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Figure 1. Comparison of the partial amino acid sequence of human and Xenopus eya1Amino acid substitutions associated with BOR and ocular defects are in larger boldface font. Italic letters indicate conserved motifs characteristic of HAD family proteins. (This Figure is modified from Rayapureddi and Hegde, 2006.) Mutation designations for human (hEYA1) as cited in the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php) are above the aligned sequences, and the corresponding numbering for Xenopus (Xeya1) is below them. OD, ocular defects.
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Figure 2. Expression of eya1 after injection of mRNA encoding eya1 proteins into Xenopus embryosEmbryos were injected (inj.) with 250Â ng of mRNA, and protein isolated from two embryos was loaded on to each lane. (A) A Western blot that shows that mRNAs encoding eya1 and four mutant variants produce detectable protein in stage 12 embryos. Anti-T7 antibody was used to detect proteins. (B) A Western blot that shows that eya1 protein made from injected mRNA persists through stage 41. Anti-T7 antibody was used to detect proteins. NI, non-injected control.
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Figure 3. Detection of otic development in Xenopus embryosOtic development was monitored by fluorescence using whole mount immunohistochemistry to detect acetylated tubulin (A, B, E, F) and F-actin using phalloidin (C, D, G, H). (AâE, G) These Figures show progressively increased magnification of a stage 32 embryo, with bracketed regions indicating the region shown in the next magnification. (A, C) These Figures were visualized using a fluorescent microscope and are shown in lateral view with heads on the right. (B, D) These Figures were visualized through the dorsalâventral plane using a confocal microscope with the anterior at the top of the images. (EâH) These Figures are confocal sections through the dorsalâlateral prospect. The anterior of the embryo is towards the top of each image. (F, H) These Figures are the otic region from a stage 40 embryo. The spcd, ciliated epithelial cells (ce), otic sensory tissue (Ost), neurites associated with otic sensory tissue (arrowheads) and cranial nerves (V, VII and VIII) are noted on panels detecting tubulin; the otic vesicle (vOt), eye and cement gland (cg) are noted on panels detecting F-actin. Scale bars in (B, D, E, F, G, H), 50 μm.
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Figure 4. Oligonucleotide-mediated loss of eya1 and rescue by injection of eya1 mRNA(A) DMED-modified oligonucleotides complementary to Xenopus eya1 reduce the levels of eya1 mRNA. Eya1 mRNA was assayed from non-injected embryos (NI), embryos injected with 1 ng of a control oligonucleotide (C) or 1, 0.5 or 0.25 ng of anti-eya1 oligonucleotide. (B) Injection of 1 ng of anti-eya1 oligonucleotide into one side of the embryo reduces the level of endogenous eya1 and six1 mRNAs shown by in situ hybridization. (C) Reduction of eya1 correlates with inability to form a proper otocyst, as assayed by whole mount immunohistochemical detection of tubulin and F-actin by confocal microscopy along the dorsalâlateral plane. Both six1 expression and normal otocyst formation are rescued by co-injection of oligonucleotide with 250 pg of rescue mRNA encoding T7âeya1 protein. The otic vesicle (vOt) and facial epibranchial placode (epVII) are identified in (B). In (C), otic sensory tissue (Ost), cranial nerves (V, VII and VIII) and neurites leading to the VIII nerve (arrowheads) are labelled. Scale bars on in situ hybridization images, 200 μm; scale bars on confocal images, 50 μm. Inj. injected.
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Figure 5. Stage 32 otic development is disrupted by eya1 mutants linked to BORStage 32 embryos were imaged using a confocal microscope from the dorsalâlateral prospect. The anterior of the embryo is towards the top of each image. Dual panels on the left show tubulin distribution; dual panels on the right detect F-actin. In each dual set, the non-injected side (NI) of the embryo is on the left and the injected side (Inj) is on the right. Material injected into the right side of the embryo is indicated for each row of panels. In panels detecting tubulin, the cranial nerves (VII and VIII) are labelled when visible. The label for VIII is placed as close to the attachment to the spcd as possible. Otic sensory tissue (Ost) is pointed out using arrows that help indicate the most anterior and posterior extents of this tissue type. Arrowheads indicate neurites that are associated with sensory tissue and VIII. The otic vesicle (vOt) is labelled where visible on panels showing F-actin. Scale bars, 50 μm. (Note: the right panel for non-injected controls are also shown enlarged in Figures 3E and 3G.) con, control.
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Figure 6. Stage 40 otic development is disrupted by eya1 mutants linked to BORStage 40 embryos were imaged using a confocal microscope from the dorsalâlateral prospect. The anterior of the embryo is towards the top of each image. Dual panels on the left show tubulin distribution; dual panels on the right show F-actin. In each dual set, the non-injected side (NI) of the embryo is on the left and the injected side (Inj) is on the right. Material injected into the right side of the embryo is indicated for each row of panels. In panels detecting tubulin the cranial nerves (VII, VIII and IX) are labelled when visible. The label for VIII is placed as close to the attachment to the spcd as possible. Otic sensory tissue (Ost) is pointed out using arrows that help indicate the most anterior and posterior extent of this tissue type and arrowheads show examples of neurites connecting to the VIII nerve. The otic vesicle (vOt) is labelled where visible on panels showing F-actin. Scale bars, 50 μm. (Note: the right panel for non-injected controls are also shown enlarged in Figures 3F and 3H). con, control.
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Figure 7. Expression of eya1 mutations that cause BOR in humans reduces otic expression of six1, dach and neuroDOne cell of a two-cell stage embryo was injected with mRNA encoding Eya1 protein. Stage 28 embryos were assayed for otic gene expression by in situ hybridization. In each pair of panels, the non-injected side (NI) of the embryo is shown on the left and the side injected with mRNA (I) is on the right. The otic vesicle (vOt), facial epibranchial placode (epVII and epIX), glossopharyngeal epibranchial placode, anterior lateral line placode (pAD), posterior lateral line placode (pP) and the eye (e) are noted. Scale bars, 200 μm.
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FigureS1 Whole mount imaging of the otic region of a stage 34 embryo
Images were captured starting from the left lateral side of the embryo and then moving medially. (A) A composite of stacks (B) and (C). (B) Each image of a confocal stack using phalloidin as a stain. (C) Each image of a confocal stack detecting acetylated α-tubulin. Enlargements of companion images show how phalloidin staining highlights the otocyst and allows an easy estimate of otic vesicle (vOt) size. The otic vesicle is coloured red in B 5. The appearance of sensory tissue in the otocyst (Ost) can be seen in C 5 and C 8 by reactivity to the anti-tubulin antibody and are selectively coloured green. In C 11 the neurites (arrowheads) connecting the VIII cranial nerve with the otocyst on the ventral medial side are coloured green. Scale bar in (A), 50 μm. An, anterior; D, dorsal; Pos, posterior; V, ventral; VII, VII cranial nerve.
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FigureS2 Whole mount imaging of the otic region of a stage 34 embryo that is expressing R435Q on its right side Images were captured starting from the dorsal-lateral area of the embryo and then moving in the ventral-medial direction. (A) A composite of stacks (B) and (C). (D) is a composite stack of (E) and (F). (B, E) Each image of a confocal stack using phalloidin. (C, F) Each image of a confocal stack detecting acetylated α-tubulin. Enlargements of companion images show how phalloidin staining highlights the otocyst (Ost) and allows an easy estimate of otic vesicle (vOt) size. The otic vesicle is coloured red in B 5 and E 5. The appearance of sensory tissue in the otocyst (Ost) can be seen in C and F 2, 5 and 8, with the greyscale image selectively inverted in C and F 5 and 11 with the inverted image showing sensory tissue as black. The position of the VII cranial nerve is indicated in C 8. In C 11 the neurites (arrowheads) between the sensory tissue and the VIII cranial nerve with the otocyst on the ventral medial side are detected in C but are barely visible in F. Arrowheads point to neurites associated with sensorial tissue and the VIII cranial nerve. Scale bars in (C) and (F), 50 μm.
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