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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 gene expression is increased in cnot8m1061 mutants.Analysis of fgf3 gene expression in cnot8m1061 mutants and WT siblings 2 dpf (A, B), and 3 dpf (C, D). All pictures show lateral views. Embryos were genotyped by PCR. Scale bar 100 µm.
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pone-0113829-g006: fgf3 gene expression is increased in cnot8m1061 mutants.Analysis of fgf3 gene expression in cnot8m1061 mutants and WT siblings 2 dpf (A, B), and 3 dpf (C, D). All pictures show lateral views. Embryos were genotyped by PCR. Scale bar 100 µm.

Mentions: The DA phenotype in the caudal hypothalamus was qualitatively different from other CA groups in that not just the th expression level was increased, but also the number of caudal hypothalamic neurons appeared increased. Therefore signaling pathways controlling DA differentiation in this anatomical region may be affected. FGF signaling namely through Fgf3 has been demonstrated to be important in caudal hypothalamus development [63]–[65]. Expression analysis of fgf3 by WISH and quantification of signal intensity revealed that cnot8m1061 mutants have a mild increase of fgf3 WISH intensity already at 2 dpf (Figure 6A, B), both in the mid-hindbrain boundary expression domain (p = 0.008) and in the caudal hypothalamus (p = 0.008). fgf3 WISH signal continues to be enhanced in both domains at 3 dpf (Figure 6C, D; MHB p = 0.008. cHyp p = 0.004). We also analyzed expression of FGF receptor encoding genes fgfr1, 2, 3 and 4 in cnot8m1061 embryos. While at 1 dpf WISH signal intensities of fgfr1, 2, 3 and 4 are not significantly altered in cnot8m1061 mutants (Figure 7A–H), at 2 dpf fgfr1 and fgfr2 showed increased WISH stain intensities in mutants (Figure 7I–P; fgfr1 ventral hindbrain domain p = 0.008; fgfr2 dorsal forebrain and midbrain p = 0.008), while fgfr3 and fgfr4 WISH signal levels were not altered in comparison to WT siblings. WISH signals of fgfr1, 2, 3, and 4 were up-regulated in cnot8m1061 mutants at 3 dpf (Figure 7Q–X; fgfr1 lower jaw p = 0.031; fgfr2 dorsal forebrain/midbrain p = 0.016; fgfr3 hindbrain p = 0.016; fgfr4 dorsal midbrain p = 0.008).


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 gene expression is increased in cnot8m1061 mutants.Analysis of fgf3 gene expression in cnot8m1061 mutants and WT siblings 2 dpf (A, B), and 3 dpf (C, D). All pictures show lateral views. Embryos were genotyped by PCR. Scale bar 100 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257555&req=5

pone-0113829-g006: fgf3 gene expression is increased in cnot8m1061 mutants.Analysis of fgf3 gene expression in cnot8m1061 mutants and WT siblings 2 dpf (A, B), and 3 dpf (C, D). All pictures show lateral views. Embryos were genotyped by PCR. Scale bar 100 µm.
Mentions: The DA phenotype in the caudal hypothalamus was qualitatively different from other CA groups in that not just the th expression level was increased, but also the number of caudal hypothalamic neurons appeared increased. Therefore signaling pathways controlling DA differentiation in this anatomical region may be affected. FGF signaling namely through Fgf3 has been demonstrated to be important in caudal hypothalamus development [63]–[65]. Expression analysis of fgf3 by WISH and quantification of signal intensity revealed that cnot8m1061 mutants have a mild increase of fgf3 WISH intensity already at 2 dpf (Figure 6A, B), both in the mid-hindbrain boundary expression domain (p = 0.008) and in the caudal hypothalamus (p = 0.008). fgf3 WISH signal continues to be enhanced in both domains at 3 dpf (Figure 6C, D; MHB p = 0.008. cHyp p = 0.004). We also analyzed expression of FGF receptor encoding genes fgfr1, 2, 3 and 4 in cnot8m1061 embryos. While at 1 dpf WISH signal intensities of fgfr1, 2, 3 and 4 are not significantly altered in cnot8m1061 mutants (Figure 7A–H), at 2 dpf fgfr1 and fgfr2 showed increased WISH stain intensities in mutants (Figure 7I–P; fgfr1 ventral hindbrain domain p = 0.008; fgfr2 dorsal forebrain and midbrain p = 0.008), while fgfr3 and fgfr4 WISH signal levels were not altered in comparison to WT siblings. WISH signals of fgfr1, 2, 3, and 4 were up-regulated in cnot8m1061 mutants at 3 dpf (Figure 7Q–X; fgfr1 lower jaw p = 0.031; fgfr2 dorsal forebrain/midbrain p = 0.016; fgfr3 hindbrain p = 0.016; fgfr4 dorsal midbrain p = 0.008).

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