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A mutation in cnot8, component of the Ccr4-not complex regulating transcript stability, affects expression levels of developmental regulators and reveals a role of Fgf3 in development of caudal hypothalamic dopaminergic neurons.

Koch P, Löhr HB, Driever W - PLoS ONE (2014)

Bottom Line: Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes.We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased.Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of developmental control genes.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Institute Biology I, Faculty of Biology University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany.

ABSTRACT
While regulation of the activity of developmental control genes at the transcriptional level as well as by specific miRNA-based degradation are intensively studied, little is known whether general cellular mechanisms controlling mRNA decay may contribute to differential stability of mRNAs of developmental control genes. Here, we investigate whether a mutation in the deadenylation dependent mRNA decay pathway may reveal differential effects on developmental mechanisms, using dopaminergic differentiation in the zebrafish brain as model system. In a zebrafish genetic screen aimed at identifying genes controlling dopaminergic neuron development we isolated the m1061 mutation that selectively caused increased dopaminergic differentiation in the caudal hypothalamus, while other dopaminergic groups were not affected. Positional cloning revealed that m1061 causes a premature stop codon in the cnot8 open reading frame. Cnot8 is a component of the Ccr4-Not complex and displays deadenylase activity, which is required for removal of the poly (A) tail in bulk mRNA turnover. Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes. We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased. Pharmacological inhibition of FGF signaling or a mutation in the fgf3 gene can compensate the gain of caudal hypothalamic dopaminergic neurons in cnot8m1061 mutants, indicating a role for Fgf3 in control of development of this dopaminergic population. The cnot8m1061 mutant phenotype provides an in vivo system to study roles of the Cnot8 deadenylase component of the mRNA decay pathway in vertebrate development. Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of developmental control genes.

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fgf3/lia FGF signaling mediates the increase in caudal hypothalamic DC7 DA neurons in cnot8m1061 mutant embryos.(A–F) Analysis of DA neurons in cnot8m1061 mutant embryos combined with a mutation in the fgf3 locus (liat24152) or pharmacological suppression of FGF signaling by SU5402. Embryos were fixed at 4 dpf, stained by anti TH immunofluorescence and DA neurons documented by confocal microscopy. Shown are Z-projections of confocal stacks representing the ventral diencephalic DA groups 1 to 7 (dorsal views). Scale bar 50 µm. (A–D) DA neurons in WT, cnot8m1061 or liat24152 single mutant, and cnot8m1061, liat24152 double mutant embryos. Double mutant embryos show loss of cnot8m1061-mediated increase of DC7 caudal hypothalamic DA neurons. (E, F) DA neurons in WT and cnot8m1061 mutant embryos treated with SU5402 from 42 to 48 hpf. Inhibition of FGF signaling by SU5402 reduces the number of DC7 caudal hypothalamic DA neurons in cnot8m1061 mutants below WT levels. (G) Quantification of effects on CA neurons by cell counting of forebrain DA neuronal clusters and locus coeruleus NA neurons in genetic and experimental conditions as indicated in the index at right. Each bar shows the average number of CA neurons in five independent embryos for each experimental condition. Error bars indicate standard deviation. Average DC7 cell numbers: cnot8+/+ lia +/+ 50.8 (WT control); cnot8-/- lia +/+ 103.2; cnot8-/- lia -/- 52.6; cnot8+/+ lia -/- (38.8); cnot8+/+ SU5402 20.6; cnot8-/-SU5402 56.2. Significance was evaluated by Mann-Whitney test. The cell count in single mutant cnot8m1061 (-/-)liat24152 (+/+) is significantly different from single mutant cnot8m1061 (+/+) liat24152 (-/-) embryos (p = 0.008). Comparison of cnot8m1061, liat24152 double mutant and WT embryos reveal no significant difference (p = 0.45). For SU5402 treatments, the number of DC7 DA neurons differs significantly between WT controls and SU5402 treated WT (p = 0.008) and between cnot8m1061 and SU5402-treated cnot8m1061 embryos (p = 0.008). For all other catecholaminergic groups, no significant differences were observed when WT was compared to single mutants, double mutants, or SU5402-treated embryos.
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pone-0113829-g009: fgf3/lia FGF signaling mediates the increase in caudal hypothalamic DC7 DA neurons in cnot8m1061 mutant embryos.(A–F) Analysis of DA neurons in cnot8m1061 mutant embryos combined with a mutation in the fgf3 locus (liat24152) or pharmacological suppression of FGF signaling by SU5402. Embryos were fixed at 4 dpf, stained by anti TH immunofluorescence and DA neurons documented by confocal microscopy. Shown are Z-projections of confocal stacks representing the ventral diencephalic DA groups 1 to 7 (dorsal views). Scale bar 50 µm. (A–D) DA neurons in WT, cnot8m1061 or liat24152 single mutant, and cnot8m1061, liat24152 double mutant embryos. Double mutant embryos show loss of cnot8m1061-mediated increase of DC7 caudal hypothalamic DA neurons. (E, F) DA neurons in WT and cnot8m1061 mutant embryos treated with SU5402 from 42 to 48 hpf. Inhibition of FGF signaling by SU5402 reduces the number of DC7 caudal hypothalamic DA neurons in cnot8m1061 mutants below WT levels. (G) Quantification of effects on CA neurons by cell counting of forebrain DA neuronal clusters and locus coeruleus NA neurons in genetic and experimental conditions as indicated in the index at right. Each bar shows the average number of CA neurons in five independent embryos for each experimental condition. Error bars indicate standard deviation. Average DC7 cell numbers: cnot8+/+ lia +/+ 50.8 (WT control); cnot8-/- lia +/+ 103.2; cnot8-/- lia -/- 52.6; cnot8+/+ lia -/- (38.8); cnot8+/+ SU5402 20.6; cnot8-/-SU5402 56.2. Significance was evaluated by Mann-Whitney test. The cell count in single mutant cnot8m1061 (-/-)liat24152 (+/+) is significantly different from single mutant cnot8m1061 (+/+) liat24152 (-/-) embryos (p = 0.008). Comparison of cnot8m1061, liat24152 double mutant and WT embryos reveal no significant difference (p = 0.45). For SU5402 treatments, the number of DC7 DA neurons differs significantly between WT controls and SU5402 treated WT (p = 0.008) and between cnot8m1061 and SU5402-treated cnot8m1061 embryos (p = 0.008). For all other catecholaminergic groups, no significant differences were observed when WT was compared to single mutants, double mutants, or SU5402-treated embryos.

Mentions: To determine whether enhanced FGF signaling pathway activity may contribute to formation of supernumerary DC7 DA neurons, we treated cnot8m1061 embryos with SU5402, an inhibitor of FGF signaling [66]. SU5402 was applied to embryos for 6 hours from 42 to 48 hpf, the developmental period during which a significant amount of DC7 DA neurons becomes postmitotic [44]. SU5402 treatment did not result in any significant changes in cell numbers of DC1-6 (compare Figure 9A and E). cnot8m1061 mutants had an average of 103.2 DC7 DA neurons, which is a 2-fold increase in DC7 cells in comparison to WT siblings (compare Figure 9A' and B'; p = 0.008). SU5402 treated cnot8m1061 mutant embryos developed an average of 56.2 DC7 neurons, while SU5402 treated WT siblings contained an average of 20.6 DC7 neurons (compare Figure 9B' and F'; p = 0.008). Thus cnot8m1061 mutants treated with SU5402 had only half the amount of DC7 neurons in comparison to non-treated m1061 mutants (p = 0.008). However, cnot8m1061 mutants treated with SU5402 still showed a 2.6 fold increase in cell number in comparison to SU5402 treated WT siblings (compare Figure 9E' and F'; p = 0.008). The significant reduction of DA neurons in both cnot8m1061 and WT embryos by SU5402 treatment by about 50% indicates that FGF signaling may be involved in formation of DC7 DA neurons. The increase in DA neuron number in cnot8m1061 may in part be mediated by enhanced FGF signaling.


A mutation in cnot8, component of the Ccr4-not complex regulating transcript stability, affects expression levels of developmental regulators and reveals a role of Fgf3 in development of caudal hypothalamic dopaminergic neurons.

Koch P, Löhr HB, Driever W - PLoS ONE (2014)

fgf3/lia FGF signaling mediates the increase in caudal hypothalamic DC7 DA neurons in cnot8m1061 mutant embryos.(A–F) Analysis of DA neurons in cnot8m1061 mutant embryos combined with a mutation in the fgf3 locus (liat24152) or pharmacological suppression of FGF signaling by SU5402. Embryos were fixed at 4 dpf, stained by anti TH immunofluorescence and DA neurons documented by confocal microscopy. Shown are Z-projections of confocal stacks representing the ventral diencephalic DA groups 1 to 7 (dorsal views). Scale bar 50 µm. (A–D) DA neurons in WT, cnot8m1061 or liat24152 single mutant, and cnot8m1061, liat24152 double mutant embryos. Double mutant embryos show loss of cnot8m1061-mediated increase of DC7 caudal hypothalamic DA neurons. (E, F) DA neurons in WT and cnot8m1061 mutant embryos treated with SU5402 from 42 to 48 hpf. Inhibition of FGF signaling by SU5402 reduces the number of DC7 caudal hypothalamic DA neurons in cnot8m1061 mutants below WT levels. (G) Quantification of effects on CA neurons by cell counting of forebrain DA neuronal clusters and locus coeruleus NA neurons in genetic and experimental conditions as indicated in the index at right. Each bar shows the average number of CA neurons in five independent embryos for each experimental condition. Error bars indicate standard deviation. Average DC7 cell numbers: cnot8+/+ lia +/+ 50.8 (WT control); cnot8-/- lia +/+ 103.2; cnot8-/- lia -/- 52.6; cnot8+/+ lia -/- (38.8); cnot8+/+ SU5402 20.6; cnot8-/-SU5402 56.2. Significance was evaluated by Mann-Whitney test. The cell count in single mutant cnot8m1061 (-/-)liat24152 (+/+) is significantly different from single mutant cnot8m1061 (+/+) liat24152 (-/-) embryos (p = 0.008). Comparison of cnot8m1061, liat24152 double mutant and WT embryos reveal no significant difference (p = 0.45). For SU5402 treatments, the number of DC7 DA neurons differs significantly between WT controls and SU5402 treated WT (p = 0.008) and between cnot8m1061 and SU5402-treated cnot8m1061 embryos (p = 0.008). For all other catecholaminergic groups, no significant differences were observed when WT was compared to single mutants, double mutants, or SU5402-treated embryos.
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pone-0113829-g009: fgf3/lia FGF signaling mediates the increase in caudal hypothalamic DC7 DA neurons in cnot8m1061 mutant embryos.(A–F) Analysis of DA neurons in cnot8m1061 mutant embryos combined with a mutation in the fgf3 locus (liat24152) or pharmacological suppression of FGF signaling by SU5402. Embryos were fixed at 4 dpf, stained by anti TH immunofluorescence and DA neurons documented by confocal microscopy. Shown are Z-projections of confocal stacks representing the ventral diencephalic DA groups 1 to 7 (dorsal views). Scale bar 50 µm. (A–D) DA neurons in WT, cnot8m1061 or liat24152 single mutant, and cnot8m1061, liat24152 double mutant embryos. Double mutant embryos show loss of cnot8m1061-mediated increase of DC7 caudal hypothalamic DA neurons. (E, F) DA neurons in WT and cnot8m1061 mutant embryos treated with SU5402 from 42 to 48 hpf. Inhibition of FGF signaling by SU5402 reduces the number of DC7 caudal hypothalamic DA neurons in cnot8m1061 mutants below WT levels. (G) Quantification of effects on CA neurons by cell counting of forebrain DA neuronal clusters and locus coeruleus NA neurons in genetic and experimental conditions as indicated in the index at right. Each bar shows the average number of CA neurons in five independent embryos for each experimental condition. Error bars indicate standard deviation. Average DC7 cell numbers: cnot8+/+ lia +/+ 50.8 (WT control); cnot8-/- lia +/+ 103.2; cnot8-/- lia -/- 52.6; cnot8+/+ lia -/- (38.8); cnot8+/+ SU5402 20.6; cnot8-/-SU5402 56.2. Significance was evaluated by Mann-Whitney test. The cell count in single mutant cnot8m1061 (-/-)liat24152 (+/+) is significantly different from single mutant cnot8m1061 (+/+) liat24152 (-/-) embryos (p = 0.008). Comparison of cnot8m1061, liat24152 double mutant and WT embryos reveal no significant difference (p = 0.45). For SU5402 treatments, the number of DC7 DA neurons differs significantly between WT controls and SU5402 treated WT (p = 0.008) and between cnot8m1061 and SU5402-treated cnot8m1061 embryos (p = 0.008). For all other catecholaminergic groups, no significant differences were observed when WT was compared to single mutants, double mutants, or SU5402-treated embryos.
Mentions: To determine whether enhanced FGF signaling pathway activity may contribute to formation of supernumerary DC7 DA neurons, we treated cnot8m1061 embryos with SU5402, an inhibitor of FGF signaling [66]. SU5402 was applied to embryos for 6 hours from 42 to 48 hpf, the developmental period during which a significant amount of DC7 DA neurons becomes postmitotic [44]. SU5402 treatment did not result in any significant changes in cell numbers of DC1-6 (compare Figure 9A and E). cnot8m1061 mutants had an average of 103.2 DC7 DA neurons, which is a 2-fold increase in DC7 cells in comparison to WT siblings (compare Figure 9A' and B'; p = 0.008). SU5402 treated cnot8m1061 mutant embryos developed an average of 56.2 DC7 neurons, while SU5402 treated WT siblings contained an average of 20.6 DC7 neurons (compare Figure 9B' and F'; p = 0.008). Thus cnot8m1061 mutants treated with SU5402 had only half the amount of DC7 neurons in comparison to non-treated m1061 mutants (p = 0.008). However, cnot8m1061 mutants treated with SU5402 still showed a 2.6 fold increase in cell number in comparison to SU5402 treated WT siblings (compare Figure 9E' and F'; p = 0.008). The significant reduction of DA neurons in both cnot8m1061 and WT embryos by SU5402 treatment by about 50% indicates that FGF signaling may be involved in formation of DC7 DA neurons. The increase in DA neuron number in cnot8m1061 may in part be mediated by enhanced FGF signaling.

Bottom Line: Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes.We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased.Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of developmental control genes.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Institute Biology I, Faculty of Biology University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany.

ABSTRACT
While regulation of the activity of developmental control genes at the transcriptional level as well as by specific miRNA-based degradation are intensively studied, little is known whether general cellular mechanisms controlling mRNA decay may contribute to differential stability of mRNAs of developmental control genes. Here, we investigate whether a mutation in the deadenylation dependent mRNA decay pathway may reveal differential effects on developmental mechanisms, using dopaminergic differentiation in the zebrafish brain as model system. In a zebrafish genetic screen aimed at identifying genes controlling dopaminergic neuron development we isolated the m1061 mutation that selectively caused increased dopaminergic differentiation in the caudal hypothalamus, while other dopaminergic groups were not affected. Positional cloning revealed that m1061 causes a premature stop codon in the cnot8 open reading frame. Cnot8 is a component of the Ccr4-Not complex and displays deadenylase activity, which is required for removal of the poly (A) tail in bulk mRNA turnover. Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes. We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased. Pharmacological inhibition of FGF signaling or a mutation in the fgf3 gene can compensate the gain of caudal hypothalamic dopaminergic neurons in cnot8m1061 mutants, indicating a role for Fgf3 in control of development of this dopaminergic population. The cnot8m1061 mutant phenotype provides an in vivo system to study roles of the Cnot8 deadenylase component of the mRNA decay pathway in vertebrate development. Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of developmental control genes.

Show MeSH
Related in: MedlinePlus