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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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The live phenotype and positional cloning of the cnot8m1061 mutation.(A) Comparison of cnot8m1061 mutant to wild-type sibling embryos at 5 dpf; cnot8m1061 mutants display edema formation in the eyes and brain (top row, arrowheads) as well as cardiac and yolk sac (bottom row, arrowhead). Top row dorsal view, bottom row lateral view, anterior to the left. Scale bar 100 µm. (B) Scheme of the m1061 genetic interval. Gene and marker positions were obtained from Ensembl Zv8. CR855270.17 p9 and BX927237 p4 define the interval which comprises the genes zgc:77151, MRP L22, gemin5, cnot8, kibra/WWC1, ARL10 and Nop16 (data not shown). Proximal SSLP markers are highlighted in blue. Distal SSLP markers are highlighted in brown. Numbers provided with SSLP markers reflect recombination events identified per number of meioses analyzed. (C) Sequencing of genomic DNA amplified by PCR from individual m1061 homozygous WT and mutant embryos. The C-to-T mutation at bp 82 of the ORF results in the formation of a premature stop codon in m1061 mutant cnot8 ORF. Numbers (79–87) indicate ORF bp position. (D) Zebrafish Cnot8 comprises 286 amino acids and contains an RNAse D domain which is truncated in cnot8m1061 mutants. (E–J) Expression analysis by WISH using cnot8 antisense probes. Sense controls processed exactly in parallel with the antisense reactions are shown as insets in E, F, G and H to evaluate background stain intensities. (E) Maternal mRNA is detected at 4 cell stage (dorsal view). (F) Ubiquitous expression of cnot8 mRNA at 4 hpf (lateral view). (G, H, I) cnot8 continues to be expressed ubiquitously at 50% epiboly, 1, 2 and 3 dpf (lateral views). Scale bars 100 µm.
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pone-0113829-g002: The live phenotype and positional cloning of the cnot8m1061 mutation.(A) Comparison of cnot8m1061 mutant to wild-type sibling embryos at 5 dpf; cnot8m1061 mutants display edema formation in the eyes and brain (top row, arrowheads) as well as cardiac and yolk sac (bottom row, arrowhead). Top row dorsal view, bottom row lateral view, anterior to the left. Scale bar 100 µm. (B) Scheme of the m1061 genetic interval. Gene and marker positions were obtained from Ensembl Zv8. CR855270.17 p9 and BX927237 p4 define the interval which comprises the genes zgc:77151, MRP L22, gemin5, cnot8, kibra/WWC1, ARL10 and Nop16 (data not shown). Proximal SSLP markers are highlighted in blue. Distal SSLP markers are highlighted in brown. Numbers provided with SSLP markers reflect recombination events identified per number of meioses analyzed. (C) Sequencing of genomic DNA amplified by PCR from individual m1061 homozygous WT and mutant embryos. The C-to-T mutation at bp 82 of the ORF results in the formation of a premature stop codon in m1061 mutant cnot8 ORF. Numbers (79–87) indicate ORF bp position. (D) Zebrafish Cnot8 comprises 286 amino acids and contains an RNAse D domain which is truncated in cnot8m1061 mutants. (E–J) Expression analysis by WISH using cnot8 antisense probes. Sense controls processed exactly in parallel with the antisense reactions are shown as insets in E, F, G and H to evaluate background stain intensities. (E) Maternal mRNA is detected at 4 cell stage (dorsal view). (F) Ubiquitous expression of cnot8 mRNA at 4 hpf (lateral view). (G, H, I) cnot8 continues to be expressed ubiquitously at 50% epiboly, 1, 2 and 3 dpf (lateral views). Scale bars 100 µm.

Mentions: During a systematic genetic screen for mutations affecting development of catecholaminergic (CA) neurons in zebrafish, we identified the m1061 mutation, which was unique in that it was the only mutation isolated which appeared to cause enhanced differentiation of selected CA neuronal groups. In the screen, which was based on detecting tyrosine hydroxylase mRNA expression as marker for CA neurons, the m1061 mutation gave rise to a stronger and expanded whole mount in situ hybridization (WISH) signal for th expression, likely reflecting higher th mRNA levels and potentially also an elevated number of DA and NA neurons. At 1 and 2 days post fertilization (dpf), (Figure 1A–F) m1061 mutants display a th expression pattern indistinguishable from wild-type siblings. At 3 dpf, a stronger in situ hybridization signal for th expression was observed in the diencephalic DA cell clusters of m1061 mutants. This phenotype was especially pronounced in the caudal hypothalamic diencephalic dopaminergic cluster 7 (DC7; Figure 1G–J, arrowhead in I, J). Most other DA groups in the forebrain developed normally up to 4 dpf, with the exception of the pretectal cluster, which showed a minor increase in stain intensity (Figure 1K, L), and the retinal amacrine DA neurons, which appear to be severely reduced in m1061 mutant embryos (Figure 1M, N). We also analyzed NA neuronal clusters, which revealed that the th signal for medulla oblongata (MO) and sympathetic NA neurons appeared to be stronger in m1061 mutant embryos (Figure 1I and asterisk in J). The NA neurons of the locus coeruleus (LC) did not appear to be affected in m1061 mutants. Whole mount immunofluorescence detection of TH was performed and confocal stacks representing the whole ventral diencephalon and rostral hindbrain were analyzed for cell numbers of diencephalic DA cell clusters and the NA cells of the LC at 4 dpf (Figure 1O–Q). As DC 4, 5 and 6 are difficult to distinguish due to close anatomical location, neurons of these groups were combined for analysis. The cell counts were analyzed for statistical significant differences in mutant embryos using the Mann-Whitney test. In m1061 mutant embryos, a strong increase of DA cell numbers was only found for the caudal hypothalamic DC7 DA group (p = 0.008). DA neurons of DC7 showed a more than 2-fold increase in number in m1061 mutants when compared to wild-type siblings (Figure 1Q). Milder increases in cell number were found in DA DC1 (p = 0.032) and 3 (p = 0.008; Figure 2C). The cell numbers in other DA cell clusters like DC2, 4, 5, 6 and the LC were not significantly different between m1061 embryos and controls.


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)

The live phenotype and positional cloning of the cnot8m1061 mutation.(A) Comparison of cnot8m1061 mutant to wild-type sibling embryos at 5 dpf; cnot8m1061 mutants display edema formation in the eyes and brain (top row, arrowheads) as well as cardiac and yolk sac (bottom row, arrowhead). Top row dorsal view, bottom row lateral view, anterior to the left. Scale bar 100 µm. (B) Scheme of the m1061 genetic interval. Gene and marker positions were obtained from Ensembl Zv8. CR855270.17 p9 and BX927237 p4 define the interval which comprises the genes zgc:77151, MRP L22, gemin5, cnot8, kibra/WWC1, ARL10 and Nop16 (data not shown). Proximal SSLP markers are highlighted in blue. Distal SSLP markers are highlighted in brown. Numbers provided with SSLP markers reflect recombination events identified per number of meioses analyzed. (C) Sequencing of genomic DNA amplified by PCR from individual m1061 homozygous WT and mutant embryos. The C-to-T mutation at bp 82 of the ORF results in the formation of a premature stop codon in m1061 mutant cnot8 ORF. Numbers (79–87) indicate ORF bp position. (D) Zebrafish Cnot8 comprises 286 amino acids and contains an RNAse D domain which is truncated in cnot8m1061 mutants. (E–J) Expression analysis by WISH using cnot8 antisense probes. Sense controls processed exactly in parallel with the antisense reactions are shown as insets in E, F, G and H to evaluate background stain intensities. (E) Maternal mRNA is detected at 4 cell stage (dorsal view). (F) Ubiquitous expression of cnot8 mRNA at 4 hpf (lateral view). (G, H, I) cnot8 continues to be expressed ubiquitously at 50% epiboly, 1, 2 and 3 dpf (lateral views). Scale bars 100 µm.
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Related In: Results  -  Collection

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pone-0113829-g002: The live phenotype and positional cloning of the cnot8m1061 mutation.(A) Comparison of cnot8m1061 mutant to wild-type sibling embryos at 5 dpf; cnot8m1061 mutants display edema formation in the eyes and brain (top row, arrowheads) as well as cardiac and yolk sac (bottom row, arrowhead). Top row dorsal view, bottom row lateral view, anterior to the left. Scale bar 100 µm. (B) Scheme of the m1061 genetic interval. Gene and marker positions were obtained from Ensembl Zv8. CR855270.17 p9 and BX927237 p4 define the interval which comprises the genes zgc:77151, MRP L22, gemin5, cnot8, kibra/WWC1, ARL10 and Nop16 (data not shown). Proximal SSLP markers are highlighted in blue. Distal SSLP markers are highlighted in brown. Numbers provided with SSLP markers reflect recombination events identified per number of meioses analyzed. (C) Sequencing of genomic DNA amplified by PCR from individual m1061 homozygous WT and mutant embryos. The C-to-T mutation at bp 82 of the ORF results in the formation of a premature stop codon in m1061 mutant cnot8 ORF. Numbers (79–87) indicate ORF bp position. (D) Zebrafish Cnot8 comprises 286 amino acids and contains an RNAse D domain which is truncated in cnot8m1061 mutants. (E–J) Expression analysis by WISH using cnot8 antisense probes. Sense controls processed exactly in parallel with the antisense reactions are shown as insets in E, F, G and H to evaluate background stain intensities. (E) Maternal mRNA is detected at 4 cell stage (dorsal view). (F) Ubiquitous expression of cnot8 mRNA at 4 hpf (lateral view). (G, H, I) cnot8 continues to be expressed ubiquitously at 50% epiboly, 1, 2 and 3 dpf (lateral views). Scale bars 100 µm.
Mentions: During a systematic genetic screen for mutations affecting development of catecholaminergic (CA) neurons in zebrafish, we identified the m1061 mutation, which was unique in that it was the only mutation isolated which appeared to cause enhanced differentiation of selected CA neuronal groups. In the screen, which was based on detecting tyrosine hydroxylase mRNA expression as marker for CA neurons, the m1061 mutation gave rise to a stronger and expanded whole mount in situ hybridization (WISH) signal for th expression, likely reflecting higher th mRNA levels and potentially also an elevated number of DA and NA neurons. At 1 and 2 days post fertilization (dpf), (Figure 1A–F) m1061 mutants display a th expression pattern indistinguishable from wild-type siblings. At 3 dpf, a stronger in situ hybridization signal for th expression was observed in the diencephalic DA cell clusters of m1061 mutants. This phenotype was especially pronounced in the caudal hypothalamic diencephalic dopaminergic cluster 7 (DC7; Figure 1G–J, arrowhead in I, J). Most other DA groups in the forebrain developed normally up to 4 dpf, with the exception of the pretectal cluster, which showed a minor increase in stain intensity (Figure 1K, L), and the retinal amacrine DA neurons, which appear to be severely reduced in m1061 mutant embryos (Figure 1M, N). We also analyzed NA neuronal clusters, which revealed that the th signal for medulla oblongata (MO) and sympathetic NA neurons appeared to be stronger in m1061 mutant embryos (Figure 1I and asterisk in J). The NA neurons of the locus coeruleus (LC) did not appear to be affected in m1061 mutants. Whole mount immunofluorescence detection of TH was performed and confocal stacks representing the whole ventral diencephalon and rostral hindbrain were analyzed for cell numbers of diencephalic DA cell clusters and the NA cells of the LC at 4 dpf (Figure 1O–Q). As DC 4, 5 and 6 are difficult to distinguish due to close anatomical location, neurons of these groups were combined for analysis. The cell counts were analyzed for statistical significant differences in mutant embryos using the Mann-Whitney test. In m1061 mutant embryos, a strong increase of DA cell numbers was only found for the caudal hypothalamic DC7 DA group (p = 0.008). DA neurons of DC7 showed a more than 2-fold increase in number in m1061 mutants when compared to wild-type siblings (Figure 1Q). Milder increases in cell number were found in DA DC1 (p = 0.032) and 3 (p = 0.008; Figure 2C). The cell numbers in other DA cell clusters like DC2, 4, 5, 6 and the LC were not significantly different between m1061 embryos and controls.

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