Journal: The EMBO Journal

Article Title: Cell cycle-driven transcriptome maturation confers multilineage competence to cardiopharyngeal progenitors

doi: 10.1038/s44318-025-00613-y

Figure Lengend Snippet: ( A – C ) Developmental dynamics of Gata4/5/6 gene expression. a. Representative confocal images showing double FISH detection of Gata4/5/6 and Depdc1b expression. Segmented cells, nuclei and transcripts shown on the right. Magenta: nuclei (GFP::PCNA); blue: Gata4/5/6 nascent RNA; green: Depdc1b mRNA. Scale bar = 5 µm; arrowhead: TVC; open arrowhead: ATM. ( B , C ) Semi-quantification of Gata4/5/6 ( B ) and Depdc1b ( C ) gene expression spanning TVC migration but prior to TVC division. ( D ) Cell cycle phases as determined by the GFP::PCNA reporter. ( E ) Confocal images of double FISH revealing Gata4/5/6 and Depdc1b mRNAs in Tyr CRISPR control and Gata4/5/6 CRISPR embryos. Scale bar = 10 µm Arrowhead: TVC; Open arrowhead: ATM. ( F – F’ , G – G’ ). Semi-quantification of Gata4/5/6 ( F – F’ ) and Depdc1b ( G – G’ ) expression in indicated conditions. ( H ) Accessibility of the Depdc1b locus during cardiopharyngeal development showing distal and proximal regulatory accessible regions upstream of the Depdc1b start site. Data from Racioppi et al, . ( I ) Systematic deletion of distal and proximal regulatory regions upstream of a 2xGFP reporter. The diagram shows control and deletion constructs. The dot plot on the right shows the level of GFP expression detected in either the mesenchyme or the TVCs upon disruption of regulatory regions, separately or in combination. Jitter is added to the graph for ease of visualizing expression changes. ( J ) Hypergeometric tests of GFP expression based on regulatory region perturbation. Color scale indicates log 10 odds ratio, and size indicates log 10 P value. ( K ) Micrographs of GFP expression as driven by constructs containing both proximal and distal regulatory regions or lacking one or both regulatory regions. ( L ) Regions of the Depdc1b regulatory region showing conservation of the Flank region, and putative GATA and FOX-binding sites, between Ciona robusta and Ciona savignyi . ( M ) Constructs containing the Flank, GATA, and FOX binding sites driving 2xGFP expression and subsequent analysis of binding site requirements for the tissue specific expression of GFP. The dot plot on the right shows proportions of GFP+ detected in either the Mesenchyme or the TVCs upon disruption of regulatory regions, separately or in combination. Jitter was added to the graph for ease of visualizing expression changes. ( N ) Hypergeometric tests of GFP expression based on regulatory region perturbation. Color scale indicates log 10 odds ratio, and size indicates log 10 P value. ( O ) Micrographs of GFP expression as driven by constructs containing both proximal and distal regulatory regions or lacking one or both regulatory regions. For both ( K , O ), arrows – First Heart Precursors (FHPs), open arrowheads – Second Heart Precursors (SHPs), solid arrowheads Atrial Siphon Muscle Founder cells (ASMF). .

Article Snippet: Adult Ciona robusta were purchased from M-Rep, USA or collected from coastal waters in Rongcheng, China, maintained in artificial seawater with constant lighting, and used for experiments within one week of arrival.

Techniques: Gene Expression, Expressing, Migration, CRISPR, Control, Construct, Disruption, Binding Assay

Journal: The EMBO Journal

Article Title: Cell cycle-driven transcriptome maturation confers multilineage competence to cardiopharyngeal progenitors

doi: 10.1038/s44318-025-00613-y

Figure Lengend Snippet: ( A ) (Top) The cardiopharyngeal lineage in Ciona robusta . The lineage of a B7.5 blastomere is shown to give rise to two founder cells, each producing a TVC and an ATM. (Bottom) Correspondence of FABA stages, post-fertilization developmental time points (18 °C), and color codes of scRNA-seq barcodes. TVC trunk ventral cell, ATM anterior tail muscle, STVC second trunk ventral cell, FHP first heart precursor, ASMF atrial siphon muscle founder cells, SHP second heart precursor. ( B ) (Top) Schematic diagram of asymmetrically oriented division of TVCs. (Bottom) Confocal images of before (left, 12 hpf) and after (right, 15 hpf) TVC division. Cyan: nuclei (NLS::LacZ); Magenta: cell membranes (hCD4::mCherry). The dashed line represents the embryo midline. M medial, L lateral. Scale bar = 5 µm. ( C ) Schematic of TVC cell cycle stages and genetic perturbations of mitotic entry. ( D – I’ ) Cell division patterns ( E , G , I ) and Tbx1/10 expression ( E’ , G’ , I’ ) following cell cycle perturbations. Control TVC division ( D , E , 3´HA), inhibition of TVC mitotic entry ( F , G , Wee1::3´HA), and induction of TVC mitotic entry ( H , I , Cdc25::3´HA) conditions are examined from 8 to 15 hpf. Perforated bars in ( G , G’ ) indicate timepoints not analyzed. Magenta: nuclei (GFP::PCNA); blue: cell membranes (hCD4::mCherry); green arrowhead: Tbx1/10 mRNA, Scale bar = 5 µm. ( J ) Schematic of variability of PCNA puncta patterns in the TVC nuclei associated with progression through the cell cycle. ( K ) Confocal images of PCNA puncta distribution in individual TVC nuclei at different stages of the cell cycle. Green: GFP::PCNA. Scale bar = 2.5 µm. ( L – N ) Determination of the S phase of TVC using PCNA. GFP::PCNA expressed in the B7.5-lineage under the Mesp enhancer at 8–12 hpf. Representative confocal images showing the G1, S, and G2 stages of TVC at 8, 10, and 12 hpf ( L ). Green: GFP::PCNA. Scale bar = 5 mm. Developmental distribution of four PCNA localization patterns ( M ). Quantification of GFP::PCNA punctæ per nucleus across developmental stages. Error bars show standard error of the mean (SEM). Data represent two biological replicates ( N ). No blinding was included in the analysis. .

Article Snippet: Adult Ciona robusta were purchased from M-Rep, USA or collected from coastal waters in Rongcheng, China, maintained in artificial seawater with constant lighting, and used for experiments within one week of arrival.

Techniques: Expressing, Control, Inhibition

Journal: bioRxiv

Article Title: A protocadherin mediates cell-cell adhesion and integrity of the oral placode in the tunicate Ciona

doi: 10.1101/2025.07.11.664433

Figure Lengend Snippet: A) Ciona robusta (intestinalis type A) embryo at Hotta Stage 25 (late tailbud III, ∼13 hours post-fertilization at 20°C), showing cell outlines and the oral siphon placode (OSP) rosette revealed by Phalloidin-AlexFluor546 staining. B ) In situ mRNA hybridization revealing Protocadherin.e ( Pcdh.e ) expression in the presumptive OSP, as well as in the Motor Ganglion Interneuron 2 (MGIN2) cells as previously reported . C ) Summary diagram of the separation of Six1/2+, future Protocadherin.e (Pcdh.e)- and Pitx- expressing oral siphon placode cells (red outline) from other Foxc+ cells (blue), mainly those contributing to the papillae. D ) Protein domain analysis diagram of Protocadherin.e (Pcdh.e) from SMART , showing presence of a signal peptide (red block), 6 extracellular cadherin repeats (CA) and a transmembrane (TM) domain, similar to the organization of vertebrate protocadherin-family proteins.

Article Snippet: Adult Ciona robusta specimens were collected in California, around San Diego (M-REP) or Los Angeles/Orange County (Marinus Scientific).

Techniques: Staining, In Situ, Hybridization, Expressing, Blocking Assay

Journal: bioRxiv

Article Title: Tunicate-specific protein Epi-1 is essential for conferring hydrophilicity to the larval tunic in the ascidian Ciona

doi: 10.1101/2024.09.30.615758

Figure Lengend Snippet: The organization of the larval tunic. (A) A tadpole larva of Ciona intestinalis Type A, lateral view. The anterior is toward the right, and the dorsal is toward the top. The larval tunic is outlined by a dotted line. (B) A schematic illustrating the layers of the larval tunic. Please note that Ciona larva possess an adult tunic near the larval body, which is omitted from the illustration for simplicity.

Article Snippet: The National BioResource Project, Japan, cultivated wild-type Ciona intestinalis Type A/ Ciona robusta individuals derived from Onagawa Bay (Miyagi, Japan) and Onahama Bay (Fukushima, Japan) as closed colonies.

Techniques:

Journal: bioRxiv

Article Title: Tunicate-specific protein Epi-1 is essential for conferring hydrophilicity to the larval tunic in the ascidian Ciona

doi: 10.1101/2024.09.30.615758

Figure Lengend Snippet: Epi-1 is a cytosolic protein. (A) A Western blot image of the Epi-1 protein. The left panel shows the Coomassie brilliant blue staining of the blotted proteins, and the right panel shows the immunostaining of Epi-1. WT, sample from wild-type larvae. Epi-1::GFP, sample from larvae overexpressing the Epi-1::GFP fusion protein. GFP, a sample from larvae overexpressing GFP. In the WT and GFP lanes, single bands at around 80 kDa can be seen, while the Epi-1::GFP lane exhibits a higher molecular-weight band representing the fusion protein. (B) Immunostaining of the Epi-1 protein in wild-type larvae. DIC, differential contrast images. The dotted lines indicate the areas shown in the enlarged image panels. Epi, epidermis; Mus, muscle; Nuc, nucleus. Epidermal cells of Ciona have multiple membranous structures, the interiors of which are immunonegative and therefore highlighted by black dots in the images. (C) Immunostaining of Epi-1 in the larvae overexpressing the Epi-1::GFP fusion protein in the epidermis. Green and red pseudocolors, respectively, represent the GFP fluorescence and immunostaining signals. (D) Epi-1::GFP fusion is localized in the cytosol. An epidermal cell is highlighted by a dotted line as an example.

Article Snippet: The National BioResource Project, Japan, cultivated wild-type Ciona intestinalis Type A/ Ciona robusta individuals derived from Onagawa Bay (Miyagi, Japan) and Onahama Bay (Fukushima, Japan) as closed colonies.

Techniques: Western Blot, Staining, Immunostaining, Molecular Weight, Fluorescence