Identification of protein precursor for thyroid hormone synthesis in basal chordate ascidian Styela clava

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: major_revision

Abstract
Thyroid hormones (THs) are essential for development, growth, and metabolism in animals. Although TH synthesis is well described in vertebrates, it remains elusive in invertebrates due to the lack of identified thyroglobulin (TG) orthologs, the protein precursor for TH synthesis. Here, we identified a functional TG-like protein in ascidian Styela clava via immunoprecipitation-mass spectrometry combined with phylogenetic and expression analyses. In vitro iodination demonstrated that ScTG-like provides hormonogenic sites for TH synthesis. In vivo ScTg-like knockdown significantly reduced THs and inhibited larval metamorphosis. An invaginated follicle-like structure in the larval trunk, deposited with ScTG-like, was identified as the location for THs synthesis and storage. Furthermore, structural analysis of ScTG-like and predicted TG-like proteins across bilaterian phyla suggests that endogenous TH synthesis may be an ancestral and synapomorphic bilaterian trait. This study reports the identification of a TH precursor outside vertebrates, shedding lights on the evolution of the TH synthesis.

Introduction
In vertebrates, thyroid hormone (TH) biosynthesis involves many essential proteins, the function of which have been well described1,2. Among them, thyroglobulin (TG) is the protein precursor of TH, serving as the highly specialized matrix for iodination of specific tyrosine residues to form monoiodotyrosine (MIT) and diiodotyrosine (DIT), and for their coupling to generate triiodothyronine (T3) or thyroxine (T4)1,2. In all vertebrates, TG is a multi-domain protein with a high-molecular-weight (approximately 330 kDa)1. It consists of a signal peptide, three types of TG type domains (Tg1-3) repeated several times and separated by different linkers and one cholinesterase-like (ChEL) domain in C-terminus3. The Tg domains are essential for TG functions, including proper folding, iodination, iodine storage, and release4,5. The ChEL domain is implicated in TG maturation and trafficking, as well as the de novo synthesis of T36–8. TG proteins, in terms of their domain distribution, structure, and key hormonogenic sites, are highly conserved in vertebrates9. The current view of TG evolution holds that lampreys may be the most ancient vertebrate species with a TG protein10,11, since the orthologs of TG have not been identified outside of vertebrates.

Outside vertebrates, THs are detectable and play crucial functions in development, especially metamorphosis12–14. Yet, how invertebrates synthesize THs remains unclear, as they lack vertebrate-like TG proteins and no functional precursor has been convincingly identified. Previous studies have reported that several marine organisms harbor the ability to synthesize TH in vivo. The sea urchin and sea hare have the ability to incorporate iodine in TH formation15. In amphioxus, a subset of endostyle proteins exhibits a similar sedimentation coefficient to vertebrate TG protein, and the iodination experiments showed that this protein fraction has the ability to synthesize THs; however, the precise hormonogenic protein has not yet identified16. In ascidian, the iodoprotein(s), with a similar sedimentation coefficient to vertebrate TG, are mainly distributed in endostyle, pharynx, and tunic17–20. Endostyle is a specialized pharyngeal organ, with zone 7-9 representing the thyroid-equivalent region in ascidian adult18,21. Antibody against mammalian TG could specifically label the thyroid-equivalent region in ascidian Styela clava endostyle22, implying that the immunoreactive protein(s) may be ascidian TG-like protein(s) potentially involved in TH synthesis. However, the exact sequences of these potential iodoproteins or TG-like protein(s) and their functions in the developmental physiology have remained unexplored.

Here, we identify a functional TG-like protein with multiple Tg1 domains in the ascidian S. clava, named ScTG-like. We show that ScTG-like provides hormonogenic sites for TH production in vitro, and that ScTg-like knockdown reduces TH levels and affects larval metamorphosis in vivo. We also discovered an invaginated follicle-like structure in the larval trunk, functionally similar to vertebrate thyroid follicles. Finally, we summarize the features of ScTG-like protein and detect the similarly organized Tg1-containing proteins in multiple bilaterian phyla, supporting the idea that endogenous TH synthesis is an ancestral bilaterian feature. Together, our funding provides the direct evidence of endogenous de novo TH synthesis outside vertebrates, and illuminates the emergence of the vertebrate thyroid endocrine system.

Results
Identification of S. clava Tg1 domain-containing proteins
To determine whether ascidian process vertebrate-like TG protein, we prepared an antibody against mammalian (Bos taurus) TG protein (anti-BtTG) and performed immunofluorescence staining on S. clava endostyle. Consistent with the previous results22, the antibody specifically labeled the thyroid-equivalent region of endostyle (Fig. 1a, b), suggesting that the TG-like protein(s) may be present in S. clava. To further identify the immunolabeled protein(s), immunoprecipitation coupled with mass spectrometry (IP-MS) was conducted to precipitate the immunolabeled protein(s) within the endostyle tissue lysates (Supplementary Fig. 1a). A total of ten Tg1 domain-containing proteins were identified from IP-MS data, including Sc.CG.S33.65, Sc.CG.S284.31, Sc.CG.S566.2, Sc.CG.S166.41, Sc.CG.S140.18, Sc.CG.S6.110, Sc.CG.S3.110, Sc.CG.S79.69, Sc.CG.S131.45, and Sc.CG.S55.56, which represent five-sixths of all predicted Tg1 domain-containing proteins in S. clava genome (Fig. 1c and Supplementary Fig. 1b–e). Furthermore, we performed a phylogenetic analysis comparing these Tg1 domain-containing proteins with vertebrate TG proteins, Tg1 domain-containing proteins in the human genome23 and predicted TG-like protein in amphioxus and sea urchin5. The result revealed that Sc.CG.S284.31 and Sc.CG.S55.56 proteins clustered within the clade of vertebrate TG proteins, suggesting that they were the closest relatives to vertebrate TG proteins among all the identified Tg1 domain-containing proteins (Fig. 1d, e).

Sc.CG.S284.31 is a TH protein precursor protein
We further investigated the expression patterns of genes encoding Sc.CG.S284.31 and Sc.CG.S55.56 proteins. Based on transcriptomic data from the embryonic and larval development of S. clava24, we revealed that Sc.CG.S284.31 gene was up-regulated from tail-bud stage with the highest expression level during larval stages, which was similar to the temporal expression profiles of the homologs of vertebrate TH-synthesis-related genes, such as thyroid peroxidase (ScTpo), Sodium/iodide symporter (ScNis), thyroid transcription factors (ScTtf1 & ScTtf2) (Fig. 2a). Distinct from the temporal expression profiles of Sc.CG.S284.31 and ScTpo, Sc.CG.S55.56 gene had the highest expression level at tail-regressed larval stage (Fig. 2a).

The in-situ hybridization showed that the Sc.CG.S284.31 transcripts were distributed in the anterior trunk and the epidermis between the trunk and tail (Fig. 2b and Supplementary Fig. 2a), exhibiting a similar distribution pattern with ScTpo and THs (Fig. 2c and Supplementary Figs. 2b, 3). In contrast, Sc.CG.S55.56 transcripts were distributed in the posterior trunk (Supplementary Fig. 2c, d). These results therefore revealed that Sc.CG.S284.31 has a similar expression dynamics and pattern as ScTpo, and might play a role in TH synthesis. We therefore prepared an antibody against this Sc.CG.S284.31 protein and performed immunofluorescence staining on the S. clava swimming larvae. The staining signals appeared at the anterior trunk and the epidermis between the trunk and tail, which was similar to the distribution pattern of immunolabeled protein(s) through the anti-BtTG staining (Fig. 2d, e). Furthermore, immunofluorescence staining with anti-TPO antibody revealed that ScTPO proteins were also present in the anterior trunk of S. clava larvae (Fig. 2f).

Given that these proteins were originally identified from the endostyle, which in ascidian adult is homologous to the thyroid follicles of vertebrates21, we investigated whether Sc.CG.S284.31 protein could be detected in the thyroid-equivalent region of S. clava endostyle. The immunostaining with an antibody specific to Sc.CG.S284.31 confirmed that this protein was specifically localized within zone 7 to 9 in endostyle (Fig. 2g), corresponding to the thyroid-equivalent region.

To determine whether Sc.CG.S284.31 can indeed serve as the matrix, providing hormonogenic tyrosine sites for TH synthesis, we performed immunoblotting assay to detect TH formation after in vitro protein iodination6 (Supplementary Fig. 4a). Given the extremely large molecular weight (630.45 kDa) of Sc.CG.S284.31 protein and the difficulty of cloning full-length sequence due to the presence of a large number of repetitive sequences, we constructed a vector to express recombinant Sc.CG.S284.31 protein containing Tg1-domain-enriched sequences (Supplementary Fig. 4b and Supplementary Dataset 1). The purified recombinant Sc.CG.S284.31 was used in iodination and subsequent immunoblotting assay. The results showed that the T3 antibody readily detected specific bands from 130 to 180 kDa (for the recombinant Sc.CG.S284.31) or >300 kDa (for the recombinant human TG) after the proteins were iodinated (Fig. 2h). However, the T3 antibody failed to detect any signal in the iodinated sample of purified Sc.CG.S55.56 (Fig. 2h).

Overall, the results of expression analyses as well as the in vitro iodination experiment clearly demonstrated that Sc.CG.S284.31 protein, rather than Sc.CG.S55.56, could be a TH protein precursor providing hormonogenic sites for de novo TH production, similar to the function of vertebrate TG proteins. Therefore, we named Sc.CG.S284.31 as ScTG-like.

ScTg-like is crucial for TH synthesis and metamorphosis
To demonstrate whether ScTg-like is involved in TH synthesis in vivo, we conducted RNA inference (RNAi) experiments to knockdown its expression and analyzed TH levels and larval metamorphosis (Fig. 3a). After treating larvae with specific siRNAs of ScTg-like, the expression of ScTg-like was decreased in both mRNA and protein levels (Fig. 3b and Supplementary Fig. 5). We simultaneously measured the THs levels by ELISA. Both T4 and T3 levels in S. clava larvae were decreased upon knockdown of ScTg-like, comparable to that of methimazole (MMI, a TPO inhibitor25)-treated larvae (Fig. 3c, d). Meanwhile, we observed that the tail regression of larvae was affected after knocking down ScTg-like (Fig. 3e, f), suggesting the crucial role of ScTg-like in tail regression, a typical event that occurs at the early metamorphosis of ascidian larvae24. We further tracked the morphological changes of S. clava larvae with or without ScTg-like knock-down, and found that the process of metamorphosis of siRNA-treated larvae was largely delayed (Supplementary Figs. 6 - 7). In addition, compared with juveniles without ScTg-like knock-down, the “juveniles” in siRNA treated groups exhibited abnormal phenotypes with inextensible siphons and immature organs (Supplementary Fig. 6-7), suggesting that the organ morphogenesis of S. clava larvae during metamorphosis was regulated by ScTg-like gene. Interestingly, we demonstrated that the role of ScTg-like in metamorphosis is effectively linked to its role in TH synthesis, since, in rescue experiments, T3 treatment effectively restored the metamorphosis of ScTg-like knockdown group to a level comparable to those of the siRNA control and seawater control groups (Fig. 3g–i and Supplementary Fig. 6). Collectively, we established that the ScTg-like was indispensable for metamorphosis by functioning as the protein precursor for TH formation in S. clava larvae.

Follicle-like structure serves as thyroid-equivalent region
In S. clava larvae, our anti-ScTG-like antibodies distinctly detected a signal in an invaginated structure consisting of three to five closely contacted cells, located at the central anterior region of the trunk (Figs. 2e and 4a, b). This invaginated structure could also be specifically labeled by the anti-TPO antibody (Figs. 2f and 4c), suggesting that the cells within this structure might function similarly to a vertebrate thyroid follicle. To validate this hypothesis, we conducted further immunostaining of THs (T3 and T4), ScDUOX1, and several transcriptional factors (including ScFOXQ1, ScPAX2/5/8, and ScTTF-2, whose vertebrate homologs are involved in thyroid morphogenesis and TH synthesis) in the larvae26,27. Notably, all the antibodies used in immunofluorescence experiment were able to distinctly label this invaginated follicle-like structure (Supplementary Fig. 8). Moreover, the peanut agglutinin (PNA) and wheat germ agglutinin (WGA) apparently labeled the follicle-like structure in S. clava larvae, indicating the glycoproteins were enriched in this structure (Supplementary Fig. 9a–c). Similar results could also be observed in the thyroid follicle of zebrafish (Supplementary Fig. 9d–f). Additionally, the anti-T3 signals were undetectable, as well as the anti-ScTG-like protein signals, in the follicle-like structure after knocking down the ScTg-like gene (Fig. 4d and Supplementary Fig. 5). The findings provide compelling evidence that this invaginated follicle-like structure, with expression of ScTG-like protein, THs, and other thyroid function-related proteins, serves as the site of TH synthesis and storage, representing a proto-thyroid-follicle in S. clava larvae.

Structural conservation implies homologs across Bilateria
Despite extensive homology searches, we did not identify a clear orthologue of ScTg-like in any other species, including other tunicates such as the closely related Styela plicata. This likely reflects the rapid evolution of tunicate genomes28 and the low primary sequence conservation typical of TG proteins. Indeed, as previously observed in vertebrates9, the conservation of TG function appears to rely more on structural architecture than on sequence similarity. ScTG-like protein shares hallmark features with vertebrate TGs, including large size (>2000 aa), multiple Tg1 domains, extensive disulfide bridges, and a C-terminal region devoid of disulfides that may fulfill a ChEL-like role. Using this domain-based signature, we identified several candidate proteins that match the predicted structural organization of ScTG-like protein, not only in other tunicates but also in cephalochordates, echinoderms, hemichordates and protostomes (Supplementary Fig. 10). Although precise sequence-based comparisons remain difficult, these findings support the existence of functional homologs of TG-like proteins across bilaterians—in accordance with the notion that endogenous TH synthesis may be an ancient and conserved feature of this clade.

Taken together, our results demonstrated the existence in S. clava, an urochordate, of a protein encoded by ScTg-like gene that play a key role as a thyroid hormonogenic protein localized in the follicle-like structure of ascidian tadpole larva. These data provide novel insights into the molecular mechanisms of TH synthesis outside vertebrates and contributes to understanding the evolutionary trajectory of the TG protein.

Methods
Sample collection
Cryosections. Endostyle tissue was isolated from S. clava adult. The head was isolated from adult zebrafish (Danio rerio). The samples were subsequently fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 2 h. Following three washes with phosphate-buffered saline (PBS), the tissues were treated with 30% sucrose solution in PBS. The samples were then embedded in Tissue-Tek OCT compound (Sakura, Torrance, CA, 4583) and snap-frozen in liquid nitrogen. The samples were stored at −80 °C before cryosection. Prior to sectioning, the OCT-embedded samples were equilibrated by placing it in a −20 °C freezing microtome for 30 min. Cryosections were collected at 10 μm intervals along the transverse plane utilizing a cryostat (CM3050, Leica). Subsequently, the sections were mounted onto adhesive slides.

Larvae. The fertilization and culture of S. clava embryos were performed according to the previously detailed procedures by Wei et al.24 and Lin et al.49. The larvae of S. clava were collected and fixed by 4% PFA at RT for 2 h. Following washing with PBS for three times to eliminate PFA, the larvae were stored at 4 °C.
All guidelines for animal experiments were approved by the Institutional Animal Care and Use Committee of Ocean University of China (OUC-IACUC), with approval numbers 2020-0032-0517 and 2023-0032-0039 for S. clava, OUC2012316 for zebrafish. S. clava is a hermaphroditic species. The thyroid follicle in zebrafish examined in this study are not sex-specific, therefore, samples were randomly selected without distinguishing between sexes.

Staining
Immunofluorescence. For immunofluorescence staining, the samples were initially permeabilized through washing with PBST (0.1% Triton X-100 in PBS) for five times within 8 h. Subsequently, blocking was performed using 10% goat serum at RT for 1 h. The samples were then incubated with primary antibodies (1:100 to 1:50) overnight at 4 °C with shaking. Following this, the samples were washed by PBST for five times within 8 h. Subsequently, samples were incubated with secondary antibody (Goat anti-Mouse IgG, Alexa Fluor™ 555 (A21422, Invitrogen) or Goat anti-Rabbit IgG, Alexa Fluor™ 568 (A11011, Invitrogen)) and Phalloidin 488 (A12379, Invitrogen) overnight at 4 °C with shaking. After repeating the washing step with PBST under the same conditions, the samples were mounted with mounting solution (Vector Laboratories) containing DAPI and observed by a confocal microscope (Zeiss, LSM900). The primary antibodies used in this study are listed in Supplementary Dataset 2.

Peanut agglutinin (PNA) staining. The samples were initially permeabilized through washing with PBST for five times within 8 h. Subsequently, blocking was performed using 10% goat serum at RT for 1 h. The samples were then incubated with biotinylated PNA (1:300) overnight at 4 °C with shaking. Following this, the samples were washed by PBST for five times within 8 h. Subsequently, samples were incubated with VariFluor 488-Streptavidin (HY-D1808, MedChemExpress) and TRITC Phalloidin (40734ES75, Yeasen) for 2 h at RT with shaking. Following this, the samples were washed by PBST for three times within 1 h. Subsequently, the samples were mounted with mounting solution (Vector Laboratories) containing DAPI and observed by a confocal microscope (Zeiss, LSM900).

Wheat germ agglutinin (WGA) staining. The samples were initially permeabilized through washing with PBST for five times within 16 h. Subsequently, samples were incubated with WGA (1:50) for 3 days at RT with shaking. Following this, the samples were washed by PBST for five times within 8 h. Subsequently, the samples were mounted with mounting solution (Vector Laboratories) containing DAPI and observed by a confocal microscope (Zeiss, LSM900).

Immunoprecipitation coupled with mass spectrometry (IP-MS)
Sample preparation. The endostyle tissues were isolated from S. clava adults, and subsequently shredded and homogenized. The homogenized tissues were incubated with RIPA lysis buffer, supplemented with 1% PMSF to effectively inhibit protease activity, for 20 min on ice. Following this, high-speed centrifugation was carried out at 13,201 × g for 5 min at 4 °C. The supernatant was then collected, and the protein concentration was determined using the Bicinchoninic Acid (BCA) Assay. Subsequently, the endostyle protein was incubated with the antibody specific to B. taurus TG protein (Supplementary Dataset 2) overnight at 4 °C with shaking, to facilitate the formation of the antigen-antibody complex. Magnetic beads were then introduced into the system and incubated for 2 h at room temperature, allowing the formation of the antigen-antibody-beads complex. Magnetic adsorption was subsequently performed using a magnetic rack to enrich the antigen-antibody-beads complex. After removing the supernatant, the complex was washed three times with PBS to ensure purity. Ultimately, the complex was collected for subsequent mass spectrometry identification. Rabbit IgG was used as a negative control. The experiments were performed with two independent biological replicates. The obtained samples were sent to Shanghai Bioprofile Technology Company Ltd. for pretreatment, data acquisition, and analysis according to the following procedures.
Gel pieces were cut from SDS-PAGE, destained for 20 min in 100 mM NH4HCO3 with 30% Acetonitrile and washed with Milli-Q water until the gels were destained. The spots were then lyophilized in a vacuum centrifuge. The in-gel proteins were reduced with dithiothreitol (10 mM DTT/ 100 mM NH4HCO3) for 30 min at 56 °C, then alkylated with iodoacetamide (200 mM IAA/ 100 mM NH4HCO3) in the dark at room temperature for 30 min. Gel pieces were briefly rinsed with 100 mM NH4HCO3 and ACN, respectively. Gel pieces were digested overnight in 12.5 ng/μl trypsin in 25 mM NH4HCO3. The peptides were extracted three times with 60% ACN/ 0.1% TFA. The extracts were pooled and dried completely by a vacuum centrifuge.

LC-MS/MS. The peptide of each sample was desalted on C18 Cartridges (Empore™ SPE Cartridges, Sigma), then concentrated by vacuum centrifugation and reconstituted in 10 µl of 0.1% (v/v) Formic acid. MS experiments were performed on a Q Exactive HF mass spectrometer that was coupled to Easy nLC (Thermo Scientific). Peptide was first loaded onto a trap column (100 μm × 20 mm, 5 μm, C18) with 0.1% formic acid, then separated by an analytical column (75 μm × 100 mm, 3 μm, C18) with a binary gradient of buffer A (0.1% Formic acid) and buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min over 60 min. The gradient was set as following: 5%–8% buffer B from 0 to 2 min, 8% to 23% buffer B from 2 to 42 min, 23% to 40% buffer B from 42 to 50 min, 40% to 100% buffer B from 50 to 52 min, 100% buffer B kept till to 60 min. MS data was acquired using a data-dependent top20 method dynamically choosing the most abundant precursor ions from the survey scan (350–1800 m/z) for HCD fragmentation. A lock mass of 445.120025 Da was used as internal standard for mass calibration. The full MS scans were acquire data resolution of 60,000 at m/z 200, and 15,000 at m/z 200 for MS/MS scan. The maximum injection time was set to for 50 ms for MS and 45 ms for MS/MS. Normalized collision energy was 27 and the isolation window was set to 1.5 Th. Dynamic exclusion duration was 30 s.

Database search. The MS data were analyzed using MaxQuant software version 1.5.8.3. MS data were searched against the database of S. clava genome. The trypsin was selected as digestion enzyme. The maximal two missed cleavage sites and the mass tolerance of 4.5 ppm for precursor ions and 20 ppm for fragment ions were defined for database search. Carbamidomethylation of cysteines was defined as fixed modification, while acetylation of protein N-terminal and Lysine, oxidation of Methionine, were set as variable modifications for database searching. The database search results were filtered and exported with <1% false discovery rate (FDR) at peptide level and protein level, respectively.

Bioinformatics analysis
Protein domain prediction for S. clava genome. HMMER was employed with profile Hidden Markov Models (pHMMs) from the Pfam database50, which contains domain-specific pHMMs representing conserved protein families, to predict the domain distribution of protein sequences in the whole genome of S. clava24. The hmmscan module was executed using default parameters (e-value ≤ 1e-5) to scan each protein sequence against the pHMM library, prioritizing domain coverage and statistical significance. The Proteins containing Tg1 domain(s) (Pfam ID: PF00086.23) were identified according to the results of protein domain prediction. Predicted domains were manually validated through cross-referencing with SMART database annotations (http://smart.embl-heidelberg.de).

Phylogenetic analysis. The information of protein sequences for phylogenetic analysis was shown in supplementary files (Supplementary Dataset 3). Sequences were aligned with MEGA software (version 11.0)51 using the ClustalW52 with default parameters, followed by manual refinement to optimize conserved motif alignment. The final alignment was used for phylogenetic analysis employing the Neighbor-Joining (NJ) method under the p-distance model. Branch support was assessed via 1000 bootstrap replicates, with nodes considered significant at >30% bootstrap values.

Downloading and preparing genomes. DNA assemblies were obtained via NCBI Datasets CLI v18.3.1. For each organism selected from the taxa Tunicata (txid 7712), Cephalochordata (txid 7735), Petromyzontiformes (txid 7745), Annelida (txid 6340), Echinodermata (txid 7586), Hemichordata (txid 10219) and Mollusca (txid 6447), the most complete assembly level available was downloaded.
Generation or collection of proteomes. When the proteome was available, the *.faa file provided by RefSeq/GenBank was used as is. If absent, GeneMarkES v4.73 predicted ORFs de novo.
Construction of HMM profiles. Thirty vertebrate sequences (25 RefSeqs in the taxa Amphibia (txid 8292), birds (txid 8782), bony fishes (txid 7898), Squamata (txid 8509), Testudines (txid 8459), and 5 mammalia (txid 40674) UniProtKB/Swiss Prot: THYG_HUMAN, THYG_MOUSE, THYG_RAT, THYG_PIG, THYG_BOVIN) were used as a reference set. The 5 annotated Uniprot sequences were used to create a first version of the HMM profile for the Tg1, Tg2, Tg3, and ChEL domains. Then, using hmmsearch (default threshold), the domains were identified in the other 25 sequences. All domain sequences were extracted and aligned with MAFFT v7.525 (--localpair --maxiterate 1000) and then converted into specific HMM profiles via hmmbuild (HMMER v3.4). These are the profiles that will be used for the rest of the pipeline.
Annotation of invertebrate proteomes. For the detection of relevant proteins, three methods were combined: (1) HMM profile search, using hmmsearch (default threshold) for the detection of Tg1, Tg2, Tg3, and ChEL domains. Only sequences with at least 5 Tg1 motifs were retained by this method. (2) Search by regex, the search patterns are Tg2-like domain (C××C×G) and Tg2-like domain (C××C××G), potential disulfide bridges C (1–20) C, and tyrosine residues Y. Blastp v2.16.0+ search against each taxon with lamprey TG protein and S. clava TG-like protein as reference sequences. HMM profile search and regex search methods were applied to the blastp results to create an annotation file. Each domtbl annotation file was converted to gff3 format.
Visualization of protein architecture. To graphically represent the architecture of candidate TG-like proteins, a Python script was developed using the Biopython (for sequence manipulation), Pandas (metadata management), and Matplotlib (visualization) libraries. It takes as input a fasta file with all the sequences (Supplementary Dataset 4), a folder containing all the annotations in gff3 format, and a tsv file listing the metadata for each sequence. For each sequence, it generates three parallel plots: positions of the Tg/ChEL domains, potential disulfide bonds, and tyrosine residues, and generates a svg vector file and a png file for each sequence.

Polymerase chain reaction (PCR) and RT-qPCR
Total RNA was extracted from the fresh samples utilizing the TRIzol method and subsequently dissolved in RNase-free ddH2O. The cDNA synthesis was performed through reverse transcription employing PrimeScript II RTase (6210A, Takara). The newly synthesized cDNA served as the template for PCR analysis, employing specific primers of targeting genes (Supplementary Dataset 5). To ensure accurate normalization, the gene expression level of Sc18s rRNA was utilized as an internal reference. PCR reactions were performed under the following conditions: pre-denaturation (3 min at 95 °C) for one cycle, denaturation (15 s at 95 °C)-annealing (15 s at 60 °C)-extension (60 s/kb at 72 °C) for 35 cycles, and complete extension (5 min at 72 °C) for one cycle. PCR products were analyzed by 1% agarose gel electrophoresis. RT-PCR reactions were performed under the following conditions: pre-denaturation (3 min at 95 °C) for one cycle, cyclic reaction (10 s at 95 °C and 30 s at 58 °C) for 40 cycles, melting curve (15 s at 95 °C, 60 s at 60 °C and 15 s at 95 °C) for 1 cycle. The RT-PCR experiment was performed using Roche LC96. Data analysis was performed through 2−ΔΔCt method. The primers for these experiments were listed in Supplementary Dataset 5.

In situ hybridization
Specific segments of the target genes were selected for the synthesis of probes. The primers utilized for amplifying specific segments were listed in Supplementary Dataset 5. The transcriptase binding sequence (SP6 RNA polymerase priming: 5’ ATTTAGGTGACACTATA 3’; T7 RNA polymerase priming: 5’ TAATACGACTCACTATAGGG 3’) was added to the 5’ end of the primer. Digoxigenin (DIG)-RNA probes were generated through in vitro transcription using either SP6 RNA polymerase (EP0131, Thermo Fisher Scientific) or T7 RNA polymerase (EP0111, Thermo Fisher Scientific), and subsequently purified using the LiCl and 100% ethanol. These DIG-RNA probes were stored at −80 °C prior to hybridization.
The in situ hybridization procedure was conducted as previously described by Yasuo et al.53. Briefly, samples were digested with proteinase K (15 μg/mL) for 30 min at 37 °C, followed by washing with PBT (0.1% Tween-20 in PBS). Samples were then refixed with 4% PFA (in PBT) for 30 min at RT and washed again with PBT. Pre-hybridization was carried out using pre-hybridization buffer (50% formamide, 5× saline sodium citrate buffer (SSC), 0.1% Tween 20) for 15 min at RT, followed by hybridization buffer (50% formamide, 5× SSC, 0.1% Tween 20, 5× Denhardt’s solution, yeast tRNA (100 μg/mL), and heparin (50 μg/mL)) for 3 h at 50 °C. Hybridization was subsequently performed with 2 ng/μL probes in hybridization buffer overnight at 50 °C. After hybridization, samples were washed four times with wash buffer I (50% formamide, 5× SSC, 0.1% Tween 20), four times with wash buffer II (50% formamide, 0.5× SSC, 0.1% Tween 20), and four times with PBT. Samples were then blocked using 1% blocking buffer for 1 h at RT. Following this, samples were incubated with DIG antibody overnight at 4 °C. Subsequently, samples were washed four times with PBT and twice with TMNT (0.1 M Tris-HCl (pH=9.5), 0.1 M NaCl, 0.02 M MgCl2, 0.1% Tween 20). Chromogenic detection was achieved using a Nitro blue tetrazolium (NBT)/bromochloroindolyl phosphate (BCIP) solution (1:50 diluted with TMNT).

Protein expression and purification
To improve the expression efficiency in mammalian cells, we synthesized the codon-optimized sequence of Sc.CG.S284.31 gene (Supplementary Dataset 1). The Tg1-enriched fragments of the Sc.CG.S284.31 gene were cloned from the codon-optimized sequences, and subsequently subcloned into a linearized pEGFP-N1 vector (pre-linearization using NheI and KpnI restriction enzymes (Thermo Fisher Scientific)). Additionally, the full-length human TG-encoding sequence was cloned from a commercial plasmid obtained from the DNASU Plasmid Repository (Clone ID: HsCD00867406) and subcloned into a linearized pcDNA3.1(+) vector (pre-linearization using NotI restriction enzymes (Thermo Fisher Scientific)). The full-length Sc.CG.S55.56 protein-encoding sequence was cloned from cDNA library of S. clava and subcloned into a linearized pcDNA3.1(+) vector (pre-linearization using NotI restriction enzymes (Thermo Fisher Scientific)). The primers for clone were listed in Supplementary Dataset 5. Purification of the resulting fragments was performed using the GeneJEL Kit (K0691, Thermo Fisher Scientific), ensuring high-quality material for subsequent experiments. Homologous recombination was carried out using the in-fusion method (C115, Vazyme). The constructed plasmids were transformed into DH5α competent cells. Bacteria successfully transformed with constructed plasmids were inoculated into LB media. Plasmid extraction and purification were performed using EndoFree Mini Plasmid Kit (DP123, Tiangen biotech). The newly generated plasmids are available upon request from the corresponding author.
HEK293T cells (SCSP-502, National Collection of Authenticated Cell Cultures) were employed for the in vitro expression of the recombinant protein. These cells were cultured in DMEM, supplemented with 10% fetal bovine serum, at 37 °C in a 5% CO2 incubator. Twenty-four hours prior to transfection, the cells were plated in 10 cm cell culture dishes. Transient transfection was performed using Lipofectamine 3000 transfection reagent (L3000001, Invitrogen) following the manufacturer’s protocol. Twenty-four hours after transfection, cells were washed with PBS and cultured with serum-free DMEM for an additional 24 h. Ultrasonication and ultracentrifugation techniques were employed to collect the recombinant Sc.CG.S284.31 protein and Sc.CG.S55.56 protein from cells. For the human TG protein, the secreted protein present in the medium of transfected cells was harvested for subsequent purification steps.
Protein purification was performed using Anti-GFP affinity gel (HY-K0229, MedChemExpress) for recombinant Sc.CG.S284.31 protein, and Ni Sepharose 6 FF (17531801, Cytiva) for Sc.CG.S55.56 protein and human TG protein. Prior to incubation, the agarose beads were thoroughly washed with PBS three times. The protein samples were then incubated with the agarose beads in gravity chromatography columns at 4 °C overnight. The protein solvent was flowed out at a controlled rate of 1 mL/min. Subsequently, the agarose beads were washed three times with wash buffer (50 mM NaHPO4, 300 mM NaCl, 20 mM imidazole, final pH=8.0, filter with 0.22 μm membrane). The recombinant proteins were then eluted using elution buffer (50 mM, NaH2PO4, 300 mM NaCl, 500 mM imidazole, final pH=8.0, filter with 0.22 μm membrane). To exchange the protein from the elution buffer to PBS, Sephadex DeSalting Gravity Columns (C500090, Sango) were employed, following the manufacturer’s protocol. Finally, the concentration of the protein sample was determined using the BCA method.

In vitro enzymatic iodination and western blotting
The in vitro iodination experiment was performed following the protocol previously outlined by Citterio et al.54. The iodination mixture comprised 30 ng/μL lactoperoxidase, 2 μg/μL glucose, 0.352 ng/μL glucose oxidase, 100 μM NaI, and 150–200 ng/μL of purified protein. All reagents were purchased from Sigma. Subsequently, the sample was incubated at 37 °C for 1.5 h, and enzymatic reaction was stopped by addition of gel sample buffer and boiling for 10 min.
Western blotting experiment was performed to detect the TH signal of the iodinated proteins. The iodinated samples were subjected to reducing SDS-PAGE, and transferred to 0.45 μm PVDF membrane (Millipore, MA, USA). Blocking was performed using 5% fat-free milk at room temperature (RT) for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with a 1:1000 dilution of T3 Monoclonal Antibody (3A6) (MA1-21669, Invitrogen). Following washes with TBST (TBS containing 0.05% Tween-20), the membranes were incubated with species-specific HRP-conjugated secondary antibodies diluted at 1:2000 in blocking buffer at RT for 2 h. After additional washes with TBST, the specific bands were visualized using a chemiluminescence digital imaging system (ImageQuant Las-4000 Mini, GE Healthcare).
To detect the signals of proteins in iodinated and non-iodinated samples, the anti-GFP rabbit polyclonal antibody (T0006, Affinity), anti-HsTG mouse polyclonal antibody (sc-365997, Santa Cruz Biotechnology) and anti-Sc.CG.S55.56 mouse polyclonal antibody (homemade) were employed (Supplementary Dataset 2).

Preparation of anti-Sc.CG.S55.56
The antibody of Sc.CG.S55.56 protein was made in our lab. The full-length Sc.CG.S55.56 protein-encoding sequence was cloned from cDNA library of S. clava (Supplementary Dataset 5), and subcloned into a linearized pET30a vector (pre-linearization using BamHI and EcoRI restriction enzymes (Thermo Fisher Scientific)). Purification of the resulting fragments was performed using the GeneJEL Kit (K0691, Thermo Fisher Scientific), and homologous recombination was carried out using the in-fusion method (C115, Vazyme). The newly generated plasmids are available upon request from the corresponding author.
To obtain the Sc.CG.55.56 protein, prokaryotic expression using bacteria was performed. The recombined plasmids were transformed into BL21 (DE3) competent cells. Bacteria successfully transformed with recombined plasmids were inoculated to LB solid media. Monoclonal colony was used for amplification culture in LB liquid media at 37 °C with 4.4 × g. When the OD value reached to 0.6, IPTG was added into bacterial fluid with the final concentration of 0.5 mM. The bacterial fluid was then developed in 20 °C environment at speed of 3.2 × g for 16 h. Ultrasonication was used for extraction of total proteins in bacteria. The coomassie blue staining was performed to labeled the Sc.CG.S55.56 protein. The gels containing Sc.CG.S55.56 protein were split, and were homogenate through ultrasonication. To obtain the antibodies of Sc.CG.S55.56, intraperitoneal injection in mice was carried out once a week for a total of four times. A total of 200 μg protein was used for intraperitoneal injection every time. Finally, the blood of mice was drawn and further separated to obtain serum. The serum contains antibodies and was used in subsequent experiments. The experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of Ocean University of China (OUC-IACUC), with approval numbers OUC-AE-2022-128.

RNA inference and rescue experiment
Fertilized eggs were cultured at 18 °C until the early tail bud stage, the embryos were gathered and placed into the wells of a 24-well plate, which had been pre-coated with 0.1% BSA. Each well contained more than 50 embryos. The siRNAs were designed and synthesized by Genecefe Biotechnology Co., Ltd (Wuxi, China). The sequences of siRNAs for this experiment were listed in Supplementary Dataset 6. The sequence of the negative control siRNA does not target any gene in the S. clava genome. The siRNAs were dissolved in RNase-free water to achieve a stock concentration of 20 μM. For transfection, the riboFECTTM CP transfection kit (C10511-05, Ribo Bio) was used according to the manufacturer’s instructions. The working concentration of the siRNAs for transfection was set at 0.4 μM, and the embryos were incubated with the siRNAs at 18 °C. For rescue experiment, the T3 was added at 18 hpf with working concentration at 50 μg/mL. Following the incubation period, the phenotypes of the larvae were recorded using a microscope, and the percentages of metamorphic larvae were analyzed. Subsequently, the larvae were collected for RNA extraction.

Enzyme linked immunosorbent assay (ELISA)
The larvae were dissociated using ultrasonication, and ultracentrifugation was performed to obtain the supernatant for ELISA detection. Standardize each group of samples by weighing. The 25 μL of sample was incubated with 100 μL TH-HRP conjugate in each well of 96-well plate for 1 h at RT. After incubation, the content of the wells was discarded, and each well was washed with 300 μL of wash buffer for five times. Subsequently, 100 μL of TMB substrate solution was added to each well, followed by incubation at RT in the dark for 15 min. Then, 100 μL of stop solution was added to each well, and the plate was gently shaken. The absorbance of the samples was measured at a wavelength of 450 nm.

Statistics and reproducibility
All values are depicted as mean ± SEM. Significant test was analyzed by two-tailed T-test. Data were estimated to be statistically significant at p-value ≤ 0.05. ****, p-value ≤ 0.0001. ***, p-value ≤ 0.001, **, p-value ≤ 0.01. *, p-value ≤ 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This article reports the identification of a functional thyroglobulin (TG) protein precursor, named ScTG-like, for thyroid hormone (TH) synthesis in the basal chordate ascidian Styela clava. The key findings include:\n1. The identification and Characterization of ScTG-like protein, encoded by the ScTg-like gene in Styela clava. This was identified through immunoprecipitation-mass spectrometry combined with phylogenetic and expression pattern analyses. This protein is a high-molecular-weight protein (630.45 kDa) containing multiple Tg1-domains. While sharing hallmark features with vertebrate TGs, such as large size and multiple Tg1 domains, it notably lacks the cholinesterase-like (ChEL) domain present in vertebrate TG proteins.\n2. The Functional verification of ScTG-like protein - In vitro experiments demonstrated that ScTG-like has the capacity to provide hormonogenic sites for TH synthesis, specifically forming T3 after iodination. In vivo gene knockdown of ScTg-like significantly inhibited larval metamorphosis and led to diminished TH (T3 and T4) levels. This inhibition was effectively restored by T3 treatment, confirming ScTG-like's crucial role in TH synthesis for metamorphosis.\n3. Discovery of a Novel Follicle-like Structure - An invaginated follicle-like structure was identified at the central anterior of the larval trunk, where both ScTG-like protein and THs were deposited. This structure is functionally similar to vertebrate thyroid follicles, serving as the location for TH synthesis and storage. Immunostaining showed the presence of other thyroid function-related proteins, such as TPO and transcriptional factors like FOXQ1, PAX2/5/8, and TTF2, within this structure.\n4. Evolutionary Implications - The study provides evidence of endogenous de novo TH synthesis outside vertebrates. The structural conservation of Tg1-containing proteins in multiple bilaterian phyla suggests that endogenous TH synthesis may be an ancestral and conserved trait of bilaterians. This work offers insights into the evolution of the TG protein and the emergence of a major vertebrate endocrine system.\nMajor concerns:\n• The authors state that their discovery resolves a long-standing mystery regarding the evolutionary \"missing\" TH protein precursor in invertebrates, where the mechanism of TH synthesis has remained elusive due to the lack of identified orthologs to vertebrate TG. First of all, the mystery about TH signaling in invertebrates is not restricted to the synthesis. With respect to synthesis however, valid efforts have been made to identify iodinated proteins in a range of organisms, including basal animals groups. For example, some sponges have been shown to contain a large percentage of iodinated protein as a structural component. So the iodination of proteins or components of proteins is not new and the authors have to do netter justice to that literature. As far as Deuterostomes are concerned, some more detailed efforts have been made to understand iodine metabolism in marine organisms (of which marine invertebrates represent a large portion). For example, learning from algae (kelp), echinoderms have been shown to organify iodine from seawater using a mechanism called peroxide facilitated (or dependent) diffusion, a process similar to what has been described in details in kelp (Miller and Heyland 2013 - JEB). Notably, this paper and others clearly outline conceptually, how iodine metabolism is different for marine organisms compared to terrestrial or freshwater organisms. Specifically, seawater contains much high concentrations of iodine and therefore, the evolved capacity of thyroid glands as we know them in vertebrates to concentrate iodine from the environment appears to play little or no role in a marine environment. Specifically, iodine - a reactive molecule that can do considerable damage to cells when in its ionic form, needs to be first and foremost de-activated as it flows in the cell by a sodium independent mechanism. The authors really need to acknowledge and discuss this body of literature as the current presentation of their study and the results can be considered somewhat chordate biased and misleading, considering the amount of literature that exists on these processes in other systems (Kupper and Carrano provide a nice review for algae - Key aspects of the iodine metabolism in brown algae: a brief critical review - PubMed. The authors also fail to cite another article on sea urchins and molluscs (Heyland et al. 2006), which for the first time showed that these species incorporate I125 and turn it into T4, using TLC. Considering that Styela clava is marine, this literature is highly relevant and needs to be considered and discussed. This is also critical, as the author's claim that there is a long standing mystery about the TH precursor in invertebrates. Based on the existing literature, this is misleading at best and should be rephrased to - an effort has been made to test the hypothesis that the precursor protein for TH synthesis is shared between mammals and ascidians. I also urge the authors to carefully review the literature on marine and freshwater fish iodine metabolism and modifications that may have occurred in vertebrates other than mammals.\n○ The authors also neglect to properly acknowledge that Monaco et al. 1981 showed thyroid hormone synthesis on thyroglobulin in a lancelet species - Thyroid hormone formation in thyroglobulin synthesized in the Amphioxus (Branchiostoma lanceolatum pallas) - ScienceDirect\n• The function of TH signaling in ascidian tadpoles remains sparse, despite efforts to understand it better. Knowledge gaps relevant to the manuscript are: the identity of the ascidian receptor, the active thyroid hormone molecule and the detailed effects of THs on the development of ascidian tadpoles. While the manuscript contributes some new data to this question, it fails to properly analyze metamorphic development in the urochordate. Ascidian tadpoles are extremely short lived, compared to some other marine invertebrate larvae. Their developmental progression from embryonic to post-embryonic and metamorphic development is happening in a few hours and therefore poses two important questions, which are not at all considered in the metamorphosis assay the authors applied: 1) what other morphogenetic effects, if any, do the hormones have on development? Considering that a large portion of the morphological transition of metamorphosis (other than tail regression) occurs after settlement, this would be an important question, considering how THs generally orchestrate the transition in chordates 2) The TGF-beta signaling has emerged as one major factor of tail regression. There is no link to TH signaling that has ever been mentioned in the literature, yet the authors are using this as a phenotypic readout. While the experimental design of this part of the experiment is generally complete (rescue experiment, knock-down, pharmacological treatment etc.) the manuscript fails to properly review and explain what we know about the function of TH in ascidian metamorphosis. What is presented in the manuscript is highly biased and forces existing information into a model that effectively suggest ascidian tadpole metamorphosis is the same as in the chordate cousins, the amphibians. This is incorrect and misleading and needs to be fixed. It would also be helpful to know for example, that T3 treatment upregulates some of the TH target genes during the critical stages. Finally, please change the y axis on the graphs in Figure 3 - you are not measuring % metamorphosis, you are measuring tail regression.\n• The authors present T4 and T3 staining in the supplement but only show T3 staining in the main text. I am confused about this. Not with standing my previous criticism that we don't actually know what the active TH is in ascidians (a fact that should be clearly stated), it would be useful to make adjustments to the current figures but more importantly, discuss and explain the distributions of T4 and T3. If we go with the mammalian model, we would expect T3 to be produce by de-iodination from T4 in target tissues. A good starting point would be to discuss whether T3 is actually found in areas and structure relevant for the metamorphic transition. Furthermore, it would be critical, to explain why there is a significant overlap between T3 and T4 presence in the tadpoles and discuss this in detail. Any TH endocrinologist will be eager to get a proper explanation of this fact. Similarly, the authors completely fail to discuss the difference/similarity between the follicular structure and the endostyle. Is this a separate organ? Why are ascidians accumulating THs in both? Are they linked and part of the same structure? What is the developmental cell lineage of that structure. There are several exciting papers from recent years showing that the endostyle 7-9 and the thyroid gland in fact originate from the same lineage and single cell studies confirm this beautifully. What about the follicular organ? Is there any information on that. This would not require more experiments but simply an expansion of existing datasets (Jiang, A. et al. Spatially resolved single-cell atlas of ascidian endostyle provides insight into the origin of vertebrate pharyngeal organs. Sci. Adv. 10, 1-16 (2024) - which is cited). I really think the authors need to address this gap, as their discussion is incredibly superficial and neglects so many important questions which will make readers who care about the evolution of TH signaling scratch their heads.\n• The authors do a great job narrowing down the identity of the putative TG-like protein, using all the right techniques and I think they show convincingly that this protein is in fact a matrix for TH production. However, I find their analysis of TGs in ascidians and among bilaterians a lot less convincing. Why, does Figure 1d show a gene phylogeny with all ascidian genes (which are relatively closely related) and compares them to vertebrate TGs. The authors appear to conclude that this reveals \" …that Sc.CG.S284.31 and Sc.CG.S55.56 proteins clustered within the clade of vertebrate TG proteins …\". I disagree, all this might indicate is that the vertebrate proteins are distantly related to ascidian proteins (support for branch is well below 90, which even for a NJ tree with 1000 replicates is bad and basically does not even support that conclusion very well). I think the authors need to go back to the dataset, which they present in the supplement (Sup fig. 7), which maps domain structures onto an animal phylogeny. This dataset should be properly analyzed phylogenetically, to see whether invertebrate TGs generally cluster together or not (or whether ascidian domains are indeed closer to vertebrates). Next the authors should conduct a synteny analysis to narrow down the evolution of TG domains and use the proper statistical framework. This would allow them to answer the question they currently state in the manuscript of 1) whether chordates (including basal chordates) are indeed different from other invertebrate or whether the separation is actually between vertebrates and invertebrates 2) it would also provide a more precise argument about the candidates they investigated here are actually evolutionarily related. Finally, it would essential that the authors provide at least a brief discussion about the presence absence of specific TG domains found across TG-like proteins in animals.\n○ The authors do not cite a relevant paper by Johnson and Moore, reviewing the carboxyesterase/cholinesterase gene family in invertebrate deuterostomes (2012).\nOther comments\n• The manuscript has some typos and grammatical errors. Please proof-read\n• While the authors establish this enrichment and draw a functional similarity between the S. clava structure and vertebrate thyroid follicles based on glycoproteins, they do not explicitly provide an explanation for the specific role of glycoproteins within the vertebrate follicle itself in the provided text. They observe their presence as a shared feature but do not elaborate on their function within the vertebrate context. Considering how little we know about the conservation of this feature across animals, we need more explanations in the text.\n• The authors mention and use transcriptomics data but fail to provide the raw data and also do not provide sufficient information on the analysis they used.", "reviewer_2": "The authors of this manuscript report on the identification of an invertebrate orthologue of thyroglobulin (TG), the protein precursor for the synthesis of thyroid hormones, in an ascidian. Previous studies have not conclusively identified this gene in invertebrates, possibly due to the need to search for structural similarities rather than by sequence alone. Here the authors identify a putative TG gene, localize its expression in the embryo and in the adult, show that it can be iodinated and demonstrate that it is required for larval metamorphosis.\nFigure 1 shows a cross-section of the adult endostyle (=thyroid gland) showing immunoreactivity for TG-like proteins, a gene tree and domain organization. Because there were several protein candidates, immunoprecipitation followed by mass-spec peptide fingerprinting identified likely candidate TG-like genes.\nIn part C the authors mention intensities, but this appears to be related to the number of peptides identified. The use of “intensities” should be changed as it implies immunofluorescent intensities (as shown in parts A-B).\nFigure 2 depicts the temporal and spatial expression pattern of the putative TG-like genes/proteins. It also shows that the protein from the Sc.CG.S248.31 gene (=TG-like) can be iodinated in vitro, suggesting that it can serve as a matrix for TH synthesis.\nFigure 3 reports the results of siRNA knockdowns on the TG-like gene and the subsequent impact on larval metamorphosis. Part B shows that siRNA treatment was able to knock-down expression by 50-60%. I am assuming that these graphs are RT-qPCR results (this should be clarified throughout figure). I would also suggest that the figure legend report the error bars as SEM (and throughout figure). Although this is mentioned in the materials and methods, it would be good to include here. The authors report an “NC” control siRNA. Although the sequence is reported in supplemental data, it is not described. Is this a scrambled siRNA? A target for a non-existent gene in the Styela genome? The authors should explain. Lastly, the authors lack a useful experiment demonstrating the outcome of their knock-down – an immunofluorescence image showing a reduction in protein levels in the larva. The authors show that they have a working antibody to TG-like, it would be very beneficial to demonstrate that the knock-down also reduces proteins levels in the siRNA-treated embryos. Lastly, they show that the siRNA phenotype can be rescued by providing exogenous T3 to rescue metamorphosis.\nIt would have been nice to show that a form the TG-like mRNA that cannot be degraded by siRNA can also rescue the knock-down. The authors should discuss why this wasn’t performed. I suspect it was because of the size of the gene and difficulty in cloning the entire product, but it would be useful to the reader to see this discussion.\nFigure 4 reports the immunolocalization of TG-like protein in the larva and depicts the putative anterior follicle-like structure. Although they show that siRNA treatment reduces the level of T3 detected by immunofluorescence, they don’t show reduction in TG-like levels, as mentioned previously. The follicle is intriguing, but it is difficult to know exactly where it is located in the larva. The SEM image isn’t particularly useful in my opinion. It would be beneficial if a drawing could be incorporated showing the location of the follicle relative to the palps and other anterior structures that the broader community is familiar with. It is located centrally? dorsally, ventrally?\nIn summary, the authors have presented a nice study identifying a TG-like gene/protein in the ascidian Styela clava. The authors should address my previous comments regarding some of the data presentation and it would be very good to show that the siRNA knock-down effectively reduces the level of TG-like protein."}, "author_response": "POINT-BY-POINT RESPONSE TO THE REVIEWERS\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis article reports the identification of a functional thyroglobulin (TG) protein precursor, named\nScTG-like, for thyroid hormone (TH) synthesis in the basal chordate ascidian Styela clava. The key\nfindings include:\n1. The identification and Characterization of ScTG-like protein, encoded by the ScTg-like gene in\nStyela clava. This was identified through immunoprecipitation-mass spectrometry combined with\nphylogenetic and expression pattern analyses. This protein is a high-molecular-weight protein\n(630.45 kDa) containing multiple Tg1-domains. While sharing hallmark features with vertebrate\nTGs, such as large size and multiple Tg1 domains, it notably lacks the cholinesterase-like (ChEL)\ndomain present in vertebrate TG proteins.\n2. The Functional verification of ScTG-like protein - In vitro experiments demonstrated that ScTG-\nlike has the capacity to provide hormonogenic sites for TH synthesis, specifically forming T3 after\niodination. In vivo gene knockdown of ScTg-like significantly inhibited larval metamorphosis and\nled to diminished TH (T3 and T4) levels. This inhibition was effectively restored by T3 treatment,\nconfirming ScTG-like's crucial role in TH synthesis for metamorphosis.\n3. Discovery of a Novel Follicle-like Structure - An invaginated follicle-like structure was identified\nat the central anterior of the larval trunk, where both ScTG-like protein and THs were deposited.\nThis structure is functionally similar to vertebrate thyroid follicles, serving as the location for TH\nsynthesis and storage. Immunostaining showed the presence of other thyroid function-related\nproteins, such as TPO and transcriptional factors like FOXQ1, PAX2/5/8, and TTF2, within this\nstructure.\n4. Evolutionary Implications - The study provides evidence of endogenous de novo TH synthesis\noutside vertebrates. The structural conservation of Tg1-containing proteins in multiple bilaterian\nphyla suggests that endogenous TH synthesis may be an ancestral and conserved trait of bilaterians.\nThis work offers insights into the evolution of the TG protein and the emergence of a major\nvertebrate endocrine system.\nResponse: We thank the reviewer for this good summary of our claims and his positive appreciation\nof the manuscript.\nMajor concerns:\n1. The authors state that their discovery resolves a long-standing mystery regarding the\nevolutionary \"missing\" TH protein precursor in invertebrates, where the mechanism of TH synthesis\n1\nhas remained elusive due to the lack of identified orthologs to vertebrate TG. First of all, the mystery\nabout TH signaling in invertebrates is not restricted to the synthesis. With respect to synthesis\nhowever, valid efforts have been made to identify iodinated proteins in a range of organisms,\nincluding basal animals groups. For example, some sponges have been shown to contain a large\npercentage of iodinated protein as a structural component. So the iodination of proteins or\ncomponents of proteins is not new and the authors have to do netter justice to that literature. As far\nas Deuterostomes are concerned, some more detailed efforts have been made to understand iodine\nmetabolism in marine organisms (of which marine invertebrates represent a large portion). For\nexample, learning from algae (kelp), echinoderms have been shown to organify iodine from\nseawater using a mechanism called peroxide facilitated (or dependent) diffusion, a process similar\nto what has been described in details in kelp (Miller and Heyland 2013 - JEB). Notably, this paper\nand others clearly outline conceptually, how iodine metabolism is different for marine organisms\ncompared to terrestrial or freshwater organisms. Specifically, seawater contains much high\nconcentrations of iodine and therefore, the evolved capacity of thyroid glands as we know them in\nvertebrates to concentrate iodine from the environment appears to play little or no role in a marine\nenvironment. Specifically, iodine - a reactive molecule that can do considerable damage to cells\nwhen in its ionic form, needs to be first and foremost de-activated as it flows in the cell by a sodium\nindependent mechanism. The authors really need to acknowledge and discuss this body of literature\nas the current presentation of their study and the results can be considered somewhat chordate biased\nand misleading, considering the amount of literature that exists on these processes in other systems\n(Kupper and Carrano provide a nice review for algae - Key aspects of the iodine metabolism in\nbrown algae: a brief critical review - PubMed. The authors also fail to cite another article on sea\nurchins and molluscs (Heyland et al. 2006), which for the first time showed that these species\nincorporate I125 and turn it into T4, using TLC. Considering that Styela clava is marine, this\nliterature is highly relevant and needs to be considered and discussed. This is also critical, as the\nauthor's claim that there is a long standing mystery about the TH precursor in invertebrates. Based\non the existing literature, this is misleading at best and should be rephrased to - an effort has been\nmade to test the hypothesis that the precursor protein for TH synthesis is shared between mammals\nand ascidians. I also urge the authors to carefully review the literature on marine and freshwater fish\niodine metabolism and modifications that may have occurred in vertebrates other than mammals.\nResponse: Thank you for the invaluable comments and suggestions. We agree that the iodine uptake\nand metabolism process in marine organisms, as the essential steps of thyroid hormones (THs)\nbiosynthesis, should be fully introduced and discussed in our manuscript. We added the related\nliterature (Eales, 1997; Heyland and Moroz, 2005; Heyland et al., 2006; Kupper and Carrano, 2019;\nMarcheggiano et al., 1985; Miller and Heyland, 2013; Silverstone et al., 1978; Tymon et al., 2017)\nto highlight the important roles of iodine metabolism in the growth and development of kelp, sea\nurchins, and mollusks in the revised manuscript (Page 2, Line 51-55; Page 6, Line 204-209). Adult\nascidians also have ability to uptake and utilize iodine from seawater, which is concentrated in\nspecific tissues including the endostyle and pharyngeal sac (Dunn, 1980; Suzuki and Kondo, 1971;\nThorpe et al., 1972) (Page 2, Line 58-60). We have also added a book chapter about the evolution\nof metamorphosis in Metazoan that provide an excellent context to our study: Heyland A, Khalturin\nK, Laudet V. Life history evolution: Complex life cycles across animal diversity. In “Life history\nevolution: traits, interactions and applied aspects” edited by Michal Segoli and Eric Wajnberg.,\nWiley, 131-153 (Reference 17).\ninvertebrates” to “Our study is the first to identify the TH protein precursor outside vertebrates by\nproviding the direct evidence of endogenous de novo TH synthesis” (Page 1-2, Line 33-36).\nWe hope these modifications will provide a clearer understanding of the significance of iodine\nmetabolism in marine organisms and avoid the misleading descriptions as the reviewer concerned.\nChanges in manuscript:\n Page 2, Line 51-55\nOutside vertebrates, THs are detectable and play crucial functions in development, especially\nmetamorphosis (Heyland et al., 2024; Heyland and Moroz, 2005; Holzer et al., 2017). Yet, how\ninvertebrates synthesize THs remains unclear, as they lack vertebrate-like TG proteins and no\nfunctional precursor has been convincingly identified. Previous studies have reported that several\nmarine organisms harbor the ability to synthesize TH in vivo. The sea urchin and sea hare have\nability to incorporate iodine in TH formation (Heyland et al., 2006).\n Page 6, Line 204-209\nIodine is a key element for THs synthesis, and it exhibits alive metabolic activity in marine\norganisms, affecting their life processes (Eales, 1997; Heyland et al., 2006; Silverstone et al., 1978).\nFor example, marine bacteria, kelp and sea urchin uptake iodine from seawater through the process\nof hydrogen peroxide-dependent diffusion (Kupper and Carrano, 2019; Miller and Heyland, 2013;\nTymon et al., 2017). Although the presence of THs and their derivatives, as well as iodoproteins, in\nmarine organisms have been demonstrated by several previous biochemical studies (Dunn, 1980;\nEales, 1997; Marcheggiano et al., 1985; Monaco et al., 1981), it has not yet conclusively established\nthat the de novo TH synthesis on a matrix protein effectively occurs in these species.\n Page 2, Line 58-60\nIn ascidian, the iodoprotein(s), with similar sedimentation coefficient to vertebrate TG, are mainly\ndistributed in endostyle, pharynx, and tunic (Dunn, 1974, 1980; Suzuki and Kondo, 1971; Thorpe\net al., 1972).\n Page 1-2, Line 33-36\nOur study is the first to identify the TH protein precursor outside vertebrates by providing the direct\nevidence of endogenous de novo TH synthesis, which offers insights into the evolution of TG protein\nand the emergence of a major vertebrate endocrine system.\n2. The authors also neglect to properly acknowledge that Monaco et al. 1981 showed thyroid\nhormone synthesis on thyroglobulin in a lancelet species - Thyroid hormone formation in\nthyroglobulin synthesized in the Amphioxus (Branchiostoma lanceolatum pallas) – ScienceDirect\nResponse: Thank you for providing this information. Monaco’s paper revealed that a subset of\nendostyle proteins in Branchiostoma lanceolatum pallas exhibits a similar sedimentation coefficient\nto vertebrate thyroglobulin (TG) protein. Their iodination experiments showed that this protein\nfraction has the ability to synthesize THs; however, the precise hormonogenic protein has not yet\n3\nidentified. We have acknowledged this study and added the descriptions of these finding in the\nIntroduction of the revised manuscript (Page 2, Line 55-58).\nChanges in manuscript:\n Page 2, Line 55-58\nIn amphioxus, a subset of endostyle proteins exhibits a similar sedimentation coefficient to\nvertebrate TG protein, the iodination experiments showed that this protein fraction has the ability\nto synthesize THs; however, the precise hormonogenic protein has not yet identified (Monaco et al.,\n1981).\n3. The function of TH signaling in ascidian tadpoles remains sparse, despite efforts to understand\nit better. Knowledge gaps relevant to the manuscript are: the identity of the ascidian receptor, the\nactive thyroid hormone molecule and the detailed effects of THs on the development of ascidian\ntadpoles. While the manuscript contributes some new data to this question, it fails to properly\nanalyze metamorphic development in the urochordate. Ascidian tadpoles are extremely short lived,\ncompared to some other marine invertebrate larvae. Their developmental progression from\nembryonic to post-embryonic and metamorphic development is happening in a few hours and\ntherefore poses two important questions, which are not at all considered in the metamorphosis assay\nthe authors applied: 1) what other morphogenetic effects, if any, do the hormones have on\ndevelopment? Considering that a large portion of the morphological transition of metamorphosis\n(other than tail regression) occurs after settlement, this would be an important question, considering\nhow THs generally orchestrate the transition in chordates 2) The TGF-beta signaling has emerged\nas one major factor of tail regression. There is no link to TH signaling that has ever been mentioned\nin the literature, yet the authors are using this as a phenotypic readout. While the experimental\ndesign of this part of the experiment is generally complete (rescue experiment, knock-down,\npharmacological treatment etc.) the manuscript fails to properly review and explain what we know\nabout the function of TH in ascidian metamorphosis. What is presented in the manuscript is highly\nbiased and forces existing information into a model that effectively suggest ascidian tadpole\nmetamorphosis is the same as in the chordate cousins, the amphibians. This is incorrect and\nmisleading and needs to be fixed. It would also be helpful to know for example, that T3 treatment\nupregulates some of the TH target genes during the critical stages. Finally, please change the y axis\non the graphs in Figure 3 - you are not measuring % metamorphosis, you are measuring tail\nregression.\nResponse: Thank you for this invaluable comment. We would like to mention that we do not claim\nin this manuscript to have solved the question of the identity of the ascidian receptor, the active\nthyroid hormone molecule and the detailed effects of THs on the development of ascidian tadpoles.\nAs nicely stated by the reviewer in its introduction our claims are strictly focused on the\nidentification of an endogenous TH-precursor in invertebrates. We agree with the reviewer that the\nother aspects are important, but they are not the goal of that study. We will carefully consider them\nfor further studies. However, we addressed the best we can the comments of the reviewer:\n(1) Regarding the thyroid hormone receptor (TR) and active THs in ascidian:\n For TR in tunicates. In our previous studies, we identified one Tr gene in the Styela clava\n4\ngenome, which is significantly upregulated after hatching and maintains a high level of\nexpression during metamorphosis (Wei et al., 2020). However, the functional role of the Sc.Tr\ngene remains unclear and requires further investigation.\n For active TH molecule in tunicates. Previous studies have demonstrated that the active TH\nmolecule in the amphioxus is TRIAC, a derivative of T3 that bind the TR and is a substrate of\ndeiodinase (Klootwijk et al., 2011; Paris et al., 2010). To date, the identification of the active\nTH molecule in tunicates remains unresolved. We added these in Discussion section (Page 7,\nLine 272-277).\nChanges in manuscript:\n Page 7, Line 272-277\nIn vertebrates, T3 is the active form of TH, and can bind to thyroid hormone receptor (TR) to activate\ndownstream genes (Laudet, 2011). However, whether a similar mechanism exists in invertebrates\nremains unknown. In amphioxus, TRIAC, a derivative of T3, is the ligand of TR, which supports\nthat TRIAC is the active form of the hormone (Klootwijk et al., 2011; Paris et al., 2010). However,\nit remains to be determined the active TH molecule in tunicates.\n(2) Regarding the detailed effects of THs on the development of ascidian tadpoles:\nWe agree that, beyond tail regression, it is important to evaluate additional morphogenetic roles of\nTHs during S. clava metamorphosis. To address this, we performed experiments to reduce TH levels\nby knocking down the ScTg-like gene and examined the developmental progress after tail regression\nin S. clava:\n At 24 hours post hatching (hph): Control animals (in FSW) contracted their tail tissues into\nthe compact trunk with four outward tunic protrusions (Supplementary Fig. 6e1 and f1),\nwhereas ScTg-like knockdown (KD) animals had not yet completed tail regression\n(Supplementary Fig. 6a1 and c1).\n At 72 hph: Control animals became spherical (Supplementary Fig. 6e2 and f2); in contrast,\nKD animals just completed tail regression (Supplementary Fig. 6a2 and c2).\n At 120 hph: Control animals exhibited a clearly recognizable juvenile body structure and\ntissues, established internal circulation, and could take up seawater (Supplementary Fig. 6e3\nand f3); however, KD animals showed little change compared with 72 hph (Supplementary\nFig. 6a3 and c3).\n At 168 hph: Control animals continued to develop (Supplementary Fig. 6e4 and f4). In KD\nanimals, siphons were visible (indicated by red arrows) but failed to extend, and the organs\nwere immature (similar to control animals at 72 hph) (Supplementary Fig. 6a4 and c4); these\nKD animals establish internal circulation and exchange with the seawater, yet their\nmorphology remains markedly divergent from controls (Supplementary Fig. 6a4 and c4).\nTaken together, these results show clearly that TH decrease markedly delay metamorphosis,\nleading to an abnormal morphology, particularly the impaired siphon outgrowth, and the immature\norgans in juveniles. Meanwhile, the abnormal phenotypes due to ScTg-like gene knockdown were\n5\nrescued by T3 in this experiment (Supplementary Fig. 6b and d). We have incorporated these results\ninto the Results (Page 4, Line 134-140) and Discussion (Page 7, Line 262-272) sections and\nSupplementary Fig. 6 of the revised manuscript.\nChanges in manuscript:\n Page 4, Line 134-140\nWe further tracked the morphological changes of S. clava larvae with or without ScTg-like knock-\ndown, and found that the process of metamorphosis of siRNA-treated larvae was largely delayed.\nIn addition, compared with juveniles without ScTg-like knock-down, the “juveniles” in siRNA\ntreated groups exhibited abnormal phenotypes with inextensible siphons and immature organs\n(Supplementary Fig. 6-7), suggesting that the organ morphogenesis of S. clava larvae during\nmetamorphosis was regulated by ScTg-like gene.\n Page 7, Line 262-272\nPrevious studies have proved that TH play crucial roles in tail regression of ascidians (Patricolo et\nal., 2001). In this study, we found that after reducing the TH levels, not only the tail regression,\nbut also the subsequent metamorphic process was impacted (Figure 3, Supplementary Fig. 6). After\nknocking down the expression of ScTg-like gene, the process of metamorphosis was significantly\ndelayed, and the morphology of \"juvenile\" was abnormal, with the inextensible siphons and\nimmature organs (Supplementary Fig. 6). Considering that the Sc.Tg-like gene contains a length of\n17,373 base pairs coding sequence (Supplementary data 1), it is difficult to clone its full-length\ncoding sequence and synthesize mRNA for rescue experiment. Instead, we performed the rescue\nexperiment by T3 treatment, which clearly showed that the rescued larvae developed normally,\nsimilar to the wild type (Figure 3, Supplementary Fig. 6). These results suggest that TH signal is of\ngreat importance in controlling the timing of metamorphosis, morphogenesis, and organ formation\nin S. clava larvae.\nclava larvae after tail regression. Two specific siRNAs, siRNA-1 (a) and siRNA-2 (c), were used\nto knock down the expression of ScTg-like gene, respectively. Negative control, larvae treated with\nNC siRNAs (e). Blank control, larvae treated with FSW (f). The concentrations of siRNAs were 0.4\nμM. The concentration of MMI was 120 mg/L. T3 was employed for rescue (b, d), with 50 μg/mL.\nThe morphologies of larvae or juveniles were observed at five time points after tail regression,\nincluding 24 hours post hatch (hph, a1-f1), 72 hph (a2-f2), 120 hph (a3-f3) and 168 hph (a4-f4).\nThe red arrows indicate the immature siphons. The fraction showed on each image indicates the\nnumber of S. clava with the phenotypes showed in the image / total number of S. clava in each\nexperimental group. Scale bars = 100 μm.\n7\n(3) Regarding TH and TGF-β signaling:\nWe agree that TGF-β signaling is an important driver of tail regression in S. clava (Shi et al., 2025).\nDisrupting either TGF-β or TH signaling produces comparable tail-regression phenotypes, implying\nthere may be a potential link between two signaling molecules. We analyzed crosstalk between the\npathways based on the transcriptome of methimazole (MMI; an inhibitor of TH synthesis)-treated\nlarvae (Reviewer-only Fig. 1a-c) (This data has been deposited in NCBI SRA database, the\naccession number is PRJNA1364819). We found that after inhibition of the TH synthesis, the\nupregulated gene is TGF-β receptor 1 (1.6 folds), and the downregulated genes includes TGF-β\nreceptor 2 (1.7 folds), Smad2 (5 folds) and E2f4/5 (1.3 folds) genes (Reviewer-only Fig. 1d). The\nexpression changes of TGF-β receptor 1 and TGF-β receptor 2 were opposite. Therefore, we further\nexamined the expression profiles of these two genes during the development of the S. clava and\nfound that the TGF-β receptor 1 gene was continuously downregulated before the larval tail\nregression, while TGF-β receptor 2 gene showed the opposite profile (Reviewer-only Fig. 1e). This\nimplied that the two receptor genes of the TGF-β signaling pathway play opposing roles during the\ntail regression of S. clava larvae, with TGF-β receptor 2 gene being positively regulated by THs.\nThese results indicate that TH depletion alters the expression of genes within the TGF-β\npathway, consistent with functional similarity between TH and TGF-β signaling in S. clava tail\nregression. Because of space limitation we cannot introduce these data in the paper, but this situation\nis reminiscent of the crosstalk between TH and TGF-β signaling during metamorphosis and will\ncertainly warrant further investigations.\npathway. (a) Workflow of MMI inhibition experiment in S. clava larva. The concentration of MMI\nis 1000 mg/L. (b-c) Inhibition of the TH synthesis led to the filature of tail regression in S. clava\nlarvae (Wei et al., 2020). The phenotypes of S. clava larvae treated with MMI and sea water\n(negative control group) and the number of larvae in each group are showed in b. The statistical\nanalysis results of the percentages of tail regression are showed in c. (d) Expression changes of key\ngenes involved in TGF-β signaling pathway after treating with MMI. The key genes involved in\nTGF-β signaling pathway and their regulatory axis are showed on the left. The expression changes\nof the genes are showed on the right. The expression level of each gene are indicated by the FPKM\nvalue. (e) The expression profiles of TGF-β receptor 1 and TGF-β receptor 1 genes during S. clava\nembryonic and larval development, including 2-8 cells (2-8 cells), gastrula (gast), neurula (neu),\ntailbud (tb), hatched swimming larval (hsl), tail regressing larval (trl), and metamorphic juvenile\n(mj) stage. Higer expression level is showed in red, and the lower expression level is showed in\nblue.\n9\n(4) For the details of annotations:\nThank you for pointing this out. The y-axis label was imprecise. We have revised Figure 3 to report\n“% of tail regressed larvae” rather than “% metamorphic larvae”.\n4. The authors present T4 and T3 staining in the supplement but only show T3 staining in the\nmain text. I am confused about this. Not with standing my previous criticism that we don't actually\nknow what the active TH is in ascidians (a fact that should be clearly stated), it would be useful to\nmake adjustments to the current figures but more importantly, discuss and explain the distributions\nof T4 and T3. If we go with the mammalian model, we would expect T3 to be produce by de-\niodination from T4 in target tissues. A good starting point would be to discuss whether T3 is actually\nfound in areas and structure relevant for the metamorphic transition. Furthermore, it would be\ncritical, to explain why there is a significant overlap between T3 and T4 presence in the tadpoles\nand discuss this in detail. Any TH endocrinologist will be eager to get a proper explanation of this\nfact. Similarly, the authors completely fail to discuss the difference/similarity between the follicular\nstructure and the endostyle. Is this a separate organ? Why are ascidians accumulating THs in both?\nAre they linked and part of the same structure? What is the developmental cell lineage of that\nstructure. There are several exciting papers from recent years showing that the endostyle 7-9 and\nthe thyroid gland in fact originate from the same lineage and single cell studies confirm this\nbeautifully. What about the follicular organ? Is there any information on that. This would not require\nmore experiments but simply an expansion of existing datasets (Jiang, A. et al. Spatially resolved\nsingle-cell atlas of ascidian endostyle provides insight into the origin of vertebrate pharyngeal\norgans. Sci. Adv. 10, 1-16 (2024) - which is cited). I really think the authors need to address this\ngap, as their discussion is incredibly superficial and neglects so many important questions which\nwill make readers who care about the evolution of TH signaling scratch their heads.\nResponse: We appreciate the reviewer’s suggestions. We added some explanations and analysis in\nthe following two aspects.\n(1) Regarding the distributions of T3 and T4 in S. clava larva:\nBased on in vivo functional experiments, our ELISA data showed a significant decrease in both T3\nand T4 levels in S. clava larvae after knocking down the ScTg-like gene (Fig. 3c-d). Whatever the\nactive form of TH is in ascidians, the changes in both T3 and T4 levels are sufficient to indicate\nwhether the TH synthesis process is affected, which is directly relevant to our study.\nAs pointed out by the reviewer, in mammals, T3 is typically produced by deiodination of T4\nin target tissues. Therefore, it is not surprising that we observe the overlap in the distributions of T4\nand T3.\n(2) Regarding the relationship between follicle-like structure in larva and endostyle in adult:\nWe agree that the relationship between the follicular-like structure and the endostyle need to be\nfurther discussed. The follicle-like structure is only present in the larval stage of S. clava\n(Supplementary Fig. 11a), while the endostyle is an organ in adult (Supplementary Fig. 11b). We\nsuggest that the follicle-like structure and the zone 7-9 regions in endostyle perform similar\nfunctions in TH synthesis at different developmental stages. Their precise relationship will be the\ntopic of future studies\nstrong signals in the follicle-like structure of the larva (Supplementary Fig. 8). Furthermore, by\nanalyzing our recently published spatially resolved single-cell transcriptomic data of the S. clava\nendostyle (Jiang et al., 2024), we observed that crucial genes involved in TH synthesis are also\nstrongly expressed in the zone 7-9 of the endostyle (Supplementary Fig. 11c). These findings\nsuggest that the zone 7-9 of the S. clava adult endostyle may share the common cellular origin with\nthe larval follicle-like structure. We now discuss fully this aspect (Page 7, Line 257-259).\nIn addition, a similar structure with the follicle-like structure in S. clava larvae has also been\nobserved in the larvae of ascidian Herdmania curvata (Eri et al., 1999), indicating the conservation\nof the follicle-like structure and functions in ascidians. We also added this point in Discussion (Page\n7, Line 259-161).\nSupplementary Figure 11 (in the revised manuscript) Response Figure 4. Distributions of\ncrucial TH synthesis-related genes in adult endostyle. (a-b) The locations and diagrams of larval\nfollicle-like structure (a) and adult endostyle (b). (c) Spatial atlas shows the distribution of cell units\nof S. clava endostyle on the transverse section colored by cell annotation (Jiang et al., 2024). The\nthyroid-like cells in S. clava endostyle are labelled by pink dots. The Distribution of ScTpo,\nScDuox1, ScDuoxa1, ScTtf1, ScTtf2 and ScPax2/5/8 genes in S. clava endostyle based on the spatial\nsingle cell transcriptomic data. Higer expression level is showed in pink, and the lower expression\nlevel is showed in green.\nChanges in manuscript:\n Page 7, Line 257-259\nFurthermore, the genes involved in TH synthesis were expressed both in the follicle-like structure\nin larvae and thyroid equivalent region of endostyle in adult (Supplementary Fig. 8 and 11).\n Page 7, Line 259-261\nIn addition, a similar structure was also detected in the larvae of ascidian Herdmania curvata,\n11\nimplying the conservation of the follicle-like structure and its functions in ascidians. (Eri et al.,\n1999).\n5. The authors do a great job narrowing down the identity of the putative TG-like protein, using\nall the right techniques and I think they show convincingly that this protein is in fact a matrix for\nTH production. However, I find their analysis of TGs in ascidians and among bilaterians a lot less\nconvincing. Why, does Figure 1d show a gene phylogeny with all ascidian genes (which are\nrelatively closely related) and compares them to vertebrate TGs. The authors appear to conclude\nthat this reveals \" …that Sc.CG.S284.31 and Sc.CG.S55.56 proteins clustered within the clade of\nvertebrate TG proteins …\". I disagree, all this might indicate is that the vertebrate proteins are\ndistantly related to ascidian proteins (support for branch is well below 90, which even for a NJ tree\nwith 1000 replicates is bad and basically does not even support that conclusion very well). I think\nthe authors need to go back to the dataset, which they present in the supplement (Sup fig. 7), which\nmaps domain structures onto an animal phylogeny. This dataset should be properly analyzed\nphylogenetically, to see whether invertebrate TGs generally cluster together or not (or whether\nascidian domains are indeed closer to vertebrates). Next the authors should conduct a synteny\nanalysis to narrow down the evolution of TG domains and use the proper statistical framework. This\nwould allow them to answer the question they currently state in the manuscript of 1) whether\nchordates (including basal chordates) are indeed different from other invertebrate or whether the\nseparation is actually between vertebrates and invertebrates 2) it would also provide a more precise\nargument about the candidates they investigated here are actually evolutionarily related. Finally, it\nwould essential that the authors provide at least a brief discussion about the presence absence of\nspecific TG domains found across TG-like proteins in animals.\nResponse: Thanks for the reviewer’s constructive suggestions for improving the understanding of\nTg and Tg-like genes in evolutionary aspects. We performed the phylogenetic analysis and added an\nanalysis of the Tg1 domain and synteny. In addition, we also supplemented the analysis of the\nstructural conservation of the Tg1 domain in vertebrate TG proteins and invertebrate TG-like\nproteins.\n(1) Regarding the phylogenetic analysis in Figure 1d:\nFor the phylogenetic tree in Figure 1d, we have made the following improvements:\n We incorporated sequences of TG proteins from a broader range of vertebrate species as\nreferences, including Homo sapiens, Macaca mulatta, Bos taurus, Canis Iupus familiaris and\nMus musculus in Mammalia, Xenopus tropicalis, Ambystoma mexicanum in Amphibian,\nGallus gallus in Aves, Danio rerio, Lepisosteus oculatus, Oryzias latipes and Paralichthys\nolivaceus in Fish, and Petromyzon marinus in Cyclostomata.\n We included five other Tg1 domain-containing proteins (Novinec et al., 2006), except TG,\nfrom H. sapiens genome to enrich the phylogenetic analysis;\n TG-like proteins in amphioxus (Branchiostoma floridae) and sea urchin (Strongylocentrotus\npurpuratus) predicted based on the number of Tg1 domains (Belkadi et al., 2012) were also\nincluded as part of the reference.\nand Sc.CG.S55.56 proteins from the ascidian S. clava still exhibit a closer relationship with\nvertebrate TG proteins compared to other Tg1 domain-containing proteins in S. clava genome (Fig.\n1d). In addition, the predicted B. floridae and S. purpuratus TG-like proteins also clustered within\nthe clade of vertebrate TGs and ascidian Sc.CG.S284.31 and Sc.CG.S55.56 proteins. However,\nother Tg1 domain-containing proteins from S. clava genome display closer evolutionary\nrelationships to different types of Tg1 domain-containing proteins in H. sapiens genome (Fig. 1d).\nWe updated the result of phylogenetic analysis in Figure 1d, and added the descriptions about this\nresult in Results (Page 3, Line 77-82).\nFigure 1d (in the revised manuscript). Phylogenetic analysis of TG proteins in vertebrates, the\nTg1 domain-containing proteins in ascidian S. clava and human, and the predicted TG-like\nprotein. The vertebrate TG proteins are labelled in red box. The predicted TG-like proteins have\n13\nbeen identified in amphioxus (B. floridae) and sea urchin (S. purpuratus) (Novinec et al., 2006).\nBootstrap values greater than 30 are indicated in the nodes of the phylogenetic tree. The blue dots\nlabel the closest relative of vertebrate TG proteins among ascidian S. clava Tg1 domain-containing\nproteins.\nChanges in manuscript:\n Page 3, Line 77-82\nFurthermore, we performed a phylogenetic analysis comparing these Tg1 domain-containing\nproteins with vertebrate TG proteins, Tg1 domain-containing proteins in human (Novinec et al.,\n2006) and predicted TG-like protein in amphioxus and sea urchin (Belkadi et al., 2012). The result\nrevealed that Sc.CG.S284.31 and Sc.CG.S55.56 proteins clustered within the clade of vertebrate\nTG proteins, suggesting that they were the closest relatives to vertebrate TG proteins among all the\nidentified Tg1 domain-containing proteins (Fig. 1d-e).\n(2) Regarding the synteny analysis between Tg and Tg-like genes:\nSynteny analysis is indeed a useful approach for investigating the evolution of certain gene in the\nspecies- to phylum-wide level. According to the reviewer’s suggestion, we performed synteny\nanalysis of vertebrate TG protein-encoding genes, as well as ascidian and amphioxus TG-like-\nencoding genes.\n Across multiple mammalian genomes, we found that the Tg locus resides within a conserved\nlocal neighborhood and the flanking genes and their arrangement are broadly retained\n(Reviewer-only Fig. 2). In contrast, this local synteny is not present in non-mammalian\nvertebrates (Reviewer-only Fig. 2).\n We examined the neighborhoods of lamprey Tg (P. marinus), ascidian Tg-like (S. clava), and\namphioxus Tg-like (B. floridae) genes and did not detect conserved gene order among these\nloci (Reviewer-only Fig. 2). The identities and relative positions of adjacent genes differ across\nthese species.\nReviewer-only Figure 2. Synteny analysis of vertebrate Tg genes and invertebrate Tg-like\ngenes. The synteny of Tg genes in H. sapiens, B. taurus, D. rerio and P. marinus, and Tg-like gene\nin S. clava and B. floridae have been analyzed. 10 genes located at upstream or downstream of\nTg/Tg-like genes have been identified. The locations of Tg/Tg-like genes are labelled in yellow\nboxes.\nsequences or domains, may have undergone frequent chromosomal rearrangements or transposition\nof DNA segment during evolution from invertebrates to mammals. Importantly, while synteny\nanalysis can support the existence of an evolutionary relationship when similarity in neighboring\ngenes is detected, it cannot be used to rule out such a relationship in the absence of synteny, as\nabsence of evidence does not constitute evidence of absence. In the present case, extensive genome\nrearrangements are well documented in urochordates, where even the structural organization of the\nHox cluster is not conserved (Blanchoud et al., 2018; Zhang et al., 2021). Therefore, additional\nfunctional studies of these proteins will be required to reach definitive evolutionary conclusions.\n(3) Regarding the conservation analysis of the Tg1 domain in TG and TG-like proteins:\nTo further investigate the conservation between TG and TG-like proteins, we analyzed the 3D\nstructure of the Tg1 domain in TG/TG-like proteins. For the structural part we added an additional\nReviewer-only Figure 3. An Alphafold prediction was performed for all sequences detected as\npredicted TG-like proteins, as well as for the TG protein in lamprey. The results show that all Tg1\ndomains share a relatively well-conserved folding pattern. The Tg2 and Tg2-like domains, although\ndefined by only 3-4 strictly conserved amino acids, also exhibit flexible folding. A common\nstructural denominator is detected for all these Tg2 domains. For Tg3, which is not detected by\nHMM searches in invertebrates, it appears that there are other domains, sufficiently different in\nsequence to be undetectable by HMMs, but which possess structural similarities with Tg3. All these\ndata confirm the validity of these proteins as likely candidates for a similar Tg-like function but we\nagree and have made clear in the paper that this require further experimental validation.\nBecause this analysis relies on Alphafold predictions, it cannot provide definitive conclusions\nabout the overall topology of invertebrate proteins. TG protein consists of many small domains\nseparated by flexible regions, only the folding of these individual domains relative to one\nanother can be meaningfully compared. As a consequence, Alphafold predictions cannot be used to\nconfirm the precise spatial placement of tyrosine residues.\nIt is nevertheless interesting that vertebrate TG is built from a limited number of domains that\nare repeated several times (with the exception of ChEL). The same pattern is observed in TG-like\nprotin of S. clava, as well as in the predicted proteins, where structural similarities emerge even\nwhen individual domains are not detected by sequence analysis alone. In addition, some repetitions\nof small domains may be present in invertebrates that do not occur in vertebrates, which would still\nbe compatible with the known structural organization of TG proteins and TG-like proteins.\nOverall, the repetition of domains likely serves as a scaffold whose primary role is to position\nspecific tyrosine residues required for TH synthesis. The remaining domains have no catalytic\nactivity themselves and function only to ensure the correct spatial arrangement of these tyrosines,\nwhich in turn explains why many of these domains are difficult to identify based solely on sequence\ninformation.\nBecause of space limitation we cannot introduce these data in the manuscript but the following\n15\nfigure is available for the reviewer.\nReviewer-only Figure 3. Comparison of thyroglobulin structural domains across species.\nDomain organization is shown for H. sapiens and Lethenteron reissneri (TG), for S. clava (protein\npreviously identified as performing a TG-like function in invertebrates), and for Branchiostoma\nbelcheri (predicted TG-like protein). Tg1, Tg2 and Tg3 domains are present in human (PDB ID:\n6SCJ) and in lamprey (AlphaFold prediction). In S. clava (AlphaFold) and B. belcheri (AlphaFold),\nTg1 domains are detected using an HMM motif search (red). This small motif, formed by two beta\nstrands and one alpha helix, shows a conserved structure across all four species, with variability\ncomparable to that observed among human TG1 repeats. In humans and lampreys, the three Tg2\ndomains consist of four short beta strands grouped into two paired elements. In S. clava, no\ncanonical Tg2 motif was detected, and only Tg2-like variants (CxxCxG) are present. B. belcheri\ndisplays a mixture of Tg2 motifs (CxxCPxG) and Tg2-like motifs (CxxCxG). In both S. clava and\nB. belcheri, AlphaFold predictions reveal the same arrangement of four short beta strands paired\nand clustered together, although the connecting loop between the two pairs varies in length. The\nTg3 domain in vertebrates consists of an alpha helix and two beta strands forming a structure similar\nto a Tg1 domain, followed by a series of loops, beta strands and alpha helices, the whole poorly\nordered that participate to flexibility to the vertebrate protein. This domain is not detected by HMM\nin invertebrates. Structural predictions indicate that the N-terminus of the S. clava TG-like protein\nexhibits a similar fold. In vertebrates, these Tg3-related structures occur near the C-terminus,\nimmediately upstream of the ChEL domain, which is entirely absent in S. clava and in other\npredicted invertebrate sequences.\n6. The authors do not cite a relevant paper by Johnson and Moore, reviewing the\ncarboxyesterase/cholinesterase gene family in invertebrate deuterostomes (2012).\nthe Discussion, and discuss the potential value of studying the ChEL domain for understanding the\nevolution of invertebrate Tg-like genes to vertebrate Tg genes (Page 6, Line 235-240).\nChanges in manuscript:\n Page 6, Line 235-240\nAdditionally, proteins containing the ChEL domain have also been identified in invertebrate\ndeuterostomes, and studies have shown that these genes undergo gene duplication and\nrearrangement, resulting in proteins with diverse functions (Johnson and Moore, 2012).\nInvestigating the interactions between the ChEL domain-containing proteins and TG-like proteins,\nas well as the potential for gene fusion, may provide insights into the evolutionary transition from\ninvertebrate Tg-like genes to vertebrate Tg gene.\nOther comments\n7. The manuscript has some typos and grammatical errors. Please proof-read\nResponse: Thank you for pointing this out. We have thoroughly proofread the manuscript and\ncorrected all identified typos and grammatical errors to ensure clarity and accuracy.\n8. While the authors establish this enrichment and draw a functional similarity between the S.\nclava structure and vertebrate thyroid follicles based on glycoproteins, they do not explicitly provide\nan explanation for the specific role of glycoproteins within the vertebrate follicle itself in the\nprovided text. They observe their presence as a shared feature but do not elaborate on their function\nwithin the vertebrate context. Considering how little we know about the conservation of this feature\nacross animals, we need more explanations in the text.\nResponse: Thanks for this question. Glycosylation in the vertebrate thyroid gland is thought to be\nessential for its function: many key proteins (such as thyrotropin and its receptor, sodium/iodide\nsymporter, pendrin, and thyroglobulin) responsible for hormone synthesis in the thyroid are\nglycosylated (Citterio et al., 2019; Marechal et al., 2022). On the other hand, high glycoprotein\ncontent reflects the high secretory activity of a tissue or structure (e.g. follicle structure) since most\nsecretory proteins need to be glycosylated before entering the secretory pathway. Vertebrate TG\nproteins are the typical glycoproteins, and their glycosylation is essential for secretion and function\n(Citterio et al., 2019; Jeso and Arvan, 2016). Thus, staining for glycoproteins can be used to reflect\nthe function of thyroid gland. A similar staining method for glycoproteins was also used to label the\nthyroid follicular structures in zebrafish (Wendl et al., 2002) and many other fish species. We have\nadded this explanation in the main text (Page 7, Line 247-252).\nChanges in manuscript:\n Page 7, Line 247-252\nGlycosylation in the vertebrate thyroid gland is thought to be essential for its function (Citterio et\nal., 2019; Marechal et al., 2022). High glycoprotein content reflects the high secretory activity of a\n17\ntissue or structure (e.g. follicle structure) since most secretory proteins need to be glycosylated\nbefore entering the secretory pathway. Vertebrate thyroglobulin proteins are the typical\nglycoproteins, and their glycosylation is essential for secretion and function (Citterio et al., 2019;\nJeso and Arvan, 2016). Thus, staining for glycoproteins can be used to reflect the function of thyroid\ngland (Wendl et al., 2002).\n9. The authors mention and use transcriptomics data but fail to provide the raw data and also do\nnot provide sufficient information on the analysis they used.\nResponse: Thank you for pointing this out. The datasets presented in this study can be found in\npublicly available repositories. The transcriptomic data of S. clava during early developmental\nprocesses are deposited in NCBI SRA database (accession number PRJNA1232216). The Stereo-\nseq datasets on the S. clava endostyle have been deposited at China National GenBank DataBase\n(CNGBdb, https://db.cngb.org/) under the accession number CNP0004228. We have added the\ninformation regarding the access to the raw data of transcriptomics used in this study in the “Data\nAvailability” section (Page 14, Line 536-541).\nBesides, the transcriptomic data of S. clava treated with MMI are deposited in NCBI SRA\ndatabase (accession number PRJNA1364819).\nChanges in manuscript:\n Page 14, Line 536-541\nData Availability\nThe datasets presented in this study can be found in publicly available repositories. The\ntranscriptomic data of S. clava during early developmental processes are deposited in NCBI SRA\ndatabase (accession number PRJNA1232216). The Stereo-seq datasets on the S. clava endostyle\nhave been deposited at China National GenBank DataBase (CNGBdb, https://db.cngb.org/) under\nthe accession number CNP0004228. Source Data are provided with this paper.\nThe authors of this manuscript report on the identification of an invertebrate orthologue of\nthyroglobulin (TG), the protein precursor for the synthesis of thyroid hormones, in an ascidian.\nPrevious studies have not conclusively identified this gene in invertebrates, possibly due to the need\nto search for structural similarities rather than by sequence alone. Here the authors identify a\nputative TG gene, localize its expression in the embryo and in the adult, show that it can be iodinated\nand demonstrate that it is required for larval metamorphosis.\n1. Figure 1 shows a cross-section of the adult endostyle (=thyroid gland) showing\nimmunoreactivity for TG-like proteins, a gene tree and domain organization. Because there were\nseveral protein candidates, immunoprecipitation followed by mass-spec peptide fingerprinting\nidentified likely candidate TG-like genes.\nIn part C the authors mention intensities, but this appears to be related to the number of peptides\nidentified. The use of “intensities” should be changed as it implies immunofluorescent intensities\n(as shown in parts A-B).\nResponse: Thank you for your valuable suggestion. The “intensity of protein” indicates the relative\nabundance of each protein in the sample. We updated the Figure 1c and added this description in its\nfigure legend (Page 19, Line 720-721).\nChanges in manuscript:\n Page 19, Line 720-721\n“Intensity of protein” represents the relative abundance of each protein among all immunolabeled\nproteins of Anti-BtTG.\n2. Figure 2 depicts the temporal and spatial expression pattern of the putative TG-like\ngenes/proteins. It also shows that the protein from the Sc.CG.S248.31 gene (=TG-like) can be\niodinated in vitro, suggesting that it can serve as a matrix for TH synthesis.\nFigure 3 reports the results of siRNA knockdowns on the TG-like gene and the subsequent\nimpact on larval metamorphosis. Part B shows that siRNA treatment was able to knock-down\nexpression by 50-60%. I am assuming that these graphs are RT-qPCR results (this should be clarified\nthroughout figure). I would also suggest that the figure legend report the error bars as SEM (and\nthroughout figure). Although this is mentioned in the materials and methods, it would be good to\ninclude here. The authors report an “NC” control siRNA. Although the sequence is reported in\nsupplemental data, it is not described. Is this a scrambled siRNA? A target for a non-existent gene\nin the Styela genome? The authors should explain. Lastly, the authors lack a useful experiment\ndemonstrating the outcome of their knock-down – an immunofluorescence image showing\na reduction in protein levels in the larva. The authors show that they have a working antibody to TG-\nlike, it would be very beneficial to demonstrate that the knock-down also reduces proteins levels in\nthe siRNA-treated embryos. Lastly, they show that the siRNA phenotype can be rescued by\n19\nproviding exogenous T3 to rescue metamorphosis.\nIt would have been nice to show that a form the TG-like mRNA that cannot be degraded by\nsiRNA can also rescue the knock-down. The authors should discuss why this wasn’t performed.\nI suspect it was because of the size of the gene and difficulty in cloning the entire product, but it\nwould be useful to the reader to see this discussion.\nResponse: Thanks for the reviewer’s suggestions, which helped us improve the rigorism of our\nresults.\n In Figure 3, we qualified the mRNA levels through RT-qPCR method, and all values in figures\nare depicted as mean ± SEM; the T-test was used for significant tests. We have added the\ndescriptions in the figure legends to clearly clarify the methods used in the experiment and\ndata analysis (Page 21-22, Line 752-756, 769-770).\n The NC siRNA used in our experiments was designed for a target for a non-existent gene in\nthe Styela clava genome, and therefore it does not interfere with the function of any genes in\nthe ascidian genome. We have explained this point in Method (Page 13-14, Line 514-515).\n To demonstrate the outcome of gene knock-down in the protein level, we performed an\nadditional experiment as described below:\nWe collected larvae with or without the knockdown of the ScTg-like gene and performed\nimmunofluorescence staining using Anti-ScTG-like antibody. We found that the\nimmunofluorescence signals in larval follicle-like structure were significantly reduced due to\nScTg-like gene knock-down, which confirms that the outcome of gene-knock is indeed a\ndecrease of ScTg-like in protein level (Supplementary Fig. 5). These results are provided in the\nSupplementary Fig. 5 (Page 4, Line 128-129).\n Due to the extremely large size of the ScTg-like gene (17,373 base pairs), it is very difficult to\namplify and subclone the entire gene into the vector. As a result, it is hard to perform a rescue\nexperiment through overexpression of the full-length ScTg-like gene. We have included a\ndiscussion of this limitation in the Discussion section of the revised manuscript (Page 7, Line\n267-271).\nknocking down the ScTg-like gene. Two specific siRNAs, siRNA-1 and siRNA-2, were used to\nknock down the expression of ScTg-like gene, respectively. Negative control, larvae treated with\nNC siRNAs. Blank control, larvae treated with FSW. The concentrations of siRNAs are 0.4 μM.\nThe presence of ScTG-like protein in the follicle-like structures was detected using antibody of\nScTG-like protein (magenta). The cell boundaries of the follicle-like structures were labeled by\nphalloidin (green) staining. The nuclei were labeled by DAPI (blue). The fraction showed on each\nimage indicates the number of S. clava with the phenotypes showed in the image / total number of\nS. clava in each experimental group. Scale bars = 10 μm.\nChanges in manuscript:\n Page 21-22, Line 752-756, 769-770\nLine 752-756: Quantifications of ScTg-like mRNA expression in gene knockdown (siRNA-1 and\nsiRNA-2, two specific siRNAs of ScTg-like are designed to treat the S. clava larvae respectively),\nnegative control siRNA treatment (NC, as the negative control), and filtered seawater treatment\n(FSW, as the blank control) groups through RT-qPCR method.\nLine 769-770: All values in this figure are depicted as mean ± SEM. Significant tests are analyzed\nby T-test.\n Page 13-14, Line 514-515\nThe sequence of negative control siRNA does not target any gene in the S. clava genome.\n21\n Page 4, Line 128-129\nAfter treating larvae with specific siRNAs of ScTg-like, the expression of ScTg-like were\nsignificantly decreased in both mRNA and protein levels (Fig. 3b, Supplementary Fig. 5).\n Page 7, Line 267-271\nConsidering that the ScTg-like gene contains a length of 17,373 base pairs coding sequence\n(Supplementary data 1), it is difficult to clone its full-length coding sequence and synthesize mRNA\nfor rescue experiment. Instead, we performed the rescue experiment by T3 treatment, which clearly\nshowed that the rescued larvae developed normally, similar to the wild type (Figure 3,\nSupplementary Fig. 6).\n3. Figure 4 reports the immunolocalization of TG-like protein in the larva and depicts the putative\nanterior follicle-like structure. Although they show that siRNA treatment reduces the level of T3\ndetected by immunofluorescence, they don’t show reduction in TG-like levels, as mentioned\npreviously. The follicle is intriguing, but it is difficult to know exactly where it is located in the\nlarva. The SEM image isn’t particularly useful in my opinion. It would be beneficial if a drawing\ncould be incorporated showing the location of the follicle relative to the palps and other anterior\nstructures that the broader community is familiar with. It is located centrally? dorsally, ventrally?\nResponse: Thanks for your valuable suggestions.\n We performed the immunofluorescence using antibody of ScTG-like on S. clava larvae with or\nwithout knockdown of Sc.Tg-like gene, which confirms the decreased level of Sc.Tg-like gene\nencoed protein in gene knock-down larvae (Supplementary Fig. 5). The results have been\nincluded in the Supplementary Fig. 5 (Page 4, Line 128-129).\n We agree with your suggestion that a schematic diagram of the follicular structure would better\nillustrate its location in S. clava larva. The follicle-like structure is located at the central anterior\nend of the trunk. We have added a schematic diagram to Figure 4b to clarify the position of the\nfollicle-like structure.\nFigure 4b (in the revised manuscript). Diagram of the follicle-like structure at the central\narterial of S. clava larval trunk.\nChanges in manuscript:\n22\n Page 4, Line 128-129\nAfter treating larvae with specific siRNAs of ScTg-like, the expression of ScTg-like were\nsignificantly decreased in both mRNA and protein levels (Fig. 3b, Supplementary Fig. 5).\nIn summary, the authors have presented a nice study identifying a TG-like gene/protein in the\nascidian Styela clava. The authors should address my previous comments regarding some of the\ndata presentation and it would be very good to show that the siRNA knock-down effectively reduces\nthe level of TG-like protein.\nResponse: We thank the reviewer for the positive overall evaluation of our study. We have carefully\naddressed all previous comments regarding data presentation, and the corresponding revisions have\nbeen made throughout the manuscript. In addition, to address the reviewer’s important suggestion\nregarding the effectiveness of siRNA knock-down at the protein level, we have now included\nadditional evidence demonstrating that siRNA treatment leads to a significant reduction of Sc.Tg-\nlike in protein level. This result has been added to the revised manuscript in Supplementary Fig. 5\nand described in the Results section (Page 4, Line 128-129). We hope that these additions help to\nfurther clarify this point."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I would like to thank the authors for thoroughly addressing the concerns raised in the original version of the manuscript. After reviewing the revision I found that concerns have been addressed satisfactorily and I have no further comments.", "reviewer_2": "The authors have sufficiently addressed my comments. Thank you for addressing these concerns."}, "author_response": ""}]

Decision letter