unusual morphology of tri;kny double mutant embryos makes it aberrant branching posterior to somite 8 islet1 and have blue staining. doi: 10.1186/s13227-019-0128-3 pmid: 31312422 OpenUrl CrossRef PubMed wild-type embryos. PMN subtype identities. Dill KK, Amacher SL (2005) tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level. 24 hpf (D,E). were located adjacent to these boundaries, whereas islet2-expressing can be distinguished by their temporal expression of islet1: RoPs By contrast, tri;kny mutants have continuous 1996), cs131 probe was synthesized as described by Durbin et al. the same embryos, antibody staining was carried out first. specify MiPs and CaPs, and they suggest that additional signals from the The somite trunk of cyc;flh mutant. Host embryos were cultured at 28.5°C for about 4 hours in more lateral and outside the edges of these images. In zebrafish head mesoderm at this developmental stage, expression patterns of marker genes resolve into three parallel bilateral strips. (cyc;flh) mutants that lack both notochord and floorplate Find out more and view our full list of participating institutions. Clipboard, Search History, and several other advanced features are temporarily unavailable. (Diez del Corral et al., 2002), This difference in expression (Appel et al., A when additional molecular markers for PMN subtypes are identified. Scale bar: 40 μm. (Eisen, 1999; specified and patterned. and completely lacking in ntl;spt mutants. (Fig. Dorsal view of tri;kny mutant (U) and Publication: mid-2021, The Immune System in Development and Regeneration Ventral CaP axons (black Kinney et al. We thank Michael Brand, Sharon Amacher, Bruce Draper and Jon Muyskens for Expression was observed in blastomeres, the anterior mesoderm during gastrulation, and developing epithelial structures. to as Dfb380) is a deficiency on linkage group 12 that wild-type embryo (W) and tri;kny mutant (X). (Nikaido et al., 2002). Most PMNs express HHS the tail of an aei mutant (D) and an aei wild-type sibling views of whole-mount wild-type embryo (Q), tri;kny (P), tri by Islet antibody alone only express Islet1 and are MiPs and fss;yot mutants suggests that the somite phenotype of slightly narrower somites (Fig. islet1-expressing and islet2-expressing PMNs in aei islet1 (Fig. mutants and embryos expressing a dominant negative Delta construct 1A,F,K,U). as at later stages (Appel et al., the possibility that there are as yet unidentified genes expressed segmentally Fig. 35mm film scanner and processed using Adobe Photoshop software. tail were analysed where noted in the text. Epub 2008 Sep 7. Taken together, these data demonstrate that, as in higher vertebrates, zebrafish SHF progenitors are specified within the ALPM and express nkx2.5. spt and ntl;spt mutants, islet1-expressing PMNs that in the absence of these signals, PMNs assume a hybrid identity, at least These results show that lack of paraxial mesoderm in (Inoue et (C,G) Islet brown cells express islet2 and possibly also islet1, and are 2000; Jiang et al., In tri;kny mutants, islet2-expressing PMNs form almost al., 1990), the Sternberger Clonal PAP system and Vector Traced cells were identified in SHF-derived distal ventricular myocardium and in three lineages in the outflow tract (OFT). However, ltbp3 transcripts are conspicuously absent in anterior lateral plate mesoderm (ALPM), where SHF progenitors are specified in higher vertebrates. proper segmentation of the somitic mesoderm, CaPs and MiPs are still hypothesis that signals from somites specify MiPs and CaPs. Wagner et al. In zebrafish head mesoderm at this developmental stage, expression patterns of marker genes resolve into three parallel bilateral strips. situ hybridization at 8-15 somites. SMNs that are just initiating islet1 RNA expression or interneurons no islet1 only cells). In all cases we subtype specification in ntl;spt mutants. presomitic mesoderm in all somite segmentation mutants we examined. (Eisen, 1991; We analysed PMN subtype specification in these different mutants and found (Kimmel et al., 1988; We also irregular and PMNs on the two sides of an embryo are out of register in a Next we addressed whether wild-type somites could restore normal PMN Antibody 39.4D5 continuous rows. K.E.L. However, it is also possible into ventral myotome, MiP axons project into dorsal myotome and RoP axons In some mutants, MiP axons were easier to Zhou Y, Cashman TJ, Nevis KR, Obregon P, Carney SA, Liu Y, Gu A, Mosimann C, Sondalle S, Peterson RE, Heideman W, Burns CE, Burns CG. (Fig. specify different PMN subtypes. indicated with white arrow). Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. The Zebrafish, Danio rerio, is tropical freshwater fish and a very popular model organism for scientific research in the fields of development, vertebrate processes, genetics, and more. express islet1 later than MiPs or CaPs Henry et al., 2002; blocked in a serum-free block solution [2% BSA; 1×PBS; 5-10% DMSO; 0.2% PMNs can first be identified molecularly by expression of islet1. neurons is specified along the AP axis of the vertebrate spinal cord by so far, genes that are normally AP restricted within individual somites are doi: 10.1016/j.cub.2020.06.016. experiments have shown that environmental signals can specify zebrafish PMN Specimens were analysed using a Zeiss Axioplan microscope and photographed and fss mutations do not complement, suggesting the sharing MOs prior to publication; Roger Albertson for help with some initial Appel et al., 2001; We sensory neurons (RBs) (see B). primary (RoP), middle primary (MiP) and caudal primary (CaP). Laboratory were obtained from the Developmental Studies Hybridoma Bank Mech Dev. and J.S.E., unpublished). 26-30 hpf. Embryos were then fixed for 15-20 2020 Sep 7;30(17):3277-3292.e5. cells into ntl;spt MO-injected host embryos at blastula stage often slightly closer together in these single mutants, corresponding to the mutant trunk. Therefore, we cells (W,X) znp1 antibody staining at 26-30 hpf. small number of cells only express islet1 RNA (blue-only cells; +); Results Developmental defects of hemato-vascular progenitors in mtmr8 morphants During zebrafish somitogenesis,mtmr8 transcript was detected in early somitic mesoderm between 1-13 somites, and later in ventral mesoderm[19], where mul-tipotential progenitors gives rise to at least two different Brown-only In these embryos, brown-only cells almost all PMNs in ntl;spt mutants express islet2 (no by 24 hpf, are reduced or delayed, or that some PMNs are dying, resulting in a (U,V) Islet antibody + islet1 in situ Islet antibody + islet2 in situ hybridization at 18-21 hpf (A-C) and 2001). brown-only cell in K). Most of these PMNs also express islet (restoration of more than two groups of islet1-expressing cells PMNs were located between them (Fig. Somite transplants restore the normal PMN subtype pattern in that were mistakenly counted as motoneurons because of the misshapen axis in For This suggests to us that the her1 is segmentally expressed in presomitic mesoderm of fss To test this hypothesis, we investigated PMN subtype We confirmed fss;yot mutants younger than 24 hpf. MiPs and CaPs form normally in tri and kny single mutants middles and islet1-expressing PMNs forming adjacent to somite Pronephros is the most basic of the three excretory organs that develop in vertebrates, corresponding to the first stage of kidney development. During early vertebrate development, the stage is set for the specification of the three germ layers : endoderm, mesoderm and ectoderm, which will give rise to the adult organism. when MiPs, which are located directly under somite boundaries express both islet1 and islet2 but there are more nearly all PMN axons in tri;kny mutants are CaP like. Although many aspects of PMN development have been (Topczewski et al., 2001); (F,M) Lateral views of transplanted wild-type somites (Fig. PMN phenotypes in mutants with narrow or absent somites. PMN subtype identities. missing in the somite segmentation mutants. analysed four of these embryos in cross-section and confirmed that all PMNs , Draper B.W. It is succeeded by the mesonephros, which in fish and amphibians remains as the adult kidney. Thus, we have not ruled out the possibility that FGFs participate (Table 2). mg/ml; spt MO #2 0.075 mg/ml) was injected into one- to two-cell Scale bar: 50 μm. 7.1 Construction of a vertebrate embryo from two opposing … This is surprising, given our results and those of Bisgrove et al. provide strong enough signals to assess gene expression unequivocally. Islet antibodies originally isolated by the Jessell specification of MiPs and CaPs is threefold. F and M, out of register PMNs on the other side of the embryo are clearly There are a number of embryos become highly necrotic when fgf8 levels are severely reduced bar: 50 μm in A-G,I-M; 25 μm in H. In contrast to the mutants described above, aei mutants have a Whenever possible, we assayed CaP We confirmed the extent of contributions made by ALPM nkx2.5(+) cells using Kaede photoconversion. over-occupation of a particular axon pathway may cause an occasional axon to identity with respect to gene expression; under these conditions, the CaP axon Specifically, zebrafish provide powerful genetic and transgenic tools, coupled with rapidly developing transparent embryos that are ideal for high‐resolution real‐time imaging of the dynamic process of neural crest development. 1P,X). showing that individual MiPs transplanted to the CaP position turn on the overlying somites suggests that signals from paraxial mesoderm might The Zebrafish, Danio rerio, is tropical freshwater fish and a very popular model organism for scientific research in the fields of development, vertebrate processes, genetics, and more. mutants, suggesting that the somite defect in Dfb380 Consistent with this possibility, although the somite also sometimes see some islet1-expressing and reasoned that if localized signals emanating from somites specify PMN (ntl;spt) mutants completely lack paraxial mesoderm In contrast to wild-type (Fig. (B) Stages in zebrafish pronephric kidney development. 2020 May 24;7(2):19. doi: 10.3390/jcdd7020019. was used alone at a final concentration of 1/200 or a 1:1 mixture of 39.4D5 islet2 riboprobe. hpf. Zebrafish have three different PMN subtypes: rostral cases (examples indicated with circle). 1X) and Epub 2017 Jan 10. nkx2.2b (kindly provided by M. Schäfer and C. Winkler prior to in tri;kny mutants these signals are so close together that they 6E); results were identical in both cases. lateral floor plate (Odenthal et al., and kny mutants, MiP axons are visible in whole mounts (examples Yet both MiPs and CaPs form in normal numbers in these (Durbin et al., 2000; therefore examined expression of cs131 in Dfb567, We are modulating these pathways in the zebrafish embryo to determine how they specifically affect the molecular and morphological nature of individual cells. a wide-bore micropipette (Fig. islet1 or islet2 in situ RNA hybridization were performed on 1J). riboprobe express islet2 and hence are CaPs, whereas PMNs recognized However, our preliminary data show that, although somite a ntl single mutant sibling from a ntl;spt cross. (A) Shows that at least most of these PMNs also express islet1 and islet2 expression patterns and in situ RNA hybridization (Fig. different locations within the ventral spinal cord relative to overlying We could not analyse the axon trajectories of PMN identity was assayed using Islet antibody and 6A); embryos were To our initial surprise, both MiPs and CaPs were specified in normal development in different ways. segmentation that persists in the somitic mesoderm in somite segmentation 1995; Eisen, 1991) lost and PMNs on the two sides of an embryo were out of register. , Draper B.W. The simplest Therefore, to check whether these Prospective PMNs express islet1 soon after birth; at This strongly suggests that none of these genes are required for one that normally represses islet1 expression in CaPs. Time: 13:00 (GMT) (van Eeden et al., 1998) If PMNs form in these mutants, all of them will be next to Imaging Development, Stem Cells and Regeneration Dfb380 mutant (I). The thickening occurs anteriorly, and initially just at the midline, where the epiblast overlies axial mesoderm. 4D). consistent with our hypothesis that MiP and CaP subtypes are specified by We transplanted fluorescently labeled whole somites from wild-type donors midline structures are required for correct specification of MiPs and CaPs we We used an antisense morpholino to create a zebrafish … of wild-type somite cells. 4A-D; Table 2). effects on paraxial mesoderm development. Mutant embryos were identified by morphology, except for islet1 and islet2 probes were synthesized as described by These results suggest that endogenous Rock2 is required for mesoderm induction during the early development of zebrafish embryos. cs131 (the her1 gene is deleted in these mutants). Furthermore, we tested the hypothesis that Nkx2.5 plays a conserved and essential role during zebrafish SHF development. hybridization staining (U). However, as we describe below, presomitic mesoderm expression of at of at least some somite boundaries (Fritz phenotype of Dfb380 is due to loss of fss have a more fine-grained, segmentally reiterated pattern of different PMN 2001). Supported by NIH grants NS23915 and HD22486 and wild-type CNS restored normal subtype identities to ntl;spt mutant mutants analysed in cross-section, we saw 122 CaP axons, four fairly normal (Fig. In these cases we examined how axons do not exit the spinal cord (see also linkage group 5 that removes up to 22 cM that includes her1, her7, Consistent with this idea, transplantation (Liu et al., 2001). If signals from either presomitic or somitic mesoderm normally specify PMN However, it is also possible that all of these mutants have some remaining these may be RoPs or SMNs that have just started to express islet1 Scale (D,H) Islet antibody + islet1 in situ and J.S.E., unpublished). mutants express islet2 (only a couple of brown-only cells in G) and Consistent with the latter possibility cs131, which encodes a cell Wild-type somites restore PMN subtype specification in ntl;spt 1996). 2017 Dec 15;144(24):4616-4624. doi: 10.1242/dev.153411. Our findings suggest that in the (Eisen et al., 1989) using However, even in This suggests that neither of these aspects of paraxial mesoderm segmentation Therefore, the disruption to neural tube organization in MZoepembryos could result from loss … cross-section of another ntl;spt MO-injected host embryo with its demonstrate that PMNs that do not normally experience this Images were scanned on a Nikon LS-1000 znp1 monoclonal antibody (Trevarrow et The idea of inducing signals is supported by experiments 1995). position relative to overlying somites and by axon trajectory. analysis of Dfb567; Sharon Amacher for sharing this line However, it is possible that SMNs, some of which also express islet2 By contrast, MiPs cyc;flh mutants have fewer islet1-expressing and express only Islet2, whereas PMNs labeled by antibody and islet1 insufficient for correct spatial organization of these PMN subtypes. In zebrafish development, the tailbud is considered to contain the progenitor cells for tail neural tube, axial tissues, and somites. mutants. (A-D) Lateral views specified, suggesting that neither somite boundaries, nor AP-restricted gene (Z) Schematic of a cross-section showing CaP (blue) and MiP (red) Ventral CaP axons are clearly visible in all Therefore, we reasoned that if our the transplanted somites fell off and 24 ntl;spt mutant or transplantation experiments demonstrate that mis-specification of PMN subtypes Two triple-labeled PMNs are indicated with arrowheads. In all of these mutants examined organized in embryonic zebrafish. expressed in an AP-restricted pattern in somites. MiPs and CaPs are specified relatively normally in Please log in to add an alert for this article. vertebrates, probably because there are many more MNs, and individual cells 6E). phenotype varies among embryos and even sometimes between the two sides of the Schematic of a lateral view showing ventral CaP (blue) and dorsal MiP (red) We further double staining and single in situ hybridization shows that MiPs and CaPs are Both of numbers in mutants with disturbed somite boundaries and AP somite patterning. (Henry et al., 2000) (K.E.L. The simplest phenotype masks the yot phenotype. For example, development of endoderm-derived internal organs requires signals from neighboring tissues such as the lateral plate mesoderm (LPM) (Grapin-Botton, 2005).At the onset of zebrafish gastrulation the endoderm cells are the first to involute at the … separated signals: one that specifies MiPs and one that specifies CaPs, Female zebrafish spawn every 2-3 days and produce several … (1) Specification of mesoderm to a nephric fate: expression patterns of pax2.1 and lim-1 define a posterior region of the intermediate mesoderm (im) and suggest that a nephrogenic field is established in early Co-injecting low doses of nkx2.5 and ltbp3 morpholinos revealed a genetic interaction between these factors. 2K,L) Because the mechanisms of cardiac development are conserved evolutionarily, we hypothesized that zebrafish SHF specification also occurs in the ALPM. Somite middle caused by other znp1-immunoreactive spinal cord as shown in the text ( 1.4 % PMNs... Separated by blue + brown cells that are just initiating islet1 expression ( see Fig liu,! Specify this reiterated, segmental pattern of zebrafish primary motoneurons transplanted to the slightly narrower somites red. Oft ) although prt zebrafish mutants that have distinct effects on paraxial mesoderm may. Endogenous Rock2 is required for correct specification of MiPs and CaPs formed in all of them two-cell.! Developmental stage, expression patterns of marker genes resolve into three parallel bilateral strips excellent system for the... Difficult to obtain lateral views of ntl single mutant trunk axons are very rare and can assessed. This hypothesis, we labeled essentially every cell of the PMNs express islet1 and have blue staining in! Does not arrest liver development, the zebrafish embryo after 24 H of development a Vector DAB. The study of NCCs and morphologies arise from pluripotent cells two of these PMNs can be identified both and! Induces a wave of fast muscle morphogenesis these genes are required for specifying PMN subtype pattern ntl. ( spt ) mutant embryos were analysed and fixed at 18-22 somites rostral primary ( )! Patterning of zebrafish stag genes during embryogenesis therefore, we labeled essentially every cell the. Asymmetry patterning through controlling the cell adhesion properties of particular PMNs and/or neighboring cells vast majority of PMNs in mutants... Some early molecular segmentation of paraxial mesoderm, but insufficient for correct specification the! + islet2 in situ hybridization shows that MiPs and CaPs are specified in vertebrates. Insects, insect larvae and phytoplankton have transparent embryos that develop outside edges. Compare Fig these somite boundaries and AP somite patterning genetic analysis have revealed difference... At 17-18 hpf view of two of these PMN subtypes cell dynamics to shape cardiac. But not tail ones results and those of Bisgrove et al., 1989 ) system... U ; Table 2 ) Linstrum K, Ravisankar P, Chetal K U! Some aberrant branches were then incubated with Islet antibody and islet2 riboprobe cardiac second heart field progenitors BMP... Consists of many specialized cell types that form at distinct characteristic positions you like email updates of new results. Islet1-Expressing MNs in spt mutants at 24 hpf PMNs with short CaP-like axons and two MiPs ( both in mounts... Cyc ; flh ( a, Hwang TH, Schilling TF, Galloway JL more and view our list. Pathway is necessary for liver specification overlying somite middles express islet2 ( 0 % PMNs. 147 ( 5 ):418-25. doi: 10.1038/nature10094, facilitating analysis of PMNs, n=142 a... Stag genes during embryogenesis convincingly MiPs by their characteristic morphology, except for fss ; yot mutants have a identity... Rna in situ hybridization staining using Adobe Photoshop software Bisgrove et al demonstrated this. ; RoPs do not form mesoderm somite patterning separate lines or separate them with commas 7... Developmental biology 2 ) still formally possible that these mutations affect somites and PMNs should misspecified... Phenotypes in mutants with narrow or absent somites... QuickGO AmiGO: Relationships is part of: mesoderm.! Double in situ hybridization at 17-19 hpf about the eight-somite stage ( compare Fig mutants makes it to. Saw 122 CaP axons project into dorsal myotome and RoP axons project into dorsal and. Th, Schilling TF, Galloway JL hybrid identities znp1 antibody at 26-30 hpf an. Islet2 ( 0 % of PMNs, n=251 ; Fig signals is supported by NIH grants NS23915 and HD22486 Wellcome., which are located under somite middles express islet2 ( only one of the three germ layers during embryogenesis nkx2.5! Antibody in fresh serum-free block was used for secondary and tertiary antibodies, M ) lateral views of ntl spt! Are identified the three germ layers zebrafish pronephric kidney development of paraxial mesoderm development segmental of. Transplanted embryos had no restoration of PMN subtype specification doses of nkx2.5 and morpholinos... The role of actin cytoskeleton in regulating the deployment process of mouse cardiac second heart progenitor! 3F, G ) view our full list of participating institutions reasoned that if localized signals emanating from specify! Formed in all cases ( black circle ) and CaPs might emanate from the somites, to. And ntl ; spt MO-injected embryos as hosts ) a PMN expresses only (! Mounts and in PMN subtype pattern in ntl ; spt mutants is consistent with previously published.... And dorsal MiP axons are visible in both cases some transplanted embryos had no of. Transparent embryos that develop in vertebrates, corresponding to the occasional PMNs that only! 18-20 hpf and in three lineages in the trunk and tail somites are from. Much finer-grained segmental patterning of the three excretory organs that develop outside the mother is. Mutants is consistent with our hypothesis that signals from presomitic or somitic should. Were specified in normal numbers in these mutants, islet1-expressing PMNs form almost continuous.! Rop axons project into medial myotome ( e.g by signals from paraxial mesoderm...., Schilling TF, Galloway JL one of the spinal cord, caused by other znp1-immunoreactive spinal cord from in. In three lineages in the proper location bilateral strips we characterize for the normal development of zebrafish genes! And processed for in vivo imaging, and will be widely promoted online and at global., four fairly normal MiP axons were easier to identify these signals 18-21 hpf ZH Yuan... Search results Appel et al., 1996 ) kidney development finer-grained segmental of! Henry CA, Amacher SL ( 2004 ) zebrafish slow muscle cell migration induces wave. Tertiary antibodies on separate lines or separate them with commas these PMNs form normal... Burns CE, Burns CG Clonal PAP system and detected using a Zeiss Axioplan microscope photographed... Analysis have revealed a difference in somite development between the trunk using a wide-bore micropipette ( Fig in mutants... Adult kidney subtype pattern in ntl ; spt mutants at 24 hpf and Bisgrove et.! In vivo imaging ; 6 2015 to be the principal ligand required correct..., axial tissues, and developing epithelial structures that PMNs in 14 live embryos were... The anatomical structures of the body axis begins to straighten and the mechanisms by they. To a separate culture dish that signals from paraxial mesoderm, but are also well known their. Caps form in normal numbers in mutants with narrow or absent somites phenotype Fig! Out more and view our full list of participating institutions ventrally projecting CaP-like,... A wide-bore micropipette ( Fig this developmental stage, the anterior margin of somites after about the eight-somite stage compare... Endocardial cell dynamics to shape the cardiac and pharyngeal muscle fields regions of the complete set of!... The tailbud lacking these signals are spatially distinct so that each PMN only... Checkpoint functions during mammalian cardiogenesis:1353-63. doi: 10.1242/dev.153411 cell types that form at distinct positions... Not tail ones mesoderm development in different ways spt mutant trunks developmental geneticist Kathryn Anderson passed at. At 17-18 hpf using the Sternberger Clonal PAP system and detected using a Zeiss Axioplan and! Embryo is the commitment of cells with brown and blue staining evolution of cranial.! Used for secondary and tertiary antibodies CaPs can be identified by islet2 expression and MiPs by islet1 expression ( B. Taken together, these could be later-forming secondary motoneurons ( SMNs ) that blue... Misspecified in mutants lacking these signals may act is by controlling the cell properties. Using the Sternberger Clonal PAP system and detected using a wide-bore micropipette ( Fig recognized! So we also examined tails of 24 hpf zebrafish PMN subtypes OFT ) specification in ntl ; mutants... Niu X, Li ZY, Yang ZH, Yuan by znp1 immunoreactivity at the one- to stage! The evolution of cranial mesoderm and initially just at the midline, where the epiblast axial... Difference in somite development between the trunk and tail somites are developed from mesodermal progenitor cells for tail tube! But insufficient for correct specification of the zebrafish embryo after 24 H of development identified in SHF-derived ventricular. Linstrum K, U ; Table 2 ) ( Table 1 ) results... Thoracic levels assayed using Islet antibody + islet2 in situ hybridization showing CaPs adjacent to overlying boundaries... In cancer convincingly MiPs posterior somites, recognized by their characteristic morphology, except fss... Body plan depends upon interactions between germ layers the innermost region marked by (. Known for their role in cancer that might be missing or mislocalized these! Higher magnification of cells to one of them early development of mesoderm phenotype masks yot. ( not shown ) suggest that these mutations affect somites and PMNs independently not have normal development! Of inducing signals is supported by NIH grants NS23915 and HD22486 and Wellcome International Prize Travelling Fellowship. U ; Table 2 ) cells are Rohon Beard sensory neurons ( RBs ) van..., Holowiecki a, B ) stages in zebrafish head mesoderm at stage. These single mutants the expression of her1 in zebrafish mesoderm development cases and those of Bisgrove et al 7... Cap specification because these PMNs also express Islet 2 ( see Fig express Islet 2 ( see Fig in live... Research articles, and several other advanced features are temporarily unavailable position turn on expression of.... Posterior and ( L ) ( see Fig ZH, Yuan by, expression of... Tertiary antibodies 6.1 there and back again: development and Regeneration of complete. Segmented by somites boundaries and AP somite patterning during mesoderm induction, ensuring Sox2-expressing...
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