Literature DB >> 36166455

Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos.

Lydvina Meister¹, Hector Escriva¹, Stéphanie Bertrand¹.

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.

Entities: Chemical

Mesh：

Substances：

Year: 2022 PMID： 36166455 PMCID： PMC9514637 DOI： 10.1371/journal.pone.0275193

Source DB: PubMed Journal: PLoS One ISSN： 1932-6203 Impact factor: 3.752

Introduction

One of the main questions in developmental biology is to understand the different behaviors adopted by the plethora of cells that are generated during embryogenesis through successive cell divisions. How, from a unique cell, a multicellular organism with a specific shape and cellular organisation is built up? Cell lineage tracing analysis refers to the study of cellular genealogies by following cell divisions and migration over developmental time. Such experiments rely on non-invasive labelling techniques that allow tracking cell shape and migration. Vital stains were the first tools to be used for such purposes, but the discovery of fluorescent proteins and of the photoactivation and photoconversion properties of some of them, as well as the development of confocal laser microscopy allowing 3D imaging of fluorescently labelled cells, has brought new opportunities to undertake fine cell-tracing analyses. The protein called Kaede, from the stony open brain coral Trachyphyllia geoffroyi, was one of the first photoconvertible molecule specifically used for cell tracking [1]. This protein emits green fluorescence when excited with blue light. After photoconversion using a pulse of UV irradiation, the protein switches irreversibly its emission from green to red. By photoconverting Kaede in a subset of cells they can be easily followed through development via time-laps imaging or by imaging the embryo at different developmental stages. Such an approach has been used for example to track cells from the hindbrain of the zebrafish embryo [2] or to study the development of lymphoid organs in mammals [3]. Although many cell-tracing experiments using Kaede are described in the literature for classical animal models [4-7], few data are available on the use of this tool in non-vertebrate marine species. Kaede was recently used in the sea urchin early embryo to distinguish pre-existing proteins from newly synthesized/imported proteins at a subcellular level [8], and in the tunicate Ciona intestinalis to follow the fate of the central nervous system cells after metamorphosis [9]. However, the use of Kaede in non-conventional models represents a true challenge in terms of sample size, embryo immobilization and imaging. Among marine animals, cephalochordates (i.e. amphioxus) represent the most basally divergent chordate clade (also comprising vertebrates and tunicates) and the best extant proxy to the ancestor of all chordates [10]. Understanding embryonic cell trajectories in amphioxus might thus give us new insights into the evolution of vertebrate morphological novelties. Although some data, using Nile blue staining to follow the fate of blastomeres at the 4, 8, 16 and 32-cell stages, were obtained in the 60’s (see [11] for a review), we are still missing a clear understanding of how the different morphological structures of the amphioxus larva develop from the two gastrula germ layers (i.e. the ectoderm and the mesendoderm). Here, we report a protocol that uses Kaede photoconversion to track cells during amphioxus embryogenesis. This protocol allows following the fate of cells from any early embryonic stage to the late neurula/larva stage and can be potentially adapted to study embryogenesis in other marine species.

Materials and methods

The protocol described in this peer-reviewed article (Fig 1) is published on protocols.io, https://dx.doi.org/10.17504/protocols.io.j8nlk46z6g5r/v1 and is included for printing purposes as S1 File.

Fig 1

Main steps for cell tracking using Kaede photoconversion in amphioxus embryos.

Expected results

We used the proposed protocol to follow the cells of the presumptive paraxial mesoderm territory. The photoconversion was undertaken in a gastrula stage embryo at the level of the dorsal paraxial mesendoderm on both sides as well as in a small region of the ectoderm on the opposite side of the embryo. This region corresponds to the presumptive ventral epidermis and Kaede was photoconverted in this territory in order to confirm that the photoconversion was undertaken in the correct position in the dorsal paraxial region. The embryo imaged at the late neurula stage showed expression in the ventral epidermis and in the somites (Fig 2). The same protocol was succesfully undertaken to trace the fate of the presumptive anterior somite territory in embryos in which the FGF signalling pathway was inhibited by using a FGFR inhibitor (SU5402) as published in [12].

Fig 2

Example of expected results for a cell tracking experiment using Kaede photoconversion in the amphioxus embryo.

The gastrula stage embryo (G4 stage) [13, 14] was imaged before and after photoconversion (pre- and post-bleach, panels on the left). The same embryo was imaged at the late neurula stage (N4 stage) [13, 14] (panels on the right). Gastrula stage pictures are blastopore views with dorsal to the top. Neurula stage images are side views with anterior to the left and dorsal to the top. Scale bar: 25 μm.

Example of expected results for a cell tracking experiment using Kaede photoconversion in the amphioxus embryo.

Step-by-step protocol, also available on protocols.io.

(PDF) Click here for additional data file. 11 Aug 2022

PONE-D-22-17287

Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos

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To my personal knowledge, the right amount of injection volume is crucial for the successful rate of microinjection experiments, thus I would suggest the authors to provide more specific description for determining this injection volume. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: No ********** 2. Has the protocol been described in sufficient detail? Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible. Reviewer #1: Yes Reviewer #2: No ********** 3. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: PONE-D-22-17287 Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos The protocol is useful for a growing cohort of researchers in this animal. The animal (amphioxus) is not new to research, nor is the protein used (Kaede) but the combination here is quite useful. “published” protocols.io is not a functional link Demonstrable success using this method is given and adequately shows the effectiveness of the protocol and goals. Has nice links to JOVE references for ease of learning the procedure. Sufficient detail is given for the protocols, and are comprehensive. Additional microscope protocols would be helpful since not everyone uses the same as described herein. Reviewer #2: The protocol described in this peer-reviewed article (Figure 1) is published already on protocols.io. The method itself is quite straight forward by injecting mRNA of fluorescent protein. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". 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9 Sep 2022 Dear editor, plesae find below our answers to the comments on our manuscript PONE-D-22-17287, “Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos”. Additional Editor Comments: 1.File S1, page 4, section 3.2, 1st step; please check the concentration of Fast Green FCF used here (18%). It seems much higher than the listed solubility (1mg/mL) on the Sigma-Aldrich catalog. Thank you for pointing out this error. The 18% correspond to the final volume percentage of a solution of fast green diluted in water. The initial solution is at 10mg/mL (the solubility being, contrary to what is indicated in the Sigma-Aldrich catalog, 20g/100ml.Please see: https://pubchem.ncbi.nlm.nih.gov/compound/Fast-Green-FCF#section=Solubility). We have modified the protocol on protocols.io accordingly. 2. File S1, page 5, section 3.2, 6th step; it should be more specific on this description of “a small volume”. To my personal knowledge, the right amount of injection volume is crucial for the successful rate of microinjection experiments, thus I would suggest the authors to provide more specific description for determining this injection volume. Thank you for the remark. We have modified as follows to be more precise: “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.” Reviewer #1: Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos The protocol is useful for a growing cohort of researchers in this animal. The animal (amphioxus) is not new to research, nor is the protein used (Kaede) but the combination here is quite useful. We would like to thank the reviewer for this kind comment. “published” protocols.io is not a functional link We have now “published” the protocol and give the correct link to the protocols.io file. Demonstrable success using this method is given and adequately shows the effectiveness of the protocol and goals. Has nice links to JOVE references for ease of learning the procedure. Sufficient detail is given for the protocols, and are comprehensive. Thanks again. Additional microscope protocols would be helpful since not everyone uses the same as described herein. We understand this concern. As requested by PloS ONE for Lab Protocols article, we only described the protocol that has been validated in our laboratory. However, we have added in step 4.1 the following paragraph that should allow researchers having a different microscope to consider undertaking an experiment of this type with their own equipment: “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.”. Reviewer #2: The protocol described in this peer-reviewed article (Figure 1) is published already on protocols.io. The method itself is quite straight forward by injecting mRNA of fluorescent protein. We are sorry we don’t understand this remark. There is no such protocol in protocols.io (nor on amphioxus injection, nor on Kaede photoconversion). Submitted filename: Point_by_point_answer.docx Click here for additional data file. 12 Sep 2022 Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos PONE-D-22-17287R1 Dear Dr. Bertrand, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jr-Kai Yu, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: 19 Sep 2022 PONE-D-22-17287R1 Exploring tissue morphodynamics using the photoconvertible Kaede protein in amphioxus embryos Dear Dr. Bertrand: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jr-Kai Yu Academic Editor PLOS ONE

14 in total

Review 1. Evolutionary crossroads in developmental biology: amphioxus.

Authors: Stephanie Bertrand; Hector Escriva
Journal: Development Date: 2011-11 Impact factor: 6.868

2. Dynamic behavior of individual cells in developing organotypic brain slices revealed by the photoconvertable protein Kaede.

Authors: T Mutoh; T Miyata; S Kashiwagi; A Miyawaki; M Ogawa
Journal: Exp Neurol Date: 2006-06-06 Impact factor: 5.330

3. Cell tracking using a photoconvertible fluorescent protein.

Authors: Kohei Hatta; Hitomi Tsujii; Tomomi Omura
Journal: Nat Protoc Date: 2006 Impact factor: 13.491

4. An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model.

Authors: Danny A Stark; Paul M Kulesa
Journal: Dev Dyn Date: 2007-06 Impact factor: 3.780

5. Ependymal cells of chordate larvae are stem-like cells that form the adult nervous system.

Authors: Takeo Horie; Ryoko Shinki; Yosuke Ogura; Takehiro G Kusakabe; Nori Satoh; Yasunori Sasakura
Journal: Nature Date: 2011-01-02 Impact factor: 49.962

6. Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate.

Authors: Lydvina Meister; Hector Escriva; Stéphanie Bertrand
Journal: Development Date: 2022-05-16 Impact factor: 6.862