TITLE: Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos
AUTHORS: Lydvina  Meister, Hector Escriva, Stephanie Bertrand

[DESCRIPTION]
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.

[STEPS]
1. Preparation of the Kaede mRNA


2. Gametes obtaining
3. Oocytes injection

Microinjection is undertaken following the protocol published previously in:  
 



4. Photoconversion
1.2. 
mRNA synthesis

Use the   and follow the manufacturer's instructions.
1. Thaw the frozen reagents.
2. Mix together:
10 µL of 2X NTP/CAP
2 µL of 10X Reaction Buffer
1 µg of the linearized plasmid
2 µL of Enzyme mix
  q.s. 20 µL 
3. Incubate at 37 °C during 120 min .
4. Add 1 µL TURBO DNase and incubate at 37 °C during 15 min .
5. Add 30 µL of   and 30 µL of LiCl Precipitation Solution.
6. Mix and incubate at -20 °C during at least 30 min .
7. Centrifuge 13000 rpm, 20 min, 4 °C .
8. Remove the supernatant and add 1 mL of   .
9. Centrifuge 13000 rpm, 10 min, 4 °C .
10. Remove the supernatant.
11. Let the pellet air dry and resuspend in 10 µL of  .
12. Mesure the concentration and check for the integrity of the RNA on a  1 Mass / % volume   gel.
13. The mRNA is either kept in small aliquots at -80 °C   for upcoming experiments or directly used for the microinjection experiment.
If the concentration is low (less than 2 µg/µL ), you can undertake several synthesis reactions,  precipitate them together and resuspend the pellet in a smaller volume.


1.1. 
Plasmid linearization

1. Mix together:
5 µg of the pCS2+ plasmid containing the coding sequence of the Kaede protein from Trachyphyllia geoffroyi (AB085641.1)
5 µL of   
20 µL of   
  q.s. 200 µL 
2. Incubate 120 min at 37 °C .
3. Add:
 20 µL of  3 Molarity (M)  
 400 µL of   
4. Incubate at -20 °C   .
5. Centrifuge 13000 rpm, 20 min, 4 °C .
6. Discard the supernatant.
7. Add 600 µL of cold  .
8. Centrifuge 13000 rpm, 10 min, 4 °C .
9. Discard the supernatant and let the pellet air  dry.
10. Disolve the pellet in 10 µL of   .
11. Run 0.5 µL in an 1 Mass / % volume   gel to test for complete linearization.
12. Measure the plasmid concentration using 0.5 µL with a 
 
or equivalent equipment.


2.1. 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 17 °C and a light/dark cycle of 840 min /600 min is applied. The seawater is changed three times per day.

2.2. Spawning induction

Gametes are obtained using the heat stimulation method published in: 
 

Amphioxus spawn at night, just after the sunset. Briefly, ripe adults kept at 17 °C are placed in a tank without sand at 23 °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.

3.1. Material to prepare in advance

1. Injection needles are prepared using   capillaries and a micropipette puller as for example: 
 

2. Poly-D-lysine coated dishes are prepared as follows:
Dilute the   in    to a final concentration of  0.1 mg/mL.
Cover the bottom of    with this solution.
Incubate at Room temperature during 5 min .
Remove the solution.
Let the dishes air dry.
Keep the dishes at 4 °C until use.

3.2. Microinjection

1. Prepare a mix containing as final concentrations:
 1.5 µg/µL of Kaede mRNA
18 % volume of    
18 % volume of  a 10 mg/mL solution of  diluted in   .
2. Load the injection needle using   . 
3. Place the needle on a micromanipulator and connect it to the microinjector: 
 

4. Deposite a line of oocytes in a   dish coated with   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. 10 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 19 °C .



5. 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 :
1038.4 millimolar (mM)    
22.2 millimolar (mM)    
20 millimolar (mM)     
49 millimolar (mM)    
51 millimolar (mM)    

Transfer the specimen with a p20 tip to a Petri dish containing 2X Artificial Sea Water. When the embryo is totally immobilised (around 10 s ) 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 5 min .

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.













4.1. Preparing the embryo for the photoconversion

1.  30 min  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)  . 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 500 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:
 
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.




4.2. 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.

  
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 1 min .

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 2 mL  of filtered seawater and incubated at 19 °C  .