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Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish.

Frohnhöfer HG, Geiger-Rudolph S, Pattky M, Meixner M, Huhn C, Maischein HM, Geisler R, Gehring I, Maderspacher F, Nüsslein-Volhard C, Irion U - Biol Open (2016)

Bottom Line: Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry.This allows us to uncouple them from events occurring later during colour patterning.Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Entwicklungsbiologie, Abteilung 3, Spemannstrasse 35, Tübingen 72076, Germany.

No MeSH data available.


Related in: MedlinePlus

Polyamine ratios in wild-type and mutant embryos. The peak area ratios (Arel) of the three polyamines measured in wild-type (n=6), idefix (ide−/−, n=4) and spermine synthase (sms−/−, n=4) mutant embryos are shown as open symbols. Peak area ratios were calculated as Ax,rel=Ax/(Aputrescine+Aspermidine+Aspermine), x=the respective polyamine. Peak area ratios were normalized to 100% for the sum of the signals in each sample to improve comparability. The mean values shown in the table are depicted as filled symbols and bars. Ionization efficiencies are: putrescine<spermine∼spermidine.
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BIO018721F8: Polyamine ratios in wild-type and mutant embryos. The peak area ratios (Arel) of the three polyamines measured in wild-type (n=6), idefix (ide−/−, n=4) and spermine synthase (sms−/−, n=4) mutant embryos are shown as open symbols. Peak area ratios were calculated as Ax,rel=Ax/(Aputrescine+Aspermidine+Aspermine), x=the respective polyamine. Peak area ratios were normalized to 100% for the sum of the signals in each sample to improve comparability. The mean values shown in the table are depicted as filled symbols and bars. Ionization efficiencies are: putrescine<spermine∼spermidine.

Mentions: To corroborate the above findings for the roles of polyamines, especially spermidine, during embryonic development and in pigment patterning we quantified the relative abundances (based on peak areas) of putrescine, spermidine and spermine in embryos. Therefore we established an analytical method based on CE-MS. Polyamine separation by CE-MS features both advantageous as well as challenging aspects compared to classical chromatographic approaches. For the analysis of fish egg samples of spermine and spermidine synthase mutants, only limited sample amounts were available, though using CE-MS allowed us to measure repetitively from sample volumes of only 15-20 µl. Furthermore, the simple extraction protocol with just hydrochloric acid and a subsequent precipitation of proteins with 70% acetonitrile, which was compatible with CE-MS analysis, proved to be straight-forward, reducing potential analyte loss during sample pretreatment to a minimum. Neither polyamine derivatization, which is commonly conducted when HPLC is used for polyamine analysis (Magnes et al., 2014); nor sample filtration (Simo et al., 2008), salt removal by e.g. solid phase extraction or other further purification steps (Lange et al., 2002) were required. We determined the ratios of the three polyamines in early embryos 4 hpf (hours post fertilization). Peak areas for each amine were determined. Fig. 8 shows the percentage for the peak area of each amine relative to the sum of all three peak areas (ionization efficiencies: putrescine<spermine∼spermidine). In the sms mutants, the spermine content was below the detection limit while the ratio between spermidine and putrescine remained unchanged compared to the wild type samples. In ide mutant embryos however, we found that the putrescine content was significantly increased due to the deactivation of spermidine synthase and accumulation of the enzyme substrate. Notably, low quantities of both spermine and spermidine are still detectable, indicating uptake of at least spermidine e.g. via food or from the water.Fig. 8.


Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish.

Frohnhöfer HG, Geiger-Rudolph S, Pattky M, Meixner M, Huhn C, Maischein HM, Geisler R, Gehring I, Maderspacher F, Nüsslein-Volhard C, Irion U - Biol Open (2016)

Polyamine ratios in wild-type and mutant embryos. The peak area ratios (Arel) of the three polyamines measured in wild-type (n=6), idefix (ide−/−, n=4) and spermine synthase (sms−/−, n=4) mutant embryos are shown as open symbols. Peak area ratios were calculated as Ax,rel=Ax/(Aputrescine+Aspermidine+Aspermine), x=the respective polyamine. Peak area ratios were normalized to 100% for the sum of the signals in each sample to improve comparability. The mean values shown in the table are depicted as filled symbols and bars. Ionization efficiencies are: putrescine<spermine∼spermidine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920196&req=5

BIO018721F8: Polyamine ratios in wild-type and mutant embryos. The peak area ratios (Arel) of the three polyamines measured in wild-type (n=6), idefix (ide−/−, n=4) and spermine synthase (sms−/−, n=4) mutant embryos are shown as open symbols. Peak area ratios were calculated as Ax,rel=Ax/(Aputrescine+Aspermidine+Aspermine), x=the respective polyamine. Peak area ratios were normalized to 100% for the sum of the signals in each sample to improve comparability. The mean values shown in the table are depicted as filled symbols and bars. Ionization efficiencies are: putrescine<spermine∼spermidine.
Mentions: To corroborate the above findings for the roles of polyamines, especially spermidine, during embryonic development and in pigment patterning we quantified the relative abundances (based on peak areas) of putrescine, spermidine and spermine in embryos. Therefore we established an analytical method based on CE-MS. Polyamine separation by CE-MS features both advantageous as well as challenging aspects compared to classical chromatographic approaches. For the analysis of fish egg samples of spermine and spermidine synthase mutants, only limited sample amounts were available, though using CE-MS allowed us to measure repetitively from sample volumes of only 15-20 µl. Furthermore, the simple extraction protocol with just hydrochloric acid and a subsequent precipitation of proteins with 70% acetonitrile, which was compatible with CE-MS analysis, proved to be straight-forward, reducing potential analyte loss during sample pretreatment to a minimum. Neither polyamine derivatization, which is commonly conducted when HPLC is used for polyamine analysis (Magnes et al., 2014); nor sample filtration (Simo et al., 2008), salt removal by e.g. solid phase extraction or other further purification steps (Lange et al., 2002) were required. We determined the ratios of the three polyamines in early embryos 4 hpf (hours post fertilization). Peak areas for each amine were determined. Fig. 8 shows the percentage for the peak area of each amine relative to the sum of all three peak areas (ionization efficiencies: putrescine<spermine∼spermidine). In the sms mutants, the spermine content was below the detection limit while the ratio between spermidine and putrescine remained unchanged compared to the wild type samples. In ide mutant embryos however, we found that the putrescine content was significantly increased due to the deactivation of spermidine synthase and accumulation of the enzyme substrate. Notably, low quantities of both spermine and spermidine are still detectable, indicating uptake of at least spermidine e.g. via food or from the water.Fig. 8.

Bottom Line: Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry.This allows us to uncouple them from events occurring later during colour patterning.Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Entwicklungsbiologie, Abteilung 3, Spemannstrasse 35, Tübingen 72076, Germany.

No MeSH data available.


Related in: MedlinePlus