(A) Overview of O. vulgaris embryonic development from Stage IX to Stage XX.2, covering early-, mid-, and late- organogenesis events and maturation. Surface renderings after DAPI staining are shown …
In situ hybridization of Ov-elav (left column) and Ov-syt (middle column) and immunoreactivity against acetylated alpha tubulin (right column) on transversal sections of embryos at Stage IX, XI, …
Phylogenetic analysis of ELAV and ELAV-like proteins shown in a maximum likelihood tree. Numbers at each branch represent bootstrap support values. The tree has been rooted using the RNA binding …
Panels on the left show representative 3D reconstructions of embryos seen from the lateral side, stained with DAPI. White dashed lines indicate the sectioning plane in respect to the dorsoventral …
Panels on the left show representative 3D reconstructions of embryos seen from the lateral side, stained with DAPI. White dashed lines indicate the sectioning plane in respect to the dorsoventral …
Expression of Ov-pcna (upper panels) and immunoreactivity against PH3 (lower panels) from Stage VII.2 to Stage XX.2. (A-D, I-L) Ov-pcna expression is broad at Stages VII.2 and IX and gets restricted …
Expression of Ov-ascl1, Ov-ngn, and Ov-neuroD on paraffin sections from Stage IX to Stage XX.2. (A-G) Ov-ascl1 is highly expressed in the lateral lips and retina at all stages. Overall, the number …
Phylogenetic analysis of SOX family proteins shown in a maximum likelihood tree. Numbers at each branch represent bootstrap support values. The tree has been rooted using the SOX transcription …
(A-b) Panels on the left show representative 3D reconstructions of embryos seen from the lateral side, stained with DAPI. White dashed lines indicate the sectioning plane in respect to the …
Phylogenetic analysis of the atonal-related NEUROD, NEUROD-like proteins, NEUROGENIN and TAP proteins shown in a maximum likelihood tree. Numbers at each branch represent bootstrap support values. …
Phylogenetic analysis of ASH and ASH-like proteins shown in a maximum likelihood tree. Numbers at each branch represent bootstrap support values. The tree has been rooted using the achaete-scute …
Panels on the left show representative 3D reconstructions of embryos seen from the lateral side, stained with DAPI. White dashed lines indicate the sectioning plane in respect to the dorsoventral …
Panels on the left show representative 3D reconstructions of embryos seen from the lateral side, stained with DAPI. White dashed lines indicate the sectioning plane in respect to the dorsoventral …
Panels on the left show representative 3D reconstructions of embryos seen from the lateral side, stained with DAPI. White dashed lines indicate the sectioning plane in respect to the dorsoventral …
Multiplex in situ hybridization (HCR v3.0) combined with immunostaining against PH3. (A) Overview image showing expression of Ov-ascl1 and Ov-ngn and presence of mitotic cells (PH3+) on a …
(A) Maximum projection after DAPI staining of a hatchling from the anterior side. Boxed area indicates the magnified region in B and dashed line indicates optical sectioning plane for C. (B-C) …
Injection of CFDA-SE in the lateral lips at Stage IX (A), Stage XII.1 (C) or Stage XV.2 (E) and tracing until Stage XIX.2 resulted in labeled cells in specific regions of the optic lobe and peduncle …
(A) Experimental setup showing CFDA-SE injection in the lateral lip at Stage XIV and embryo sampling at Stage XV.2. Dashed lines indicate optical sectioning planes for D-R. (B-C) Volumetric …
This panel shows the location of the CFDA-SE injection sites at Stage XIV, with each domain representing a single experimental condition. The lateral lips are pseudo-colored in green. Abbreviations …
Multiplex in situ hybridization (HCR v3.0) on a transversal section of a Stage XV.1 embryo that was injected 48 hr before with CFDA-SE in the dorsal lateral lip. (A) Overview image showing …
(A) The expression domains of Ov-ngn (purple), Ov-ascl1 (green), Ov-neuroD (red), and Ov-elav (pink) are depicted during O. vulgaris neurogenesis. Areas indicate high level expression in most cells, …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Anti-Tubulin, Acetylated antibody (Mouse Monoclonal) | Sigma | Cat#: T6793 | (1:300) |
Antibody | Anti-phospho-Histone H3 (Ser10) Antibody (Rabbit Polyclonal) | Millipore | Cat#: 06–570 | (1:300) |
Antibody | Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 | Life Tech (Invitrogen) | Cat#: A-21202 | (1:300) |
Antibody | Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Life Tech (Invitrogen) | Cat#: A-31572 | (1:300) |
Antibody | Fluorescein Antibody (Goat Polyclonal) | Novus Biologicals | Cat#: NB600-493 | (1:300 on sections, 1:500 whole mount) |
Antibody | Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | Cat#: A-11055 | (1:300) |
Strain, strain background (Escherichia coli) | JM109 chemocompetent cells | Promega | Cat#: L2005 L1001 | |
Chemical compound, drug | Mowiol 4–88 | Sigma | Cat#: 81381 | |
Chemical compound, drug | TRI Reagent Solution | Invitrogen | Cat#: AM9738 | |
Chemical compound, drug | Eukitt quick-hardening mounting medium | Sigma | Cat#: 03989 | |
Chemical compound, drug | Poly(dimethylsiloxane-co-methylphenylsiloxane) viscosity 125 cSt | Sigma-Aldrich | Cat#: 378488 | |
Chemical compound, drug | Mineral oil | Sigma-Aldrich | Cat#: M8410 | |
Chemical compound, drug | Silicone Elastomer, 2 Part, 1:1 Mix, Sylgard 170, Black / White, Container, 2 kg | DOWSIL | Cat#: 101693 | |
Commercial assay or kit | RNeasy Micro Kit | Qiagen | Cat#: 74004 | |
Commercial assay or kit | SMARTer PCR cDNA Synthesis Kit | Takara Bio Inc | Cat#: 634925 | |
Commercial assay or kit | NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module | New England BioLabs. | Cat#: E6421S | |
Commercial assay or kit | Superscript III Reverse Transcriptase | Invitrogen | Cat#: 18080–044 | |
Commercial assay or kit | TOPO TA Cloning Kit, Dual Promoter, without competent cells (25 reactions). | Invitrogen | Cat#: 450640 | |
Commercial assay or kit | Micro Bio-Spin P-30 Gel Columns, Tris Buffer (RNase-free) | BioRad Lab. | Cat#: 7326250 | |
Commercial assay or kit | Ribomap Kit | Roche | Cat#: 5266190001 | |
Commercial assay or kit | Bluemap detection kit | Roche | Cat#: 5266327001 | |
Other | Proteinase K, recombinant, PCR Grade | Roche | Cat#: 3115887001 | ISH (1:1000) HCR (1:3000) |
Other | Tissue-Tek Biopsy 6-Chamber Cassette | Sakura | Cat#: 4073 Biopsy 6 Chamber Cassette White 1.000pcs | For paraffin embedding |
Other | DAPI | Sigma Aldrich | Cat#: 32670–5 MG-F | (1:1000) |
Other | HCR Amplifier B1, Alexa Fluor 546 | Molecular Instruments (US) | HCR Amplifier | |
Other | HCR Amplifier B2, Alexa Fluor 647 | Molecular Instruments (US) | HCR Amplifier | |
Other | HCR Amplifier B3, Alexa Fluor 488 | Molecular Instruments (US) | HCR Amplifier | |
Other | Glass Capillaries, 3.5’, For All Model Nanoject Models | Drummond | Cat#: 3-000-203-G/X | For microinjection |
Other | CFDA-SE | SanBio | Cat#: 14456–10 | (1 mM for trajectory mapping, 0.1 mM for long term tracing in filtered seawater) |
Other | Fast Green FGF | Sigma-Aldrich | Cat#: F7252 | (0.3 mg/ml) |
Software, algorithm | Fiji: ImageJ | DOI: 10.1038/nmeth.2019 | ||
Software, algorithm | SMRT Link v. 9.0.0 | Pacific Biosciences (PACBIO) | ||
Software, algorithm | IsoSeq 3.3 | Pacific Biosciences (PACBIO) | ||
Software, algorithm | ARIVIS Vision4D Zeiss Edition 3.1.4 | ARIVIS AG | ||
Software, algorithm | Adobe Photoshop | Adobe | ||
Software, algorithm | Blast2GO | DOI: 10.1093/bioinformatics/bti610 | ||
Software, algorithm | insitu_probe_generator | DOI: 10.5281/zenodo.4086058 | ||
Software, algorithm | tBLASTn, BLASTp | NCBI: https://blast.ncbi.nlm.nih.gov/Blast.cgi | ||
Software, algorithm | UCSC Genome Browser on Euprymna scolopes Euprymna scolopes Assembly (eupSco1) | DOI: 10.1073/pnas.1817322116 | ||
Software, algorithm | ORFfinder | NCBI (RRID:SCR_016643) | ||
Software, algorithm | MUSCLE Alignment | DOI: 10.1093/bioinformatics/bth090 | ||
Software, algorithm | MegaX | DOI: 10.1093/molbev/msy096 | ||
Software, algorithm | TrimAl | DOI: 10.1093/bioinformatics/btp348 | ||
Software, algorithm | IQTree | DOI: 10.1093/molbev/msu300 | ||
Software, algorithm | FigTree v.1.4.4 | DOI:http://tree.bio.ed.ac.ukSoftware/Figtree/ | ||
Software, algorithm | CDD Search Tool | DOI: 10.1093/nar/gkz991 |
Number of biological replicates (unless specified otherwise) for immunohistochemistry and in situ hybridization experiments.
N/A, not applicable; TR, technical replicate.
Nucleotide sequence of primers used to amplify gene fragments for ISH probes.
Nucleotide sequence of probes for colorimetric in situ hybridization.
Nucleotide sequences of HCR probes.
Accession numbers of protein sequences used for phylogenetic tree construction.
Protein sequences of O. vulgaris homologs used in phylogenetic tree reconstruction.