Discovery of methylfarnesoate as the annelid brain hormone reveals an ancient role of sesquiterpenoids in reproduction

5 figures and 2 tables

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 …

https://doi.org/10.7554/eLife.17126.003
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
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 …

https://doi.org/10.7554/eLife.17126.006
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 …

https://doi.org/10.7554/eLife.17126.007
Figure 2 with 2 supplements
Methylfarnesoate, not protein, constitutes the main nereidin/brain hormone activity.

(AC) Nereidin activity elutes in lipophilic fractions of head extracts but is not proteinaceous as previously hypothesised. (A) Schematised fractionation of premature Platynereis head extracts, …

https://doi.org/10.7554/eLife.17126.008
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 …

https://doi.org/10.7554/eLife.17126.010
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 …

https://doi.org/10.7554/eLife.17126.011
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 …

https://doi.org/10.7554/eLife.17126.012
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
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 …

https://doi.org/10.7554/eLife.17126.015
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 …

https://doi.org/10.7554/eLife.17126.016
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 …

https://doi.org/10.7554/eLife.17126.017
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 …

https://doi.org/10.7554/eLife.17126.018
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; …

https://doi.org/10.7554/eLife.17126.019
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 …

https://doi.org/10.7554/eLife.17126.021
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 …

https://doi.org/10.7554/eLife.17126.022
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.

https://doi.org/10.7554/eLife.17126.024

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.

https://doi.org/10.7554/eLife.17126.025

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

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