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Figure 2 - Foxj1 morpholinos inhibit ciliogenesis in Xenopus skin cells.
From the following article. (a–d) Xenopus embryos injected with both Foxj1 morpholinos or a control morpholino were stained at stage 26 with antibodies to ZO-1 (red) and acetylated tubulin (AT, green) to label cell borders and cilia, respectively (a,b), or were injected with RNAs encoding a membrane-localized RFP (red) and a centrin2-GFP fusion protein (green) to label cell membranes and basal bodies, respectively (c,d). (e) Control or Foxj1 morphants as above were co-injected with RNA encoding a centrin2-GFP fusion protein (green), fixed at stage 26 and stained with rhodamine-phalloidin (red) to label the apical actin network. Bottom panels show a 2-mum Z-scan through the apical domain. Scale bars, 20 um in a,b; 10 um in c–e.
Image redisplayed with permission from Macmillan Publishers Ltd
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Figure 3 - Xenopus Foxj1 RNA misexpression in surface epithelial cells induces ectopic cilia formation. (a–f) Shown is a confocal image of the superficial epithelium in Xenopus embryos at the indicated stage, stained with antibodies to ZO-1 (red) and acetylated tubulin (green) to label cell borders and cilia, respectively. Embryos were injected at the two-cell stage with RFP RNA alone (a,c), with Foxj1 and RFP RNA (b,d), with ICD and RFP RNA (e) or with Foxj1, ICD and RFP RNA (f). Average cilia length in microns is indicated in b and d (plusminus s.d., n = 45–60 cilia from three embryos). Scale bars, 20 mum. (g,h) Transmission electron micrographs of a cilium in a multiciliated cell (g) or of an ectopic cilium (h) induced by Foxj1 RNA in an ICD background (as in f). Arrows indicate the central pair; arrowheads indicate outer dynein arms. Scale bars, 100 nm.
Image redisplayed with permission from Macmillan Publishers Ltd
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Figure 5 - Validation of gene expression regulated by Foxj1. (a–f) Shown is the roof of the gastrocoel in stage 17 embryos after staining for the expression of Tekt2 (a,d), Spag6 (b,e) and Dnah9 (c,f) RNA using whole-mount in situ hybridization. Expression (red-blue stain) in the posterior GRP is marked with an arrow. Panels a,b and c show staining in uninjected embryos, whereas panels d,e and f show staining in embryos injected twice in one blastomere at the two-cell stage with Foxj1 RNA. Injected side is oriented to the right. (g) Embryos were injected at the two-cell stage with the indicated RNAs or with Foxj1 or control morpholinos. At stage 10, the ectoderm was isolated, cultured on fibronectin-coated glass to stage 22, and then extracted for total RNA. The expression of Tekt2, Spag6 or Dnah9 RNA was measured in each sample using quantitative PCR and normalized relative to a ubiquitously expressed control RNA, ODC. The value for each experimental condition is an average of three measurements and is expressed on a logarithmic plot as a ratio to the average value obtained with a control. Uninjected controls were used for the RNA injected samples, and a control morpholino sample was used as a control for Foxj1 morpholino injection. Error bars, s.d.
Image redisplayed with permission from Macmillan Publishers Ltd
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Figure 2. XFoxJ1 morpholinos inhibits ciliogenesis in Xenopus skin cells.(A-D)
Xenopus embryos injected with both XFoxJ1 morpholinos, or a control morpholino were stained at stage 26 with ZO-1 (red) and anti-acetylated tubulin (green) to label cell borders and cilia, respectively (A,B), or were injected with RNAs encoding a membrane-localized RFP (red) and a GFP-centrin2 fusion protein (green), to label cell membranes and basal bodies, respectively (C,D). (E) Control or FoxJ1 morphants as above were co-injected with RNA encoding a GFP-centrin2 fusion protein (green), fixed at stage 26 and stained with rhodamine-phallodin (red) to label the apical actin network. Bottom panels show a 2μm Z-scan through the apical domain. Scale bars represent 20μm in A-B and 10μm in C-E.
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Figure 3. XFoxJ1 RNA misexpression in surface epithelial cells induces ectopic cilia formation.(A-F) Shown is a confocal image of the superficial epithelium in Xenopus embryos at the indicated stage, stained with antibodies to ZO-1 (Red) and acetylated-tubulin (green) to label cell borders and cilia, respectively. Embryos were injected at the two-cell stage with RFP RNA alone (A,C) with FoxJ1 and RFP RNA (B,D), with ICD and RFP RNA (E) or with FoxJ1, ICD and RFP RNA (F). Average cilia length in microns is indicated in B and D (n=15-20 cilia from 3 embryos). Scale bars are 20μm. (G,H) Transmission electron micrographs of a cilium in a multi-ciliate cell (G) or of an ectopic cilium (H) induced by FoxJ1 RNA in an ICD background (as in panel F). Arrows indicate the central pair and arrowheads indicate outer dynein arms. Scale bars are 100nm.
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Figure 4. Bi-ciliate cells on the GRP and induced ectopically by XFoxJ1(A-B) Shown is a confocal image of the skin at stage 26 of embryos injected with RFP RNA (A) or with both FoxJ1 and RFP RNA (B), and stained with an antibody to γ-tubulin (green). Arrows denote centriole number and position. (C-D) Confocal image of the uninjected GRP at stage 17, either (C) stained with antibodies to ZO-1 (Red) and acetylated-tubulin (green) or (D) with antibodies to ZO-1 (Red) and γ-tubulin (green). Arrows indicate cilia number (C) or centriole position (D). Scale bars = 10μm. (E-F) Quantification of cilia number (E) and split centrioles (F) in the uninjected GRP at stage 17 (St.17 GRP, n=100 cilia or centriole pairs from each of 4 embryos), or in the outer epithelial cells (OCs) of stage 26 embryos that were injected with FoxJ1 RNA (St26/OCs/FoxJ1) or with just RFP RNA as a control (St26/OCs). For stage 26 n=200 cilia or centriole pairs from each of 4 embryos. Error bars represent S.D.
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Figure 5. Validation of gene expression regulated by FoxJ1.(A-C) Shown is the roof of the gastrocoel in stage 17 embryos after staining for the expression of Tektin-t (A,A′), PF16 (B,B′) and L-R dynein (C,C′) RNA using whole-mount, in situ hybridization. Expression (Red-Blue stain) in the posterior GRP is marked with an arrow. Top panels show staining in uninjected embryos while lower panels shows that in embryos injected twice in one blastomere at the two-cell stage with XFoxJ1 RNA. Injected side is oriented to the right. (D) Embryos were injected at the two-cell stage with the indicated RNAs or with XFoxJ1 or control morpholinos. At stage 10, the ectoderm was isolated, cultured on fibronectin-coated glass to stage 22, and then extracted for total RNA. The levels of Tektin-t, PF16 (Spag6), or L-R dynein RNA was measured in each sample using quantitative PCR, and normalized relative to a ubiquitously expressed control RNA, ODC. Values for each experimental condition is an average of three measurements, and are expressed on a logarithmic plot as a ratio to the average value obtained with a control. Uninjected controls were used for the RNA injected samples and a control morpholino sample was used as a control for FoxJ1 morpholino injection.
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Figure 6. Model for cilia subtype specification.Epithelial cells (EC) extend non-motile primary cilia via a default pathway2. In response to low levels of FoxJ1 epithelial cells extend a motile monocilia that can mediate flow required for left-right patterning. With increased levels of FoxJ1, ECs can be induced to form bi-ciliate cells. FoxJ1 also regulates the expression of genes required for the formation of motile cilia in multiciliated cells, whose differentiation requires additional unknown factor(s).
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