Citation: P :
Type: Journal_article
Literature: xenopus
Accession (PMID): 25715760

" White pigment cells " are derived from melanophore precursors and contain both melanophore-specific and iridophore-specific pigment organelles . Whereas melanophores differentiate in the wild type regenerating tail , white pigment cells appear in the regenerating tail in the periodic albino mutant ( a ( p ) / a ( p ) ) of Xenopus laevis . The localization and density of white pigment cells in the mutant regenerating tail are similar to those of melanophores in the wild type regenerating tail . Here , white pigment cells in the mutant regenerating tail have been compared with melanophores in the wild type regenerating tail in the presence of phenylthiourea ( PTU ) , which inhibits melanosome maturation in melanophores but does not affect reflecting platelet formation in white pigment cells . Ultrastructural analysis shows that reflecting platelet formation in white pigment cells is different from that in iridophores . Reflecting platelets in iridophores are formed from spherical vesicles with electron-dense material , whereas they are formed from stage II melanosomes characteristic of melanophore precursors in white pigment cells . Ultrastructural features of pigment organelles , except reflecting platelets , are similar between mutant melanophores and white pigment cells . In an attempt to identify specific genes in white pigment cells , a subtracted cDNA library enriched for mutant cDNAs has been prepared . Subtracted cDNA fragments have been cloned and selected by whole mount in situ hybridization . Among cDNA fragments examined so far , the ferritin H subunit gene is specifically expressed in white pigment cells , but not in melanophores . Pigment organellogenesis and specific gene expression in white pigment cells are also discussed .

SECTION: abstract. Here , white pigment cells in the mutant regenerating tail have been compared with melanophores in the wild type regenerating tail in the presence of phenylthiourea ( PTU ) , which inhibits melanosome maturation in melanophores but does not affect reflecting platelet formation in white pigment cells . [Field: abstract, subscore: 1.00]
SECTION: introduction. Using a tail-regenerating system , we have compared white pigment cells that differentiate in the mutant regenerating tail with differentiated melanophores in the wild type regenerating tail in the presence of phenylthiourea ( PTU ) , an inhibitor of melanogenesis ( Gross et al . 2002 ; Sims 1962 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. Here , white pigment cells in the mutant regenerating tail have been compared with melanophores in the wild type regenerating tail in the presence of phenylthiourea ( PTU ) , which inhibits melanosome maturation in melanophores but does not affect reflecting platelet formation in white pigment cells . [Field: introduction, subscore: 1.00]
SECTION: references. Development 127 : 3031 3044 Pietzsch-Rohrschneider I ( 1977 ) The influence of phenylthiourea ( PTU ) on the pigment epithelium and the photoreceptor cells in the developing retina of Haplochromis burtoni ( Cichlidae , Teleostei ) . [Field: references, subscore: 1.00]
SECTION: results. Bar1 m Table 1 Effect of phenylthiourea ( PTU ) on melanosome genesis in wild type melanophores Treatment Stage I + IIa / total Stage IIIa / total Stage IVa / total Controlb 0 % 0 % 100 % PTUc 28 . 1 % * 69 . 8 % * 2 . 1 % * * P < 0 . 001 ( 2 test ) aPercentage of melanosomes at each stage among total melanosomes . [Field: results, subscore: 1.00]
SECTION: results. Fig . 1 Expression of pigment cells in the 6-day regenerating tail in the absence ( a , c ) or presence ( b , d ) of phenylthiourea ( PTU ) ; amputated at stage 50 . a , b Wild type regenerating tail observed under transmitted light . c , d Mutant regenerating tail observed under incident light . [Field: results, subscore: 1.00]

Citation: V : 7 P : 56
Type: Journal_article
Literature: xenopus
Accession (PMID): 17521450

BACKGROUND : After amputation of the Xenopus tadpole tail , a functionally competent new tail is regenerated . It contains spinal cord , notochord and muscle , each of which has previously been shown to derive from the corresponding it issue in the stump . The regeneration of the neural crest derivatives has not previously been examined and is described in this paper . RESULTS : Labelling of the spinal cord by electroporation , or by orthotopic grafting of transgenic it issue expressing GFP , shows that no cells emigrate from the spinal cord in the course of regeneration . There is very limited regeneration of the spinal ganglia , but new neurons as well as fibre tracts do appear in the regenerated spinal cord and the regenerated tail also contains abundant peripheral innervation . The regenerated tail contains a normal density of melanophores . Cell labelling experiments show that melanophores do not arise from the spinal cord during regeneration , nor from the mesenchymal it issues of the skin , but they do arise by activation and proliferation of pre-existing melanophore precursors . If tails are prepared lacking melanophores , then the regenerates also lack them . CONCLUSION : On regeneration there is no induction of a new neural crest similar to that seen in embryonic development . However there is some regeneration of neural crest derivatives . Abundant melanophores are regenerated from unpigmented precursors , and , although spinal ganglia are not regenerated , sufficient sensory systems are produced to enable essential functions to continue .

SECTION: materials. Tail regeneration in Phenylthiourea ( PTU ) treated tadpoles Figure 9 Tail regeneration in Phenylthiourea ( PTU ) treated tadpoles . [Field: materials, subscore: 2.00]
SECTION: materials. To inhibit melanin synthesis 0 . 10 . 2 mM phenylthiourea ( PTU ) was used . [Field: materials, subscore: 1.00]
SECTION: results. To distinguish whether the regenerated melanophores come from pre-existing pigmented melanophores or from un-pigmented melanophore precursors , we used the tyrosinase inhibitor phenylthiourea ( PTU ) to block melanin synthesis [ 30 ] . [Field: results, subscore: 1.00]

Citation: V : 146 I : 6 P : 1265-76
Type: Journal_article
Literature: xenopus
Accession (PMID): 10491390

Previously , we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner , and that myosin V is bound to purified melanosomes ( Rogers , S . , and V . I.Gelfand . 1998 . Curr . Biol . 8 : 161-164 ) . In the present study , we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport . To examine whether myosin V was regulated in a cell cycle-dependent manner , purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments . Motility of organelles treated with mitotic extract was found to decrease dramatically , as compared with untreated or interphase extract-treated melanosomes . This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes , but the activity of soluble motor remained unaffected . Furthermore , we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts . We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles , and that this binding is likely regulated by phosphorylation of myosin V during mitosis .

SECTION: materials. Cultures isolated in this manner were grown in standard growth medium , supplemented with 1 mM phenylthiourea . [Field: materials, subscore: 1.00]
SECTION: materials. Further melanin production was inhibited by treating the cells for 2 wk with 1 mM phenylthiourea . [Field: materials, subscore: 1.00]
SECTION: results. Any residual melanin was eliminated by culturing the cells in 1 mM phenylthiourea , a potent tyrosinase inhibitor , for two weeks . [Field: results, subscore: 1.00]

Citation: V : 156 I : 5 P : 855-65
Type: Journal_article
Literature: xenopus
Accession (PMID): 11864991

Many cellular components are transported using a combination of the actin- and microtubule-based transport systems . However , how these two systems work together to allow well-regulated transport is not clearly understood . We investigate this question in the Xenopus melanophore model system , where three motors , kinesin II , cytoplasmic dynein , and myosin V , drive aggregation or dispersion of pigment organelles called melanosomes . During dispersion , myosin V functions as a " molecular ratchet " to increase outward transport by selectively terminating dynein-driven minus end runs . We show that there is a continual tug-of-war between the actin and microtubule transport systems , but the microtubule motors kinesin II and dynein are likely coordinated . Finally , we find that the transition from dispersion to aggregation increases dynein-mediated motion , decreases myosin V--mediated motion , and does not change kinesin II--dependent motion . Down-regulation of myosin V contributes to aggregation by impairing its ability to effectively compete with movement along microtubules .

SECTION: introduction. * Abbreviations used in this paper : MSH , melanocyte-stimulating hormone ; PTU , phenylthiourea . [Field: introduction, subscore: 1.00]
SECTION: results. To solve this problem , we reduced the number of pigment granules by treating cells with the tyrosinase inhibitor phenylthiourea ( PTU ) . [Field: results, subscore: 1.00]
SECTION: results. Phenylthiourea treatment allows quantitative characterization of individual melanosome motion To understand the role of individual motors in organelle transport and how transport is regulated during aggregation versus dispersion , we combined tracking of individual melanosomes with quantitative analysis of movement . [Field: results, subscore: 1.00]

Citation: V : 153 I : 1-3 P : 13-4
Type: Journal_article
Literature: xenopus
Accession (PMID): 17666201

SECTION: introduction. Propylthiouracil , phenylthiourea , and thiourea inhibit thyroperoxidase and 5 -deiodinase . [Field: introduction, subscore: 1.00]
SECTION: introduction. One of these model Table 2 Summary of lowest observed effect concentrations ( mg / L ) of thyroid-disrupting compounds in zebrafish , fathead minnow and stickleback Compound Species Hormone levels , molecular effects Histology Teratogenicity Toxicity Perchlorate ZF 0.011 ; colloidal follicular structure , angiogenesis ZF 6 ; follicle structure , angiogenesis ZF 10 ; T4 reduced in females and embryos 10 ; thyroidal hypertrophy , jar malformation 10 ; reduced weight in female ZF 10 collidal area , eipthelial cell height ZF 250 ; T4 reduced 250 ( 2500 ) ; delayed hatch ( retarded , less motile ) 25000 ; cell death FM 1 ; thyroid hyperblasia 10 ; retardation , reduced pigmentation SB a 30 ( 100 ) ; male nuptial coloration ( intersexual gonads ) 30 ; reduced survival Propylthiouracil ZF 10 ; T3 , T4 decreased 1 ; thyroid follicle synthetic activity 100 ; body weight F1 generation 100 ; reduced body weight ZF 100 ; T4 reduced 100 ; retarded , curved body 1000 ; reduced survival Phenylthiourea ZF 30 ; T4 reduced 30 ( 300 ) ; no pigmentation ( retarded growth , curved body ) 300 ; reduced survival Thiourea ZF 300 ; T3 , T4 decreased Methimazole ZF 117 ; T4 reduced 117 ( 234 ) ; delayed hatch ( retarded.less motile , curved body shape ) 1170 ; reduced survival ZF 34 mg / L ; various morphometric parameters reduced 34 , 000 ; reduced length ZF 34 ; RagI , IgL-C-expression , 34 ; reduced length FM 0.032 ; T3 ( 28 days inhibition , 56 induction ) 0.032 ; delayed hatching ( not at 0.32 mg / L ) Arsenite ZF 10 ; Colloid area and height ( not in all concentrations and time points ) Thyroxine ( T4 ) ZF 0.008 ; TR , TR gene expression increased 0.023 ; deflation of swim bladder 0.023 ; reduced length ZF 23.3 ; RagI-expression 23.3 ; Thymus structure Perchlorate is an inhibitor of thyroidal iodine uptake . [Field: introduction, subscore: 1.00]
SECTION: references. Phenylthiourea disrupts thyroid function in developing zebrafish . [Field: references, subscore: 1.00]

Citation: V : 90 I : 1 P : L07-9
Type: Journal_article
Literature: xenopus
Accession (PMID): 16284273

We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm . The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm , in agreement with the step size of myosin-V determined in vitro . In contrast , trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps . The step duration was in the range 20-80 ms , slower than what it would be expected from in vitro results . We speculate that the cytoplasm high viscosity may affect significantly the melanosomes ' ' motion .

SECTION: materials. Cells grown in the presence of phenylthiourea were incubated at 0C for 30 min with 10 mM nocodazole . [Field: materials, subscore: 1.00]
SECTION: materials. The number of melanosomes in the cell was reduced by treatment with phenylthiourea ( 14 ) to track the movement of individual organelles . [Field: materials, subscore: 1.00]

Citation: V : 8 I : 5 P : e62846
Type: Journal_article
Literature: xenopus
Accession (PMID): 23667529

The zebrafish ( Danio rerio ) has become one of the major animal models for in vivo examination of sensory and neuronal computation . Similar to Xenopus tadpoles neural activity in the optic tectum , the major region controlling visually guided behavior , can be examined in zebrafish larvae by optical imaging . Prerequisites of these approaches are usually the transparency of larvae up to a certain age and the use of two-photon microscopy . This principle of fluorescence excitation was necessary to suppress crosstalk between signals from individual neurons , which is a critical issue when using membrane-permeant dyes . This makes the equipment to study neuronal processing costly and limits the approach to the study of larvae . Thus there is lack of knowledge about the properties of neurons in the optic tectum of adult animals . We established a procedure to circumvent these problems , enabling in vivo calcium imaging in the optic tectum of adult zebrafish . Following local application of dextran-coupled dyes single-neuron activity of adult zebrafish can be monitored with conventional widefield microscopy , because dye labeling remains restricted to tens of neurons or less . Among the neurons characterized with our technique we found neurons that were selective for a certain pattern orientation as well as neurons that responded in a direction-selective way to visual motion . These findings are consistent with previous studies and indicate that the functional integrity of neuronal circuits in the optic tectum of adult zebrafish is preserved with our staining technique . Overall , our protocol for in vivo calcium imaging provides a useful approach to monitor visual responses of individual neurons in the optic tectum of adult zebrafish even when only widefield microscopy is available . This approach will help to obtain valuable insight into the principles of visual computation in adult vertebrates and thus complement previous work on developing visual circuits .

SECTION: introduction. This problem can , in principle , be avoided by using mutations with pigmentation defects [ 12 , 13 ] or by treatment with the melanin synthesis inhibitor phenylthiourea [ 14 ] . [Field: introduction, subscore: 1.00]
SECTION: references. Li Z , Ptak D , Zhang L , Walls EK , Zhong W , et al . ( 2012 ) Phenylthiourea specifically reduces zebrafish eye size . [Field: references, subscore: 1.00]

Citation: V : 43 I : 10 P : 916-23
Type: Journal_article
Literature: xenopus
Accession (PMID): 23911547

The Southern house mosquito , Culex quinquefasciatus--a vector of West Nile virus--is equipped with 130 odorant receptors ( ORs ) , which enable young females to locate plants and blood-meal sources and older females to find suitable sites for oviposition . In our attempts to de-orphanize ORs expressed in female antennae , we identified CquiOR37 and CquiOR99 , which were narrowly tuned to two phenolic compounds , 4-methylphenol and 4-ethylphenol . When tested in the Xenopus oocyte recording system the observed EC50s for 4-methylphenol and 4-ethylphenol were 6 . 4 and 18 . 2 M for CquiOR37 and 14 . 4 and 0 . 74 M for CquiOR99 ( goodness of fit , R = 0 . 88-0 . 99 ) , respectively . Indoor behavioral assays demonstrated that gravid female mosquitoes laid significantly more eggs in water trays spiked with these compounds than in control water trays . Field studies with gravid traps corroborated that 4-ethylphenol is active in a wide range of doses from 0 . 1 to 10 g / l , as required for practical applications . A dsRNA construct based on the two genes , CquiOR37 / 99-dsRNA was stable in pupa hemolymph for up to 3 h . Pupae injected with CquiOR37 / 99-dsRNA , -galactosidase-dsRNA or water had more than 40 % survival rate at the peak of oviposition ( day-9 ) . qPCR analysis showed individual variation , but significant mean reduction in CquiOR37 and CquiOR99 transcript levels in CquiOR37 / 99-dsRNA-treated mosquitoes . Water-injected females and those treated with the control gene laid significantly more eggs in trays containing 4-ethylphenol than in water trays , whereas CquiOR37 / 99-dsRNA-treated mosquitoes laid normal number of eggs , but could not discriminate treatment from control . This study linked for the first time specific receptors for 4-ethylphenol with increased oviposition in the important vector Cx . quinquefasciatus .

SECTION: acknowledgments. Phenylthiourea , an effective inhibitor of the insect hemolymph melanization reaction , interferes with the detection of lipoprotein hydroper- oxide . [Field: acknowledgments, subscore: 1.00]
SECTION: materials. Ex vivo determination of dsRNA stability Hemolymph was collected from day-0 pupae , combined in chilled 1 . 5 ml microcentrifuge tube containing phenylthiourea ( PTU ) to prevent melanization ( Arakawa , 1995 ) . [Field: materials, subscore: 1.00]

Citation: V : 13 I : 22 P : 2983-95
Type: Journal_article
Literature: xenopus
Accession (PMID): 10580005

The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study . Here we present evidence from both loss- and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development . We demonstrate that zebrafish Gata5 is encoded by the faust locus . Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors and the expression of normal levels of several myocardial genes including nkx2.5 . Later , Gata5 is necessary for the elaboration of ventricular it issue . We further demonstrate that Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis . Significantly , overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial it issue . Together , these results implicate zebrafish Gata5 in controlling the growth , morphogenesis , and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5 .

SECTION: materials. Whole-mount in situ hybridization and immunohistochemistry We performed in situ hybridization as described ( Alexander et al.1998 ) ; in certain cases , embryos older than 28 hpf were incubated in 0.003 % phenylthiourea to inhibit pigmentation . [Field: materials, subscore: 1.00]

Citation: V : 127 I : 2 P : 225-35
Type: Journal_article
Literature: xenopus
Accession (PMID): 10603341

Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors , but the extracellular signals specifying heart precursor gene expression are not known . We describe here that the secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors , particularly during initiation of cardiac gene expression.fgf8 is mutated in acerebellar ( ace ) mutants , and homozygous mutant embryos do not establish normal circulation , although vessel formation is only mildly affected . In contrast , heart development , in particular of the ventricle , is severely abnormal in acerebellar mutants . Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells : fgf8 is expressed in cardiac precursors and later in the heart ventricle . Fgf8 is required for the earliest stages of nkx2.5 and gata4 , but not gata6 , expression in cardiac precursors . Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 RNA , or by implanting a Fgf8-coated bead into the heart primordium . Pharmacological inhibition of Fgf signalling during formation of the heart primordium phenocopies the acerebellar heart phenotype , confirming that Fgf signaling is required independently of earlier functions during gastrulation . These findings show that fgf8 / acerebellar is required for induction and patterning of myocardial precursors .

SECTION: materials. In some cases , 0.2 mM phenylthiourea ( PTU ) was added to prevent melanization . [Field: materials, subscore: 1.00]

Citation: V : 97 I : 6 P : 2615-20
Type: Journal_article
Literature: xenopus
Accession (PMID): 10706622

The direct developing anuran , Eleutherodactylus coqui , lacks a tadpole , hatching as a tiny frog . We investigated the role of the metamorphic trigger , thyroid hormone ( TH ) , in this unusual ontogeny . Expression patterns of the thyroid hormone receptors , TRalpha and TRbeta , were similar to those of indirect developers . TRbeta mRNA levels increased dramatically around the time of thyroid maturation , when remodeling events reminiscent of metamorphosis occur . Treatment with the goitrogen methimazole inhibited this remodeling , which was reinitiated on cotreatment with TH . Despite their radically altered ontogeny , direct developers still undergo a TH-dependent metamorphosis , which occurs before hatching . We propose a new model for the evolution of anuran direct development .

SECTION: results. T TS79 embryos caused precocious tail regression in E.coqui ( 12 ) , and treatment of E.martinicensis with the goitrogen phenylthiourea ( PTU ) inhibited tail resorption ( 8 ) . [Field: results, subscore: 1.00]

Citation: V : 101 I : 1-2 P : 249-53
Type: Journal_article
Literature: xenopus
Accession (PMID): 11231085

The Roundabout ( Robo ) family of receptors and their extracellular ligands , the Slit protein family , play important roles in repulsive axon guidance . First identified in Drosophila , Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin ( Ig ) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain , a transmembrane domain , and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling ( Cell 92 ( 1998 ) 205 ; Cell 101 ( 2000 ) 703 ) . Robo family members have now been identified in C . elegans , Xenopus , rat , mouse , and human ( Cell 92 ( 1998 ) 205 ; Cell 92 ( 1998 ) 217 ; Cell 96 ( 1999 ) 807 ; Dev . Biol . 207 ( 1999 ) 62 ) . Furthermore , multiple robo genes have been described in Drosophila , rat , mouse and humans , raising the possibility of potential redundancy and diversity in robo gene function . As a first step in elucidating the role of Robo receptors during vertebrate development , we identified and characterized two Robo family members from zebrafish . We named these zebrafish genes robo1 and robo3 , reflecting their amino acid sequence similarity to other vertebrate robo genes . Both genes are dynamically expressed in the developing nervous system in distinct patterns . robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina . robo1 nervous system expression appears later in development and is more restricted . Moreover , both genes are expressed in non-neuronal it issues consistent with additional roles for these genes during development .

SECTION: materials. To facilitate visualization of RNA in situ hybridization in embryos older than 36 h , 0 . 2 mM phenylthiourea was added to the sh water at approximately 22 h . [Field: materials, subscore: 1.00]

Citation: V : 233 I : 2 P : 329-46
Type: Journal_article
Literature: xenopus
Accession (PMID): 11336499

Ectopic expression by injection of plasmid DNA is rarely used in zebrafish embryos due to a low frequency of cells expressing a transgene of interest at detectable levels . Furthermore , the mosaic nature of ectopic expression by plasmid injection requires the direct detection of transgene-expressing cells . We have used the transcriptional activator Gal4-VP16 to amplify transgene expression in living zebrafish embryos . In comparison to conventional expression vectors , Gal4-VP16-amplified expression results in a significant higher number of cells which express a transgene at detectable levels . The Gal4-VP16-activator and the Gal4-VP16-dependent transgene can be placed on a single expression vector . Using it issue-specific regulatory elements , we show that expression of a Gal4-VP16-dependent transgene can be reliably restricted to muscle , notochordal , or neuronal it issues . Furthermore , Gal4-VP16 can drive the expression of two or more transgenes from the same construct resulting in simultaneous coexpression of both genes in virtually all expressing cells . The reported expression system works effectively not only in zebrafish embryos but also in Xenopus embryos , chicken , mouse , and human cultured cells and is thus applicable to a broad variety of vertebrates . The high frequency of transgene expression together with the linked coexpression of more than one transgene opens the possibility of easily monitoring the behavior of individual transgene-expressing cells in real time by labeling them with the fluorescent reporter GFP . The combinatorial nature of the expression system greatly facilitates changing the it issue-specificity , the transgene expressed , or the cell compartment-specific GFP reporter , making it simpler to address a gene ' ' s function in different it issues as well as its cell biological consequences .

SECTION: materials. To block pigmentation , phenylthiourea was added to Danieau medium when the embryos reached the 10-somite stage ( ca . [Field: materials, subscore: 1.00]

Citation: V : 129 I : 7 P : 1645-55
Type: Journal_article
Literature: xenopus
Accession (PMID): 11923201

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities . In mouse , Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes . We show that spiel ohne grenzen ( spg ) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape . Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed . Segment-specific expression of hoxa2 , hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 ( Krox20 ortholog ) and valentino ( val ; kreisler ortholog ) expression is impaired.spg mutants carry loss-of-function mutations in the pou2 gene.pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val.Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants , but does not induce ectopic expression of these genes . This suggests that spg / pou2 acts in a permissive manner and is essential for normal expression of krx20 and val.We propose that spg / pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories .

SECTION: materials. Some embryos were incubated with the addition of 0.2 mM phenylthiourea to prevent pigmentation . [Field: materials, subscore: 1.00]

Citation: V : 182 I : 2 P : 273-85
Type: Journal_article
Literature: xenopus
Accession (PMID): 15283688

Several hypotheses have been proposed to explain the increase and sustained expression of pituitary thyrotropin ( TSH ) in the presence of elevated plasma thyroid hormone ( TH ) concentrations at metamorphic climax in amphibians . It has been proposed that the negative feedback of TH on TSH is inoperative until metamorphic climax , and that it is established at this time by the upregulation of pituitary deiodinase type II ( DII ) ; DII converts thyroxine ( T ( 4 ) ) to 3 , 5 , 3 ' ' -triiodothyronine ( T ( 3 ) ) . However , earlier investigators , using indirect measures of TSH , reported that TH negative feedback on TSH was functional in premetamorphic tadpoles . In an effort to understand pituitary TSH regulation during amphibian metamorphosis , we analyzed multiple pituitary genes known or hypothesized to be involved in TSH regulation in tadpoles of Xenopus laevis . Tadpole pituitary explant cultures were used to examine direct negative feedback on TSH mRNA expression . Negative feedback is operative in the early prometamorphic tadpole pituitary and both T ( 3 ) and T ( 4 ) can downregulate TSH mRNA expression throughout metamorphosis . The expression of both DII and TH receptor betaA mRNAs increased during development and peaked at climax ; however , these increases coincided with similar increases in deiodinase type III , which inactivates TH . Moreover , corticotropin-releasing factor ( CRF ) receptors , CRF binding protein and thyrotropin-releasing hormone receptor type 2 mRNA expression also peaked at climax . Our data suggest that the regulation of TSH is more complex than the timing of DII expression , and likely involves a balance between stimulation of TSH synthesis and secretion by neuropeptides ( e._DSQPRD_ CRF ) of hypothalamic or pituitary origin , increased pituitary sensitivity to neuropeptides through upregulation of their receptors , and intrapituitary TH levels .

SECTION: references. Hanoaka Y 1967 Effect of phenylthiourea upon posterior hypothalectomized tadpoles of Rana pipiens . [Field: references, subscore: 1.00]

Citation: V : 90 I : 1 P : 318-27
Type: Journal_article
Literature: xenopus
Accession (PMID): 16214870

Xenopus melanophores have pigment organelles or melanosomes which , in response to hormones , disperse in the cytoplasm or aggregate in the perinuclear region . Melanosomes are transported by microtubule motors , kinesin-2 and cytoplasmic dynein , and an actin motor , myosin-V.We explored the regulation of melanosome transport along microtubules in vivo by using a new fast-tracking routine , which determines the melanosome position every 10 ms with 2-nm precision . The velocity distribution of melanosomes transported by cytoplasmic dynein or kinesin-2 under conditions of aggregation and dispersion presented several peaks and could not be fit with a single Gaussian function . We postulated that the melanosome velocity depends linearly on the number of active motors . According to this model , one to three dynein molecules transport each melanosome in the minus-end direction . The transport in the plus-end direction is mainly driven by one to two copies of kinesin-2 . The number of dyneins transporting a melanosome increases during aggregation , whereas the number of active kinesin-2 stays the same during aggregation and dispersion . Thus , the number of active dynein molecules regulates the net direction of melanosome transport . The model also shows that multiple motors of the same polarity cooperate during the melanosome transport , whereas motors of opposite polarity do not compete .

SECTION: materials. To track the movement of individual organelles , the number of melanosomes in the cell was reduced by treatment with phenylthiourea ( 17 ) . [Field: materials, subscore: 1.00]

Citation: V : 297 I : 1 P : 26-43
Type: Journal_article
Literature: xenopus
Accession (PMID): 16860306

The expression of zebrafish hoxb3a and hoxb4a has been found to be mediated through five transcripts , hoxb3a transcripts I-III and hoxb4a transcripts I-II , driven by four promoters . A " master " promoter , located about 2 kb downstream of hoxb5a , controls transcription of a pre-mRNA comprising exon sequences of both genes . This unique gene structure is proposed to provide a novel mechanism to ensure overlapping , it issue-specific expression of both genes in the posterior hindbrain and spinal cord . Transgenic approaches were used to analyze the functions of zebrafish hoxb3a / hoxb4a promoters and enhancer sequences containing regions of homology that were previously identified by comparative genomics . Two neural enhancers were shown to establish specific anterior expression borders within the hindbrain and mediate expression in defined neuronal populations derived from hindbrain rhombomeres ( r ) 5 to 8 , suggesting a late role of the genes in neuronal cell lineage specification . Species comparison showed that the zebrafish hoxb3a r5 and r6 enhancer corresponded to a sequence within the mouse HoxA cluster controlling activity of Hoxa3 in r5 and r6 , whereas a homologous region within the HoxB cluster activated Hoxb3 expression but limited to r5 . We conclude that the similarity of hoxb3a / Hoxa3 regulatory mechanisms reflect the shared descent of both genes from a single ancestral paralog group 3 gene .

SECTION: materials. Whole mount in situ hybridization Embryos were obtained from our breeding colony , raised at 28.4C in system water containing 0.003 % phenylthiourea ( PTU ) to prevent melanization . [Field: materials, subscore: 1.00]

Citation: V : 371 I : 1-3 P : 99-109
Type: Journal_article
Literature: xenopus
Accession (PMID): 17011022

The narcotic and lethal concentrations of organic compounds have been compiled for several tadpole species ( Rana temporaria , Rana pipiens , Rana japonica , Xenopus laevis and Rana brevipoda porosa ) . The narcotic and lethal concentrations have been correlated using the Abraham solvation parameter model to yield an equation that can be used to predict the narcotic concentrations of additional nonpolar and polar narcotic compounds to R.temporaria , and a more general correlation that should be applicable to different species of tadpoles . The more general equation is based on 240 experimental data points . A training set of 123 compounds could be fitted with the Abraham solvation parameters with R ( 2 ) = 0.931 and S . D . = 0.343 log units . The training equation predicted the test set of 122 values with AE = -0.022 log units , S . D . = 0.300 log units and an average absolute error , AAE , of 0.227 log units . The structural features that are important in narcosis of tadpoles have been examined ; it is concluded that hydrogen bond basicity reduces narcotic activity of compounds and that compound size increases narcotic activity . The solvation parameter model enables narcosis of tadpoles to be compared to various other biological processes and to physicochemical processes that might be used as models for narcosis .

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Citation: V : 37 I : 1-2 P : 117-61
Type: Journal_article
Literature: xenopus
Accession (PMID): 17364707

Metamorphosis of the amphibian tadpole is a thyroid hormone ( TH ) -dependent developmental process . For this reason , the tadpole is considered to be an ideal bioassay system to identify disruption of thyroid function by environmental contaminants . Here we provide an in-depth review of the amphibian thyroid system with particular focus on the role that TH plays in metamorphosis . The amphibian thyroid system is similar to that of mammals and other tetrapods . We review the amphibian hypothalamic-pituitary-thyroid ( HPT ) axis , focusing on thyroid hormone synthesis , transport , and metabolism . We also discuss the molecular mechanisms of TH action , including the role of TH receptors , the actions of TH on organogenesis , and the mechanisms that underlie the pleiotropic actions of THs . Finally , we discuss methods for evaluating thyroid disruption in frogs , including potential sites of action , relevant endpoints , candidate protocols for measuring thyroid axis disruption , and current gaps in our knowledge . The utility of amphibian metamorphosis as a model for evaluating thyroid axis disruption has recently led to the development of a bioassay using Xenopus laevis .

SECTION: references. Effect of phenylthiourea upon posterior hypothalactomized tadpoles of Rana pipiens . [Field: references, subscore: 1.00]

Citation: V : 38 I : 2 P : 224-35
Type: Journal_article
Literature: xenopus
Accession (PMID): 18455430

Insect nicotinic acetylcholine receptors ( nAChRs ) are targets for insecticides . Despite the importance of the nAChR as a major target for insecticide action , modulators of nAChRs in insects remain unidentified . Here we describe the cloning and identification of a nAChR modulator gene in an insect . This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library , and the gene itself encodes a protein 120 amino acids in length , named Pr-lynx1 . Pr-lynx1 shares all the features , including a cysteine-rich consensus motif and common gene structure , of the Ly-6 / neurotoxin superfamily . The recombinant Pr-lynx1 , which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells , is normally present at the cell surface as a GPI-anchored protein . Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various it issues , such as the ganglion , brain , mandibular muscle , proventriculus , leg muscle , and epidermis . This expression pattern is similar to the distribution of nAChRs as assayed by alpha3 nAChR immunoreactivity . Co-expression of Pr-lynx1 in Xenopus oocytes expressing alpha3beta4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents , indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs . This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species .

SECTION: materials. A few crystals of phenylthiourea were added to the test tubes to prevent melanization . [Field: materials, subscore: 1.00]

Citation: V : 49 I : 9 P : 3812-20
Type: Journal_article
Literature: xenopus
Accession (PMID): 18502992

PURPOSE : During vertebrate phototransduction 11-cis-retinal is isomerized to all-trans-retinal.Light sensitivity is restored by recombination of apo-opsin with 11-cis-retinal to regenerate visual pigments . The conversion of all-trans retinal back to 11-cis-retinal is known as the visual cycle . Within the retina , cellular retinal-binding protein ( CRALBP ) is abundantly expressed in the retinal pigment epithelium ( RPE ) and Mller glia . CRALBP expressed in the RPE is known to facilitate the rate of the rod visual cycle . Recent evidence suggests a role for Mller glia in an alternate cone visual cycle . In this study , the role of RPE- and Mller-CRALBP in cone vision was characterized . METHODS : The CRALBP orthologues rlbp1a and rlbp1b were identified in zebrafish by bioinformatic methods . The spatial and developmental expression of rlbp1a and rlbp1b was determined by in situ hybridization and immunohistochemistry . Depletion of the expression of the corresponding Cralbp a and Cralbp b proteins was achieved by microinjection of antisense morpholinos . Visual function was analyzed in 5-day post fertilization ( dpf ) larvae using the optokinetic response assay . RESULTS : The zebrafish genome contains two CRALBP ohnologues , rlbp1a and rlbp1b . These genes have functionally diverged , exhibiting differential expression at 5 dpf in RPE and Mller glia , respectively . Depletion of CRALBP in the RPE or Mller glia results in abnormal cone visual behavior . CONCLUSIONS : The results suggest that cone photoreceptors incorporate 11-cis-retinoids derived from the rod and cone visual cycles into their visual pigments and that Mller-CRALBP participates in the cone visual cycle .

SECTION: materials. Wild-type Tu bingen zebrafish embryos were obtained by natural spawning and raised in embryo medium.31 Larvae used for in situ hybridization were raised in embryo medium supplemented with 0.003 % phenylthiourea ( Sigma-Aldrich ) to inhibit pigmentation . [Field: materials, subscore: 1.00]

Citation: V : 52 I : 3 P : 191-201
Type: Journal_article
Literature: xenopus
Accession (PMID): 19002657

The observation that several cargoes move bidirectionally along microtubules in vivo raised the question regarding how molecular motors with opposed polarity coordinate during transport . In this work , we analyzed the switch of microtubule motors during the transport of melanosomes in Xenopus melanophores by registering trajectories of these organelles moving along microtubules using a fast and precise tracking method . We analyzed in detail the intervals of trajectories showing reversions in the original direction of transport and processive motion in the opposite direction for at least 250 nm . In most of the cases , the speed of the melanosome before the reversion slowly decreases with time approaching zero then , the organelle returns over the same path moving initially at a very high speed and slowing down with time . These results could be explained according to a model in which reversions are triggered by an elastic collision of the cargo with obstacles in the cytosol . This interaction generates a force opposed to the movement of the motor-driven organelle increasing the probability of detaching the active motors from the track . The model can explain reversions in melanosome trajectories as well as other characteristics of in vivo transport along microtubules observed by other authors . Our results suggest that the crowded cytoplasm plays a key role in regulating the coordination of microtubules-dependent motors .

SECTION: materials. In order to track the movement of 192 Cell Biochem Biophys ( 2008 ) 52 : 191201 individual organelles , the number of melanosomes in the cell was reduced by treatment with phenylthiourea [ 11 ] . [Field: materials, subscore: 1.00]

Citation: V : 80 I : 1 Pt 1 P : 011912
Type: Journal_article
Literature: xenopus
Accession (PMID): 19658734

Intracellular transport of large cargoes , such as organelles , vesicles , or large proteins , is a complex dynamical process that involves the interplay of adenosine triphosphate-consuming molecular motors , cytoskeleton filaments , and the viscoelastic cytoplasm . In this work we investigate the motion of pigment organelles ( melanosomes ) driven by myosin-V motors in Xenopus laevis melanocytes using a high-spatio-temporal resolution tracking technique . By analyzing the obtained trajectories , we show that the melanosomes mean-square displacement undergoes a transition from a subdiffusive to a superdiffusive behavior . A stochastic theoretical model , which explicitly considers the collective action of the molecular motors , is introduced to generalize the interpretation of our data . Starting from a generalized Langevin equation , we derive an analytical expression for the mean square displacement , which also takes into account the experimental noise . By fitting theoretical expressions to experimental data we were able to discriminate the exponents that characterize the passive and active contributions to the dynamics and to estimate the " global " motor forces correctly . Then , our model gives a quantitative description of active transport in living cells with a reduced number of parameters .

SECTION: acknowledgments. In order to track the movement of individual organelles , the number of melanosomes in the cell was reduced by treatment with phenylthiourea 5 . [Field: acknowledgments, subscore: 1.00]

Citation: V : 97 I : 6 P : 1548-57
Type: Journal_article
Literature: xenopus
Accession (PMID): 19751659

The organization of the cytoplasm is regulated by molecular motors , which transport organelles and other cargoes along cytoskeleton tracks . In this work , we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores . Melanophores have pigment organelles or melanosomes , which , in response to hormones , disperse in the cytoplasm or aggregate in the perinuclear region . We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion , focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge . These data could not be explained by previous models that only consider active transport . We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion . This model predicts that organelles spend approximately 70 % and 10 % of the total time in active transport during dispersion and aggregation , respectively . Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V.

SECTION: materials. To track the movement of individual organelles , the number of melanosomes in cells was reduced by treatment with phenylthiourea ( 16 ) . [Field: materials, subscore: 1.00]

Citation: V : 29 I : 44 P : 13981-91
Type: Journal_article
Literature: xenopus
Accession (PMID): 19890008

Adhesion controls growth cone motility , yet the effects of axon guidance cues on adhesion site dynamics are poorly understood . Here we show that ephrin-A1 reduces retinal ganglion cell ( RGC ) axon outgrowth by stabilizing existing adhesions and inhibiting new adhesion assembly . Ephrin-A1 activates focal adhesion kinase ( FAK ) in an integrin- and Src-dependent manner and the effects of ephrin-A1 on growth cone motility require FAK activation . We also find that FAK is expressed in a high temporal to low nasal gradient in RGCs , similar to EphA receptors , and that balanced FAK activation is necessary for optimal axon outgrowth . Last , we find that FAK is required for proper topographic positioning of retinal axons along the anterior-posterior axis of the optic tectum in both Xenopus and zebrafish , a guidance decision mediated in part by A-type ephrins . Together , our data suggest that ephrin-A1 controls growth cone advance by modulating adhesive point contacts through FAK activation and that graded FAK signaling is an important component of ephrin-A-mediated retinotopic mapping .

SECTION: materials. Embryos were raised to stages 45 47 in 0 . 1 mM phenylthiourea in 0 . 1 modified Ringer ' s solution to inhibit pigment synthesis and facilitate dye injection . [Field: materials, subscore: 1.00]

Citation: V : 44 I : 7-8 P : 707-15
Type: Journal_article
Literature: xenopus
Accession (PMID): 20036484

This study was designed to develop a zebrafish experimental model to examine defects in retinoic acid ( RA ) signaling caused by embryonic ethanol exposure . RA deficiency may be a causative factor leading to a spectrum of birth defects classified as fetal alcohol spectrum disorder ( FASD ) . Experimental support for this hypothesis using Xenopus showed that effects of treatment with ethanol could be partially rescued by adding retinoids during ethanol treatment . Previous studies show that treating zebrafish embryos during gastrulation and somitogenesis stages with a pathophysiological concentration of ethanol ( 100mM ) produces effects that are characteristic features of FASD . We found that treating zebrafish embryos with RA at a low concentration ( 10 ( -9 ) M ) and 100mM ethanol during gastrulation and somitogenesis stages significantly rescued a spectrum of defects produced by treating embryos with 100mM ethanol alone . The rescued phenotype that we observed was quantitatively more similar to embryos treated with 10 ( -9 ) M RA alone ( RA toxicity ) than to untreated or 100mM ethanol-treated embryos . RA rescued defects caused by 100mM ethanol treatment during gastrulation and somitogenesis stages that include early gastrulation cell movements ( anterior-posterior axis ) , craniofacial cartilage formation , and ear development . Morphological evidence also suggests that other characteristic features of FASD ( e._DSQPRD_ , neural axis patterning ) are rescued by RA supplement .

SECTION: materials. For some experiments , 0 . 2 mM phenylthiourea was added to embryo medium to prevent melanization . [Field: materials, subscore: 1.00]

Citation: V : 6 I : 4 P : e18332
Type: Journal_article
Literature: xenopus
Accession (PMID): 21483765

The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks . Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2 , respectively . In this work , we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules . We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track . The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment . A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is one order smaller than that determined for motor proteins in vitro . This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex . We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules .

SECTION: materials. 0018332 . g006 Mechanical Properties of Motor-Attached Organelles PLoS ONE | www . plosone . org 6 April 2011 | Volume 6 | Issue 4 | e18332 organelles , the number of melanosomes in cells was reduced by treatment with phenylthiourea [ 54 ] . [Field: materials, subscore: 1.00]

Citation: V : 2011 P : 376243
Type: Journal_article
Literature: xenopus
Accession (PMID): 21860775

In the past , various approaches have been developed to detect adverse effects of pollutants on the thyroid of vertebrates , most of these with special emphasis on the South African clawed frog , Xenopus laevis . Although fish are primarily affected by thyroid-disrupting chemicals , studies into alterations of the thyroid of fish are scarce . Therefore , effects of the reference compound propylthiouracil on histopathology of the thyroid axis were analyzed in a modified early life-stage test with zebrafish ( Danio rerio ) exposed to propylthiouracil . The test substance induced dose-dependent alterations of thyroidal it issue concomitant with increases in the number of surrounding blood vessels . Despite this massive proliferation of the thyroid , zebrafish were not able to maintain thyroxin concentrations . The pituitary was affected displaying significant alterations in thyroid-stimulating hormone cell counts . Quantitative evaluation of pituitary surface areas revealed a dose-dependent increase of adenohypophyseal it issue . Distinct histopathological effects may contribute to a more easy identification and interpretation of alterations induced by thyroid-disrupting chemicals .

SECTION: references. [ 35 ] O . A . Elsalini and K . B . Rohr , " Phenylthiourea disrupts thyroid function in developing zebrafish , " Development Genes and Evolution , vol . 212 , no . 12 , pp . [Field: references, subscore: 1.00]

Citation: V : 50 I : 3 P : 307-15
Type: Journal_article
Literature: xenopus
Accession (PMID): 22084035

We have developed a method of injecting bacterial artificial chromosome ( BAC ) DNA into Xenopus embryos that is simple and efficient , and results in consistent and it issue-specific expression of transgenes cloned into BAC vectors . Working with large pieces of DNA , as can be accommodated by BACs , is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression . We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60 % of injected embryos express the reporter in a manner consistent with endogenous expression . The behavior of these BACs , which are replicated after injection , contrasts with that of smaller plasmids , which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome .

SECTION: materials. Embryos were cultured in 0 . 002 % phenylthiourea ( PTU ) to prevent pigment formation and enable observation of fluorescent transgene expression in retinal it issues ( Sims , 1962 ) . rax capped mRNA was synthesized from pCS2-Xrx1 ( a kind gift from Massimiliano Andreazzoli ; Andreazzoli et al . , 2003 ) using mMESSAGE mMACHINE1 SP6 Kit ( Invitrogen ) and 20 pg mRNA was injected into one dorsal blastomere at early fourcell stage of embryos ( Xenopus tropicalis ) to observe overexpression phenotype . [Field: materials, subscore: 1.00]

Citation: V : 116 I : 7 P : 2306-13
Type: Journal_article
Literature: xenopus
Accession (PMID): 22235949

The behavior of a fluorophore near a gold nanoparticle is rationalized by a theoretical description of the parameters that modify the fluorescence emission : nanoparticle-fluorophore distance , fluorescence quantum yield ( ( 0 ) ) , and fluorophore absorption and emission spectra , to find optimum conditions for designing fluorophore-nanoparticle probes . The theoretical maximum gain in brightness of the nanoparticle-fluorophore system with respect to the isolated molecule increases almost inversely proportional to ( 0 ) . The brightness enhancement in imaging experiments in vitro was assessed by using Au-SiO ( 2 ) core-shell nanoparticles deposited on glass . A 13-fold emission brightness enhancement for weakly fluorescent molecules was observed . A significant increase in fluorophore photostability , rendering longer imaging times , was obtained for fluorophores interacting with gold nanoparticles incorporated by endocytosis in cells . Our results illustrate a way to increase imaging times and to study molecules in the vicinity of a metallic nanoparticle after photobleaching of background fluorescence .

SECTION: materials. Xenopus laevis melanophores were cultured as previously described 9 in the presence of phenylthiourea ( PTU ) to inhibit melanin biosynthesis . [Field: materials, subscore: 1.00]

Citation: V : 65 I : 1 P : 1-11
Type: Journal_article
Literature: xenopus
Accession (PMID): 22814950

Molecular motors are responsible of transporting a wide variety of cargos in the cytoplasm . Current efforts are oriented to characterize the biophysical properties of motors in cells with the aim of elucidating the mechanisms of these nanomachines in the complex cellular environment . In this study , we present an algorithm designed to extract motor step sizes and dwell times between steps from trajectories of motors or cargoes driven by motors in cells . The algorithm is based on finding patterns in the trajectory compatible with the behavior expected for a motor step , i . . , a region of confined motion followed by a jump in the position to another region of confined motion with similar characteristics to the previous one . We show that this algorithm allows the analysis of 2D trajectories even if they present complex motion patterns such as active transport interspersed with diffusion and does not require the assumption of a given step size or dwell period . The confidence on the step detection can be easily obtained and allows the evaluation of the confidence of the dwell and step size distributions . To illustrate the possible applications of this algorithm , we analyzed trajectories of myosin-V driven organelles in living cells .

SECTION: materials. In order to track the movement of individual organelles , the number of melanosomes in cells was reduced by treatment with phenylthiourea [ 33 ] . [Field: materials, subscore: 1.00]

Citation: V : 33 I : 25 P : 10384-95
Type: Journal_article
Literature: xenopus
Accession (PMID): 23785151

The RNA-binding protein Hermes [ RNA-binding protein with multiple splicing ( RBPMS ) ] is expressed exclusively in retinal ganglion cells ( RGCs ) in the CNS , but its function in these cells is not known . Here we show that Hermes protein translocates in granules from RGC bodies down the growing axons . Hermes loss of function in both Xenopus laevis and zebrafish embryos leads to a significant reduction in retinal axon arbor complexity in the optic tectum , and expression of a dominant acting mutant Hermes protein , defective in RNA-granule localization , causes similar defects in arborization . Time-lapse analysis of branch dynamics reveals that the decrease in arbor complexity is caused by a reduction in new branches rather than a decrease in branch stability . Surprisingly , Hermes depletion also leads to enhanced early visual behavior and an increase in the density of presynaptic puncta , suggesting that reduced arborization is accompanied by increased synaptogenesis to maintain synapse number .

SECTION: materials. 5858-12 . 2013 Copyright 2013 the authors 0270-6474 / 13 / 3310384-12 $ 15 . 00 / 0 10384 The Journal of Neuroscience , June 19 , 2013 33 ( 25 ) : 10384 10395 ( Danio rerio ) embryos were obtained from wild-type ( WT ) AB and TL strains , mutant Lakritz strains , and Tg ( atoh7 : gap43-RFP ) ( Zolessi et al . , 2006 ) maintained and bred at 26 . 5C . Embryos were raised at 28 . 5C in embryo medium supplemented with 0 . 003 % phenylthiourea ( Sigma ) for pigment reduction . [Field: materials, subscore: 1.00]

Citation: V : 1830 I : 11 P : 5095-103
Type: Journal_article
Literature: xenopus
Accession (PMID): 23872153

Organelle transport is driven by the action of molecular motors . In this work , we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment . We used single particle tracking to obtain trajectories of melanosomes ( pigmented organelles in Xenopus laevis melanophores ) . In response to certain hormones , melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors . Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent ( ) provided information regarding the trajectories ' ' topography and thus of the processes causing it . During aggregation , the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments ( IF ) . Depolymerization of IF significantly reduced values of small organelles during aggregation but slightly affect their directionality during dispersion . Our results could be interpreted considering that the number of copies of active motors increases with organelle size . Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors . Also , we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment . The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs . dynein at the single molecule level .

SECTION: materials. In order to track the movement of individual organelles , the number of melanosomes in cells was reduced by treatment with phenylthiourea [ 21 ] . [Field: materials, subscore: 1.00]

Citation: V : 106 I : 12 P : 2625-35
Type: Journal_article
Literature: xenopus
Accession (PMID): 24940780

The cytoskeleton is involved in numerous cellular processes such as migration , division , and contraction and provides the tracks for transport driven by molecular motors . Therefore , it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization . It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations . However , tracking individual filaments in living cells is extremely complex due , for example , to the high and heterogeneous background . We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with 9 nm precision in in vitro conditions . To illustrate potential applications of this algorithm , we studied the curvature distributions of fluorescent microtubules in living cells . By performing a Fourier analysis of the microtubule shapes , we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of 20 m , a value significantly smaller than that measured in vitro . We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value ; however , the disruption of intermediate filaments decreased the persistence length . Also , we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells , and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network . Moreover , the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region , and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells .

SECTION: materials. Melanin production was inhibited by treatment with phenylthiourea ( 25 ) . [Field: materials, subscore: 1.00]

Citation: P :
Type: Journal_article
Literature: xenopus
Accession (PMID): 26247430

The mechanisms involved in bidirectional transport along microtubules remain largely unknown . We explored the collective action of kinesin-2 and dynein motors during transport of melanosomes in Xenopus laevis melanophores . These motors are attached to organelles through accessory proteins establishing a complex molecular linker . We determined both the stiffness of this linker and the organelles speed and observed that these parameters depended on the organelle size and cargo direction . Our results suggest that melanosome transport is driven by two dissimilar teams : whereas dynein motors compete with kinesin-2 affecting the properties of plus-end directed organelles , kinesin-2 does not seem to play a similar role during minus-end transport .

SECTION: non-sectioned. Cells were grown in 70 % L-15 medium ( Sigma Aldrich ) supplemented with phenylthiourea [ 25 ] to reduce the amount of melanosomes for tracking experiments . [Field: body, subscore: 1.00]

Citation: V : 36 I : 27 P : 7325-39
Type: Journal_article
Literature: xenopus
Accession (PMID): 27383604

Fragile X mental retardation protein ( FMRP ) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis , but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking . Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity . We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging ( FUNCAT ) . Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil . Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals , but plasticity degrades over 24 h . These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity . Fragile X syndrome ( FXS ) is the most common form of inherited intellectual disability . Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein ( FMRP ) is thought to underlie cognitive deficits in FXS , but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals . Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner . We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes . Furthermore , induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown , but performance rapidly deteriorated in the absence of FMRP . These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning-induced behavioral plasticity in vivo .

SECTION: materials. To reduce interference from pigment cells in the skin , we raised tadpoles in 0 . 001 % phenylthiourea ( Sigma ) starting 23 d after fertilization until stage 47 / 48 when they were used for experiments . [Field: materials, subscore: 1.00]

Citation: P :
Type: Journal_article
Literature: xenopus
Accession (PMID): 27619407

Thyroid hormones ( TH ) have been mainly associated with post-embryonic development and adult homeostasis but few studies report direct experimental evidence for TH function at very early phases of embryogenesis . We assessed the outcome of altered TH signaling on early embryogenesis using the amphibian Xenopus as a model system . Precocious exposure to the TH antagonist NH-3 or impaired thyroid receptor beta function led to severe malformations related to neurocristopathies . These include pathologies with a broad spectrum of organ dysplasias arising from defects in embryonic neural crest cell ( NCC ) development . We identified a specific temporal window of sensitivity that encompasses the emergence of NCCs . Although the initial steps in NCC ontogenesis appeared unaffected , their migration properties were severely compromised both invivo and invitro . Our data describe a role for TH signaling in NCCs migration ability and suggest severe consequences of altered TH signaling during early phases of embryonic development .

SECTION: references. Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos . [Field: references, subscore: 1.00]

Citation: V : 36 I : 50 P : 12697-12706
Type: Journal_article
Literature: xenopus
Accession (PMID): 27974617

The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets . In the vertebrate visual system , such topography is seen clearly in the optic tract ( OT ) and in the optic radiations . However , the molecular mechanisms involved in pretarget axon sorting are poorly understood . Here , we show in zebrafish that the RNA-binding protein Hermes , which is expressed exclusively in retinal ganglion cells ( RGCs ) , is involved in this process . Using a RiboTag approach , we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs . One of these targets is the guidance cue receptor Neuropilin 1 ( Nrp1 ) , which is sensitive to the repellent cue Semaphorin 3A ( Sema3A ) . Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1 . Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT . Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1 . SIGNIFICANCE STATEMENT : An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract ; however , the molecular mechanisms involved in this process remain largely unknown . The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell ( RGC ) axons in the optic tract and tectum . We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs , with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections . We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting .

SECTION: materials. Embryos to be collected for fluorescence imaging had the embryo medium supplemented with 0 . 003 % phenylthiourea ( Sigma-Aldrich ) for pigment reduction . [Field: materials, subscore: 1.00]

Citation: V : 7 I : 8 P : 2493-501
Type: Journal_article
Literature: xenopus
Accession (PMID): 3612250

Regulation of the number and size of neurons presumably plays a role in the matching of a group of neurons to their target . In this paper the relationship of the cardiac ganglion neurons of the frog to their target is examined . Neurons in this ganglion first appear in the embryo and continue to accumulate for several months , even after the animal has completed metamorphosis , and eventually reach a fixed number of cells in the adult . This prolonged period of neuron production has provided an opportunity to manipulate development and test various mechanisms of neuronal regulation . Manipulation of animal culture conditions and hormone levels has shown that the addition of neurons to the ganglion continues up to the characteristic adult number and depends upon neither the chronological age nor the developmental stage of the animal.The size of neurons also changes markedly during development . The average cell body size initially decreases due to the addition of many smaller cells to the ganglion . After metamorphosis neuron size increases dramatically . The changes in size and number complement one another such that the total volume of neuronal cell bodies increases in proportion with the size of both the target and the entire body . The relationship holds for changes in animal size that extend over 4 orders of magnitude and follows a power function of the form y = bxm . Regulation of cardiac ganglion size can be divided into 3 overlapping phases : ( 1 ) the arrival of neurons and precursors from the neural crest , ( 2 ) an increase in neuron number , ( 3 ) and an increase in neuron size . A common denominator for all phases is that the size of the ganglion is , in a coherent way , precisely matched to the size of its target .

SECTION: materials. Oversized larvae were produced by blocking the production of thyroxine with 0.0 1 % phenylthiourea ( PTU ) in 10 % Holtfreter ' s solution ( Hoskins and Grobstein , 1984 ) . [Field: materials, subscore: 1.00]

Citation: V : 36 I : 2 P : 330-42
Type: Journal_article
Literature: xenopus
Accession (PMID): 4131093

SECTION: references. The effects of phenylthiourea on the synthesis and deposition of melanin in developing oocytes of Xenopus laevis . [Field: references, subscore: 1.00]