Concentrations ◽  
Full Effect ◽  
20 Nm ◽  
Otic Vesicles

The effects of retinoic acid (RA) on the early development of the inner ear were studied in vitro using isolated chick embryo vesicles. Low concentrations of RA (1–50 nM) inhibited vesicular growth in stage 18 otic vesicles that were made quiescent and then reactivated by either serum or bombesin. Growth inhibition was concentration-dependent and was paralleled by a reduction in the rate of DNA synthesis as measured by [3H]thymidine incorporation. Half-inhibition occurred between 1 and 10 nM RA, and the full effect at 20 nM. Retinoic acid, in the presence of serum, induced the precocious differentiation of (1) secretory epithelium, the tegmentum vasculosum and endolymphatic sac and (2) early sensory and supporting epithelia. These structures were positioned in their corresponding normal presumptive areas. The overall direction of growth was reversed by RA and the ratio of the internal to the external vesicular surface area increased with RA concentration. The expression of the nuclear proto-oncogene c-fos in the developing otic vesicle was transient and stage-dependent. High levels of c-fos mRNA were positively correlated with cell proliferation. Incubation of growth-arrested otic vesicles with bombesin plus insulin at concentrations that induced cell proliferation produced a strong induction of c-fos. This mitogen-induced expression was suppressed by 25 nM RA. The results suggest (1) a role for retinoic acid in controlling the early development of the inner ear and (2) that this control is effected through the regulation of the proto-oncogene c-fos.

A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro

Development ◽  
1997 ◽  
Vol 124 (7) ◽  
pp. 1313-1322 ◽  
Author(s):  
H. Sasaki ◽  
C. Hui ◽  
M. Nakafuku ◽  
H. Kondoh
Keyword(s):  
Binding Site ◽  
Neural Tube ◽  
Floor Plate ◽  
Signalling Pathway ◽  
Axonal Guidance ◽  
Critical Event ◽  
Enhancer Activity ◽  
Gli Proteins

The floor plate plays important roles in ventral pattern formation and axonal guidance within the neural tube of vertebrate embryos. A critical event for floor plate development is the induction of a winged helix transcription factor, Hepatocyte Nuclear Factor-3beta (HNF-3beta). The enhancer for floor plate expression of HNF-3beta is located 3′ of the transcription unit and consists of multiple elements. HNF-3beta induction depends on the notochord-derived signal, Sonic hedgehog (Shh). Genetic analysis in Drosophila has led to the identification of genes involved in the Hh signalling pathway, and cubitus interruptus (ci), encoding a protein with five zinc finger motifs, was placed downstream. In the present work, we test the involvement of Gli proteins, the mouse homologues of Ci, in activation of the floor plate enhancer of HNF-3beta. Transgenic analysis shows that a Gli-binding site is required for the activity of the minimal floor plate enhancer of HNF-3beta in vivo. Three Gli genes are differentially expressed in the developing neural tube. Gli expression is restricted to the ventral part, while Gli2 and Gli3 are expressed throughout the neural tube and dorsally, respectively. Strong Gli and Gli2, and weak Gli3 expressions transiently overlap with HNF-3beta at the time of its induction. Consistent with ventrally localized expression, Gli expression can be up-regulated by Shh in a cell line. Finally, the Gli-binding site acts as a Shh responsive element, and human GLI, but not GLI3, can activate this binding site in tissue culture. Taken together, these findings suggest that Gli, and probably also Gli2, are good candidates for transcriptional activators of the HNF-3beta floor plate enhancer, and the binding site for Gli proteins is a key element for response to Shh signalling. These results also support the idea that Gli/Ci are evolutionary conserved transcription factors in the Hedgehog signalling pathway.

Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons

Development ◽  
2000 ◽  
Vol 127 (7) ◽  
pp. 1397-1410 ◽  
Author(s):  
R. Imondi ◽  
C. Wideman ◽  
Z. Kaprielian
Keyword(s):  
Spinal Cord ◽  
Floor Plate ◽  
Plate Boundaries ◽  
Eph Receptors ◽  
Axon Pathfinding ◽  
Mouse Spinal Cord ◽  
Commissural Axon ◽  
Ligand Interactions ◽  
Ventral Funiculus

In the developing spinal cord, axons project in both the transverse plane, perpendicular to the floor plate, and in the longitudinal plane, parallel to the floor plate. For many axons, the floor plate is a source of long- and short-range guidance cues that govern growth along both dimensions. We show here that B-class transmembrane ephrins and their receptors are reciprocally expressed on floor plate cells and longitudinally projecting axons in the mouse spinal cord. During the period of commissural axon pathfinding, B-class ephrin protein is expressed at the lateral floor plate boundaries, at the interface between the floor plate and the ventral funiculus. In contrast, B-class Eph receptors are expressed on decussated commissural axon segments projecting within the ventral funiculus, and on ipsilaterally projecting axons constituting the lateral funiculus. Soluble forms of all three B-class ephrins bind to, and induce the collapse of, commissural growth cones in vitro. The collapse-inducing activity associated with B-class ephrins is likely to be mediated by EphB1. Taken together, these data support a possible role for repulsive B-class Eph receptor/ligand interactions in constraining the orientation of longitudinal axon projections at the ventral midline.

scholarly journals MO069HIF-1Α C-TERMINAL TRANSCRIPTIONAL ACTIVATION DOMAIN MEDIATED KLF5 SIGNALING DRIVES AKI TO CKD PROGRESSION

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Zuolin Li ◽  
Jia-ling Ji ◽  
Linli Lv ◽  
Yan Yang ◽  
Tao-tao Tang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Ischemic Injury ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Mice Model ◽  
Ckd Progression ◽  
Transcriptional Activation Domain ◽  
Tubular Cells

Abstract Background and Aims Acute kidney injury (AKI) is increasingly recognized as a major risk factor for progression to CKD. However, the mechanisms governing AKI to CKD progression are poorly understood. Hypoxia is a key player in the pathophysiology of the AKI to CKD transition. Thus, we aimed to investigate the exact mechanisms of AKI to CKD progression mediated by hypoxia. Method Mild ischemic injury and severe ischemic injury (AKI-to-CKD transition) were established by clamping renal pedicle for 30 and 40 minutes, respectively. Meanwhile, the mice model of AKI-to-CKD transition was treated with HIF-1α inhibitor, PX-478. In vitro, PHD inhibition and combined PHD with FIH inhibition mimic the HIF-1α activation caused by mild or severe hypoxia, respectively. Besides the human proximal tubular epithelial cell line HK-2, tubular cells were isolated from mice for primary culture. KLF5 knockdown, FIH and HIF-1α C-terminal transcriptional activation domain (C-TAD) overexpression in tubular cells were achieved by Lentiviral transfection. Immunocoprecipitation was used to explore the relationship between the HIF-1α and FIH-1. Luciferase reporter assay was used to investigate whether KLF5 was regulated transcriptionally by HIF-1α C-TAD. To explore the roles of FIH-1 and HIF-1α C-TAD in vivo, FIH-1 and HIF-1α C-TAD overexpression (Lentivirus-mediated) was given after severe ischemic injury or mild ischemic injury via tail vein injection, respectively. Results AKI to CKD progression was highly associated with the time-course expression of tubular HIF-1α in severe ischemia/reperfusion injury. Interestingly, ameliorated AKI-to-CKD transition was observed by treating PX-478, which destabilized HIF-1α. In vitro, fibrogenesis could be induced by combined PHD with FIH inhibitor treatment in TEC. More interestingly, alleviated fibrogenesis could be achieved by knockdown of KLF5 and overexpression of FIH, respectively, while HIF-1α C-TAD overexpression promoted fibrogenesis in tubular cells. Immunocoprecipitation results indicated that HIF-1α and FIH-1 are interactive. Furthermore, we demonstrated that KLF5 could be regulated transcriptionally by HIF-1α C-TAD by luciferase reporter assay. In vivo, AKI to CKD progression was ameliorated significantly when mice model of AKI-to-CKD transition intervened with FIH-1 overexpression (Lentivirus-mediated). However, treatment of HIF-1α C-TAD (Lentivirus-mediated) in mild ischemic injury model could promote progression of CKD significantly. Conclusion FIH-1 mediated HIF-1α C-TAD activation was the key mechanism of AKI to CKD transition by transcriptionally regulating the KLF5 pathway in tubules. Blockade of FIH-1 mediated HIF-1α C-TAD in tubules may serve as a novel therapeutic approach to ameliorate AKI to CKD progression.

scholarly journals Modulation of FoxO1 Expression by miR-21 to Promote Growth of Pancreatic Ductal Adenocarcinoma

2015 ◽  
Vol 35 (1) ◽  
pp. 184-190 ◽  
Author(s):  
Weifeng Song ◽  
Qi Li ◽  
Lei Wang ◽  
Liwei Wang
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Underlying Mechanism ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Primary Tumors ◽  
Protein Levels ◽  
Infusion System

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal primary tumors in humans, with undetermined tumorigenesis. Although previous work by us, and by others, has clearly demonstrated an involvement of miR-21 in the growth of PDAC, the underlying mechanism has not been clarified. Methods: Here we analyzed the regulation of FoxO1 by miR-21 in vitro and in vivo, using luciferase-reporter assay and pancreatic intraductal infusion of antisense of miR-21, respectively. Results: We found that overexpression of miR-21 in PDAC cells decreased FoxO1 protein levels, whereas inhibition of miR-21 increased FoxO1 levels. Further, miR-21 bound to FoxO1 mRNA to prevent its translation through its 3'UTR. Moreover, administration of antisense of miR-21 through an intraductal infusion system significantly decreased miR-21 levels and increased FoxO1 levels in implanted PDAC, resulting in a significant decrease in PDAC growth. Conclusion: Taken together, our data highlight miR-21/FoxO1 axis as a novel therapeutic target for inhibiting the growth of PDAC.

Differentiation-independent retinoid induction of folate receptor type β, a potential tumor target in myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3529-3536 ◽  
Author(s):  
Hui Wang ◽  
Xuan Zheng ◽  
Frederick G. Behm ◽  
Manohar Ratnam
Keyword(s):  
Retinoic Acid ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Transforming Growth Factor ◽  
Retinoic Acid Receptor ◽  
Folate Receptor ◽  
Leukemia Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Construct

Abstract Folate receptor (FR) type β is expressed in the myelomonocytic lineage, predominantly during neutrophil maturation and in myeloid leukemias. FR-β expression was elevated up to 20-fold by all-trans retinoic acid (ATRA) in KG-1 myeloid leukemia cells in a dose-dependent and reversible manner in the absence of terminal differentiation or cell growth inhibition. ATRA also increased FR-β expression in vitro in myeloid leukemia cells from patient marrow. FR-β was not up-regulated in KG-1 cells treated with phorbol ester, dexamethasone, 1,25-dihydroxy vitamin D3, or transforming growth factor β. ATRA did not induce FR-β expression in receptor negative cells of diverse origin. The ATRA-induced increase in FR-β expression in KG-1 cells occurred at the level of messenger RNA synthesis, and in 293 cells containing a stably integrated FR-β promoter–luciferase reporter construct, ATRA induced expression of the reporter. From experiments using retinoid agonists and antagonists and from cotransfection studies using the FR-β promoter and expression plasmids for the nuclear receptors retinoic acid receptor (RAR)α, RARβ, or RARγ, it appears that the retinoid effect on FR-β expression could be mediated by ligand binding to RARs α, β, or γ, but not to retinoid X receptors. Furthermore, there was apparent cross-talk between RARα and RARγ selective agonists or antagonists, suggesting a common downstream target for RAR isoforms in inducing FR-β expression. Thus, blocks in the RARα-specific pathway of retinoid-induced differentiation may be bypassed during retinoid induction of FR-β expression. The results suggest that to facilitate FR-targeted therapies, retinoids may be used to modulate FR-β expression in myeloid leukemia cells refractory to retinoid differentiation therapy.

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