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Figure 3. Loss of znf367 function interferes with the expression of neuronal differentiation markers. (A) Embryos injected with gfp (250 pg) and either ZNF367-MO or Control morpholino (Co-MO) (9 ng) at one dorsal blastomere at the four-cells stage showed fluorescence only in the neural plate at neurula stage (st. 18). (B–E’) mRNA distribution of N-tubulin and elrC in znf367 morphants and controls. (B,C) dorsal view and (B’,C’) frontal view of neurula morphants showing a clear loss of differentiation markers expression in primary neurons (arrows). (D,E) dorsal view and (D’,E’) frontal view of neurula embryos injected with Co-MO. (F) Quantification of the data in A and B. (G) qRT-PCR analysis. The levels of expression for the indicated mRNAs were evaluated for Co-MO or ZNF367-MO populations by qRT-PCR, and normalized to that of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression. The mean of the Control-MO was set to 1. For each gene, three independent RNA samples from morphants and controls were analysed. (H–H’) mRNA distribution of neurogenin in znf367 morphants. (I) TUNEL staining. ZNF367-MO injection did not lead to an increase of TUNEL positive cells compared to the un-injected side. Abbreviations: n, number of independent experiments; N, number of evaluate embryos in total; Error bars indicate standard error of the means (s.e.m); *p ≤ 0,05; ***p ≤ 0,001. P-value were calculated by Student’s t-test.
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Figure 4. znf367 morphants analysis and control rescue experiments. (A) mRNA distribution of sox2 and rx1 in znf367 morphants and controls. (B) Statistical analysis of the data in A. (C) The levels of expression for the indicated mRNAs were evaluated for Co-MO or ZNF367-MO populations by qRT-PCR, and normalized to that of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression, and the mean of the Control-MO was set to 1. For each gene, three independent RNA samples from morphants and controls were analysed. (D) The morphants phenotype can be rescued by the co-injection of morpholino plus full length Xenopus znf367 mRNA as shown by the recovered expression of sox2 and elrC. Abbreviations: n, number of independent experiments; N, number of evaluated embryos in total; Error bars indicate standard error of the means (s.e.m); *p ≤ 0,05; **p ≤ 0,01. P-value were calculated by Student’s t-test. |
