Sep 08, 2022

Public workspaceExploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.j8nlk46z6g5r/v1
  • 1[Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins, BIOM, F-66650, Banyuls-sur-Mer, France ]
Stephanie Bertrand
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk46z6g5r/v1
External link: https://doi.org/10.1371/journal.pone.0275193
Protocol Citation: Lydvina Meister, Hector Escriva, Stephanie Bertrand 2022. Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk46z6g5r/v1
Manuscript citation:
Meister L, Escriva H, Bertrand S (2022)Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos. PLOS ONE 17(9): e0275193. https://doi.org/10.1371/journal.pone.0275193
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 28, 2021
Last Modified: September 08, 2022
Protocol Integer ID: 49501
Keywords: Kaede, photoconversion, development, cell tracing, amphioxus, cephalochordate, embryo, fluorescent protein, confocal, FRAP wizard
Funders Acknowledgement:
Agence Nationale de la Recherche
Grant ID: ANR-19-CE13-0011-01
Agence Nationale de la Recherche
Grant ID: ANR-16-CE12-0008-01
H2020-INFRAIA-1-2016- 2017
Grant ID: 730984
Abstract
Photoconvertible proteins are powerful tools widely used in cellular biology to study cell dynamics and organelles. Over the past decade, photoconvertible proteins have also been used for developmental biology applications to analyze cell lineage and cell fate during embryonic development. One of these photoconvertible proteins called Kaede, from the stony coral Trachyphyllia geoffroyi, undergoes irreversible photoconversion from green to red fluorescence when illuminated with UV light. Undertaking a cell tracing approach using photoconvertible proteins can be challenging when using unconventional animal models. In this protocol, we describe the use of Kaede to track specific cells during embryogenesis of the cephalochordate Branchiostoma lanceolatum. This protocol can be adapted to other unconventional models, especially marine animals.
Protocol materials
ReagentThin-Wall Capillary 4 1.0mm (fil)World Precision InstrumentsCatalog #TW100F-4
Step 3.1
ReagentFalcon™ Easy-Grip Tissue Culture DishesFisher ScientificCatalog #10038820
In 2 steps
ReagentGlycerol - for molecular biology, ≥99%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
Step 3.2
ReagentEppendorf™ Microloader™ Pipette TipsEppendorfCatalog #5242956003
Step 3.2
ReagentMagnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2773
Step 5
ReagentCalcium chloride dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #C5080
Step 5
ReagentEthanol 70%
In 2 steps
ReagentNotI - 500 unitsNew England BiolabsCatalog #R0189S
Step 1.1
ReagentPOLY-D-LYSINE HYDROBROMIDE MOL WT 70000 - 5MGMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6407-5MG
In 2 steps
ReagentFast Green FCFMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7252
Step 3.2
ReagentPotassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Step 5
ReagentAgaroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9539
In 2 steps
ReagentMilliQ water
In 2 steps
Reagent Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #746398
Step 5
ReagentNunc™ Glass Bottom Dishes, 12mmThermo FisherCatalog #150680
Step 4.1
ReagentEthanol
Step 1.1
Reagentammonium acetate
Step 1.1
ReagentCutSmart® BufferNew England BiolabsCatalog #B7204S
Step 1.1
ReagentMagnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)
Step 5
ReagentNuclease-free Water
In 5 steps
ReagentmMESSAGE mMACHINE™ SP6 Transcription KitThermo FisherCatalog #AM1340
Step 1.2
Preparation of the Kaede mRNA



Plasmid linearization

1. Mix together:
  • Amount5 µg of the pCS2+ plasmid containing the coding sequence of the Kaede protein from Trachyphyllia geoffroyi (AB085641.1)
  • Amount5 µL of ReagentNotI - 500 unitsSigma AldrichCatalog #R0189S
  • Amount20 µL of ReagentCutSmart® BufferSigma AldrichCatalog #B7204S
  • ReagentNuclease-free WaterSigma Aldrich q.s. Amount200 µL
2. Incubate Duration02:00:00 at Temperature37 °C .
3. Add:
  • Amount20 µL of Reagentammonium acetateSigma Aldrich Concentration3 Molarity (M)
  • Amount400 µL of ReagentEthanolSigma Aldrich
4. Incubate at Temperature-20 °C DurationOvernight .
5. Centrifuge Centrifigation13000 rpm, 4°C, 00:20:00 .
6. Discard the supernatant.
7. Add Amount600 µL of cold ReagentEthanol 70% Sigma Aldrich .
8. Centrifuge Centrifigation13000 rpm, 4°C, 00:10:00 .
9. Discard the supernatant and let the pellet air dry.
10. Disolve the pellet in Amount10 µL of ReagentNuclease-free WaterSigma Aldrich .
11. Run Amount0.5 µL in an Concentration1 Mass / % volume ReagentAgaroseSigma AldrichCatalog #A9539 gel to test for complete linearization.
12. Measure the plasmid concentration using Amount0.5 µL with a
Equipment
Nanodrop 2000C
NAME
Thermo Scientific
BRAND
TSC-ND2000C
SKU
or equivalent equipment.


2h 30m

mRNA synthesis

Use the ReagentmMESSAGE mMACHINE™ SP6 Transcription KitSigma AldrichCatalog #AM1340 and follow the manufacturer's instructions.
1. Thaw the frozen reagents.
2. Mix together:
  • Amount10 µL of 2X NTP/CAP
  • Amount2 µL of 10X Reaction Buffer
  • Amount1 µg of the linearized plasmid
  • Amount2 µL of Enzyme mix
  • ReagentNuclease-free WaterSigma Aldrich q.s. Amount20 µL
3. Incubate at Temperature37 °C during Duration02:00:00 .
4. Add Amount1 µL TURBO DNase and incubate at Temperature37 °C during Duration00:15:00 .
5. Add Amount30 µL of ReagentNuclease-free WaterSigma Aldrich and Amount30 µL of LiCl Precipitation Solution.
6. Mix and incubate at Temperature-20 °C during at least Duration00:30:00 .
7. Centrifuge Centrifigation13000 rpm, 4°C, 00:20:00 .
8. Remove the supernatant and add Amount1 mL of ReagentEthanol 70% Sigma Aldrich .
9. Centrifuge Centrifigation13000 rpm, 4°C, 00:10:00 .
10. Remove the supernatant.
11. Let the pellet air dry and resuspend in Amount10 µL ofReagentNuclease-free WaterSigma Aldrich .
12. Mesure the concentration and check for the integrity of the RNA on a Concentration1 Mass / % volume ReagentAgaroseSigma AldrichCatalog #A9539 gel.
13. The mRNA is either kept in small aliquots at Temperature-80 °C for upcoming experiments or directly used for the microinjection experiment.
If the concentration is low (less than Concentration2 µg/µL ), you can undertake several synthesis reactions, precipitate them together and resuspend the pellet in a smaller volume.


3h 15m
Gametes obtaining
Adult collection

Ripe Branchiostoma lanceolatum adults are obtained by sieving the sand collected at a 5 m depth near the Racou beach (Argelès sur Mer, France) during the months of May and June.
They are kept in small seawater tanks with few centimeters of sand. The temperature is maintained at Temperature17 °C and a light/dark cycle of Duration14:00:00 /Duration10:00:00 is applied. The seawater is changed three times per day.

1d
Spawning induction

Gametes are obtained using the heat stimulation method published in:
CITATION
Fuentes M, Benito E, Bertrand S, Paris M, Mignardot A, Godoy L, Jimenez-Delgado S, Oliveri D, Candiani S, Hirsinger E, D'Aniello S, Pascual-Anaya J, Maeso I, Pestarino M, Vernier P, Nicolas JF, Schubert M, Laudet V, Geneviere AM, Albalat R, Garcia Fernandez J, Holland ND, Escriva H (2007). Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum).. Journal of experimental zoology. Part B, Molecular and developmental evolution.

Amphioxus spawn at night, just after the sunset. Briefly, ripe adults kept at Temperature17 °C are placed in a tank without sand at Temperature23 °C the day before spawning, with the same day/night cycle. Two hours before the light is turned off the spawning day, animals are individualized in plastic cups filled with a small volume of filtered seawater and placed on a black background in order to facilitate the vizualisation of the gametes as they are released. After one hour in the dark, the gametes are collected: the sperm diluted in the seawater is conserved at 4°C and can be used during several hours. The eggs are collected by pipetting and placed in small scratched Petri dishes to avoid sticking to the plastic.

Oocytes injection

Microinjection is undertaken following the protocol published previously in:
CITATION
Hirsinger E, Carvalho JE, Chevalier C, Lutfalla G, Nicolas JF, Peyriéras N, Schubert M (2015). Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.. Journal of visualized experiments : JoVE.
LINK
https://doi.org/10.3791/52042



Material to prepare in advance

1. Injection needles are prepared using ReagentThin-Wall Capillary 4 1.0mm (fil)Sigma AldrichCatalog #TW100F-4 capillaries and a micropipette puller as for example:
Equipment
P-97 micropipette puller
NAME
Flaming/Brown Micropipette Puller
TYPE
Sutter instrument
BRAND
N/A
SKU
https://www.sutter.com/MICROPIPETTE/p-97.html
LINK

2. Poly-D-lysine coated dishes are prepared as follows:
  • Dilute the ReagentPOLY-D-LYSINE HYDROBROMIDE MOL WT 70000 - 5MGSigma AldrichCatalog #P6407-5MG in ReagentMilliQ waterSigma Aldrich to a final concentration of Concentration0.1 mg/mL .
  • Cover the bottom of ReagentFalcon™ Easy-Grip Tissue Culture DishesSigma AldrichCatalog #10038820 with this solution.
  • Incubate at TemperatureRoom temperature during Duration00:05:00 .
  • Remove the solution.
  • Let the dishes air dry.
  • Keep the dishes at Temperature4 °C until use.

5m
Microinjection

1. Prepare a mix containing as final concentrations:
  • Concentration1.5 µg/µL of Kaede mRNA
  • Concentration18 % volume of ReagentGlycerol - for molecular biology, ≥99%Sigma AldrichCatalog #G5516
  • Concentration18 % volume of a Concentration10 mg/mL solution of ReagentFast Green FCFSigma AldrichCatalog #F7252 diluted in ReagentMilliQ waterSigma Aldrich .
2. Load the injection needle using ReagentEppendorf™ Microloader™ Pipette TipsSigma AldrichCatalog #5242956003 .
3. Place the needle on a micromanipulator and connect it to the microinjector:
Equipment
Picospritzer III Intracellular Microinjection Dispense System
NAME
Injection system
TYPE
Picospritzer
BRAND
052-0500-900
SKU
https://ph.parker.com/us/12051/en/picospritzer-iii-intracellular-microinjection-dispense-systems-picospritzer-micro-dispense-system/052-0500-900
LINK
100 psi, 2 channel
SPECIFICATIONS

4. Deposite a line of oocytes in a ReagentFalcon™ Easy-Grip Tissue Culture DishesSigma AldrichCatalog #10038820 dish coated withReagentPOLY-D-LYSINE HYDROBROMIDE MOL WT 70000 - 5MGSigma AldrichCatalog #P6407-5MG and filled with filtered seawater.
5. Cut the end of the needle using fine forceps under the binocular. Use a binocular that allows for 200X magnification.
6. Insert the needle into the oocyte and inject a small volume of injection mix (1/100 to 1/50 of the volume of the oocyte). Depending on the size of the needle after cutting, several injection pulses might be necessary to inject a sufficient volume.
7. When all the oocytes are injected, proceed to fertilization. Amount10 µL of diluted sperm are sufficient to fertilize 50 injected oocytes. The embryos are detached from the poly-D-lysine coated dish by delicate water waves pushes after fertilization membrane raising, transferred into a clean Petri dish filled with filtered seawater and kept at Temperature19 °C .



Photoconversion
Preparing the embryo for the photoconversion

1. Duration00:30:00 before the photoconversion, observe the embryos under a fluorescent binocular using the green filter.
2. Individualize the fluorescent well-developed embryos into glass bottom culture dishes (35 mm) ReagentNunc™ Glass Bottom Dishes, 12mmSigma AldrichCatalog #150680 . Transfer the embryos using a P200 tip to minimize the risk of damage.
3. Maintain the embryo in the smallest possible volume of filtered seawater. In the center of the Petri dish, a volume of Amount500 µL of seawater is sufficient for the embryo survival and photoconversion.
4. Place the embryo in the desired orientation (blastopore view in our example) using a 10 cm steel syringe needle tip under the binocular.
5. Transfer the Petri dish under the confocal inverted microscope for the photoconversion. Here we will describe the procedure using the following microscope:
Equipment
White Light Laser Confocal Microscope Leica TCS SP8 X
NAME
Confocal Inverted Microscope
TYPE
Leica
BRAND
NA
SKU
However, any confocal inverted microscope equipped with a UV laser and lasers to image the fluorescence emitted by the Kaede protein can be used. The microscope must also allow scan zoom and ROI scanning in order to effectively target a specific region using this protocol for photoconversion. The UV laser intensity and scan time must be adjusted. If a FRAP module or a photoconversion/photoactivation module is available on the microscope, it can be used following the manufacturer's instructions.

6. Visualize the embryo under the 20x/NA 0.8 objective by setting the white laser excitation wavelength at 508nm.
7. Scan the embryo all along the z-axis in order to see if the specimen is properly positioned.
8. Activate the FRAP-Wizard (Fluorescent Recovery After Photobleaching) module of the LAS X software platform.




30m
Photoconversion settings

At the top of the FRAP-wizard interface, several buttons corresponding to the different steps are available : “Overview”, “Set Up”, “Bleach”, “Time course”, “Evaluation”. For photoconversion, the "Evaluation" step is not used.

1. Step 1: Settings for pre- and post- bleach imaging
  • Click on the "Set Up" button and adjust the parameters for imaging the embryo before and after photoconversion.
  • Set up the laser and detectors parameters to excite and detect emission of the Kaede in its two states: before (uncleaved, green, Ex508/Em518) and after (cleaved, red, Ex572/Em582) photoconversion.

Asset URL:
  • Adjust the intensity of the laser depending on the amount of Kaede produced by the embryo (correlated to the amount of mRNA injected in the oocyte). Usually, an intensity of 15% is sufficient for imaging the photoconversion.
  • Choose the "xyt" acquisition mode for photoconvertion.

2. Step 2: Parameters for photobleaching
  • Click on the "Bleach" button.
  • Set the intensity of the 405nm (UV) diode laser at 85%.
  • Turn off the white laser.
  • Draw the Regions Of Interest (ROI) on the image of the embryo obtained before the photoconversion. In our example, three circles are drawn, two at the position of the presumptive paraxial mesoderm and one in the putative ventral ectoderm territory.
  • Activate the Zoom-In mode (more light is applied to the ROI).
  • Turn on the following options: « Set Background to Zero », « Delete Bleach Images after Scan » and « Use laser Settings for all ROIs ».

3. Step 3: Time Course - Defining the number of prebleach, bleach and postbleach intervals
  • Select the "Time-Course" menu.
  • One pre-bleach and one post-bleach intervals are imposed. For photoconversion, use only one repetition. The photoconversion time depending on the total size of ROIs, adjust the photoconversion session in order to program a total bleaching time of Duration00:01:00 .

After setting all these parameters, the photobleaching can be started. If the photoconversion is successful, the ROI previously fluorescent after an excitation at 508 nm becomes fluorescent under an excitation at 572 nm. The Petri dish is then filled with Amount2 mL of filtered seawater and incubated at Temperature19 °C .

1m
Imaging embryos at later stages

1. Keep the embryo in the incubator until the desired developmental stage.
2. After 15 hours post fertilization, the embryo starts moving thanks to cilia beating and becomes difficult to scan at high magnification. To immobilise it, incubate the embryo into 2X Artificial Sea Water :
  • Concentration1038.4 millimolar (mM) Reagent Sodium chlorideSigma AldrichCatalog #746398
  • Concentration22.2 millimolar (mM) ReagentPotassium chlorideSigma AldrichCatalog #P9333
  • Concentration20 millimolar (mM) ReagentCalcium chloride dihydrateSigma AldrichCatalog #C5080
  • Concentration49 millimolar (mM) ReagentMagnesium chloride hexahydrateSigma Aldrich
  • Concentration51 millimolar (mM) ReagentMagnesium sulfate heptahydrateSigma AldrichCatalog #M2773

Transfer the specimen with a p20 tip to a Petri dish containing 2X Artificial Sea Water. When the embryo is totally immobilised (around Duration00:00:10 ) and sinks to the bottom of the dish, put it back in the initial glass-bottom dish using the same tip. The embryo will remain immobilised for Duration00:05:00 .

3. Proceed to embryo imaging (z-stack) using excitation at 508 nm (green) and at 572 nm (red) and using the sequential mode. Depending on the developmental stage, the embryo can be imaged on both sides by turning it over delicately after the first scan using a 10 cm steel syringe needle.













5m 10s
Citations
Step 2.2
Fuentes M, Benito E, Bertrand S, Paris M, Mignardot A, Godoy L, Jimenez-Delgado S, Oliveri D, Candiani S, Hirsinger E, D'Aniello S, Pascual-Anaya J, Maeso I, Pestarino M, Vernier P, Nicolas JF, Schubert M, Laudet V, Geneviere AM, Albalat R, Garcia Fernandez J, Holland ND, Escriva H. Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum).
Step 3
Hirsinger E, Carvalho JE, Chevalier C, Lutfalla G, Nicolas JF, Peyriéras N, Schubert M. Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.
https://doi.org/10.3791/52042