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Figure 2. MCI induces MCC differentiation(a,b) Confocal images of the skin in embryos injected at the two-cell stage with MT-MCI and mRFP RNA (b) or mRFP RNA alone (a), fixed at stage 28, stained with an acetylated tubulin antibody to label cilia (green). Asterisks in b denote unusually large cells. (c,d) Confocal images of the skin in embryos injected with MCI-HGR RNA along with mRFP RNA as a tracer treated with DEX at stage 11.5 (d), and then fixed at stage 28 and stained for cilia (green). (eâg) The outer epithelium from donor embryos injected with mRFP RNA (red) was transplanted onto host embryos injected with mGFP RNA (green) as illustrated in Fig. 1g. Host (f) and donor (g) embryos were also injected with MCI-HGR RNA. Embryos were treated with DEX at stage 11.5 and fixed at stage 28. Shown are confocal images of a control transplant (e), a transplant onto a host injected with MCI-HGR RNA (f), or a transplant from a donor injected with MCI-HGR RNA (g), after staining for cilia (blue). (h) Cell types in the skin were scored as MCCs (green with blue cilia staining), PSCs (green with no cilia staining), outer cells (red), or outer cells with cilia (red with blue cilia). Data is presented as an average of 10â15 fields (±s.d.) obtained from at least three transplants where the asterisks denote experimental values significantly different from control values (p<.005). Scale bars =20microns
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Figure 3. MCI acts downstream of Notch to promote centriole assembly and motile cilia extension via FoxJ1(aâe) Shown are confocal images of the skin in embryos injected with varying amounts of MCI-HGR RNA (a,d), ICD RNA (c), or both RNAs (b,e), along with mRFP (red) and Centrin4-GFP RNA (green) as a tracer and to mark centrioles, respectively. Embryos were treated with DEX at stage 11.5, fixed at stage 28 and stained with acetylated tubulin to visualize cilia (blue). In embryos injected with ICD and 37pgs of MCI-HGR RNA (e), MCC differentiation was sometimes incomplete, resulting in cells extending cilia but failing to undergo centriole assembly (arrowhead) or vice versa (arrow). Average number of centrioles (lower right, ±s.d.) is statistically the same with or without ICD at 150pg of MCI-HGR but different at 37pg of MCI-HGR RNA (p=6Ã10â6). Scale bars=20 microns. (f) The average percentage of MCCs (multiple centrioles and cilia) per field (±s.d) is plotted based on data obtained from three fields from four embryos (n=12). Values marked with an asterisk are highly significant (p<.005). (gâh) Shown are confocal images of the skin of embryos injected with MCI-HGR RNA, Centrin4-RFP and mRFP RNA, followed by a FoxJ1 (h) or a control morpholino (g)7. Embryos were treated with DEX at stage 11.5, fixed at stage 28 and stained with an acetylated tubulin antibody to label cilia (green). Scale bars=20microns. (i) Cilia number per cell was scored from two fields from five embryos (n=10) for each condition, and plotted as the average fraction of cells (±s.d.) with different cilia number as indicated per field.
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Figure 4. MCI induced centriole assembly(aâj) Shown are confocal images of the skin of control (aâe) or MCI-HGR RNA injected embryos (fâj), also expressing SAS6-GFP (green) and mRFP(red). Embryos were treated with DEX at stage 11.5, followed by fixation at the time intervals indicated. In control embryos, Sas6-GFP lightly labels centrioles (a,c arrowhead) in outer cells and basal bodies/centrioles in MCCs (d,e, arrow). By contrast, large bright Sas6-GFP foci are induced in outer cells by MCI-HGR (g,i arrowhead). Scale bar (a)=20microns (k) Plot showing the average fraction of cells (±s.d.) per field with different SAS-GFP foci number at the indicated time point, based on twelve fields taken from four embryos injected with MCI-HGR RNA. In control embryos, every outer cell scored contained 0â2 centrioles labeled with Sas6-GFP (data not shown). (l) Shown is a TEM image taken of an embryo injected with MCI-HGR RNA and treated with DEX for 8hrs. Structures similar to deuterosomes (arrows) in a pigmented (arrowhead) outer cell are located apical to the nucleus. Scale bar=500nm. (mân) Shown are confocal images of the skin in embryos injected with mRFP and Hyls1-GFP RNA to mark cell boundaries (red) and centrioles (green), respectively, either alone (m) or with MCI-HGR RNA (n). At stage 11.5, embryos were treated with Dex, and then fixed at stage 28. Scale bars= 20microns (o) The average centriole number per cell (±s.d.) is plotted for control and MCI-HGR expressing embryos, using data obtained by scoring cells in at least three fields from five embryos. The slight increase in basal body number obtained with 150pg is significantly different (p<0.05) from control MCCs.
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Figure 5. MCI activates MCC gene expression(a–h) Shown is the expression of α-tubulin (a–d) or for FoxJ1 (e–h) in embryos injected with a control morpholino (a,e), with MCI-MOspl (b, f) or with MCI-HGR RNA along with nlacz RNA as a tracer (d,h) or alone as a control (c,g). RNA injected embryos were treated with DEX at stage 11.5, and all embryos were fixed at stage 26, and stained with X-gal (light blue). Scale bar=0.5mm. Insert shows a higher power (i) Animal caps were isolated at stage 10 from embryos injected with ICD RNA, MCI-HGR RNA (MCI), or the two RNAs together, treated with DEX at st11, and extracted for total RNA at stage 12, approximately two hours later. Shown is the log level of expression (±s.d.) relative to ICD values set at zero, as measured in triplicate using QT-PCR and after normalization against a ubiquitously expressed RNA, ODC. (j–m) Single animal blastomeres were injected with MCI-HGR RNA at the two-cell stage. At stage 11, half of the injected and uninjected control embryos were treated with cyclohexemide (CHX) for one hour, and then additionally treated with DEX for another hour to induce MCI-HGR activity. CHX is sufficient to block the onset of α-tubulin expression in the controls (compare k to j), indicating that the CHX treatment is effective, but does not block the activation of α-tubulin by MCI-HGR (m). Scale bar (j)=0.2mm.
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Figure 6. Domains required for MCI function(a) Diagram of MCI. (bâe) Shown are embryos injected with RNA encoding different MCI mutants as indicated, fixed at stage 13/14, and stained for α-tubulin RNA expression (fâi) Confocal images of the skin of embryos injected with RNA encoding different MCI mutants, fixed at stage 28, and stained with ZO-1 (red) and acetylated tubulin (green) antibodies to label cell boundaries and cilia, respectively. Scale bars=0.5mm (bâe), 10 microns (fâh), or 20 microns (i).
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Figure 7. Mouse MCI governs MCC differentiation in MTEC cultures(aâc) MTEC cultures were established, infected with lentivirus expressing MT-MCI, MT-MCIÎCC or GFP as a control, and switched to ALI culture conditions. At 4 days post-ALI, cultures were fixed, stained for infected cells (green) using an antibody to the myc-tag or GFP, to pericentrin (red) to identify MCCs and to E-cadherin (blue) to label cell boundaries. Shown are examples of cultures infected with a virus expressing GFP (a), MT-MCI (b) or MT-MCI-ÎCC (c). Arrows and asterisks denote infected cells that are pericentrin positive or negative, respectively. The insert in panel b shows a higher power image of pericentrin staining that marks clusters of nascent basal bodies. Scale bars = 50 microns (d) Percentage of infected cells that were ciliated versus nonciliated based on FoxJ1 or pericentrin staining under each condition, where n=total # of infected cells scored under each condition (±s.d.). Values marked with an asterisk are statistically significant (p<1Ã10 â10) from the values obtained for the GFP controls.
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