Discovery of methylfarnesoate as the annelid brain hormone reveals an ancient role of sesquiterpenoids in reproduction
- University of Vienna, Austria
Figures
Figure 1 with 2 supplements
Vitellogenin expression in eleocytes is a quantitative measure for the maturation stage of Platynereis, allowing for the establishment of a bioassay to purify the enigmatic brain hormone, nereidin.
(A) Scheme summarising the critical role of the brain hormone nereidin in energy expenditure, as derived from classical experiments: Before maturation (left), high nereidin levels sustain somatic …
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Figure 1—source data 1
Data for the graphs in Figure 1C, Figure 1—figure supplement 2A, Figure 1—figure supplement 2B (vtg expression levels over the time course of maturation).
- https://doi.org/10.7554/eLife.17126.004
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Figure 1—source data 2
Alignment file for the phylogenetic tree of Vitellogenins (Figure 1—figure supplement 1B).
- https://doi.org/10.7554/eLife.17126.005
Figure 1—figure supplement 1
Identification of a Platynereis Vitellogenin orthologue.
(A) Domain analysis for the protein encoded by the identified Platynereis cDNA (top) as well as chicken Vtg2 (middle) and mosquito Vit1 (bottom) reveals that Platynereis Vtg shares three conserved …
Figure 1—figure supplement 2
Irrespective of the chosen reference gene, vitellogenin is regulated over the course of maturation.
The figure shows the same data as Figure 1C, but with the results for the individual reference genes plotted separately. In the range relevant for the bioassay (early premature stage), the observed …
Figure 2 with 2 supplements
Methylfarnesoate, not protein, constitutes the main nereidin/brain hormone activity.
(A–C) Nereidin activity elutes in lipophilic fractions of head extracts but is not proteinaceous as previously hypothesised. (A) Schematised fractionation of premature Platynereis head extracts, …
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Figure 2—source data 1
Data for Figure 2B,C,E,F,G (vtg expression levels normalised to the respective control treatment) and Figure 2—figure supplement 2 (stability of Ct values for rps9).
- https://doi.org/10.7554/eLife.17126.009
Figure 2—figure supplement 1
Identification of MF as the principal component in peak 15.
(A,B) The panels shows the selected ion currents (SIC) in the range of m/z 251.0–251.5 (top panels) and the selected MS2-spectrum of the precursor ion at m/z 251.1 (lower panels). (A) Peak 15. Note …
Figure 2—figure supplement 2
Stability of the reference gene rps9 in coelomocyte culture.
Ct values for rps9 were normalised to the mean of each experimental series. The box shows the first and third quartile and the median (solid line). Whiskers denote the 1.5x fold range of the …
Figure 3 with 4 supplements
Levels of MF as well as the sesquiterpenoid receptor orthologue Met drop over the course of maturation.
(A) Significant decrease of MF levels between premature and mature heads; boxplots show amounts of MF normalised to the total protein content of the respective head sample. Heads of premature …
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Figure 3—source data 1
Data for the graphs in Figure 3A (MF content in pg normalised to mg protein per head), Figure 3C (met expression levels normalised to premature eleocytes) and Figure 3D (met expression levels normalised to the control treatment); data for the graphs in Figure 3—figure supplement 2 and Figure 3—figure supplement 3 (MF content in pg per head), Figure 3—figure supplement 4A and Figure 3—figure supplement 4B(met expression levels quantified against individual reference genes).
- https://doi.org/10.7554/eLife.17126.013
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Figure 3—source data 2
Alignment file for the phylogenetic tree of Met homologs (Figure 3B).
- https://doi.org/10.7554/eLife.17126.014
Figure 3—figure supplement 1
Quantification of MF in the heads of Platynereis by GC-MS.
Representative Selected Ion Mode (SIM) chromatograms from premature Platynereis heads and authentic MF (upper panel). The lower panel shows the selected ions at m/z 114.0, 136.0 and 219.0. (A) SIM …
Figure 3—figure supplement 2
The levels of MF per head drop over the course of maturation.
Significant decrease of MF levels between premature and mature heads; the boxplot shows the quantified amounts of MF normalised per head. Heads of premature animals contain on average ~ 34.6 ± 8.0 …
Figure 3—figure supplement 3
Methylfarnesoate is found in the heads of earthworms.
The head of earthworms contain on average ~ 5.3 ± 1.9 (S.E.M) pg MF per head. The data range is between ~2.1 and ~9.8 pg MF per head. The amounts are corrected for a MF recovery rate of 85%. The box …
Figure 3—figure supplement 4
The choice of reference genes does not impact on the observed down-regulation of met.
The figure shows the same data as Figure 3C, but with the results for the individual reference genes plotted separately. The observed relative expression of met in eleocytes is independent of the …
Figure 4 with 1 supplement
MF also represses vtg expression in vivo, and sustains expression of a marker for caudal growth upon posterior amputation.
(A) Setup of experiment testing the ability of MF to interfere with vitellogenesis in vivo. Following decapitation, female individuals are known to start vitellogenesis, reflecting loss of nereidin; …
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Figure 4—source data 1
Data for the graphs in Figure 4B and Figure 4C (vtg/hox3 expression levels normalised to the respective control treatment).
- https://doi.org/10.7554/eLife.17126.020
Figure 4—figure supplement 1
The choice of reference genes does not impact on the observed down-regulation of vtg.
The figure shows the same data as Figure 4B, but with the results for the individual reference genes plotted separately. The observed relative expression of vtg in whole animals treated with either …
Figure 5
Vitellogenin expression in worm coelomocytes is suppressed by the hormone agonists methoprene and pyriproxyfen that are considered to act exclusively on insects.
Expression levels of vtg in coelomocytes are significantly down-regulated by treatment with 10 nM methoprene (Meth) and 10 nM pyriproxyfen (Pyr) in 0.01% DMSO. The graph shows qRT-PCR quantification …
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Figure 5—source data 1
Data for the graph in Figure 5 (vtg expression levels normalised to the control treatment).
- https://doi.org/10.7554/eLife.17126.023
Tables
Table 1
List of primers used for qRT-PCR, including predicted melting temperature.
Name | Sequence 5’→3’ | Tm / °C | Efficiency |
|---|---|---|---|
Pdu vtg qPCR1 F | ACAGGCCATCACATTCACAA | 56.4 | 101% |
Pdu vtg qPCR1 R | TCTGCTCACGTCTCTTTCCA | 58.4 | |
Pdu met qPCR1 F | GGATGATTATGATGTATACCTGCAAC | 62.9 | 102% |
Pdu met qPCR1 R | AGACCGAACTGGCGTTTG | 56.3 | |
Pdu hox3 qPCR1 F | CTACCCCTGGATGAGGGAAT | 60.5 | 95% |
Pdu hox3 qPCR1 R | ACTTCCGGTTCCTGGTCC | 58.4 | |
Pdu rps9 F | CGCCAGAGAGTTGCTGACT | 59.5 | 102% |
Pdu rps9 R | ACTCCAATACGGACCAGACG | 60.5 | |
Pdu sams qPCR1 F | CAGCAACGGTGAAATAACCA | 56.4 | 101% |
Pdu sams qPCR1 R | CATCACTCACTTGATCGCAAA | 57.5 | |
Pdu cim6pr qPCR1 F | ACTTCCCCTGCTGATGAGTG | 60.5 | 99% |
Pdu cim6pr qPCR1 R | TTCGTAAGTCAGGTTTCCATTG | 58.4 | |
Pdu cdc5 F | CCTATTGACATGGACGAAGATG | 60.1 | 100% |
Pdu cdc5 R | TTCCCTGTGTGTTCGCAAG | 57.5 |
Table 2
List of primers used for cloning and sequence validation.
Name | Sequence 5’→3’ | Tm / °c |
|---|---|---|
Pdu vtg 1F | ATGAAGACTCTCCTGATCTTCG | 60.1 |
Pdu vtg 1R | CTAGTAGTAGAATCTTGGTCCTTCAC | 64.6 |
Pdu vtg_seq 1F | AGCCCTAGAAGCTGCCTCTG | 62.5 |
Pdu vtg_seq 2F | ATTGCTCAATCTGAACTCCCATGC | 63.6 |
Pdu vtg_seq 3F | GCTGTTCCACAGGAAATTGC | 58.4 |
Pdu vtg_seq 4F | GCTTTGGTCAGTGGACTTCC | 60.5 |
Pdu vtg_seq 1R | GGCAATCCTCTGATGTAAACATTCTC | 64.6 |
Pdu vtg_seq 2R | CAAGCGTTTCACGACCAAGAGG | 64.2 |
Pdu vtg_seq 3R | GAAGAGCTTCTTGCTGGAGC | 60.5 |
Pdu vtg_seq 4R | AAGACCAGCTGGCGCGTTATG | 63.2 |
Pdu met F | ATGGAGCCGAATTCGGAGCAGAATTCGG | 71.8 |
Pdu met R | TCAACATGTCTCAGTTTCTTTTTGAGCG | 65.6 |
Pdu hox3 F | CCCCGGGGCTCTTGGTTTT | 61.6 |
Pdu hox3 R | GCCATCTCTATTCTCCTCGGCCG | 68.3 |
