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| Gene |
Clone |
Species |
Stages |
Anatomy |
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ednra.L
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laevis
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NF stage 12.5
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ectoderm
,
neural plate border
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ednra.L
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laevis
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NF stage 15
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neural crest
,
dorsal
,
trunk neural crest
,
trunk
,
neural plate border
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ednra.L
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laevis
|
NF stage 24
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otic vesicle
,
mandibular crest
,
hyoid crest
,
neural crest
,
trunk neural crest
,
[+]
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ednra.L
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laevis
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NF stage 29 and 30
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otic vesicle
,
mandibular crest
,
hyoid crest
,
neural crest
,
pharyngeal arch
,
[+]
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Display additional annotations [+]
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Fig. 1. Xenopus laevis Endothelin-1 receptor type A features and expression pattern. (A) Molecular phylogenetic analysis of Ednra shows the relationship with Xenopus ETAX, Xenopus Ednrb and with Ednra from different vertebrate species. The protein sequences of different Ednra were analyzed using ClustalW 1.81, and an unrooted tree was constructed by neighbour-joining analysis. Xenopus laevis Ednrb, Danio rerio EdnrA1, and Danio rerio EdnrA2 branches are collapsed 50%. (B) RT-PCR analysis of temporal expression pattern of Ednra, Ppet-1, and ECE-1 during different developmental stages. Total RNA isolated from stages 1, 10.5, 12, 14, 18, and 21 was isolated, retrotranscribed and amplified as described in Materials and methods. Contamination of genomic DNA was examined by experiments without RT reactions (−RT) using total RNA from stage 18 embryos. EF1α expression was used as loading control. (C–L) Expression pattern of Xenopus laevis Ednra. (C) Ednra transcripts are first detected from late gastrula stage (St. 12.5) in the lateral domains of neural plate (arrows). (D) During neurulation Ednra is expressed in the prospective cephalic (asterisk) and trunk neural crest (arrows). (E) In situ hybridization for FoxD3. (F) Double in situ hybridization for Ednra (purple) and Sox2 (turquoise). (G) Transverse section of (F). Asterisk, prospective neural crest. (H) Tailbud-stage (St. 24) embryo showing expression in the migrating cephalic neural crest (arrowheads), trunk region (arrow), crest cells migration into dorsal fin (white arrowheads), and otic vesicle (OtV). (I) Frontal section of (H), Ednra expression in the otic vesicle (OtV) and the neural crest hyoid migratory stream (arrowheads). NT, neural tube; n, notochord. (J) A St. 30-embryo showing Ednra expression in the branchial arches (black arrowheads), cells into the dorsal fin (white arrowheads), and otic vesicle (OtV). OpV, optic vesicle. (K) Section showing Ednra expression in the dorsomedial and ventromedial region of the otic vesicle (arrowheads). (L) Horizontal section showing Ednra expression (arrowheads) in the first and second pharyngeal arches surrounding the central mesodermal core (asterisks). |
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Fig. 2. Ednra is required for neural crest development. (A–I) Efficiency of Ednra antisense morpholino oligonucleotide. (A) EdnraMO inhibits in vitro translation of Ednra in a dose-dependent fashion. Arrow indicates Ednra protein product. (B–I) EdnraMO inhibits in vivo expression of Ednra–GFP. (B,C) Embryos injected with mRNA encoding Ednra–GFP (1 ng/embryo) showing GFP fluorescence. (D,E) Embryos injected with Ednra–GFP mRNA (1 ng/embryo) and CoMO (30 ng/embryo). (F–I) Embryos injected with Ednra–GFP mRNA (1 ng/embryo) and EdnraMO (F–G, low dose, (l) 10 ng/embryo; H–I, high dose (h), 20 ng/embryo). No embryo shows GFP fluorescence at high dose. White arrows indicate the injected side. (J–S) Analysis of the formation of neural crest derivatives. Normal melanocyte formation (J, arrows) and Trp2 expression (M, arrows). EdnraMO-injected embryos show inhibition in melanocyte development (K) or Trp2 expression (N). The melanocyte formation was completely rescued by the coinjection of EdnraMO and Ednra′ mRNA (L). Induction of melanocytes in vitro by conjugating animal caps with prospective paraxial mesoderm cultured until the equivalent of St. 38. Control conjugates show melanocytes (O, arrows). Conjugates prepared with a 10 μM BQ123-soaked bead show no melanocyte formation (P). EdnraMO-injected (Q, arrowhead) embryos present a reduction of Meckel's and ceratohyal cartilages. (R) Schematic representation of EdnraMO effects on Xenopus head cartilages. M, Meckel's cartilage; Ir, infrarrostral; CH, ceratohyal; BH, basihyal; CB, ceratobranchial. (S) Normal cartilage formation was completely rescued by the coinjection of EdnraMO and Ednra′ mRNA. |
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Display additional annotations [+]
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Fig. 9. Ednra in the genetic cascade that specifies the neural crest. Ednra lies downstream Msx1 and upstream Sox9–Sox10. (A–O) Representative examples of embryos injected with the indicated reagents. The graphs at the right side represent the percentage of embryos with the indicated phenotypes; the letter in each column correspond to the treatment described for each figure in panels A–O. (A, B) Embryos injected with Msx1-GR show increased expression of Ednra and neural crest marker FoxD3. (C) Msx1-GR construct failed to increase the expression of FoxD3 marker when was coinjected with EdnraMO. (C') EdnraMO leads to a reduction of FoxD3 expression in the injected side (arrowhead). (D, E) Msx1-dominant negative inducible construct (HDMsx1-GR) inhibited the expression of Ednra and FoxD3. (F) The coinjection of dominant negative HDMsx1-GR construct and Ednra mRNA rescue the expression of FoxD3 in the neural crest. (F') Ednra overexpression leads to an increased FoxD3 expression in the injected side (arrowhead). (G) No changes in the expression of Msx1 was observed in embryos microinjected with Ednra mRNA. (H, I) Ednra mRNA-microinjected embryos show expanded expression of Sox9 and Sox10 in the injected side (arrowhead). (J–L) EdnraMO-microinjected embryos show no effect on Msx1 (J), but inhibition of Sox9 (K) and Sox10 (L) expression in the injected side (arrowhead). (M–O) Sox9 mRNA and Sox10 mRNA rescue FoxD3 expression in EdnraMO-injected embryos. (N') Expansion of FoxD3 in embryo injected with Sox9. (O') Expansion of FoxD3 in embryo injected with Sox9. |
