Identification of neural progenitor cells and their progeny reveals long distance migration in the developing octopus brain

  1. Astrid Deryckere  Is a corresponding author
  2. Ruth Styfhals
  3. Ali Murat Elagoz
  4. Gregory E Maes
  5. Eve Seuntjens  Is a corresponding author
  1. Laboratory of Developmental Neurobiology, Department of Biology, KU Leuven, Belgium
  2. Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Italy
  3. Center for Human Genetics, Genomics Core, UZ-KU Leuven, Belgium
  4. Centre for Sustainable Tropical Fisheries and Aquaculture, College of Science and Engineering, James Cook University, Australia
  5. Laboratory of Biodiversity and Evolutionary Genomics, Department of Biology, KU Leuven, Belgium
9 figures, 1 table and 7 additional files

Figures

Overview of the developing O. vulgaris embryo and its nervous system.

(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 …

Figure 2 with 3 supplements
Formation of the O. vulgaris brain.

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, …

Figure 2—figure supplement 1
Phylogeny Ov-ELAV.

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 …

Figure 2—figure supplement 2
Complementary panels to Figure 2 of Ov-elav expression in the head region of developing O. vulgaris embryos.

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 …

Figure 2—figure supplement 3
Complementary panels to Figure 2 of Ov-syt expression in the head region of developing O. vulgaris embryos.

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 …

Cell division in the developing O. vulgaris embryo.

Expression of Ov-pcna (upper panels) and immunoreactivity against PH3 (lower panels) from Stage VII.2 to Stage XX.2. (A-D, I-LOv-pcna expression is broad at Stages VII.2 and IX and gets restricted …

Figure 4 with 7 supplements
Expression of O. vulgaris proneuronal and neuronal differentiation bHLH genes during embryonic development.

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 …

Figure 4—figure supplement 1
Phylogeny Ov-SOXB1.

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 …

Figure 4—figure supplement 2
Expression of Ov-soxB1 during O. vulgaris embryonic development.

(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 …

Figure 4—figure supplement 3
Phylogeny of Ov-NEUROD and Ov-NGN.

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. …

Figure 4—figure supplement 4
Phylogeny of Ov-ASCL1.

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 …

Figure 4—figure supplement 5
Complementary panels to Figure 4 of Ov-ascl1 expression in the head region of developing O. vulgaris embryos.

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 …

Figure 4—figure supplement 6
Complementary panels to Figure 4 of Ov-ngn expression in the head region of developing O. vulgaris embryos.

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 …

Figure 4—figure supplement 7
Complementary panels to Figure 4 of Ov-neuroD expression in the head region of developing O. vulgaris embryos.

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 …

Cell proliferation profiles of the progenitor populations in the lateral lips.

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 …

CFDA-SE lineage tracing from the lateral lips to the supra- and subesophageal mass.

(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) …

CFDA-SE lineage tracing from the lateral lips to the optic lobe and peduncle complex.

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 …

Figure 8 with 2 supplements
Trajectory mapping of migrating cells from the lateral lips to the central brain.

(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 …

Figure 8—figure supplement 1
Summary of short-term CFDA-SE lineage tracing.

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 …

Figure 8—figure supplement 2
Migrating cells through the PTZ are neurogenic.

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 …

Overview of the expression of neurogenic genes and hypothetical neurogenesis process in O. vulgaris.

(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, …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-Tubulin, Acetylated antibody (Mouse Monoclonal)SigmaCat#: T6793(1:300)
AntibodyAnti-phospho-Histone H3 (Ser10) Antibody (Rabbit Polyclonal)MilliporeCat#: 06–570(1:300)
AntibodyDonkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488Life Tech (Invitrogen)Cat#: A-21202(1:300)
AntibodyDonkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555Life Tech (Invitrogen)Cat#: A-31572(1:300)
AntibodyFluorescein Antibody (Goat Polyclonal)Novus BiologicalsCat#: NB600-493(1:300 on sections, 1:500 whole mount)
AntibodyDonkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488InvitrogenCat#: A-11055(1:300)
Strain, strain background (Escherichia coli)JM109 chemocompetent cellsPromegaCat#: L2005 L1001
Chemical compound, drugMowiol 4–88SigmaCat#: 81381
Chemical compound, drugTRI Reagent SolutionInvitrogenCat#: AM9738
Chemical compound, drugEukitt quick-hardening mounting mediumSigmaCat#: 03989
Chemical compound, drugPoly(dimethylsiloxane-co-methylphenylsiloxane) viscosity 125 cStSigma-AldrichCat#: 378488
Chemical compound, drugMineral oilSigma-AldrichCat#: M8410
Chemical compound, drugSilicone Elastomer, 2 Part, 1:1 Mix, Sylgard 170, Black / White, Container, 2 kgDOWSILCat#: 101693
Commercial assay or kitRNeasy Micro KitQiagenCat#: 74004
Commercial assay or kitSMARTer PCR cDNA Synthesis KitTakara Bio IncCat#: 634925
Commercial assay or kitNEBNext Single Cell/Low Input cDNA Synthesis and Amplification ModuleNew England BioLabs.Cat#: E6421S
Commercial assay or kitSuperscript III Reverse TranscriptaseInvitrogenCat#: 18080–044
Commercial assay or kitTOPO TA Cloning Kit, Dual Promoter, without competent cells (25 reactions).InvitrogenCat#: 450640
Commercial assay or kitMicro Bio-Spin P-30 Gel Columns, Tris Buffer (RNase-free)BioRad Lab.Cat#: 7326250
Commercial assay or kitRibomap KitRocheCat#: 5266190001
Commercial assay or kitBluemap detection kitRocheCat#: 5266327001
OtherProteinase K, recombinant, PCR GradeRocheCat#: 3115887001ISH (1:1000)
HCR (1:3000)
OtherTissue-Tek Biopsy 6-Chamber CassetteSakuraCat#: 4073 Biopsy 6 Chamber Cassette White 1.000pcsFor paraffin embedding
OtherDAPISigma AldrichCat#: 32670–5 MG-F(1:1000)
OtherHCR Amplifier B1, Alexa Fluor 546Molecular Instruments (US)HCR Amplifier
OtherHCR Amplifier B2, Alexa Fluor 647Molecular Instruments (US)HCR Amplifier
OtherHCR Amplifier B3, Alexa Fluor 488Molecular Instruments (US)HCR Amplifier
OtherGlass Capillaries, 3.5’, For All Model Nanoject ModelsDrummondCat#: 3-000-203-G/XFor microinjection
OtherCFDA-SESanBioCat#: 14456–10(1 mM for trajectory mapping, 0.1 mM for long term tracing in filtered seawater)
OtherFast Green FGFSigma-AldrichCat#: F7252(0.3 mg/ml)
Software, algorithmFiji: ImageJDOI: 10.1038/nmeth.2019
Software, algorithmSMRT Link v. 9.0.0Pacific Biosciences (PACBIO)
Software, algorithmIsoSeq 3.3Pacific Biosciences (PACBIO)
Software, algorithmARIVIS Vision4D Zeiss Edition 3.1.4ARIVIS AG
Software, algorithmAdobe PhotoshopAdobe
Software, algorithmBlast2GODOI: 10.1093/bioinformatics/bti610
Software, algorithminsitu_probe_generatorDOI: 10.5281/zenodo.4086058
Software, algorithmtBLASTn, BLASTpNCBI: https://blast.ncbi.nlm.nih.gov/Blast.cgi
Software, algorithmUCSC Genome Browser on Euprymna scolopes Euprymna scolopes Assembly (eupSco1)DOI: 10.1073/pnas.1817322116
Software, algorithmORFfinderNCBI (RRID:SCR_016643)
Software, algorithmMUSCLE AlignmentDOI: 10.1093/bioinformatics/bth090
Software, algorithmMegaXDOI: 10.1093/molbev/msy096
Software, algorithmTrimAlDOI: 10.1093/bioinformatics/btp348
Software, algorithmIQTreeDOI: 10.1093/molbev/msu300
Software, algorithmFigTree v.1.4.4DOI:http://tree.bio.ed.ac.ukSoftware/Figtree/
Software, algorithmCDD Search ToolDOI: 10.1093/nar/gkz991

Additional files

Supplementary file 1

Number of biological replicates (unless specified otherwise) for immunohistochemistry and in situ hybridization experiments.

N/A, not applicable; TR, technical replicate.

https://cdn.elifesciences.org/articles/69161/elife-69161-supp1-v1.docx
Supplementary file 2

Nucleotide sequence of primers used to amplify gene fragments for ISH probes.

https://cdn.elifesciences.org/articles/69161/elife-69161-supp2-v1.docx
Supplementary file 3

Nucleotide sequence of probes for colorimetric in situ hybridization.

https://cdn.elifesciences.org/articles/69161/elife-69161-supp3-v1.docx
Supplementary file 4

Nucleotide sequences of HCR probes.

https://cdn.elifesciences.org/articles/69161/elife-69161-supp4-v1.docx
Supplementary file 5

Accession numbers of protein sequences used for phylogenetic tree construction.

https://cdn.elifesciences.org/articles/69161/elife-69161-supp5-v1.docx
Supplementary file 6

Protein sequences of O. vulgaris homologs used in phylogenetic tree reconstruction.

https://cdn.elifesciences.org/articles/69161/elife-69161-supp6-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/69161/elife-69161-transrepform-v1.pdf

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