Figures and data
Gq and Gs pathways are required for Ciona metamorphosis.
(A) Schematic illustration of Ciona metamorphosis.
(B) A larva knocked down with Gαq using the antisense morpholino oligonucleotide (MO). Metamorphosis did not initiate at 2 days post-fertilization (2 dpf).
(C) A Gαs knockdown larva.
(D) A control animal injected with the standard (STD) MO. Metamorphosis initiated, as indicated by the completion of tail regression.
(E) Effect of Gαq knockdown on the percentage of metamorphosis initiation (indicated by the initiation of tail regression), shown as box-and-whisker plots. Dots indicate experiment replicates. ***, p<0.001 (Fisher’s exact test). n, number of examined larvae in total.
(F) Effect of Gαs knockdown.
(G-I) Effects of PLCβ1/2/3, PLCβ4, and IP3R knockdowns.
(J-M) Results of the experiments where adhesion was prevented by tail amputation and laying larvae on agar-coated plates.
(J) A control larva overexpressing the Kaede reporter in the entire nervous system using the PC2 cis element.
(K) A larva overexpressing constitutive active (ca)PLCβ1/2/3.
(L) Effect of caGαq overexpression.
(M) Effect of caPLCβ1/2/3 overexpression.
(N-O) Live imaging of Ca2+ transient, monitored by injecting GCaMP8 mRNA.
(N) Snapshot of the control larva injected with GCaMP8 mRNA. An increase in the fluorescence intensity in the adhesive papillae (arrow) was observed. Rod, the position of the glass rod used to stimulate papillae. The percentage and number exhibit the rate of animals showing Ca2+ transient in the papillae.
(O) A larva injected with Gαq MO plus GCaMP8 mRNA. An increase in the fluorescence intensity in the papillae (arrow) was not observed even though the brightness of the whole body was raised. See also Figure S1.
cAMP initiates Ciona metamorphosis.
(A) An adhesion-prevented control (uninjected) larva at 2 dpf. Metamorphosis did not initiate.
(B) An animal with adhesion prevention and overexpression of caGαs in the entire nervous system. Metamorphosis initiated.
(C) Effect of caGαs overexpression.
(D) A control (uninjected) larva.
(E) A theophylline-treated animal. Metamorphosis initiated.
(F) Effect of theophylline treatment.
(G) Effect of bPAC overexpression in the nervous system.
(H) Effect of bPAC overexpression over time. In this experiment, adhesion was permitted. Control groups (overexpressed with mCherry) exhibited a slower initiation of metamorphosis than bPAC-overexpressed groups.
Gq and Gs pathways are activated in the adhesive papillae to initiate metamorphosis.
(A) A theophylline-treated animal. Metamorphosis initiated.
(B) A papilla-amputated larva treated with theophylline. Metamorphosis did not initiate.
(C) Effect of papilla amputation on theophylline treatment.
(D) Effects of papilla amputation on caGαq, caPLCβ1/2/3, caGαs, and bPAC overexpression.
(E) Measurement of cAMP concentration, as indicated by the fluorescence of Pink Flamindo. Left, the quantification of Pink Flamindo fluorescent intensity is shown for a larva that initiated metamorphosis at the time indicated by an arrowhead. The time of stimulation of the adhesive papillae with a glass rod is denoted by a bar. Right, the position of the region of interest (ROI). See also Figure S3.
Relationships between Gq and Gs pathways in metamorphosis.
(A) A Gαq knockdown larva.
(B) Theophylline ameliorated the effect of Gαq knockdown.
(C) Effect of theophylline on Gαq knockdown.
(D) Effect of theophylline on PLCβ knockdown. In this experiment, PLCβ1/2/3 and PLCβ4 were knocked down simultaneously.
(E) Effect of theophylline on IP3R knockdown.
(F) Effect of caGαs overexpression on Gαq knockdown.
(G) Gαs is not necessary for Ca2+ transient in the adhesive papillae (arrow) upon adhesion.
(H) A Gαq knockdown and Kaede-overexpressed larva.
(I) A Gαq knockdown and caPLCβ1/2/3-overexpressed larva.
(J) A Gαs knockdown and caPLCβ1/2/3-overexpressed larva.
(K) Effect of caPLCβ1/2/3 overexpression on Gαq knockdown.
(L) Effect of caPLCβ1/2/3 overexpression on Gαs knockdown.
(M) Effect of caGαq overexpression on Gαs knockdown.
(N) Effect of theophylline treatment on Gαs knockdown.
GABA functions in the adhesive papillae to induce metamorphosis.
(A) A GABABR1 knockdown and mCherry-overexpressed larva.
(B) A GABABR1 knockdown and caPLCβ1/2/3-overexpressed larva. Metamorphosis initiated.
(C) Effect of caPLCβ1/2/3 overexpression on GABABR1 knockdown.
(D) Effect of theophylline on GABABR1 and GAD knockdowns.
(E) GABA is necessary for Ca2+ transient in the adhesive papillae (arrow).
(F) Effect of GABA on Gαq knockdown.
(G) Effect of GABA on PLCβ knockdown.
(H) Effect of GABA on Gαs knockdown. See also Figure S4.
Gi is required for metamorphosis initiation.
(A) Effect of Gαi knockdown.
(B) A control animal injected with STD MO.
(C) A dvGαi_Chr2 knockdown larva.
(D) A dvGαi_Chr2 knockdown and theophylline-treated animal.
(E) Effects of GABA and theophylline on dvGαi_Chr2 knockdown.
(F) Effects of wtGαi and wtGαs overexpression.
(G) Effect of dnGαi overexpression.
(H) Effect of GABABR1 and Gαq simultaneous knockdown.
(I) Effects of caPLCβ1/2/3 and caGαs overexpressions on GABABR1 and Gαq simultaneous knockdown.
(J) GIRK knockdown promoted metamorphosis initiation.
(K) Effect of U0126 on GABA treatment.
(L) Effect of U0126 on theophylline treatment. See also Figure S5.
Schematic illustration of the signaling cascades for initiating Ciona metamorphosis. AC, adenylate cyclase; ER, endoplasmic reticulum; GPCR, G-protein coupled receptor; PKA, protein kinase A.
Ciona phospholipase β proteins
Phylogenetic relationships between PLCβ proteins, as revealed by the maximum likelihood method. The number beside each branch indicates the percentage of times that a node was supported in 1,000 bootstrap pseudoreplications. Scores equal to or exceeding 50% are shown. Ciona PLCβs are indicated by black circles. Human homologs are indicated by accession numbers in the Uniprot database (https://www.uniprot.org) and “HS” for Homo sapiens.
Ciona phosphodiesterase repertoire
(A) Phylogenetic relationships between PDEs. Black circles and gene models indicate Ciona protein models in the Ghost database (http://ghost.zool.kyoto-u.ac.jp/default_ht.html). The numbers and letters in parentheses indicate the PDE groups and their suspected substrates. A, cAMP; G, cGMP; D, dual substrate. Human and insect proteins are shown by Uniprot (https://www.uniprot.org) accession numbers, as well as “HS” for Homo sapiens and “DM” for Drosophila melanogaster proteins.
(B) Expression profiles of the genes related to Gq and Gs pathways, as revealed by RNA-seq of the papilla region. RPKM, reads per kilobase of exon per million mapped reads. See also Table S1.
cAMP increase in the papillae is coupled with metamorphosis initiation
Measurement of cAMP concentration in the larva that did not initiate metamorphosis upon stimulation of the papillae, as indicated by the fluorescence of Pink Flamindo. Left, the quantification of Pink Flamindo fluorescent intensity of a larva that did not initiate metamorphosis; right, the position of the region of interest (ROI).
GABA stimulates the adhesive papillae to initiate metamorphosis.
(A) A GABA-treated animal initiated metamorphosis.
(B) A papilla-amputated larva treated with GABA. Metamorphosis was not initiated.
(C) Effect of papilla amputation on GABA treatment.
(D-H) The sensory vesicle is not required for the anterior region to respond to GABA.
(D) Schematic of the experiment.
(E) A control anterior fragment at 2 dpf.
(F) Anterior fragment treated with GABA. The percentage indicates the proportion of the fragments exhibiting the morphological signature of metamorphosis.
(G) Anterior fragment treated with theophylline.
(H) Expression levels of two GABA-responsive genes in the control- and GABA-treated anterior fragments, as revealed by quantitative RT-PCR.
Ciona has atypical Gαi proteins.
(A) Phylogenetic relationships between Gα proteins, as revealed by the maximum likelihood method. The three atypical Gα proteins are highlighted in red.
(B) Comparison of the five C-terminal amino acid sequences of Gα proteins that are shown to be essential for their functions, including two additional amino acids for dvGαi_Chr2. Residues conserved with human Gαi1 are shown in red.
Autonomous activity of wild-type Gαq.
(A) Effect of wtGαq overexpression in the adhesion-permitted condition.
(B) Effect of wtGαq overexpression in the adhesion-prevented condition.
(C) Effect of wtGαq overexpression on Gαs knockdown.
(D) Morphology of caGαq-overexpressed larva at 1 dpf (before initiation of metamorphosis).
(E) Morphology of wtGαq-overexpressed larva at 1 dpf.
(F) Effect of dnGαq overexpression in the adhesion-permitted condition.
Expression levels of genes related to G-protein signaling during metamorphosis, as revealed by RNA-seq of the larval anterior tip including papillae
