Corazonin signaling integrates energy homeostasis and lunar phase to regulate aspects of growth and sexual maturation in Platynereis

Abstract

The molecular mechanisms by which animals integrate external stimuli with internal energy balance to regulate major developmental and reproductive events still remain enigmatic. We investigated this aspect in the marine bristleworm, Platynereis dumerilii, a species where sexual maturation is tightly regulated by both metabolic state and lunar cycle. Our specific focus was on ligands and receptors of the gonadotropin-releasing hormone (GnRH) superfamily. Members of this superfamily are key in triggering sexual maturation in vertebrates but also regulate reproductive processes and energy homeostasis in invertebrates. Here we show that 3 of the 4 gnrh-like (gnrhl) preprohormone genes are expressed in specific and distinct neuronal clusters in the Platynereis brain. Moreover, ligand-receptor interaction analyses reveal a single Platynereis corazonin receptor (CrzR) to be activated by CRZ1/GnRHL1, CRZ2/GnRHL2, and GnRHL3 (previously classified as AKH1), whereas 2 AKH-type hormone receptors (GnRHR1/AKHR1 and GnRHR2/AKHR2) respond only to a single ligand (GnRH2/GnRHL4). Crz1/gnrhl1 exhibits a particularly strong up-regulation in sexually mature animals, after feeding, and in specific lunar phases. Homozygous crz1/gnrhl1 knockout animals exhibit a significant delay in maturation, reduced growth, and attenuated regeneration. Through a combination of proteomics and gene expression analysis, we identify enzymes involved in carbohydrate metabolism as transcriptional targets of CRZ1/GnRHL1 signaling. Our data suggest that Platynereis CRZ1/GnRHL1 coordinates glycoprotein turnover and energy homeostasis with growth and sexual maturation, integrating both metabolic and developmental demands with the worm's monthly cycle.

Keywords: GnRH; corazonin; lunar periodicity; regeneration; reproduction.

Fig. 1.

P. dumerilii possesses both CrzR and AKHR receptor orthologs that are differentially activated by 4 GnRH superfamily ligands. (A) Maximum likelihood phylogeny supporting the assignment of the 3 Platynereis receptors (arrowheads) of the GnRHR superfamily as members of the CrzR and AKHR subfamilies. Non-Platynereis sequences were retrieved from ref. and complemented from the National Center for Biotechnology Information sequence repository. Bootstrap values, where not shown, are ≥55. (B) Receptor–ligand interactions as determined by systematic calcium mobilization assays performed with all receptor–ligand combinations. The table lists half maximal effective concentrations (EC₅₀ values) for significant interactions. See SI Appendix, Fig. S3, for individual analyses.

Fig. 2.

Transcript levels of Platynereis gnrh-like preprohormones are regulated by maturation, lunar phase, and food intake. (A) Schemes of premature worms, mature males, and mature females. (B) Relative expression levels of gnrh-like genes in premature (black), mature male (magenta), and mature female (orange) worm heads. n = 4 to 8 biological replicates. Statistical significance was tested using Kruskal–Wallis test followed by a Dunn’s multiple comparison test to adjust the P value for multiple testing. *P < 0.05. (C) Scheme showing the different sampling time points across the monthly lunar cycle (FM, full moon; FM+1, week after full moon; NM, new moon; FM-1, week preceding full moon). During the FM period, animals were exposed to 6 nights of a dimmer nocturnal light mimicking moonlight (lower bar). Black vertical bars schematize numbers of spawning wild-type animals (PIN strain) across a lunar month. Animals have been sampled between ZT7-9 in the days corresponding to the middle of the lunar phase/week. (D–G) Crz1/gnrhl1, crz2/gnrhl2, gnrhl3, and crzr are significantly up-regulated in the heads of late premature worms sampled during FM compared to animals collected in week FM − 1. n = 8 to 13 biological replicates. Statistical significance was tested using Kruskal–Wallis test followed by a Dunn’s multiple comparison test to adjust the P value for multiple testing. *P < 0.05; **P < 0.01; ***P < 0.001. (H) gnrh-like gene expression in the head of fed (spinach) or unfed (control) premature worms. Crz1/gnrhl1 is significantly more abundant in the heads of fed animals compared to unfed controls. gnrhl3 and gnrh2/gnrhl4 show only a mild trend. n = 7 to 8 biological replicates. Statistical significance was tested using Mann–Whitney U test. *P < 0.05. For all experiments, 4 to 5 heads were pooled for single biological replicates. Relative expression was calculated using cdc5 as reference gene, and data were normalized to the expression of premature worms (B), premature worms sampled during NM (D–G), and unfed worms (H).

Fig. 3.

Engineered crz1 mutants exhibit maturation delay, dampened growth rate, and posterior regeneration, while food intake is unaffected. (A) Schematized crz1/gnrhl1 locus highlighting the site in the first coding exon (exon 2) selected for targeted mutagenesis and the alignment of wild-type and crz1/gnrhl1 mutant gene sequences (Bottom). Black rectangles represent exons, and yellow sections highlight the binding sites for TALENs. Blue arrows schematize primers used for genotyping, and gray areas correspond to the restriction site around which TALENs were designed. In the DNA sequence alignment, the resulting amino acid sequences of wild-type peptide (blue) and the corresponding aberrant peptide (magenta) are boxed. (B and C) In crz1 mutants (KO), maturation is delayed compared to wild-type siblings. Data are presented as cumulative curves based on the percentage of mature animals over time (weeks) (B), as well as plotting the age at which worms reached maturation (C). Wild types are shown in blue, and mutants are shown in magenta. n ≥ 213. Statistical significance was tested using t test. ****P < 0.0001. (D and E) crz1 mutants (KO) exhibit a reduction in growth rate. (D) Length in mm of 2-mo-old young worms does not differ between crz1^−/− and ^+/+. n ≥ 232. (E) Length of 5-mo-old premature crz1^−/− and ^+/+ worms. Age-matched mutant worms are significantly smaller compared to wild-type controls. n ≥ 75. Statistical significance was tested using t test. ****P < 0.0001. (F and G) Posterior regeneration is dampened in crz1 mutants (KO). (F) Schematic representation of the experimental design. Fifteen segments have been amputated from age-matched ∼50-segment-long premature crz1^−/− and ^+/+ worms, and regenerated segments (red area) have been counted weekly for 4 wk. (G) Mutants regenerate a reduced number of segments per time unit (week) compared to wild types. Data are shown as cumulative number of regenerated segments for all of the 4 wk. n ≥ 48. Statistical significance was tested using t test. *P < 0.05; **P < 0.01. (H–J) Food intake is not affected in crz1 mutants (KO). (H) Representative spinach circle provided in the feeding experiments and representative spinach circles after a 1-d experiment. (Scale bar, 18 mm.) The eaten area was calculated subtracting the residual leaf area (Bottom) from the averaged area of 6 control leaves (Top). (I) Eaten leaf area reported for crz1^−/− and ^+/+ worms in the 2 time points (after 1 and 3 d). n = 12. (J) Food intake rate is not affected in crz1^−/− mutants. Data are reported as average time (days) required to eat completely a single spinach circle. n ≥ 25.

Fig. 4.

CRZ1/GnRHL1 contributes to glycoprotein turnover and carbohydrate homeostasis. (A) Down-regulation of lysosomal α-mannosidase in heads of crz1^−/− (KO) specimens (Right) compared to wild-type controls (Left). Plot extracted from proteomic comparison, represented as LFQ intensity. Lysosomal α-mannosidase is 1.6-fold enriched in the male ^+/+ proteome compared to the ^−/− counterpart. n = 6 to 7. (B and C) Relative mRNA levels in metabolically active tissues (trunk) of the lysosomal α-mannosidase identified by mass spectrometry analysis. This gene was strongly down-regulated in both crz1^−/− (KO) mature males and females (B), as well as in crz1^−/− (KO) premature worms (C) compared to wild-type counterparts. (D–G) crz1 knockout (KO) affects the expression of enzymes involved in glucose homeostasis at the transcript level. Relative expression levels of tobi-like 1 (D) and tobi-like 2 (E) α-glucosidases are significantly down-regulated in crz1 mutants (KO), as well as the identified pepck (F). Inversely, mRNA levels of glycogen synthase were significantly up-regulated in crz1 mutants (KO) (G). For all qRT-PCR experiments, 4 to 5 trunks were used for single biological replicate. Relative expression was calculated using sams as reference gene, and data were normalized to the expression of crz1^+/+ (wt) controls. n = 10 to 13 biological replicates. Statistical significance was tested using Mann–Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001. (H) Graphical summary of the presented data and the proposed contribution of Platynereis CRZ1/GnRHL1 signaling to the regulation of the energy balance according to developmental and environmental cues and the coordination of processes such as developmental progression and growth.