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1.
Mol Cell Neurosci ; 129: 103932, 2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38679247

ABSTRACT

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are widely expressed by many types of retinal neurons and are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin and FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases acting through retinal neurons and MG "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

2.
Glia ; 71(7): 1729-1754, 2023 07.
Article in English | MEDLINE | ID: mdl-36971459

ABSTRACT

Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein, we investigate the expression and functions of S-adenosylhomocysteine hydrolase (SAHH; AHCY) and histone methyltransferases (HMTs) during the formation of Müller glia (MG)-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1 and AHCYL2, and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to convey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the differentiation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.


Subject(s)
Chromatin , Signal Transduction , Animals , Mice , Signal Transduction/physiology , Chromatin/metabolism , Stem Cells/metabolism , Ependymoglial Cells/metabolism , Retina , Neuroglia/metabolism , Chickens/genetics , Transcription Factors/metabolism , Cell Proliferation/physiology
3.
bioRxiv ; 2023 Dec 12.
Article in English | MEDLINE | ID: mdl-38168320

ABSTRACT

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin in FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

4.
J Comp Neurol ; 530(8): 1213-1230, 2022 06.
Article in English | MEDLINE | ID: mdl-34729776

ABSTRACT

The regenerative potential of Müller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Müller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina. Accordingly, we sought to characterize the patterns of expression of NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single-cell RNA-sequencing (scRNA-seq) and immunolabeling, we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), upregulated in E8 RPCs, further upregulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further downregulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.


Subject(s)
NFI Transcription Factors , Signal Transduction , Animals , Cell Proliferation/physiology , Mammals , Mice , NFI Transcription Factors/metabolism , Neuroglia/metabolism , Neurons/metabolism , Primates/metabolism , Retina , Signal Transduction/physiology , Stem Cells , Swine
5.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: mdl-33830943

ABSTRACT

A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.


Subject(s)
Cell-Derived Microparticles/immunology , Immune Tolerance/radiation effects , Keratinocytes/immunology , Ultraviolet Rays , Animals , Cell Line , Cell-Derived Microparticles/genetics , Female , Humans , Mice , Mice, Knockout , Platelet Activating Factor/genetics , Platelet Activating Factor/immunology , Platelet Membrane Glycoproteins/genetics , Platelet Membrane Glycoproteins/immunology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/immunology , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/immunology
6.
J Immunol ; 205(1): 193-201, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32434939

ABSTRACT

Thermal burn injuries are an important environmental stressor that can result in considerable morbidity and mortality. The exact mechanism by which an environmental stimulus to skin results in local and systemic effects is an area of active research. One potential mechanism to allow skin keratinocytes to disperse bioactive substances is via microvesicle particles, which are subcellular bodies released directly from cellular membranes. Our previous studies have indicated that thermal burn injury of the skin keratinocyte in vitro results in the production of the lipid mediator platelet-activating factor (PAF). The present studies demonstrate that thermal burn injury to keratinocytes in vitro and human skin explants ex vivo, and mice in vivo generate microvesicle particles. Use of pharmacologic and genetic tools indicates that the optimal release of microvesicles is dependent upon the PAF receptor. Of note, burn injury-stimulated microvesicle particles do not carry appreciable protein cytokines yet contain high levels of PAF. These studies describe a novel mechanism involving microvesicle particles by which a metabolically labile bioactive lipid can travel from cells in response to environmental stimuli.


Subject(s)
Burns/immunology , Cell-Derived Microparticles/immunology , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Skin/pathology , Animals , Biopsy , Burns/pathology , Cell Line , Cell-Derived Microparticles/metabolism , Disease Models, Animal , Female , Humans , Keratinocytes/immunology , Keratinocytes/metabolism , Lipid Metabolism/immunology , Mice , Mice, Knockout , Platelet Membrane Glycoproteins/genetics , Primary Cell Culture , Receptors, G-Protein-Coupled/genetics , Skin/immunology
7.
Genesis ; 58(3-4): e23354, 2020 03.
Article in English | MEDLINE | ID: mdl-31909537

ABSTRACT

To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR-dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc-tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3'UTRs identified miR-199a-3p ("miR-199") as a candidate regulator of EFTFs, and miR-199 was shown to regulate rax1 in vivo. Targeted overexpression of miR-199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir-199 and the rax1 3'UTR reversed the small eye phenotype. Although targeted knockdown of miR-199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR-199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR-199 knockdown. Our results indicate that miR-199 plays both positive and negative regulatory roles in eye development.


Subject(s)
Eye/embryology , Eye/metabolism , Gene Expression Regulation, Developmental , MicroRNAs/genetics , RNA Interference , Xenopus laevis/embryology , Xenopus laevis/genetics , Animals , Eye Proteins/genetics , Eye Proteins/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , Genetic Association Studies , Loss of Function Mutation , Organogenesis/genetics , Phenotype , Protein Binding , Receptor Protein-Tyrosine Kinases/genetics , Reproducibility of Results , Transcription Factors/genetics , Transcription Factors/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolism
8.
Int J Mol Sci ; 20(1)2018 Dec 21.
Article in English | MEDLINE | ID: mdl-30577630

ABSTRACT

Studies, including ours, have shown that pro-oxidative stressors, such as chemotherapeutic agents, generate oxidized lipids with agonistic platelet-activating factor (PAF) activity. Importantly, recent reports have implicated that these PAF-agonists are transported extracellularly via microvesicle particles (MVPs). While the role of PAF-receptor (PAF-R) has been implicated in mediating chemotherapy effects, its significance in chemotherapy-mediated MVP release in pancreatic cancer has not been studied. The current studies determined the functional significance of PAF-R in gemcitabine chemotherapy-mediated MVP release in human pancreatic cancer cells. Using PAF-R-expressing (PANC-1) and PAF-R-deficient (Hs766T) cells, we demonstrate that gemcitabine induces MVP release in a PAF-R-dependent manner. Blocking of PAF-R via PAF-R antagonist or inhibition of MVP generation via inhibitor of acid sphingomyelinase (aSMase) enzyme, significantly attenuated gemcitabine-mediated MVP release from PANC-1 cells, however, exerted no effects in Hs766T cells. Notably, MVPs from gemcitabine-treated PANC-1 cells, contained a measurable amount of PAF-agonists. Mechanistically, pretreatment with ERK1/2 or p38 inhibitors significantly abrogated gemcitabine-mediated MVP release, indicating the involvement of mitogen-activated protein kinase (MAPK) pathway in PAF-R-dependent gemcitabine-mediated MVP release. These findings demonstrate the significance of PAF-R in gemcitabine-mediated MVP release, as well as the rationale of evaluating PAF-R targeting agents with gemcitabine against pancreatic cancer.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cell-Derived Microparticles/metabolism , Deoxycytidine/analogs & derivatives , MAP Kinase Signaling System/drug effects , Pancreatic Neoplasms/metabolism , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Cell Line, Tumor , Deoxycytidine/pharmacology , Humans , Platelet Membrane Glycoproteins/agonists , Platelet Membrane Glycoproteins/antagonists & inhibitors , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/metabolism , Gemcitabine
9.
Dev Dyn ; 247(11): 1199-1210, 2018 11.
Article in English | MEDLINE | ID: mdl-30311321

ABSTRACT

BACKGROUND: The retinal homeobox (rx/rax) gene is a transcription factor expressed in the developing eye field that is necessary for normal eye development. rax is necessary for retinal specification and stem cell development. The genetic program of early retinal development, including rax expression, can be induced in naïve ectoderm by activation of insulin-like growth factor (IGF) signaling. We have undertaken a microarray-based approach to identify rax-dependent IGF-induced genes. RESULTS: We identified 21 IGF-induced genes that exhibit at least a two-fold decrease in expression when rax expression is knocked down. Ten of these genes were expressed in the developing eye, eight were expressed in the ciliary marginal zone of the mature tadpole retina, and four could significantly rescue the rax knockdown phenotype. One of these, the nei endonuclease VIII-like 3 (neil3) gene, rescued the rax knockdown phenotype to a remarkable degree. We found that neil3 is necessary for normal retinal lamination and retinal neuron differentiation. CONCLUSIONS: We have identified neil3 as a component of the rax genetic pathway necessary for normal retinal progenitor cell development. neil3 is involved in the base excision DNA repair pathway, suggesting that this pathway is essential for normal rax-dependent progenitor cell development in the mature retina. Developmental Dynamics 247:1199-1210, 2018. © 2018 Wiley Periodicals, Inc.


Subject(s)
Eye Proteins/genetics , N-Glycosyl Hydrolases/genetics , Xenopus Proteins/genetics , Animals , Eye Proteins/analysis , Homeodomain Proteins/genetics , Larva/growth & development , Protein Array Analysis , Retina/chemistry , Retina/cytology , Stem Cells , Xenopus Proteins/analysis , Xenopus laevis/embryology
10.
Methods Mol Biol ; 1797: 325-335, 2018.
Article in English | MEDLINE | ID: mdl-29896701

ABSTRACT

In situ hybridization performed using whole fixed embryos provides accurate and detailed visualization of gene expression patterns. These patterns are useful for investigating spatial patterns of gene expression in normally developing embryos but can also be useful in investigating the effects of genetic or environmental changes on expression of genetic markers characteristic of particular tissues, organs, or genetic pathways. Our lab's protocol for whole-mount in situ hybridization is presented.


Subject(s)
Gene Expression Regulation, Developmental , In Situ Hybridization , Transcriptome , Xenopus laevis/embryology , Xenopus laevis/genetics , Animals , Embryo, Nonmammalian , Gene Expression Profiling/methods , In Situ Hybridization/methods , Phenotype
11.
J Invest Dermatol ; 138(11): 2461-2469, 2018 11.
Article in English | MEDLINE | ID: mdl-29857067

ABSTRACT

Thermal burn injuries in patients who are alcohol-intoxicated result in greater morbidity and mortality. Murine models combining ethanol and localized thermal burn injury reproduce the systemic toxicity seen in human subjects, which consists of both acute systemic cytokine production with multiple organ dysfunction, as well as a delayed systemic immunosuppression. However, the exact mechanisms for these acute and delayed effects are unclear. These studies sought to define the role of the lipid mediator platelet-activating factor in the acute and delayed effects of intoxicated burn injury. Combining ethanol and thermal burn injury resulted in increased enzymatic platelet-activating factor generation in a keratinocyte cell line in vitro, human skin explants ex vivo, as well as in murine skin in vivo. Further, the acute increase in inflammatory cytokines, such as IL-6, and the systemic immunosuppressive effects of intoxicated thermal burn injury were suppressed in mice lacking platelet-activating factor receptors. Together, these studies provide a potential mechanism and treatment strategies for the augmented toxicity and immunosuppressive effects of thermal burn injury in the setting of acute ethanol exposure, which involves the pleotropic lipid mediator platelet-activating factor.


Subject(s)
Burns/immunology , Ethanol/metabolism , Keratinocytes/physiology , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/genetics , Receptors, G-Protein-Coupled/genetics , Acute Disease , Alcoholic Intoxication , Animals , Cell Line , Cytokines/metabolism , Female , Hot Temperature , Humans , Inflammation Mediators/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Up-Regulation
12.
Mol Vis ; 22: 1421-1428, 2016.
Article in English | MEDLINE | ID: mdl-28003732

ABSTRACT

PURPOSE: The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. METHODS: X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. RESULTS: We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. CONCLUSIONS: vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system.


Subject(s)
Eye Proteins/genetics , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/genetics , Photoreceptor Cells/metabolism , Regulatory Elements, Transcriptional/genetics , Transcription Factors/genetics , Xenopus Proteins/genetics , Animals , Electrophoretic Mobility Shift Assay , Gene Amplification , Genes, Homeobox , In Situ Hybridization , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Transcription, Genetic , Xenopus laevis
13.
Genesis ; 54(11): 562-567, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27696680

ABSTRACT

The Retinal homeobox (rax) gene is expressed in vertebrate retinal progenitor and stem cells and is essential for retinal development. In frogs, rax is expressed in the ciliary marginal zone (CMZ), a region containing retinal progenitor and stem cells at the anterior of the eye. Little is known regarding regulation of rax transcription and regulation of transcription of rax targets. We found that three ultra-conserved genomic elements (UCEs) flanking the rax coding region regulate expression of a rax promoter-GFP transgene in Xenopus tadpoles. One of these elements, UCE1, regulates expression of the transgene in the dorsal CMZ. UCE1 contains a Rax binding site, PCE-1. We demonstrate that rax regulates expression of the transgene through the PCE-1 site found in UCE1. Therefore, rax transcription in the CMZ is controlled, in part, by autoregulatory mechanisms.


Subject(s)
Eye Proteins/genetics , Regulatory Sequences, Nucleic Acid/genetics , Retina/growth & development , Xenopus Proteins/genetics , Xenopus/genetics , Animals , Binding Sites , Conserved Sequence/genetics , Eye Proteins/biosynthesis , Eye Proteins/metabolism , Gene Expression Regulation, Developmental , Larva/genetics , Larva/growth & development , Promoter Regions, Genetic , Retina/metabolism , Xenopus/growth & development , Xenopus Proteins/biosynthesis , Xenopus Proteins/metabolism
14.
Dev Genes Evol ; 226(6): 423-428, 2016 11.
Article in English | MEDLINE | ID: mdl-27392729

ABSTRACT

Nkx5 family members are homeobox transcription factors important for sensory organ development. Several members of the Nkx5 family are expressed in the eye, brain, developing ear, and lateral line. Members of this family have been previously identified in medaka, chick, and mouse. Here, we characterize two members of the Nkx5 family, Nkx5.3 and SOHo, in Xenopus laevis. We verify the identity of X. laevis Nkx5.3 and SOHo by phylogenetic comparison to chicken, medaka, and zebrafish orthologs. Both Nkx5.3 and SOHo are expressed in the developing eye, ear, lateral line system, and cranial neurons as determined by in situ hybridization.


Subject(s)
Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Sense Organs/growth & development , Transcription Factors/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/growth & development , Amino Acid Sequence , Animals , Ganglia/metabolism , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , In Situ Hybridization , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Phylogeny , Sense Organs/metabolism , Sequence Alignment , Transcription Factors/chemistry , Transcription Factors/genetics , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus laevis/anatomy & histology , Xenopus laevis/metabolism
15.
Methods Mol Biol ; 884: 41-51, 2012.
Article in English | MEDLINE | ID: mdl-22688697

ABSTRACT

Analysis of transcription factor-target interactions in vivo is important to the study of transcriptional regulation of gene expression. A key experiment involves analysis of the functional interaction between a trans-acting factor and its corresponding cis-acting element in the context of a target promoter in vivo. We describe a method for this analysis in transgenic Xenopus tadpoles in which expression of the trans-acting factor is knocked down using an shRNA-mediated approach.


Subject(s)
Animals, Genetically Modified , Response Elements , Transcription Factors/genetics , Transcription Factors/metabolism , Xenopus laevis/genetics , Animals , Cell Nucleus , Female , Gene Expression Regulation , Gene Knockdown Techniques , Gene Targeting , Male , Nuclear Transfer Techniques/instrumentation , RNA, Small Interfering/metabolism , Spermatozoa/cytology , Trans-Activators/metabolism , Xenopus laevis/metabolism
16.
Dev Biol ; 353(1): 10-8, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21334323

ABSTRACT

The Retinal Homeobox (Rx) gene is essential for vertebrate eye development. Rx function is required for the specification and maintenance of retinal progenitor cells (RPCs). Loss of Rx function leads to a lack of eye development in a variety of species. Here we show that Rx function is also necessary during retinal regeneration. We performed a thorough characterization of retinal regeneration after partial retinal resection in pre-metamorphic Xenopus laevis. We show that after injury the wound is repopulated with retinal progenitor cells (RPCs) that express Rx and other RPC marker genes. We used an shRNA-based approach to specifically silence Rx expression in vivo in tadpoles. We found that loss of Rx function results in impaired retinal regeneration, including defects in the cells that repopulate the wound and the RPE at the wound site. We show that the regeneration defects can be rescued by provision of exogenous Rx. These results demonstrate for the first time that Rx, in addition to being essential during retinal development, also functions during retinal regeneration.


Subject(s)
Drosophila Proteins/metabolism , Homeodomain Proteins/metabolism , Regeneration , Retina/physiology , Transcription Factors/metabolism , Xenopus laevis/embryology , Animals , Drosophila Proteins/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Larva/physiology , Retina/cytology , Stem Cells/physiology , Transcription Factors/genetics , Transgenes
17.
Gene ; 467(1-2): 13-24, 2010 Nov 01.
Article in English | MEDLINE | ID: mdl-20627122

ABSTRACT

The retinal homeobox (Rx/rax) gene is essential for the development of the eye. Rax is among the earliest genes expressed during eye development, beginning in the prospective eye fields in the anterior neural plate. Additionally Rax expression persists in retinal progenitor cells and in differentiated photoreceptors. We have isolated and characterized a 2.8 kb genomic DNA fragment that regulates expression of Rax in the developing and maturing retina. We have discovered and characterized cis-acting elements that function to specifically control spatial and temporal Rax expression during retinal development. We have found that the regulation of Rax2A promoter activity requires cooperative interactions between positive and negative regulatory elements. Further, a highly conserved genomic element containing SOX, OTX, and POU transcription factor binding sites is necessary but not sufficient for promoter activity in retinal progenitor or stem cells. Finally, a putative binding element for forkhead transcription factors is necessary for promoter activity and can cooperate with other cis-acting elements to drive Rax2A promoter activity.


Subject(s)
Eye Proteins/genetics , Gene Expression Regulation, Developmental , Genes, Homeobox , Retina/embryology , Xenopus Proteins/genetics , Xenopus laevis/embryology , Xenopus laevis/genetics , Animals , Base Sequence , Binding Sites , Conserved Sequence , Female , Male , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Deletion , Time Factors , Transcription, Genetic
18.
Dev Biol ; 339(2): 494-506, 2010 Mar 15.
Article in English | MEDLINE | ID: mdl-20060393

ABSTRACT

The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type.


Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Photoreceptor Cells/metabolism , Rhodopsin/genetics , Rod Opsins/genetics , Xenopus Proteins/genetics , Animals , Embryo, Nonmammalian/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Homeodomain Proteins/genetics , Retinaldehyde , Rhodopsin/metabolism , Rod Opsins/metabolism , Xenopus Proteins/metabolism , Xenopus laevis
19.
Int J Dev Biol ; 53(1): 29-36, 2009.
Article in English | MEDLINE | ID: mdl-19123124

ABSTRACT

Forkhead transcription factors comprise a large family of proteins with diverse functions during development. Recently, there has been accumulating evidence that several members of this family of proteins play an important role in the development of the vertebrate retina. Here, we summarize the cumulative data which demonstrates the integral role that forkhead factors play in cell cycle control of retinal precursors, as well as in cell fate determination, during retinal development. The expression patterns for 14 retinal expressed forkhead transcription factors are presented with an emphasis on comparing the expression profiles across species. The functional data regarding forkhead gene products expressed within the retina are discussed. As presented, these data suggest that forkhead gene products contribute to the complex regulation of proliferation and differentiation of retinal precursors during vertebrate eye development.


Subject(s)
Eye/metabolism , Forkhead Transcription Factors/metabolism , Animals , Eye/embryology , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Developmental , Humans , Organ Specificity , Stem Cells/metabolism
20.
Genesis ; 47(1): 19-31, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19006070

ABSTRACT

The aristaless-related gene, Arx, plays a fundamental role in patterning the brain in humans and mice. Arx mutants exhibit lissencephaly among other anomalies. We have cloned a Xenopus aristaless homolog that appears to define specific regions of the developing forebrain. xArx2 is transcribed in blastula through neurula stages, and comes to be restricted to the ventral and lateral telencephalon, lateral diencephalon, neural floor plate of the anterior spinal cord, and somites. In this respect, Arx2 expresses in regions similar to Arx with the exception of the somites. Overexpression enlarges the telencephalon, and interference by means of antisense morpholino-mediated translation knockdown reduces growth of this area. Overexpression and inhibition studies demonstrate that misregulation of xArx2 imposes dire consequences upon patterns of differentiation not only in the forebrain where the gene normally expresses, but also in more caudal brain territories and derivatives as well. This suggests that evolutionary changes that expanded Arx-expression from ventral to dorsal prosencephalon might be one of the determinants that marked development and expansion of the telencephalon.


Subject(s)
Brain/embryology , Brain/metabolism , Transcription Factors/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Xenopus laevis/metabolism , Amino Acid Sequence , Animals , Brain/growth & development , Conserved Sequence , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Molecular Sequence Data , Phylogeny , Protein Biosynthesis/genetics , RNA, Messenger/genetics , Sequence Alignment , Transcription Factors/chemistry , Transcription Factors/classification , Transcription Factors/genetics , Xenopus Proteins/chemistry , Xenopus Proteins/classification , Xenopus Proteins/genetics , Xenopus laevis/genetics , Xenopus laevis/growth & development
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