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Figure 1. Multiple sequence alignments of znf367 amino acid sequences. The gray boxes highlighted the conserved zinc-fingers binding domains of znf367 from X. laevis (both splicing variants znf367a and znf367b), human (both splicing variants ZFF29a and ZFF29b) and N. furzeri (n) obtained using Clustal Omega.
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Figure 2. Znf367 gene expression pattern during Xenopus laevis development. (A,B) Znf367 expression at blastula stage (stage 3) using sense control probe (A) and antisense probe (B). (C,D) At neurula (C) and at tadpole stages (D) znf367 is expressed in the neural tube (black arrow), the developing eye (black arrowhead), the neural crest cells (white arrowhead) in the otic vesicle (white arrow) and in the most anterior regions of the nervous system. (E,F) Stage 40 embryo is shown in lateral view (E) and in a transversal section at the level of the hindbrain (F). MB, midbrain; NCC, skeletogenic neural crest cells; RE, retina.
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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.
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Figure 5. znf367 morphants analysis for proliferating and differentiating precursors (A) mRNA distribution of pcna in znf367 morphant and control. (B) Statistical analysis of the data in A. (C) RT-PCR analysis revealed a significant increase of pcna. (D,E) Znf367 depletion leads to a significant reduction of proliferating cells compared to the un-injected side. pH3 positive cells were counted in the areas defined by the black rectangles. Statistical evaluation of the data is shown (E). (F) RT-PCR analysis of cyclinB1 (ccnb1). The levels of expression for the indicated mRNAs were evaluated for Co-MO or ZNF367-MO embryos by qRT-PCR in triplicate, and normalized to glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression. (G–G’) p27 is down regulated at stage 18 in znf367 morphants (arrowheads). (G) dorsal view; (G’) frontal view. (H–H’). p27 expression in control embryo, (H) dorsal view; (H’) frontal view. (I–L) rx1 and elrD expression at tailbud stages confirmed the phenotype observed at neurula stages: increased expression of rx1 and downregulation of a neuronal differentiation marker (elrD) (arrowheads). 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.
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Network and molecular analysis of znf367 neighbors. (A) Distribution of membership values for the analyzed module in human cells and N. furzeri brain. The vertical line indicates the rank of znf367 and the horizontal line its membership value (black for N. furzeri and red for H. sapiens). Please note due to the high convexity of the human distribution ZNF367 has very high membership despite its rank. (B) Central part of the gene module containing ZNF367. Only genes showing topological overlap >0.3 are shown. ZNF367 is in red, while genes used for further analysis are in orange (SMC2, SKA3 and FANCD2). (C) qRT-PCR analysis of fancd2, ska3 and smc2. The levels of expression for the indicated mRNAs were evaluated for Co-MO or ZNF367-MO populations by qRT-PCR in triplicate and normalized to, glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression. (D) mRNA distribution of fancd2, ska3 and smc2 in znf367 morphants. Neurula stage embryos are shown in frontal (D,E,F) and dorsal view (D’,E’,F’). Arrowheads indicated the expanded domain of the mRNA distribution of each gene in the injected side of the embryo. Abbreviations: n, number of independent experiments; Error bars indicate standard error of the means (s.e.m); n.s., not significant. *p ≤ 0,05; **p ≤ 0,01. P-value were calculated by Student’s T-test.
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znf367(zinc finger protein 367) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, horizontal view, animal up.
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znf367(zinc finger protein 367) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior down
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znf367(zinc finger protein 367) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior right, dorsal up.
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znf367 (zinc finger protein 367) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior right, dorsal up.
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Figure 6. Network and molecular analysis of znf367 neighbors. (A) Distribution of membership values for the analyzed module in human cells and N. furzeri brain. The vertical line indicates the rank of znf367 and the horizontal line its membership value (black for N. furzeri and red for H. sapiens). Please note due to the high convexity of the human distribution ZNF367 has very high membership despite its rank. (B) Central part of the gene module containing ZNF367. Only genes showing topological overlap >0.3 are shown. ZNF367 is in red, while genes used for further analysis are in orange (SMC2, SKA3 and FANCD2). (C) qRT-PCR analysis of fancd2, ska3 and smc2. The levels of expression for the indicated mRNAs were evaluated for Co-MO or ZNF367-MO populations by qRT-PCR in triplicate and normalized to, glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression. (D) mRNA distribution of fancd2, ska3 and smc2 in znf367 morphants. Neurula stage embryos are shown in frontal (D,E,F) and dorsal view (D’,E’,F’). Arrowheads indicated the expanded domain of the mRNA distribution of each gene in the injected side of the embryo. Abbreviations: n, number of independent experiments; Error bars indicate standard error of the means (s.e.m); n.s., not significant. *p ≤ 0,05; **p ≤ 0,01. P-value were calculated by Student’s T-test.
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