June 22, 2021

Dear Dr. Iyer,

Thank you very much for your review of our manuscript “Light induced GFP expression in zebrafish embryos using the optogenetic TAEL/C120 system" (manuscript ID JoVE62818). Please find attached a revised version of this manuscript for publication in JoVE. Revised portions are highlighted in blue text.

We thank the editors and reviewers for their thoughtful and constructive evaluation of our work. We have revised the manuscript as completely as possible to address their concerns. A point-by-point response to their comments is appended to this letter.

As suggested by the editor and reviewers, we have:

Replaced references to “TAEL 2.0” with the more general “TAEL/C120 system.”

Revised the Protocol section to use the imperative tense and number each step

Revised the Discussion section to include discussion of critical steps, troubleshooting, limitations of the technique, significance with respect to existing methods, and future applications.

Added data to Table 1 to show low-level activation of the TAEl/C120 system by ambient room light.

Revised the Protocol, Discussion, Fig. 2A, and Materials Table to emphasize the importance of light power and add details for measuring light power.

We believe that these revisions have improved and strengthened our manuscript, which we now hope is suitable for publication in Zebrafish.

Thank you again for your time and consideration.

Sincerely,

Stephanie Woo, Ph.D.

Assistant Professor, Dept. of Molecular Cell Biology

University of California Merced

Point-by Point Response to editorial and reviewer comments (comments are italicized, reponses are nonitalicized).

Editorial comments:
Changes to be made by the Author(s):
1. Please take this opportunity to thoroughly proofread the manuscript to ensure that there are no spelling or grammar issues. Please define all abbreviations at first use.

We have made every effort to ensure there are no spelling or grammar issues and to define all abbreviations.

2. Please replace “with the TAEL 2.0 system” in your title with something sounding less commercial.

We have revised the title to make it more accessible to a general audience. The revised title is “Light induced GFP expression in zebrafish embryos using the optogenetic TAEL/C120 system.”

3. Please provide an email address for each author.

We have provided email addresses for all authors

4. Increase the word count of your abstract to150-300 words.

We have revised the abstract and increased the word count to approximately 180 words.

5. JoVE policy states that the video narrative is objective and not biased towards a particular product featured in the video. The goal of this policy is to focus on the science rather than to present a technique as an advertisement for a specific item. To this end, we ask that you please reduce the number of instances of "TAEL 2.0" within your text. The term may be introduced but please use it infrequently and when directly relevant. Otherwise, please refer to the term using generic language.

Our optogenetic expression system consists of two major components: 1) a light-sensitive transcription factor called TAEL and 2) a TAEL-responsive regulatory DNA sequence called C120. “TAEL 2.0” refers to collective modifications we have made to both the transcription factor and regulatory elements that improve performance (LaBelle et al., 2021). The term “TAEL 2.0” is used to distinguish these new constructs from the unmodified versions described in our initial publication (Reade et al., 2017) and does not refer to any commercial product. However, to minimize confusion, we have replaced instances of “TAEL 2.0” with a more general “TAEL/C120” (similar to “Gal4/UAS”). Unfortunately, we do not feel that a more generic term would be appropriate. Although there are other optogenetic expression systems in addition to TAEL/C120 (Shimizu-Sato et al., 2002; Liu et al., 2012; Wang et al., 2012), each system is unique in their activating light wavelengths, activation half-life, and other properties. Thus, the specific system used should be specified when describing optogenetic gene expression experiments.

6. Please revise the text, especially in the protocol, to avoid the use of any personal pronouns (e.g., "we", "you", "our" etc.).

We have made every effort to minimize the use of personal pronouns in the text.

7. Please ensure that all text in the protocol section is written in the imperative tense as if telling someone how to do the technique (e.g., “Do this,” “Ensure that,” etc.). The actions should be described in the imperative tense in complete sentences wherever possible. Avoid usage of phrases such as “could be,” “should be,” and “would be” throughout the Protocol. Any text that cannot be written in the imperative tense may be added as a “Note.” However, notes should be concise and used sparingly. Please include all safety procedures and use of hoods, etc.

We have revised the Protocol section to use the imperative tense.

8. The Protocol should contain only action items that direct the reader to do something. Please move the discussion about the protocol to the Discussion. The Protocol should be made up almost entirely of discrete steps without large paragraphs of text between sections. Please simplify the Protocol so that individual steps contain only 2-3 actions per step and a maximum of 4 sentences per step.

We have revised the Protocol section accordingly and moved any discussion of the steps to the Discussion section.

9. Please number the steps in the protocol under sections: 1., 1.1., 1.1.1., 1.1.1.1.

We have revised the Protocol section to number each step as suggested.

10. Please note that your protocol will be used to generate the script for the video and must contain everything that you would like shown in the video. Please add more details to your protocol steps. Please ensure you answer the “how” question, i.e., how is the step performed? Alternatively, add references to published material specifying how to perform the protocol action (e.g., step 3.1: how is RNA extracted; step 3.2: what were the conditions for qRT-PCR?). There should be enough detail in each step to supplement the actions seen in the video so that viewers can easily replicate the protocol.

We have revised the Protocol section to add more detail and/or references as needed.

11. As we are a methods journal, please revise the Discussion to explicitly cover the following in detail in 3-6 paragraphs with citations:
a) Critical steps within the protocol
b) Any modifications and troubleshooting of the technique
c) Any limitations of the technique
d) The significance with respect to existing methods
e) Any future applications of the technique

We have revised the discussion section to address these issues.

12. Please include a scale bar for all images taken with a microscope to provide context to the magnification used. Define the scale in the appropriate Figure Legend.

We have added scale bars to all images and defined them in the figure legend.

13. Please sort the Materials Table alphabetically by the name of the material.

We have sorted the Materials Table alphabetically.
____________________________________
Reviewers' comments:
Reviewer #1:
Manuscript Summary:
This article details the basic experimental approaches needed to induce global gene expression using the blue-light activated TAEL 2.0 optogenetics system
Minor Concerns:
The protocol is for "a simple LED panel to activate 45 the TAEL 2.0 system and induce ubiquitous expression of a reporter gene, GFP". Some of the main challenges of using optogenetic systems in zebrafish are related to achieving specific light patterning. Is there scope within this Jove article to describe how the authors best achieve patterned light illumination? I think the article is useful without this extra part - this is a suggestion for improvement only since the authors do already reference their publications that use patterned light.

We thank the reviewer for their suggestion. In fact, we have previously demonstrated several methods for achieving specific light patterning (Reade et al., 2017). However, because these approaches are subject to many variables that depend on lab’s exact setup, we felt it was beyond the scope of this particular JoVE protocol.
Figure 2A. the figure included doesn't give much information. A schematic and/or specs of the LED panel would allow other users to better reproduce the set up.
We have revised Fig. 2A to emphasize that the distance between the LED panel and petri dishes should be adjusted so that the received power of light is approximately 1.5 mW/cm2. We thank the reviewer for this helpful comment.
Reviewer #2:
Manuscript Summary:
This study by LaBelle and Woo describes the use of optogenetically-induced transcription using TAEL 2.0 which is well0suited for in vivo applications. This study nicely presents how to do these experiments in a living animal; however, the authors need to add a bit more detail to make it more useful to a new audience. With additional details, the manuscript is suitable for publication. Concerns are listed below:
Major Concerns:
Given the contribution of blue light to white light sources, how sensitive is TAEL 2.0 to standard room light or microscopy brightfield? How careful will someone have to be with room light exposure if utilizing TAEL 2.0 for their experiments? Controls for the experiments showing room light compared to dark would be useful but not necessary.
We thank the reviewer for bringing this issue to our attention. We measured GFP induction in embryos exposed to ambient room light and found that indeed, the TAEL/C120 system can be activated by ambient light but to a smaller extent compared to using the blue light LED panel (approximately 16-fold in room light versus 130-fold under blue light). These results have been added to Table 1, and we have included a note of caution to minimize ambient light exposure to the Discussions section.

The authors talk about how light is advantageous for spatial control in the intro and discussion but do not demonstrate how to carry out those experiments. Given the fluorescent reporter, it would be an easy experiment to visualize. This would significantly increase the utility of this protocol to other scientists.
We thank the reviewer for their suggestion. As noted above in response to Reviewer #1, we have previously demonstrated multiple methods to deliver spatially patterned light to achieve spatial control of TAEL activation (Reade et al., 2017). However, we felt this was beyond the scope of this particular JoVE protocol as these light patterning methods are subject to many variables that depend on lab’s exact setup.

How is light intensity measured/calculated? For first-time users, they would need to know this process so they can optimize their illuminations accordingly.
Light power can be measured with a power and energy meter. We used a Thorlabs PM100D Laser and Power and Energy Meter Console. We have added this information the Protocol section (Step 2.2) and the Materials Table.

Minor Concerns:
None

References cited:

LaBelle, J., Ramos-Martinez, A., et al. TAEL 2.0: An Improved Optogenetic Expression System for Zebrafish. Zebrafish 18 (1), 20–28, doi:10.1089/zeb.2020.1951 (2021).

Liu, H., Gomez, G., Lin, S., Lin, S. & Lin, C. Optogenetic control of transcription in zebrafish. PloS one 7 (11), e50738, doi:10.1371/journal.pone.0050738 (2012).

Reade, A., Motta-Mena, L. B., Gardner, K. H., Stainier, D. Y., Weiner, O. D. & Woo, S. TAEL: a zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control.

Shimizu-Sato, S., Huq, E., Tepperman, J. M. & Quail, P. H. A light-switchable gene promoter system. Nature biotechnology 20 (10), 1041–1044, doi:10.1038/nbt734 (2002).

Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nature methods 9 (3), 266–269, doi:10.1038/nmeth.1892 (2012).