1. Cell Biology
  2. Developmental Biology

Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish

  1. Mylene Lancino
  2. Sara Majello
  3. Sebastien Herbert
  4. Fabrice De Chaumont
  5. Jean-Yves Tinevez
  6. Jean-Christophe Olivo-Marin
  7. Philippe Herbomel  Is a corresponding author
  8. Anne Schmidt  Is a corresponding author
  1. Institut Pasteur, France
  2. CNRS, UMR 3738, France
  3. Sorbonne Université, UPMC Paris 06, Complexité du Vivant, France
  4. CNRS, UMR3691, France
https://doi.org/10.7554/eLife.37355
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Cite this article
  1. Mylene Lancino
  2. Sara Majello
  3. Sebastien Herbert
  4. Fabrice De Chaumont
  5. Jean-Yves Tinevez
  6. Jean-Christophe Olivo-Marin
  7. Philippe Herbomel
  8. Anne Schmidt
(2018)
Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish
eLife 7:e37355.
https://doi.org/10.7554/eLife.37355
  2. Download BibTeX
  3. Download .RIS
12 figures, 3 videos, 1 table and 3 additional files

Figures

Figure 1 with 4 supplements
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Sequential steps and morphological changes during the EHT

(A–B) The EHT is variable in space and time. Schematic representations of (A) a zebrafish embryo at 48 hpf; a yellow rectangle shows the region of imaging. (B) Left, transversal sections of the …

https://doi.org/10.7554/eLife.37355.003
Figure 1—figure supplement 1
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The time-length of the EHT is very heterogeneous (see text also).

(A) Optical sections (Z-planes) extracted from a spinning-disk confocal TL sequence performed on a 48 hpf Tg(kdrl:Ras-mCherry) embryo and showing the progression of the EHT, starting from a flat …

https://doi.org/10.7554/eLife.37355.004
Figure 1—video 1
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EHT at high spatio-temporal resolution.

Confocal TL sequence obtained from a 48 hpf Tg(kdrl:Ras-mCherry; kdrl:eGFP) embryo showing a 3D-rendering view of a portion of the dorsal aorta and the underlying axial vein (see also Figure 1C–G …

https://doi.org/10.7554/eLife.37355.005
Figure 1—video 2
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Luminal/apical membrane invagination of an EHT undergoing cell.

Single confocal Z-plane obtained from a 48 hpf Tg(kdrl:Ras-mCherry; kdrl:eGFP) embryo showing a cell undergoing EHT and emerging from the dorsal aorta (see also Figure 1H–L for a decomposition of …

https://doi.org/10.7554/eLife.37355.006
Figure 1—video 3
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The EHT- from the flat hemogenic endothelium to the emergence in the sub-aortic space.

Maximum projection of Z-planes (main field) and single Z-plane (top left white rectangle) from a scanning confocal TL sequence performed on a 48 hpf Tg(kdrl:Ras-mCherry) embryo showing a cell …

https://doi.org/10.7554/eLife.37355.007
Figure 2 with 2 supplements
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Apical constriction undergoes pulsatile activity.

(A) Maximum projection of Z-planes extracted from a spinning-disk confocal TL sequence performed on a 48 hpf Tg(fli1:Gal4; UAS:RFP; UAS:eGFP-ZO1) embryo. Yellow arrowheads: regions enriched with …

https://doi.org/10.7554/eLife.37355.008
Figure 2—figure supplement 1
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Bbiomechanical parameters of the apical closure.

Magnification of the upper panel of Figure 2C illustrating: (1) a contraction phase (shaded area, left part of the graph) with the steep decrease of the A-P luminal distance (green line), made of …

https://doi.org/10.7554/eLife.37355.009
Figure 2—video 1
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Dynamics of the apical constriction followed by eGFP-ZO1.

Maximum projection of Z-planes from a spinning-disk confocal TL sequence performed on a 48 hpf Tg(fli1:Gal4; UAS:RFP; UAS:eGFP-ZO1) embryo showing a cell undergoing EHT. White arrowheads point at …

https://doi.org/10.7554/eLife.37355.010
Figure 3 with 1 supplement
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The EHT cellular landscape unravelled by 2D-projection.

Schematic representations of: (A) Successive steps leading to the 2D projection (see text, Materials and methods and Source code 2), (B) The angle at which the Z-projections in the top panels (C–E) …

https://doi.org/10.7554/eLife.37355.011
Figure 3—video 1
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Dynamics of the EHT in the aortic landscape using 2D-maping-1.

Top panel; maximum projection of Z-planes from a spinning-disk confocal TL sequence performed on a 48 hpf Tg(fli1:Gal4; UAS:RFP; UAS:eGFP-ZO1) embryo. The bottom panel was obtained after duplication …

https://doi.org/10.7554/eLife.37355.012
Figure 4 with 1 supplement
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Intercellular contacts between hemogenic cells are reinforced.

(A) Top panel: maximum projection of Z-planes extracted from a confocal TL sequence performed on a 48 hpf Tg(fli1:Gal4; UAS:RFP; UAS:eGFP-ZO1) embryo. e1; EHT cell, red arrow. Cell three localises …

https://doi.org/10.7554/eLife.37355.013
Figure 4—source data 1

Figure 4D Numerical data (width, length and area) and corresponding 2D-Maps with the contours of EHT cells (red), hemogenic cells (blue) and endothelial cells (green).

https://doi.org/10.7554/eLife.37355.014
Figure 4—video 1
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Dynamics of the EHT in the aortic landscape using 2D-maping-2.

Top panel; maximum projection of Z-planes from a confocal TL sequence performed on a 48 hpf Tg(fli1:Gal4; UAS:RFP; UAS:eGFP-ZO1) embryo. The bottom panel was obtained after duplication of the …

https://doi.org/10.7554/eLife.37355.015
Figure 5 with 1 supplement
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Actin localization and dynamics.

(A–B) Lifeact-eGFP expressing 48 hpf embryos were imaged by spinning-disk confocal microscopy to obtain maximum projections of Z-planes (A, top and B), single Z planes (A, middle), or 2D-map …

https://doi.org/10.7554/eLife.37355.017
Figure 5—source data 1

Figure 5A: Z-projections (on top) and full 2D-Maps (bottom) from which the images presented in the bottom panels of Figure 5A were extracted.

https://doi.org/10.7554/eLife.37355.018
Figure 5—video 1
Download asset
Actin dynamics during the EHT.

Spinning-disk TL confocal sequence performed on a 48 hpf Tg(fli1:Lifeact-eGFP; kdrl:Ras-mCherry) embryo. The top panel shows a maximum projection of Z-planes. The white rectangle surrounds an EHT …

https://doi.org/10.7554/eLife.37355.019
Figure 6 with 3 supplements
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Blood flow influences the morphology as well as the cytoskeletal and junctional organization of hemogenic and emerging cells.

Lifeact-eGFP (A,B) or eGFP-ZO1 (C) expressing 48 hpf embryos (A,B) were imaged by confocal microscopy after injection of sih or control morpholinos. (A) Transversal views of control and sih …

https://doi.org/10.7554/eLife.37355.020
Figure 6—video 1
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Aortic phenotype in sih morphants.

Single Z-stacks obtained by scanning confocal microscopy of 48 hpf Tg(fli1:Lifeact-eGFP) embryos. Control embryos (top panel) or injected embryos with sih morpholinos (three bottom panels) showing …

https://doi.org/10.7554/eLife.37355.021
Figure 6—video 2
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Morphodynamics of the emergence in sih morphants.

TL sequence obtained with scanning confocal microscopy performed on a 48 hpf Tg(kdrl:Ras-mCherry; kdrl:eGFP) embryo injected with sih morpholinos showing the dynamics and orientation of cell …

https://doi.org/10.7554/eLife.37355.022
Figure 6—video 3
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Alteration of ZO1 localization in sih morphants.

3D-rendering, using the Imaris software, of the aortic regions of 48 hpf Tg(fli1:Gal4; UAS:RFP) embryo injected with UAS:eGFP-ZO1 encoding plasmid for transient expression and, in addition, either a …

https://doi.org/10.7554/eLife.37355.023
Figure 7 with 1 supplement
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Modeling of the EHT process.

Left panels: Lifeact-eGFP expressing 48 hpf embryo imaged by spinning-disk confocal microscopy to obtain single Z planes. Images were extracted from Figure 7—video 1. Grey arrows: evidence for the …

https://doi.org/10.7554/eLife.37355.024
Figure 7—video 1
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Evidence for forces deployed during the EHT.

Single Z-plane of a spinning-disk confocal TL sequence performed on a 48 hpf Tg(fli1:Lifeact-eGFP; kdrl:Ras-mCherry) embryo, showing evidence for pushing forces deployed from an endothelial …

https://doi.org/10.7554/eLife.37355.025
Figure 8 with 2 supplements
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Myosin regulatory light chains 9a and 9b are required for definitive hematopoiesis.

(A) In situ hybridizations of 48 hpf embryos. Left panels, whole mount; right panel, transversal section. a, aorta; n, notochord; nt, neural tube; s, somites; v, vein; ye, yolk extension; ys, yolk …

https://doi.org/10.7554/eLife.37355.026
Figure 8—figure supplement 1
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Quantitative analysis of the effects of myl9a and b splicing morpholinos (see also text).

(A) Protein sequence alignment of zebrafish Myl9a, 9b, 12.1 and 12.2 (Myl9az, Myl9bz, Myl12.1z and Myl12.2z, respectively), human and mouse Myl9 (referred to as Human and Mouse, respectively). Grey …

https://doi.org/10.7554/eLife.37355.027
Figure 8—figure supplement 2
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Myl9a and myl9b morpholinos phenocopy of hematopoietic phenotype and myl9b morpholino rescue.

(A) CD41:eGFP positive cell numbers in the AGM (top panel) and the CHT (bottom panel) after myl9a and myl9b morpholinos at the concentrations of 4, 8 and 12 ng. (B) CD41:eGFP positive cell numbers …

https://doi.org/10.7554/eLife.37355.028
Figure 9 with 3 supplements
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The amino-terminal phosphorylation site on Myl9b is essential for definitive hematopoiesis.

(A) Maximum projection of Z-planes obtained from 40 hpf and 50 hpf embryos expressing either Myl9b-eGFP or Myl9bA2A3-eGFP under the control of the runx’1 + 23’ enhancer that allows expression in …

https://doi.org/10.7554/eLife.37355.031
Figure 9—figure supplement 1
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The runx’1 + 23’ enhancer allows expression of Myl9 in hematopoietic stem cells (see also text).

(A) Top panels; maximum projection of Z-planes of the head, AGM and CHT (left to right) performed on a 40 hpf embryo expressing the runx’1 + 23’:eGFP transgene. Black arrows point at ectopic runx'1 +…

https://doi.org/10.7554/eLife.37355.032
Figure 9—video 1
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Phenotype of Myl9b phosphorylation mutant −1.

Maximum projection of Z-planes of spinning-disk confocal TL sequences performed on 40 hpf Tg(kdrl:Ras-mCherry) embryos expressing either Myl9b-eGFP (left panels) or Myl9bA2A3-eGFP (right panels), …

https://doi.org/10.7554/eLife.37355.033
Figure 9—video 2
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Phenotype of Myl9b phosphorylation mutant −2.

Maximum projection of Z-planes of a spinning-disk confocal TL sequence performed on a 40 hpf Tg(kdrl:Ras-mCherry) embryo expressing the runx’1 + 23’:Myl9bA2A3-eGFP transgene. Note the bursting of …

https://doi.org/10.7554/eLife.37355.034
Author response image 1
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Runx1 morpholino analysis.

This figure shows the several experiments that were performed to characterize the runx1 morpholino that was used in this study (first submission). The panel C was originally in the previous Figure …

https://doi.org/10.7554/eLife.37355.039
Author response image 2
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Analysis of Myl9b and Myl9a expression after deletion of part of the Runx1 sequence encoded by exon 4 (Runx1 crispants).

This is the first of the 2 strategies that we have used to challenge the phenotypes obtained using the Runx1 morpholino.

https://doi.org/10.7554/eLife.37355.040
Author response image 3
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Analysis of Myl9b and Myl9a expression after expression of a deleted form of Runx1.
https://doi.org/10.7554/eLife.37355.041

Videos

Video 1
Download asset
Dynamic 2D-maping of the apical constriction followed by eGFP-ZO1.

Spinning-disk TL confocal sequence performed on a 48 hpf Tg(fli1:Gal4; UAS:RFP; UAS:eGFP-ZO1) embryo, showing a cell undergoing the EHT and processed with the 2D-algorithm. Only the eGFP channel is …

https://doi.org/10.7554/eLife.37355.016
Video 2
Download asset
Dynamics of Myl9a and Myl9b during the EHT.

Top panels: Maximum projection of Z-planes of a laser scanning confocal TL sequence performed on a 48 hpf Tg(kdrl:Ras-mCherry; kdrl:Myl9a-eGFP) embryo focusing on the localization of Myl9a-eGFP at …

https://doi.org/10.7554/eLife.37355.029
Video 3
Download asset
Dynamics of the contractile and anisotropic actin and myosin rings.

Spinning-disk confocal TL sequence showing 3D rendering ‘en face’ views of EHT cells from 48 hpf Tg(fli1:Lifeact-eGFP) (top panel), Tg(kdrl:Myl9a-eGFP) (middle panel) and Tg(kdrl:Myl9b-eGFP) (bottom …

https://doi.org/10.7554/eLife.37355.030

Tables

Key resources table
Reagent type
(species) or
resource		DesignationSource or referenceIdentifiersAdditional information
Strain, strain
background
(Zebrafish)		Zebrafish: ABZebrafish International
Resource Center (ZIRC)		ZFIN:
ZDB-GENO-960809–7
Strain, strain
background
(Zebrafish)		Zebrafish Tg(kdrl:HsHRAS
-mCherry): s916Tg		Chi et al. (2008)ZFIN ID:
ZDB-ALT-090506–2		referred to as Tg(kdrl:Ras-mCherry)
Strain, strain
background
(Zebrafish)		Zebrafish:
Tg(fli1a:LIFEACT-eGFP): zf495Tg		Phng et al. (2013)ZFIN ID:
ZDB-ALT-140610–8
Strain, strain
background
(Zebrafish)		Zebrafish:
Tg(5xUAS:RFP): nkuasrfp1aTg		Asakawa et al. (2008)ZFIN ID:
ZDB-ALT-080528–2
Strain, strain
background
(Zebrafish)		Zebrafish:
Tg(fli1ep:Gal4ff): ubs3Tg		Zygmunt et al. (2011)ZFIN ID:
ZDB-ALT-120113–6
Strain, strain
background
(Zebrafish)		Zebrafish:
Tg(UAS:EGFP-ZO1): ubs5Tg		Herwig et al. (2011)ZFIN ID:
ZDB-ALT-120113–7
Strain, strain
background
(Zebrafish)		Zebrafish:
Tg(kdrl:EGFP): s843Tg		Jin et al. (2005)ZFIN ID:
ZDB-ALT-050916–14
Strain, strain
background
(Zebrafish)		Zebrafish: Tg(kdrl:gal4;UAS:RFP)NANA
Strain, strain
background
(Zebrafish)		Zebrafish Tg(−6.0itga2b:EGFP):
la2Tg		Lin et al., 2005
Traver		ZFIN ID:
ZDB-ALT-051223–4		referred to as Tg(CD41:eGFP)
Strain, strain
background
(Zebrafish)		Zebrafish
Tg(kdrl:myl9a-eGFP): ip5Tg		This PaperN/A
Strain, strain
background
(Zebrafish)		Zebrafish
Tg(kdrl:myl9b-eGFP): ip6Tg		This paperN/A
AntibodySheep Anti-Digoxigenin
Fab fragments,
POD Conjugated		RocheCat# 11207733910;
RRID: AB_514500		1:5000
Recombinant
DNA reagent		Transposase pCS-zT2TPSuster et al. (2011)N/A
Recombinant
DNA reagent		pUAS:eGFP-hs-ZO1Herwig et al. (2011)N/A
Recombinant
DNA reagent		pEGFP-C1ClontechN/A
Recombinant
DNA reagent		pmKate2-f-memEvrogenCat#: FP186
Recombinant
DNA reagent		pG1-flk1-MCS-tol2Jin et al. (2005)Addgene
plasmid # 26436
Recombinant
DNA reagent		PG1-flk-MCS-tol2-kdrl-
myl9a-eGFP		This paperN/A
Recombinant
DNA reagent		PG1-flk-MCS-tol2-kdrl-
myl9b-eGFP		This paperN/A
Recombinant
DNA reagent		p5E-Runx1 + 23-betaglobinTamplin et al. (2015)Addgene
plasmid # 69602
Recombinant
DNA reagent		pTol2-Runx1 + 23×1-
betaglobin:eGFP		This paperN/A
Recombinant
DNA reagent		pTol2-Runx1 + 23×1-betaglobin
-myl9b-eGFP		This paperN/A
Recombinant
DNA reagent		pTol2-Runx1 + 23×1-betaglobin
-myl9bA2A3-eGFP		This paperN/A
Recombinant
DNA reagent		pTol2-Runx1 + 23×1-betaglobin
-myl9a-mKate2		This paperN/A
Chemical
compound, drug		TricaineSigma AldrichCat#: A5040
Chemical
compound, drug		Methylen BlueSigma AldrichCat#: M4159
Chemical
compound, drug		N-Phenylthiourea (PTU)Sigma AldrichCat#: P7629
Chemical
compound, drug		Low Melting Point,
Analytical Grade		PromegaCat#: V2111
Chemical
compound, drug		Formaldehyde (FA)PolysciencesCat#:04018–1
Chemical
compound, drug		Proteinase KAmbionCat#: AM2546
Chemical
compound, drug		Digoxigenin labeled
nucleotides		Jena BioscenceCat#: NU-803-DIGX
Chemical
compound, drug		Wester Blotting
Reagent (WBR)		RocheCat#: 11921673001
Chemical
compound, drug		NBTSigma AldrichCat#: N6639
Chemical
compound, drug		BCIPSigma AldrichCat#: B-8503
Chemical
compound, drug		Tissue Freezing
Medium, Blue		Electron Microscopy SciencesCat#: 72592-B
Chemical
compound, drug		Aqua-Poly/MountBiovalleyCat#: 18606–5
Chemical
compound, drug		Immersol W 2010, oilZeissCat#:
444969-0000-000
Chemical
compound, drug		AquaSound Clear
ultrasound gel		Free-MedCat#: FU00071
Commercial
assay or kit		Cloning Gibson AssemblyNEBCat#: E2611S
Commercial
assay or kit		cDNA synthesis for
cloning Super Script III		Thermo FisherCat#: 8080093
Commercial
assay or kit		Expand High
Fidelity polymerase		Sigma AldrichCat#:11732641001
Commercial
assay or kit		RNA synthesis mMESSAGE
mMACHINE SP6
Transcription		AmbionCat#: AM1340
Commercial
assay or kit		Poly(A) Tailing KitAmbionCat#: AM1350
Commercial
assay or kit		DNA purification for
injection NucleoBond
Xtra Midi EF		Macherey NagelCat#: 740420.10
Commercial
assay or kit		T7 RNA polymerasePromegaCat#: P2075
Commercial
assay or kit		RNA extraction RNeasy
Plus Mini Kit		QIagenCat#: 74134
Commercial
assay or kit		cDNA synthesis for qPCR
M-MLV Reverse Transcriptase		InvitrogenCat#: 28025013
Commercial
assay or kit		RNAse OutInvitrogenCat#: 10777019
Commercial
assay or kit		qPCR Takyon Rox SYBR
Master mix blue dTTP		EurogenetecCat#: UF-RSMT-B0701
Commercial
assay or kit		NucAwayTM d’AmbionTMInvitrogenCat#: AM10070
Software,
algorithm		Acquisition-Imaging
analysis-LAS X		Leicahttp://www.Leica-microsystems.com
Software,
algorithm		Acquisition-VolocityPerkin Elmerhttp://www.perkinelmer.com/
Software,
algorithm		Imaging analysis-Imaris
8.4.1		Bitplanehttp://www.bitplane.com/
Software,
algorithm		Imaging
analysis-Matlab R2017a/R2017b		MathWorkshttps://fr.mathworks.com
Software,
algorithm		Image Analysis-Prism 6Graph Padhttps://www.graphpad.com/
Software,
algorithm		Image Analysis-FijiNIHhttps://imagej.net/Fiji
Software,
algorithm		Image Analysis
TrackMate plugin for Fiji		Tinevez et al. (2017)https://imagej.net/TrackMate
Software,
algorithm		Image Analysis Icyde Chaumont et al., 2012http://icy.bioimageanalysis.org/
Software,
algorithm		Image Analysis
TubeSkinner plugin
for Icy		This paperThis Paper
Software,
algorithm		Figures-Photoshop
CC 2017.1.1		Adobehttp://www.adobe.com/cn/
Software,
algorithm		Figures-Illustrator
CC 2017.1.1		Adobehttp://www.adobe.com/cn/
Recombinant
DNA reagent		Morpholino
Standard Control		Gene ToolsN/ACCTCTTACCTCAGTTACAATTTATA
Recombinant
DNA reagent		Morpholino sihSehnert et al. (2002)ZDB-MRPHLNO
-060317–4		CATGTTTGCTCTGATCTGACACGCA
Recombinant
DNA reagent		Morpholino
Myl9aspe2i2		Gene Tools; This paperN/AATTCAGCTTGTATATCTCTCACCCA
Recombinant
DNA reagent		Morpholino
Myl9aspi3e4		Gene Tools; This paperN/ACCCTGGTACAAACACACCGCAGATT
Recombinant
DNA reagent		Morpholino
Myl9bspe2i2		Gene Tools; This paperN/ACTATTATTTCACCCAGACTCACCCA
Recombinant
DNA reagent		Morpholino
Myl9bspe3i3		Gene Tools; This paperN/ATTGTTGTTGTTTTTACCAGATCCCT
Recombinant
DNA reagent		Oligo:
Rescue-SP6-myl9b
forward		This paperN/AatttaggtgacactatagaagngATGT
CCAGCAAAAGAGCAAAGGGG
Recombinant
DNA reagent		Oligo: Rescue-myl9b
reverse		This paperN/ACTACATGTCGTCTTTGTCTTT
GGCTCCG
Recombinant
DNA reagent		Oligo: qPCR myl9a
forward		This paperN/ACACCTGCTTCGATGAGGACGCCA
Recombinant
DNA reagent		Oligo: qPCR myl9a
reverse		This paperN/ACCGAGATCTGCTGACCACTATGG
GCGATC
Recombinant
DNA reagent		Oligo: qPCR myl9b
forward		This paperN/AGCCTGCTTCGATGAGGAGGGATC
Recombinant
DNA reagent		Oligo: qPCR myl9b
reverse		This PaperN/ACTGACCACCATGGGAGACC
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9asp22
forward		This paperN/ACACGTCCAATGTCTTCGCCA
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9asp22
reverse		This paperN/ATGGGTTCTTCCCCAGAGAGG
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9asp34
forward		This PaperN/AGGACGCCACTGGGTTCATCC
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9asp34
reverse		This paperN/AGTTTCCTTTCTTGTCGATGGGCGC
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9bsp22
forward		This paperN/AGCATCTTTGGGTAAGAACCCGTCTG
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9bsp22
reverse		This paperN/ATCAGCCGCTCTCCAAACATG
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9bsp33
forward		This paperN/AGAGGAGGGATCTGGTTTCATCC
Recombinant
DNA reagent		Oligo: qPCR
splicemyl9bsp33
reverse		This paperN/ACCCTTCTTGTCAATGGGAGCC
Recombinant
DNA reagent		Oligo: Probe
myl9a forward		This paperN/AGATAAGGAGGATCTGCATGA
CATGCTCG
Recombinant
DNA reagent		Oligo: Probe T7
myl9a reverse		This paperN/Agaaattaatacgactcactatagg
GTGAAGCGATCGCCCATAGTGG
Recombinant
DNA reagent		Oligo: Probe
myl9b forward		This paperN/AGCACGATATGCTAGCATCTTTGGG
Recombinant
DNA reagent		Oligo: Probe
T7-myl9b reverse		This paperN/Agaaattaatacgactcactataggg
CATGGTGGTCAGCAGCTCCC
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9a-
eGFP-myl9a-for		This paperN/ATATTTTAACAGACAAGGGCGATGT
CTGCAGCCAAACGCGCCAAAGGAAAG
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9a-
eGFP-myl9a-rev		This paperN/ACCTTGCTCACCATGTCGTCCTTG
TCTTTGGCTCCGTGCTTTAG
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9a-
eGFP-eGFP-for		This paperN/AGGACGACATGGTGAGCAAGGGCG
AGGAGCTGTTCACCGGG
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9a-
eGFP-eGFP-rev		This paperN/ACGATATCCTCGAGGGTACCGTTATCTA
GATCCGGTGGATCCCGGGCCCGC
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9b-
eGFP-myl9b-for		This paperN/AGACGTTATTTTAACAGACAAGGGCG
ATGTCCAGCAAAAGAGCAAAGGGG
AAGACC
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9b-
eGFP-myl9b-rev		This paperN/ACGCCCTTGCTCACCATGTCGTCTTTGT
CTTTGGCTCCGTGTTT
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9b-
eGFP-eGFP-for		This paperN/ACAAAGACGACATGGTGAGCAAGGGCG
AGGAGCTGTTCACCGGG
Recombinant
DNA reagent		Oligo for Cloning:
PG1-flk-MCS-tol2-myl9b-
eGFP-eGFP-rev		This paperN/AGGATCCGATATCCTCGAGGGTACCG
TTATCTAGATCCGGTGGATCCCG
GGCCCGC
Recombinant
DNA reagent		Oligo for Cloning:
pTol2-Runx1 + 23-betaglobin-
myl9a-mKate2-myl9a-for		This paperN/AATCCCGCGGTGGAGCTCCAGAATTCAT
GTCTGCAGCCAAACGCGCCAAAGG
Recombinant
DNA reagent		Oligo for Cloning:
pTol2-Runx1 + 23-betaglobin-
myl9a-mKate2-myl9a-rev		This paperN/AGCATGTTCTCCTTAATCAGCTCGCTC
ACCATGTCGTCCTTGTCTTTGGCTCC
Recombinant
DNA reagent		Oligo for Cloning:
pTol2-Runx1 + 23-betaglobin-
myl9a-mKate2-mKate2-for		This paperN/AGAGCCAAAGACAAGGACGACATGGT
GAGCGAGCTGATTAAGGAGAACATGC
Recombinant
DNA reagent		Oligo for Cloning:
pTol2-Runx1 + 23-betaglobin-
myl9a-mKate2-mKate2-rev		This paperN/AGGATCCGATATCCTCGAGGGTACCGTC
ATCTGTGCCCCAGTTTGCTAGGG
Recombinant
DNA reagent		Oligo for Cloning:
pTol2-Runx1 + 23×1-
betaglobin-myl9b-eGFP-
myl9a-for		This paperN/ACAGACATCCCGCGGTGGAGCTCC
AGGTCGCCACCATGTCCAGCAAAAG
AGCAAAGGGGAAGACCACCAAG
Recombinant
DNA reagent		Oligo for Cloning:
pTol2-Runx1 + 23×1-
betaglobin-myl9b-eGFP-
myl9a-rev		This paperN/ACGCCCTTGCTCACCATggtggcgacGAA
TTCCATGTCGTCTTTGTCTTTGG
CTCCGTG
Recombinant
DNA reagent		Oligos for mutagenesis:
pTol2-Runx1 + 23×1-
betaglobin-myl9b-eGFP-
myl9aA2A3-for		This paperN/ACAGACATCCCGCGGTGGAGCTCCA
GgtcgccaccAtggccgccaaaagagca
aaggggaagaccaccaag
Recombinant
DNA reagent		Oligo for mutagenesis:
pTol2-Runx1 + 23×1
betaglobin- myl9b-eGFP-

myl9aA2A3-rev		This paperN/ACGCCCTTGCTCACCATggtggcgac
GAATTCcatgtcgtctttgtctttg
gctccgtg

Additional files

Source code 1

Matlab code for tracking, A-P distances through time and closing speeds.

https://doi.org/10.7554/eLife.37355.035
Source code 2

Code of the plugin AortaTracker (used for Figures 3, 4 and 5a-bottom panel, and see Method section).

https://doi.org/10.7554/eLife.37355.036
Transparent reporting form
https://doi.org/10.7554/eLife.37355.037

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