Basement membranes are crucial for proper olfactory placode shape, position and boundary with the brain, and for olfactory axon development

  1. Pénélope Tignard
  2. Karen Pottin
  3. Audrey Geeverding
  4. Mohamed Doulazmi
  5. Mélody Cabrera
  6. Coralie Fouquet
  7. Mathilde Liffran
  8. Jonathan Fouchard
  9. Marion Rosello
  10. Shahad Albadri
  11. Filippo Del Bene
  12. Alain Trembleau  Is a corresponding author
  13. Marie Anne Breau  Is a corresponding author
  1. Sorbonne Université, Centre National de la Recherche Scientifique (CNRS UMR7622), Institut de Biologie Paris-Seine (IBPS), Developmental Biology Laboratory, France
  2. Sorbonne Université, Centre National de la Recherche Scientifique (CNRS UMR8246), Inserm U1130, Institut de Biologie Paris-Seine (IBPS), Neuroscience Paris Seine (NPS), France
  3. Imaging Facility, Institut de Biologie Paris-Seine (IBPS), France
  4. Sorbonne Université, Centre National de la Recherche Scientifique (CNRS UMR8256), Institut de Biologie Paris-Seine (IBPS), Adaptation Biologique et Vieillissement, France
  5. Sorbonne Université, INSERM, CNRS, Institut de la Vision, France
  6. Institut National de la Santé et de la Recherche Médicale (INSERM), France
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 4 supplements
Expression profile of Laminin in relation with the development of the olfactory system.

(A–D) Immunostaining for Laminin magenta on Tg(neurog1:GFP) embryos (green) at 18 and 22 hpf (A-B’, dorsal views), and on Tg(omp:meYFP) embryos (green) at 28 and 36 hpf (C-D’, frontal views). In A, …

Figure 1—video 1
BM assembly around the brain and the OP during OP coalescence, related to Figure 1.

Same embryo as the z-section shown in Figure 1J. Confocal live imaging performed on a TgBAC(lamC1:lamC1-sfGFP);Tg(cldnb:Gal4;UAS:RFP) embryo in which Laminin γ1-sfGFP is expressed under the control …

Figure 1—video 2
Formation of the exit and entry points in the BMs surrounding the OP and the brain, related to Figure 1.

Same embryo as the z-sections shown in Figure 1E-I and in Figure 1—video 4. Confocal live-imaging performed on a TgBAC(lamC1:lamC1-sfGFP);Tg(cldnb:Gal4;UAS:RFP) embryo in which Laminin γ1-sfGFP is …

Figure 1—video 3
Formation of the exit and entry points in the BMs surrounding the OP and the brain visualised with a mosaic labelling of OP axons, related to Figure 1.

Confocal live-imaging performed on a TgBAC(lamC1:lamC1-sfGFP);Tg(cldnb:Gal4;UAS:lyn-TagRFP) embryo in which Laminin γ1-sfGFP is expressed under the control of its own promoter (green), and OP and …

Figure 1—video 4
3D z-stacks showing the exit and entry points, related to Figure 1.

Same embryo as the z-sections shown in Figure 1E-I and Figure 1—video 2. Confocal z-stacks performed on a live TgBAC(lamC1:lamC1-sfGFP);Tg(cldnb:Gal4;UAS:RFP) embryo in which Laminin γ1-sfGFP is …

Figure 2 with 1 supplement
The integrity of the BMs of OP and brain tissues is strongly affected in sly mutants.

(A–I) Immunostaining for Laminin (A–C), Nidogen (D–F) and Collagen IV (G–I) (magenta) on sly mutants and control siblings at 22 (dorsal view), 28, and 36 hpf (frontal view). For Laminin and Nidogen, …

Figure 2—source data 1

Thickness of the intercellular space in sly mutants (between OP and brain cells) and control siblings (between NCC and brain or OP cells).

https://cdn.elifesciences.org/articles/92004/elife-92004-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Electron microscopy analysis of intercellular spaces in the OP and brain tissues.

A, A’ and C, C’ Examples of images of the intercellular space located between OP cells in control siblings (A, C) and in sly mutants (A’, C’), at 24 and 32 hpf. Arrows = plasma membranes. (B, D) …

Figure 2—figure supplement 1—source data 1

Thickness of the intercellular space within OP and brain tissues in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 2 supplements
Analysis of OP coalescence in sly mutants and control siblings.

(A, B) Images (dorsal views, 1 z-section) of representative OPs from a Tg(neurog1:GFP); sly -/- mutant (right) and a control Tg(neurog1:GFP) sibling (left) at the end of OP coalescence (22 hpf). The …

Figure 3—source data 1

OP dimensions at 22 hpf, and MSD analysis and cell displacements during OP coalescence in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Analysis of proliferation and apoptosis in the OPs of sly mutants and control siblings.

(A-D’) Immunostaining for Phospho-histone H3 (magenta) to label dividing cells on Tg(neurog1:GFP) embryos (green) at 16 hpf and 21 hpf (A-B’ show images at 21 hpf, dorsal view), and on Tg(omp:meYFP) …

Figure 3—figure supplement 1—source data 1

Analysis of OP proliferation and apoptosis and total number of YFP+ cells in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig3-figsupp1-data1-v1.xlsx
Figure 3—video 1
OP coalescence in a control embryo and a sly mutant, related to Figure 3.

Same embryos as the images and analyses shown in Figure 3F-I. Confocal live imaging showing OP coalescence movements in sly mutant and control Tg(neurog1:GFP) embryos, in which OP and brain neurons …

Figure 4 with 3 supplements
Analysis of OP and brain morphogenesis in sly mutants and control siblings during the forebrain flexure.

(A, B) Images (frontal view, 1 z-section) of representative placodes from a Tg(omp:meYFP); sly -/- mutant (right) and a control Tg(omp:meYFP) sibling (left) at 36 hpf. Laminin immunostaining in …

Figure 4—source data 1

OP dimensions at 36 hpf, and MSD analysis and angles of OP cell trajectories from 22 hpf in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Additional results on the OP morphogenesis defects observed in sly mutants.

(A-L) Analysis of OP dimensions at 24, 28, and 32 hpf. (A, A’, E, E’ and I, I’) Images (frontal views, 1 z-section) of representative OPs from Tg(omp:meYFP); sly -/- mutants (right) and control Tg(om…

Figure 4—figure supplement 1—source data 1

OP dimensions at 24, 28, and 32 hpf in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig4-figsupp1-data1-v1.xlsx
Figure 4—video 1
Behaviour of the OP neurons during the brain flexure movement in a control embryo and a sly mutant, related to Figure 4.

Same embryos as the images and analyses shown in Figure 4I-L. Confocal live imaging showing OP cell movements and axon behaviours in sly mutant and control Tg(omp:meYFP) embryos, in which …

Figure 4—video 2
3D stacks showing the axonal defects and ectopic OP cells observed in sly mutants, related to Figure 4, Figure 6—figure supplement 1.

The embryos are at 28 hpf and carry the Tg(omp:meYFP) transgene to visualise the membrane of ompb-expressing OP neurons and their axons (green). In control embryos, the bundle of axons exit the OP …

Figure 5 with 5 supplements
Analysis of brain width and brain/placode boundary in sly mutants and control siblings.

(A, A’) Immunostaining for HuC (cyan) at 36 hpf on Tg(cldnb:Gal4; UAS:RFP) (magenta) control and sly mutant embryos (frontal view). Similar immunostainings performed at 28 hpf are shown in Figure …

Figure 5—source data 1

Brain width measurements and distortion index of the OP/brain boundary in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Additional results for the analysis of brain shape and proliferation/apoptosis in sly mutants and control siblings.

(A, A’) Immunostaining for HuC (cyan) at 28 hpf performed on Tg(cldnb:Gal4; UAS:RFP) (magenta) control and sly mutant embryos (frontal view). Similar immunostainings performed at 36 hpf are shown in …

Figure 5—figure supplement 1—source data 1

Brain width measurements and analysis of brain proliferation and apoptosis in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
NCC defects in sly mutants.

(A, A’) In situ hybridisation for the NCC marker crestin on 32 hpf controls and sly mutants. Arrowheads = crestin-positive NCC cluster at the OP/brain interface, which is absent in sly mutants. (B) …

Figure 5—figure supplement 3
Olfactory system development in foxd3 mutants.

(A, B) In situ hybridisation for the NCC marker crestin on 32 hpf controls and foxd3 mutants. Arrowheads = crestin-positive NCC cluster at the OP/brain interface, which is absent in foxd3 mutants. (C…

Figure 5—figure supplement 3—source data 1

OP dimensions and length of the axon bundle in foxd3 mutants and controls siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig5-figsupp3-data1-v1.xlsx
Figure 5—video 1
3D stacks showing the organisation of brain and OP neurons in control embryos and sly mutants, related to Figure 5, Figure 5—figure supplement 1.

sly mutant and control Tg(cldnb:Gal4;UAS:RFP) embryos, in which OP and peridermal skin cells are labelled in with cytoplasmic RFP (magenta), were immunostained for HuC (cyan) at 28 hpf and 36 hpf to …

Figure 5—video 2
NCC migration in sly mutants and control siblings, related to Figure 5—figure supplement 2.

Confocal live imaging from 16 to 32 hpf showing OP and NCC cell movements in sly mutant and control Tg(neurog1:GFP);Tg(UAS:RFP) embryos injected with the sox10(7.2):KalTA4 plasmid. The DNA injection …

Figure 6 with 1 supplement
Quantitative live imaging of axonal behaviours in sly mutants and control siblings.

(A, B) Images extracted from confocal live imaging on control (A-A’’’’) and sly mutant (B-B’’’’) embryos injected with the omp:meYFP plasmid to obtain a mosaic labelling of OP neurons and their …

Figure 6—source data 1

Mean speed and persistence of growth cones in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig6-data1-v1.xlsx
Figure 6—source data 2

Angles of growth cone trajectories in sly mutants and control siblings.

https://cdn.elifesciences.org/articles/92004/elife-92004-fig6-data2-v1.xlsx
Figure 6—figure supplement 1
Additional results on olfactory axon development in sly mutants and control siblings.

(A, B) Images extracted from confocal live imaging experiments performed during OP coalescence on Tg(cldnb:Gal4; UAS:RFP) embryos injected with the neurog1:GFP plasmid, allowing a sparse labelling …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain
(Danio rerio)
Zebrafish wild-type hybrid
(TL x AB) strains
IBPS aquatic facility, ParisN/A
Strain
(Danio rerio)
slywi390 (sly/lamc1)Wiellette et al., 2004; PMID:15593329ZDB-ALT-050317–6
Strain
(Danio rerio)
foxd3zdf10Stewart et al., 2006; PMID:16499899ZDB-ALT-060519–4
Strain
(Danio rerio)
Tg(–8.4neurog1:GFP)sb1Blader et al., 2003; PMID:12559493ZDB-ALT-030904–6
Strain
(Danio rerio)
Tg(–2.0ompb:gapYFP)rw032Sato et al., 2007; PMID:17301169ZDB-ALT-050513–2
Strain
(Danio rerio)
Tg(–4.0cldnb:GalTA4, cry:RFP)nim11Breau et al., 2013; PMID:24082091ZDB-ALT-130822–6
Strain
(Danio rerio)
Tg(14XUAS:mRFP,Xla.Cryg:GFP)tpl2Balciuniene et al., 2013; PMID:24034702ZDB-ALT-131119–25
Strain
(Danio rerio)
TgBAC(lamc1:lamc1-sfGFP,cryaa:Cerulean)sk116TgYamaguchi et al., 2022; PMID:35165417ZDB-ALT-241010–5
Strain
(Danio rerio)
Tg(eltC:GFP)zf199TgStedman et al., 2009; PMID:19152797ZDB-ALT-101103–3
Strain
(Danio rerio)
Tg(UAS:lyn-tagRFP)This studyLab of Filippo Del Bene,
Institut de la Vision, Paris
AntibodyRabbit anti-Laminin polyclonalSigma-AldrichCat# L9393; RRID:AB_4771631/200
AntibodyRabbit anti-Nidogen polyclonalAbcamCat# ab14511, RRID:AB_3012901/200
AntibodyRabbit anti-Collagen IV polyclonalAbcamCat# ab6586, RRID:AB_3055841/200
AntibodyChicken anti-GFP polyclonalAves labsCat# GFP-1020, RRID:AB_100002401/200
AntibodyRabbit anti-DsRed polyclonalTakara BioCat# 632496; RRID:AB_100134831/300
AntibodyRabbit anti phospho-Histone H3
polyclonal
MilliporeCat# 06–570; RRID:AB_3101771/200
AntibodyRabbit anti Caspase 3, active form polyclonalR and D SystemsCat# AF835; RRID:AB_22439521/200
AntibodyMouse anti-HuC/HuD clone 16A11ThermoFisher scientificCat# A-21271, RRID:AB_2214481/200
AntibodyMouse anti-Dlx3b monoclonalZIRCCat# anti-DLX3b, RRID:AB_100137711/500
recombinant DNA reagent10XUAS:lyn-tagRFP plasmidThis studyGateway cloning,
Lab of Filippo Del Bene,
Institut de la Vision, Paris
recombinant DNA reagent–2.0ompb:gapYFP plasmidMiyasaka et al., 2005; PMID:15716341ZDB-TGCONSTRCT-070117–121
recombinant DNA reagentsox10(7.2):KalTA4 plasmidAlmeida and Lyons, 2015; PMID:26485616ZDB-TGCONSTRCT-170418–9
sequence-based reagentFW genotyping primer for slywi390This studyCATGACGGCAAAGTTGGTGA
sequence-based reagentRV1 genotyping primer for slywi390This studyCCATGCCTTGCAAAATGGCGTTA CTTAA
sequence-based reagentRV2 genotyping primer for slywi390This studyTGTAGGAGAGAAGTCGCGAG
sequence-based reagentcrestin PCR amplification for ISH probe synthesis
FW primer
This studyAAGCCCTCGAAACTCACCTG
sequence-based reagentcrestin PCR amplification for ISH probe synthesis
RV primer
This studyCCACTTGATTCCCACGAGCT
Commercial assay or kitMultisite Gateway system kitInvitrogenCat# 12537–023
software, algorithmLASXLeicaRRID:SCR_013673
software, algorithmFijihttps://imagej.net/Fiji/DownloadsRRID:SCR_002285
software, algorithmIlastikhttps://www.ilastik.org/RRID:SCR_015246
software, algorithmPrismGraphPad https://www.graphpad.com/RRID:SCR_002798
software, algorithmNumpy library, PythonHarris et al., 2020RRID:SCR_008633
software, algorithmMatlabThe Mathworks, Inc.
https://fr.mathworks.com/products/matlab.html
RRID:SCR_001622

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