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Figure 3. The size of the kidney field is reduced in Fry-depleted embryos. (a–d) Pax8 expression in S21 (stage 21) embryos, lateral views. Arrow points to the otic vesicle. (a) Uninjected embryo. (b) Random-MO injected embryo. (c) Fry-MO injected embryo. (d) Fry-MO + FD + LZ mRNA (400 pg) coinjected embryo. (e) Percentage of embryos with reduced (RE), absent (AE) or not affected (NA) pax8 expression. Uninjected (N = 2, 35), Random-MO (N = 2, 23), Fry-MO 5 ng (N = 2, 18), 15 ng (N = 3, 53), 20 ng (N = 4, 70), Fry-MO 20 ng + FD + LZ mRNA 200 pg (N = 2, 39), Fry-MO 20 ng + FD + LZ mRNA 400 pg (N = 2, 51), FD + LZ mRNA 400 pg (N = 2, 46). Data on graph is presented as mean. (f–h) wt1 expression in S24 (stage 24) embryos. (f) Uninjected (n = 20), (g) control and (h) injected (n = 20) sides of the same embryo are shown. (i–k) 3G8 immunostaining. (i) Uninjected (n = 25), (j) control and (k) injected (n = 28) sides of the same embryo. Magnifications of the boxed areas are shown in each panel. (l–n) β1-NaK-ATPase expression in S39 (stage 39) embryos. (l) Uninjected (n = 22). (m) Control and (n) injected (n = 23) sides of the same embryo, arrow points to pronephros positive staining. Representative embryos are shown. Embryos at 8-cell were injected 1x V2 with 15 or 20 ng of the morpholino or as indicated. |
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Figure 5. Synergistic interaction of Fry and Lhx1 to pattern the kidney field. (a–d) Pax8 expression in S21 (stage 21) embryos, lateral views. (e) Percentage of S21 embryos with abnormal pax8 expression under different treatments. Uninjected (N = 3, 41), Fry-MO (N = 3, 56), Lhx1-AS (N = 3, 65), Fry-MO + Lhx1-AS (N = 3, 75) (N = number of experiments, number of embryos). (f–i) 3G8 immunostaining of S32 (stage 32) embryos. (j) Percentage of S32 embryos with reduced or absent 3G8 staining. Uninjected (N = 3, 122), Fry-MO (N = 3, 88), Lhx1-AS (N = 3, 79), Fry-MO + Lhx1-AS (N = 3, 81). (k–n) β1-NaK-ATPase expression in S39 (stage 39) embryos. (o) Percentage of embryos with abnormal β1-NaK-ATPase expression. Uninjected (N = 6, 168), Fry-MO (N = 6, 165), Lhx1-AS (N = 6, 193), Fry-MO + Lhx1-AS (N = 6, 183). Reduced (RE), absent (AE) or not affected (NA) expression/staining. 8-cell embryos were injected 1x V2 with 2.5 ng of Fry-MO and/or 50 pg of Lhx1-AS. Representative embryos are shown. Data in the graphs is presented as means. Statistical significance was evaluated using Fisher’s exact test ****p < 0.0001.
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Figure 9. Overexpression of miR-199a/214 and miR-23b/27b reduce the kidney field size. (a–i) Pax8 expression in S21 (stage 21) embryos injected with LNA mimics. (a,b,f and g) Injected with LNA control. (a,f) Uninjected side (ctrl). (b,g) Injected side with LNA control. (c,d) Embryo injected with LNA miR-199a + miR214. Uninjected (c) and injected (d) sides of the same embryo. (h,i) Embryos injected with LNA miR-27b + miR23b. Uninjected (h) and injected (i) sides of the same embryo. (e) Percentage of embryos with abnormal pax8 expression field. Uninjected (N = 3, 63); LNA ctrl. (N = 3, 64); LNA miR-199a + miR-214 (N = 3, 76, 49% reduced or absent expression) (N = number of experiments, number of embryos, % of affected embryos). (j) Percentage of embryos with abnormal pax8 expression field. Uninjected (N = 3, 66); LNA ctrl. (N = 3, 65); LNA miR-27b + miR-23b (N = 3, 59, 64% reduced or absent expression). Reduced (RE), absent (AE) or not affected (NA) field of expression. Data in graphs is presented as means. ****p < 0.0001, **p < 0.01 Fisher’s exact test |
