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Dev Dyn
2019 Oct 01;24810:969-978. doi: 10.1002/dvdy.98.
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Overexpression of Lin28a delays Xenopus metamorphosis and down-regulates albumin independently of its translational regulation domain.
Gundermann DG
,
Martínez J
,
De Kervor G
,
González-Pinto K
,
Larraín J
,
Faunes F
.
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BACKGROUND: Lin28 regulates stem cell biology and developmental timing. At the molecular level Lin28 inhibits the biogenesis of the micro RNA let-7 and directly controls the transcription and translation of several genes. In Xenopus, Lin28 overexpression delays metamorphosis and affects the expression of genes of the thyroid hormone (TH) axis. The TH carrier albumin, synthesized by the liver, is down-regulated in limbs and tail after Lin28 overexpression. The molecular mechanisms underlying the interaction between Lin28, let-7, and the hypothalamus-pituitary-thyroid gland (HPT) axis are unknown.
RESULTS: We found that precursor and mature forms of let-7 increase during Xenopus metamorphosis. In the liver, lin28b is down-regulated and albumin is up-regulated during metamorphosis. Overexpression of a truncated form of Lin28a (Lin28aΔC), which has been shown not to interact with RNA helicase A to regulate translation, delays metamorphosis, indicating that the translational regulation domain is not required to inhibit the HPT axis. Importantly, both full length Lin28a and Lin28aΔC block the increase of albumin mRNA in the liver independently of changes in TH signaling.
CONCLUSIONS: These results suggest that Lin28 delays metamorphosis through regulation of let-7 and that the decrease of the TH carrier albumin is one of the early changes after Lin28 overexpression.
Figure 1
Pri‐let7 and let‐7 increase during metamorphosis in the central nervous system. RT‐qPCR for pri‐let7a (A), pri‐let7c (B) in brain RNA samples. Expression was normalized to eef1α and compared to st.50 (y‐axis; average ± SEM, n = 3 independent experiments). *P < .05, t‐test against hypothetical value = 1 (no change compared to st.50). Tadpoles were fixed at stage 54 (C–E, I–K) and 58 (F–H, L–N) and in situ hybridization was performed in transversal sections of spinal cord. Specific locked nucleic acid (LNA) probes for let‐7a (C, F, J, M), let‐7c (D, G, K, N), and let‐7f (E, H) were used. Scramble LNA probe was used as a control (I, L)
Figure 2
lin28b is down‐regulated in the liver during metamorphosis. Liver samples were isolated at different Nieuwkoop–Faber (NF) stages (x‐axis) and analyzed by RT‐qPCR for lin28b (A), thrβA (B), albumin (alb‐b; C), transthyretin (ttr; D), pri‐let7a (E), pri‐let7c (F), and mature let‐7a (G; average ± SEM, n = 3–4 independent experiments). For lin28b, ttr, pri‐let‐7a, and pri‐let‐7c expression was normalized to eef1α and compared to st.50 (y‐axis). *P < .05; **P < .01; t‐test against hypothetical value = 1 (no change compared to st.50). For thrβA and alb‐b expression was normalized to eef1α. *P < .05; **P < .01; ***P < .001, ANOVA with Dunnett's multiple comparisons tests to stage 50. For let‐7a, expression was normalized to 5S rRNA. *P < .05; ***P < .001; ANOVA with Dunnett's multiple comparisons tests to stage 50
Figure 3
Overexpression of Lin28ΔC. A, Protein sequences of Lin28a and Lin28aΔC‐flag highlighting the cold shock domain in green, the zinc‐knuckle domains in blue, the C‐terminal domain in brown and the flag epitope in red. B, Western blot with the anti‐flag epitope in tail samples of nontransgenic (−) and Lin28ΔC transgenic animals heat‐shocked for 3 weeks (three times per week). The specific band of Lin28ΔC‐flag is observed under 25 kDa. Nonspecific bands were detected above 75 kDa (asterisks) and were considered as loading control
Figure 4
C‐terminal domain of Lin28a is not required to delay metamorphosis. A, Metamorphosis progress (stages, y‐axis) of F1 nontransgenic siblings (control, green line) and transgenic animals (lin28a+) with HS from day 0 to day 18 (red bar) starting at st.50 (days, x‐axis). ***P < .001, unpaired t‐test for days 26, 28, 33, and 41. No significant differences were detected for days 11 and 19. B, Metamorphosis progress (stages, y‐axis) of F1 nontransgenic siblings (control, green line) and transgenic animals (lin28a+, blue line) with HS from day 0 to day 11 (red bar, 2 weeks) starting at st.57 (days, x‐axis). No significant differences were detected for days 9, 11, and 16
Figure 5
Overexpression of Lin28a or Lin28aΔC blocks the increase of albumin RNA in the liver. F1 nontransgenic siblings (control, green line) and transgenic animals (lin28a+, blue line) were heat shocked from day 0 to day 18 (three times per week for 3 weeks) starting at st.50. RNA was isolated at days 0, 9, and 18 and RT‐qPCR was performed for lin28a (A), thrβa (B), and alb‐b (C). The same experimental scheme was used to overexpress Lin28aΔC (lin28aΔC+, blue line) and compared the gene expression to control animals (control, green line) for lin28a (D), thrβA (E), and alb‐b (F). Expression was normalized to eef1α and compared to day 0 (y‐axis; average ± SEM, n = 3 independent experiments). *P < .05, two‐way ANOVA plus Sidak's multiple comparisons test
