Funding for this protocol was provided by the NICHD Intramural Program to KWR (ZIA HD009002-01). We thank Jeff Farrell and his lab for their illuminating feedback, Will Anderson for excellent technical support, Leanne Iannucci for stress testing the protocol and measuring irradiance, and the NIH Shared Zebrafish facility for their hard work keeping the zebrafish healthy.

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

The authors have nothing to disclose.

Injection of mRNA is the current strategy to deliver bOpto-BMP/Nodal to zebrafish embryos. This method has several drawbacks. First, the appropriate amount of mRNA varies between labs. The amount used should be sufficient to activate signaling robustly with light exposure, but without inadvertent dark activation. It is a good idea to test several amounts to find optimal mRNA levels, and once established, create aliquots of a master mix to reproducibly introduce the same amount of mRNA. Second, uneven distribution of injected mRNA may lead to uneven signaling activation. Injecting into the center of the cell (not the yolk) is thought to promote even mRNA distribution. Finally, because injected mRNA degrades over time, this approach may not be suitable for experiments in older embryos. In the future, these problems could be addressed by transgenic zebrafish lines ubiquitously expressing bOpto-BMP/Nodal with a maternal or drug-inducible promoter. Although working with potentially light-sensitive adult zebrafish may be a challenge in this context, zebrafish61,62 and Drosophila22,34,35,63 transgenics harboring optogenetic tools have been successfully developed.
Avoiding inadvertent photoactivation is a general challenge with optogenetic tools. For simplicity, treat injected embryos older than 1.5 hpf as light sensitive. Inadvertent light exposure can often be avoided by simply wrapping plates or dishes with aluminum foil. However, for experiments requiring visual observation of live embryos older than 1.5 hpf, it is possible to use red light sources or to cover white light sources with inexpensive gel filter paper that blocks LOV-dimerizing wavelengths (Table of Materials).
The light box described here is designed for specific applications requiring precise control over light irradiance levels, dynamics, and wavelengths (Figure 2). Other benefits of this light box include uniform light exposure, negligible inadvertent sample heating, ample space for multiple 6-well plates, and long-lived, spectrally well-characterized light sources. However, different light exposure strategies may be preferable depending on the research application. Many labs have developed simpler and more cost-effective uniform light exposure systems with smaller footprints, including lining incubators with LED strips, suspending LED panels over samples, or incorporating LEDs in culture dish lids32,38,39,40,64,65,66. Importantly, the light box used in this protocol does not allow users to independently regulate individual wells (in contrast to Bugaj et al.52) or provide spatial control over light exposure. Spatially localized optogenetic activation has been demonstrated with bOpto-BMP38 and bOpto-Nodal39 using lasers in SPIM or confocal systems, respectively, and has also been realized with many other optogenetic strategies in a variety of model systems (discussed in Rogers and M&#252;ller12). Some approaches have even achieved sub-cellular spatial resolution29,30,31. Although the implementation of spatially localized light exposure systems is outside of the scope of this protocol, spatial activation experiments with bOpto-BMP/Nodal are theoretically possible with specialized equipment such as digital micromirror devices or masking approaches. Readers are encouraged to explore the extensive literature on DIY light boxes for optogenetic experiments before committing to a light exposure strategy (see e.g., Gerhardt et al.51, Bugaj et al.52, Kumar and Khammash53 and more at&#160;https://www.optobase.org/materials/).
Molecular optogenetic strategies often offer a higher degree of spatiotemporal control over biological processes compared to historical approaches such as mutants, ectopic gene expression, recombinant proteins, and drugs. Readers who are interested in the benefits of optogenetic approaches may explore other published tools available in zebrafish and other organisms. These include tools to manipulate additional signaling pathways32,65,67,68, regulate gene expression61,64,66,69,70,71, alter protein localization31,72, and activate apoptosis62. These tools and many others are conveniently cataloged at OptoBase, a curated web resource for molecular optogenetics approaches28. For those inspired to create novel optogenetic tools, the resource also features useful descriptions of light-responsive proteins that have been employed in a wide range of strategies, including light-responsive proteins that respond to green, red, and near-infrared wavelengths. We are excited for the scientific community to realize the full potential of molecular optogenetic approaches.

Signaling pathways allow cells to respond to their environment and coordinate activities at tissue- and organism-wide scales. Signals crucial for embryonic development include the TGF-beta superfamily members&#160;bone morphogenetic protein (BMP)&#160;and Nodal1,2,3. During embryogenesis, the pathways regulated by these signals and others pattern the body plan by controlling gene expression and additional processes to ensure that diverse tissues and organs develop and interface properly. Pathologies, including birth defects and cancer, can occur when signaling or responses to signaling are perturbed4,5,6,7. Despite&#160;rigorous investigation into signaling, much more remains to be discovered about how levels and dynamics are decoded in a variety of contexts8,9,10,11, especially during development12,13,14,15,16,17,18,19.
To understand how signaling is decoded, an ideal experiment would be to manipulate signaling levels, timing, and/or dynamics-with a high degree of spatial and temporal control-and assess outcomes. For example, precise spatial signaling gradients are proposed to pattern developing tissues20,21. Altering signaling gradient spatial distributions would help test this hypothesis22. Additionally, the importance of signaling dynamics in generating diverse cellular responses is becoming clearer: The same signaling pathway can instruct cells to differentiate or proliferate depending on signaling frequency, for example9,23. Experimental paradigms in which signaling dynamics can be easily manipulated will be valuable to explore the relationship between dynamics and cell fate decisions8,12,13,14,15.
Historically, multiple methods have been used to manipulate signaling in developmental contexts, leading to fundamental discoveries1,2,3. Signaling can be blocked using pathway loss-of-function mutants, ectopic inhibitor expression, or antagonist drugs. Methods to activate signaling include agonist drugs, recombinant ligands, ectopic expression of ligands or constitutively active receptors, and pathway inhibitor loss-of-function mutants. These methods range along a continuum of experimental control. For example, mutants and ectopic expression may fall on the sledgehammer side of the continuum: With these approaches, dramatic, systemic changes in pathway activity may cause early death and preclude investigations at later stages, or over time may result in pleiotropic effects that are difficult to disentangle. Additionally, it is often challenging to independently manipulate one signaling feature at a time, such as level or duration. Toward the other end of the continuum, some methods offer more precise experimental control, such as microfluidic devices that expose samples to drugs or recombinant proteins with temporal and sometimes spatial control18,24,25, or genetic methods, including heat shock-inducible and tissue-specific promoters that can offer similar benefits16,26,27. However, these methods can be difficult to execute, may not be reversible, may have relatively slow kinetics or poor resolution, and may be unavailable in some model systems.
Molecular optogenetic approaches are a powerful addition to this toolkit. These approaches use proteins that respond to different light wavelengths to manipulate biological processes, including signaling8,12,13,14,15, and have been developed over decades for use in a variety of systems from cell culture to whole animals12,13,28. Compared to historical approaches, molecular optogenetics can often offer a higher degree of spatiotemporal control over biological processes: The controller in optogenetic systems is light, and control of light wavelength, intensity, duration, and exposure frequency is relatively straightforward. With sophisticated systems such as confocal and two-photon microscopes, spatial control in the subcellular range is possible29,30,31. Tools to optogenetically manipulate signaling have been developed and applied in several systems, including those described in Johnson et al.22, &#268;apek et al.32, Krishnamurthy et al.33, and Huang et al.34. For example, exploiting the spatial control afforded by optogenetics, this strategy was recently used to modify a signaling gradient in Drosophila embryos, demonstrating that fly embryogenesis is surprisingly robust to changes in this gradient22. The reversibility and fast on/off kinetics of optogenetic signaling activators have also made them attractive tools for investigating the decoding of signaling dynamics8,12,13,14,15,34,35,36.
The early zebrafish embryo is an in vivo system well-suited for optogenetic studies because it is externally fertilized, transparent, microscopy-friendly, and genetically tractable. Light exposure is easier to deliver to embryos that develop outside of the mother, light can penetrate and access their non-opaque tissues, live zebrafish embryos tolerate imaging well (in addition to being transparent), and existing genetic methods provide straightforward opportunities for knockdown and overexpression experiments, in addition to the development of useful transgenics37.
Recently, optogenetic tools were developed to activate BMP38 and Nodal39 signaling in zebrafish embryos with blue light exposure (Figure 1). We refer to these tools as bOpto-BMP and bOpto-Nodal (b for blue light-activated and Opto for optogenetic). bOpto-BMP/Nodal are based on similar pathway activation mechanisms. The binding of BMP or Nodal ligands to their respective receptor serine-threonine kinases drives receptor kinase domain interactions that lead to the phosphorylation of signaling effectors (Smad1/5/9 for BMP and Smad2/3 for Nodal). Phosphorylated signaling effectors then translocate to the nucleus and regulate target gene expression3 (Figure 1A,D). These receptor kinase interactions can be made light-responsive by coupling receptor kinases to light-responsive dimerizing proteins: With light exposure, these chimeric proteins should dimerize, causing the receptor kinase domains to interact and activate signaling (Figure 1B,C,E,F). Importantly, in contrast to endogenous receptors, bOpto-BMP/Nodal do not contain extracellular ligand-binding domains, ensuring ligand-independent activity (Figure 1C,F). This optogenetic activation strategy was first achieved with receptor tyrosine kinases40,41,42 and then applied to receptor serine-threonine kinases.
bOpto-BMP/Nodal use the blue light-responsive (~450 nm) homodimerizing light-oxygen-voltage sensing (LOV) domain from the algae Vaucheria fridiga AUREO1 protein (VfLOV)43,44. These constructs consist of a membrane-targeting myristoylation motif followed by either BMP or Nodal receptor kinase domains, fused to a LOV domain (Figure 1B,E). Blue light exposure should cause LOV homodimerization, resulting in receptor kinase domain interactions that lead to respective Smad phosphorylation and pathway activation (Figure 1C,F). For bOpto-BMP, a combination of constructs with the type I receptor kinase domains from Acvr1l (also known as Alk8) and BMPR1aa (also known as Alk3) and the type II receptor kinase domain from BMPR2a was found to optimally activate signaling38 (Addgene #207614, #207615, and #207616). For bOpto-Nodal, a combination of constructs with the type I receptor kinase domain from Acvr1ba and the type II receptor kinase domain from Acvr2ba is used39.
bOpto-BMP/Nodal have been introduced into early zebrafish embryos by injecting mRNA at the one-cell stage, and used to investigate the role of signaling duration in Nodal interpretation39, to determine why zebrafish lose the ability to respond to Nodal45, and to examine how BMP target genes respond to different BMP signaling levels38. It is likely that these tools will continue to be useful in a diverse range of future investigations. However, the strength of optogenetic signaling activators is also their weakness: light-sensitive samples must be treated with care to avoid inadvertent ectopic signaling activity. Exposure to room light or sunlight can activate bOpto-BMP/Nodal.
This protocol provides practical suggestions for using mRNA-encoded LOV-based BMP and Nodal activators in early zebrafish embryos. It begins by detailing one strategy to build a light box to control uniform light exposure and temperature (Figure 2, Supplementary File 1, Supplementary File 2, Supplementary File 3, Supplementary File 4, Supplementary File 5, Supplementary File 6, Supplementary File 7, Supplementary File 8). It then describes two key control experiments that determine whether an optogenetic signaling activator is behaving as expected-i.e., activating pathway activity only when exposed to light (Figure 3). The first control assay involves examining phenotypes at one day post-fertilization in light-exposed and unexposed embryos (Figure 3A). mRNA-injected light-exposed embryos, but not unexposed embryos, should phenocopy BMP or Nodal overexpression (Figure 4A,B; BMP phenotypes in particular are clearly distinguishable at this time point46). This assay provides a fast activity readout. In the second control assay, to determine whether phenotypes are caused specifically by excess BMP or Nodal signaling and to directly observe the change in signaling levels, immunofluorescence staining is used to detect phosphorylated signaling effectors (pSmad1/5/9 or pSmad2/3, respectively) after a 20 min light exposure around late blastula / early gastrulation stage, when signaling activity has been well described12,16,17,47,48,49,50 (Figure 3B and Figure 4C). (Note that, although spatially localized activation has been demonstrated for both bOpto-BMP38 and bOpto-Nodal39, this protocol only describes uniform light exposure and signaling activation strategies.) It is advisable to execute these control experiments prior to applying bOpto-BMP/Nodal to specific research questions in order to determine ideal local experimental conditions.

<table><tbody><tr><td>&lt;strong&gt;Building a light box &amp;amp; Light exposure protocol&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>#8 x 1&quot; Hex Self-drilling Screw</td><td>McMaster-Carr</td><td>99663A222</td><td>1.4.5</td></tr><tr><td>Digital Optical Power and Energy Meter</td><td>ThorLabs</td><td>PM100D</td><td>1.7&lt;br/&gt; 4</td></tr><tr><td>Incubator (142 liters)</td><td>Boekel Scientific</td><td>139400</td><td>1.3.1</td></tr><tr><td>Incubator Panel Mount (1/4&quot; thick cast black acrylic)</td><td>Custom part / Piedmont Plastics</td><td>Incubator_panel</td><td>1.4.4</td></tr><tr><td>Large HSS Spiral Groove Step Drill Bit</td><td>CO-Z</td><td>SDB0001TA</td><td>1.3.2</td></tr><tr><td>LED lens gasket, Incubator gasket; 1/32&quot; thick black silicone</td><td>McMaster-Carr</td><td>5812T12</td><td>1.4.3&lt;br/&gt; 1.4.4</td></tr><tr><td>LED microplate illuminator</td><td>Prizmatix</td><td>NA</td><td>1.1&lt;br/&gt; 1.4.3</td></tr><tr><td>M3 10mm Cube Standoff</td><td>Newark Eletronics</td><td>005.60.533</td><td>1.4.1</td></tr><tr><td>M3 x 10mm 316SS Flat Head Screw</td><td>McMaster-Carr</td><td>91801A156</td><td>1.4.1</td></tr><tr><td>M6 x 10mm 316SS Flat Head Screw</td><td>McMaster-Carr</td><td>91801A305</td><td>1.4.3</td></tr><tr><td>Memory card thermometer</td><td>Fisherbrand</td><td>15-081-111</td><td>1.9&lt;br/&gt; 3.2.1</td></tr><tr><td>Microscope Slide Power Meter Sensor Head (150 mW)</td><td>ThorLabs</td><td>S170C</td><td>1.7&lt;br/&gt; 4</td></tr><tr><td>Red gel filter paper #E106</td><td>Rosco / B&amp;amp;H Foto &amp;amp; Electronics</td><td>110084014805-E106</td><td>4.2.1</td></tr><tr><td>Side Brackets (1/4&quot; thick cast black acrylic)</td><td>Custom part / Piedmont Plastics</td><td>Side_bracket</td><td>1.4.2</td></tr><tr><td>Vertical Bracket (1/4&quot; thick cast black acrylic)</td><td>Custom part / Piedmont Plastics</td><td>Vertical_bracket</td><td>1.4.1</td></tr><tr><td>Weather stripping: Light duty EPDM foam, 1/2&quot; wd 1/4&quot; tk</td><td>McMaster-Carr</td><td>8694K12</td><td>1.8</td></tr><tr><td>&lt;strong&gt;Generating mRNA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EZNA MicroElute Cycle Pure Kit</td><td>Omega</td><td>D6293-02</td><td>2.4</td></tr><tr><td>GeneJET Miniprep Kit (250 rxns)</td><td>Thermo Scientific</td><td>K0503</td><td>2.2</td></tr><tr><td>Microsample incubator (Hybex)</td><td>SciGene</td><td>1057-30-0</td><td>2</td></tr><tr><td>Microsample incubator 1.5 ml tube block (Hybex)</td><td>SciGene</td><td>1057-34-0</td><td>2</td></tr><tr><td>Nanodrop One Spectrophotometer</td><td>Thermo Scientific</td><td>ND-ONE-W</td><td>2.4</td></tr><tr><td>NotI-HF restriction enzyme</td><td>New England Biolabs (NEB)</td><td>R3189L</td><td>2.1</td></tr><tr><td>pCS2-Opto-Alk3</td><td>Addgene</td><td>207614</td><td>2</td></tr><tr><td>pCS2-Opto-Alk8</td><td>Addgene</td><td>207615</td><td>2</td></tr><tr><td>pCS2-Opto-BMPR2a</td><td>Addgene</td><td>207616</td><td>2</td></tr><tr><td>RNeasy Mini Kit (250 rxns)</td><td>Qiagen</td><td>74106</td><td>2.3</td></tr><tr><td>&lt;strong&gt;Injecting mRNA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose (UltraPure)</td><td>Invitrogen / Thermo Fisher</td><td>16500500</td><td>3.1.1</td></tr><tr><td>250 ml glass beakers</td><td>Fisherbrand</td><td>FB100250</td><td>3.3.2</td></tr><tr><td>6-well dishes (case of 50)</td><td>Falcon</td><td>08 772 1B</td><td>3.1.6</td></tr><tr><td>B-8A ball joint</td><td>Narishige</td><td>B-8A</td><td>3.3</td></tr><tr><td>Back pressure unit (microinjection rig component)</td><td>Applied Scientific Instrumentation (ASI)</td><td>BPU</td><td>3.3</td></tr><tr><td>Foot switch (microinjection rig component)</td><td>Applied Scientific Instrumentation (ASI)</td><td>FWS</td><td>3.3</td></tr><tr><td>GJ-1 magnetic stand</td><td>Narishige</td><td>GJ-1</td><td>3.3</td></tr><tr><td>Glass capillaries (4 in, OD 1 mm, filament)</td><td>World Precision Instruments</td><td>1B100F-4</td><td>3.1.11</td></tr><tr><td>Glass petri dish bottoms (for dechorionating)</td><td>Pyrex</td><td>08-748A</td><td>3.3.2</td></tr><tr><td>Glass pipettes (5 3/4&quot; with wide tip)</td><td>Kimble-Chase</td><td>63A53WT</td><td>3.1.9</td></tr><tr><td>Injection dish molds</td><td>Adaptive Science Tools</td><td>tu1</td><td>3.1.3</td></tr><tr><td>IP iron plate</td><td>Narishige</td><td>IP</td><td>3.3</td></tr><tr><td>M-152 micromanipulator</td><td>Narishige</td><td>M-152</td><td>3.3</td></tr><tr><td>Micro pipette holder kit (microinjection rig component)</td><td>Applied Scientific Instrumentation (ASI)</td><td>MIMPH-MPIP-Kit</td><td>3.3</td></tr><tr><td>Micrometers</td><td>Meiji Techno America</td><td>MA285</td><td>3.3</td></tr><tr><td>MPPI-2 pressure injector (microinjection rig component)</td><td>Applied Scientific Instrumentation (ASI)</td><td>MPPI-3</td><td>3.3</td></tr><tr><td>Needle puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>3.1.11</td></tr><tr><td>Petri dishes (100 mm x 15 mm, case of 500)</td><td>Falcon</td><td>08-757-100D</td><td>3.1.2</td></tr><tr><td>Pipettor (10 ml, green)</td><td>Bel-Art</td><td>F37898-0000</td><td>3.3</td></tr><tr><td>Pronase</td><td>Roche</td><td>11459643001</td><td>3.3.2</td></tr><tr><td>Squeeze bottles (500 ml)</td><td>Nalgene / Thermo Scientific</td><td>2402-0500</td><td>3.3</td></tr></tbody></table>

optogenetic signaling activation in zebrafish embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

The goal of the two control experiments described here is to determine whether bOpto-BMP/Nodal activate their respective pathways in response to blue light exposure without affecting signaling in the absence of light, as expected. Use these controls to establish the appropriate experimental workflow in your lab before applying bOpto-BMP/Nodal to your research questions of interest.
The phenotyping assay can be completed in only 2 days and provides a useful indication of signaling activity and phototoxicity (Figure 3A). Injected, blue light-exposed embryos should phenocopy excess BMP signaling (ventralization46; Figure 4A, left panel) or Nodal signaling (developmental defects related to extra mesendoderm1,3,47,57,58,59,60 (Figure 4A, right panel)). If injected, light-exposed embryos are aphenotypic, test the quality of the mRNA and consider injecting more, and double-check the light exposure strategy to ensure constant exposure to bright light (~450 nm light with an irradiance of 45 W/m2 should strongly activate signaling). In contrast, injected, unexposed embryos should look identical to non-injected siblings. If injected, unexposed embryos exhibit phenotypes, reduce the amount of mRNA injected and reassess the experimental set up to ensure that unexposed embryos are protected from light exposure. The data shown in Figure 4B shows the results from typical phenotyping experiments with appropriate mRNA amounts and exposure conditions: strong signaling activity is evident in injected, light-exposed embryos, with only a small fraction of injected, unexposed embryos exhibiting phenotypes.
The phenotyping assay also provides an opportunity to assess phototoxicity. If phototoxicity is negligible, non-injected, light-exposed embryos should appear wild type, similar to non-injected, unexposed embryos. If non-injected, light-exposed embryos have defects, but not non-injected, unexposed embryos, consider decreasing the light irradiance. An irradiance of 45 W/m2 robustly activates signaling without obvious phototoxicity. The data shown in Figure 4B shows no concerning differences between non-injected, light-exposed and non-injected, unexposed embryos, indicating negligible phototoxicity.
Although the immunofluorescence assays require more time and effort (~1 week) compared to the phenotyping assay (2 days), immunofluorescence staining provides a direct readout of signaling pathway activity and may reveal subtle signaling changes that might not be reflected by gross morphology. Immunofluorescence is especially important to assess responses to bOpto-Nodal, because excess Nodal signaling often results in embryos lysing by 1 dpf-which can have many causes-in contrast to the specific ventralization phenotypes characteristic of excess BMP signaling46 (Figure 4A). Injected, blue light-exposed embryos should exhibit a uniform increase in Smad1/5/9 or Smad2/3 phosphorylation compared to non-injected, light-exposed embryos. If levels are not increased, or only weakly increased, test the quality of the mRNA and consider injecting more, and double-check the light exposure strategy. A 20 min exposure to blue light with an irradiance of 45 W/m2 around 40% epiboly should strongly activate signaling. If pSmad staining is non-uniform, try injecting mRNA into the center of the cell (rather than the yolk), which may result in more uniform mRNA distribution.
Injected, unexposed embryos should have pSmad levels comparable to non-injected embryos. Anecdotally, we have observed leakier Smad phosphorylation with bOpto-Nodal than bOpto-BMP. If pSmad levels are increased in injected, unexposed embryos, reduce the amount of mRNA injected. In addition, re-assess the experimental set up to ensure 1) unexposed embryos are not inadvertently exposed to light, and 2) light exposure during fixation is minimal. During the fixation step, it is critical to allow no more than 45 s to elapse between removal from the light box and immersion in formaldehyde. In addition, during this step minimize exposure to room light and sunlight by closing window blinds, turning off white light sources, using red lights, or covering white light sources with blue light-blocking gel filter paper (Table of Materials).
The data in Figure 4C shows the results from typical immunofluorescence staining experiments with appropriate mRNA amounts and light exposure conditions: pSmad levels are similar in non-injected and unexposed embryos, whereas injected, light-exposed embryos exhibit higher levels of Smad phosphorylation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65733/65733fig01.jpg" /> 
Figure 1: bOpto-BMP and -Nodal signaling activation strategy. (A) The endogenous BMP signaling pathway is activated by BMP ligand binding, leading to formation of a type I/II receptor complex, phosphorylation of Smad1/5/9, and expression of BMP target genes. The type I receptors BMPR1aa and Acvr1l are also known as Alk3 and Alk8, respectively. BMPR2a is a type II receptor. (B) bOpto-BMP constructs38. Putative kinase domains from BMPR1aa and Acvr1l are fused to LOV; the BMPR2a-LOV fusion contains the putative kinase domain and receptor C-terminal domain (CTD). All fusions are membrane-targeted with a myristoylation motif (Myr). Domains are separated by glycine-serine (GS) linkers. Constructs are tagged at the CTD with an HA epitope tag. This combination of three constructs was found to optimally activate BMP signaling. (C) bOpto-BMP-mediated BMP signaling activation. When exposed to blue light, LOV domains dimerize, which is thought to trigger complex formation and signaling activation. (D) The endogenous Nodal signaling pathway is activated by Nodal ligand binding, leading to formation of a type I/II receptor complex, phosphorylation of Smad2/3, and expression of Nodal target genes. The type I receptor Acvr1ba and the type II receptor Acvr2ba are also known as Acvr1b and Acvr2b, respectively. (E) bOpto-Nodal constructs39. Putative kinase domains from Acvr1ba and Acvr2ba are fused to LOV. All fusions are membrane-targeted with a myristoylation motif (Myr). Domains are separated by GS linkers. Constructs are tagged at the CTD with an HA epitope tag. (F) bOpto-Nodal-mediated Nodal signaling activation. When exposed to blue light, LOV domains dimerize, which is thought to trigger complex formation and signaling activation. <a href="https://www.jove.com/files/ftp_upload/65733/65733fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65733/65733fig02.jpg" /> 
Figure 2: Temperature controlled light box for optogenetic experiments. (A) An LED microplate illuminator is mounted to the top of an incubator using a custom-built LED holder. Zebrafish embryos in a 6-well plate on the first shelf are exposed to light through a hole drilled into the top of the incubator. The lower shelf holds a second set of unexposed control embryos in an aluminum foil-wrapped 6-well plate. The incubator door is lined with weather stripping to prevent inadvertent exposure to room light or sunlight. (B) Detail of procedure to create a hole in the incubator using a step drill. The incubator model used here has an internal panel that required drilling a second, larger hole (Table of Materials). (C) Detail of the custom LED holder designed for a three-wavelength illumination system. <a href="https://www.jove.com/files/ftp_upload/65733/65733fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65733/65733fig03.jpg" /> 
Figure 3: bOpto-BMP/Nodal experiment workflow. Phenotype assay and pSmad immunofluorescence staining to test activity of bOpto-BMP/Nodal. Embryos are injected with mRNA at the one-cell stage and transferred to a light box no later than 1.5 h post-fertilization (hpf). (A) Phenotype assay. Injected embryos and non-injected siblings are reared in the dark or exposed to uniform blue light starting at 1.5 hpf until 1-day post-fertilization (dpf). Optogenetic signaling activity can be evaluated by scoring embryos for phenotypes consistent with excess pathway activity. (B) pSmad immunofluorescence staining. Injected embryos and non-injected siblings are reared in the dark until 40% epiboly (~6 hpf). Half of the injected and half of the non-injected embryos are then exposed to uniform blue light for 20 min. After exposure, all embryos are fixed and subjected to immunofluorescence staining for pSmad. Elevated levels of pSmad1/5/9 or pSmad2/3 reflect optogenetic activation of BMP or Nodal signaling, respectively. <a href="https://www.jove.com/files/ftp_upload/65733/65733fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65733/65733fig04.jpg" /> 
Figure 4: Assessing light-activated signaling responses in zebrafish embryos. Zebrafish embryos were injected at the one-cell stage with mRNA encoding bOpto-BMP/Nodal. (A) Embryos were either reared in the dark or exposed to uniform blue light starting at 1.5 h post fertilization (hpf). Phenotypes were scored at 1 day post fertilization (dpf). Representative phenotypes are shown. Excess BMP signaling leads to ventralization (left panel), while excess Nodal signaling causes developmental defects associated with extra mesendoderm (right panel). Scale bar = 500 &#181;m. (B) Phenotype quantification. Injected embryos and non-injected siblings were reared in the dark starting at 1.5 hpf (black bulb). Half of the injected and half of the non-injected embryos were exposed to uniform blue light (blue bulb). (C) Injected embryos and non-injected siblings were reared in the dark starting at 1.5 hpf (black bulb). At 40% epiboly (~6 hpf), half of the injected and half of the non-injected embryos were exposed to uniform blue light (blue bulb). After 20 min, all embryos were fixed and subjected to immunofluorescence staining for either phosphorylated Smad1/5/9 or Smad2/3. Higher pSmad intensities indicate increased BMP/Nodal signaling, respectively. Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65733/65733fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Light box full assembly. 3D PDF file showing a 3D view of the full light box assembly. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_1_Light_box_full_assembly_2023-08-15.PDF" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Light box exploded view. 3D PDF file showing a 3D view of the exploded light box assembly. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_2_Light_box_exploded_view_2023-08-15.PDF" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Large light gasket. CAD drawing file (.DWG format) to fabricate the large light gasket for the LED holder using a laser cutter. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_3_Large_light_gasket_2023-08-15.DWG" target="_blank">Please click here to download this File.</a>
Supplementary File 4: Small light gasket. CAD drawing file (.DWG format) to fabricate the small light gasket for the LED holder using a laser cutter. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_4_Small_light_gasket_2023-08-15.DWG" target="_blank">Please click here to download this File.</a>
Supplementary File 5: Acrylic platform base. CAD drawing file (.DWG format) to fabricate the LED holder acrylic platform base using a laser cutter. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_5_Acrylic_platform_base_2023-08-15.DWG" target="_blank">Please click here to download this File.</a>
Supplementary File 6: Acrylic platform vertical. CAD drawing file (.DWG format) to fabricate the LED holder acrylic platform vertical using a laser cutter. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_6_Acrylic_platform_vertical_2023-08-15.DWG" target="_blank">Please click here to download this File.</a>
Supplementary File 7: Acrylic support left. CAD drawing file (.DWG format) to fabricate the LED holder acrylic left support using a laser cutter. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_7_Acrylic_support_left_2023-08-15.DWG" target="_blank">Please click here to download this File.</a>
Supplementary File 8: Acrylic support right. CAD drawing file (.DWG format) to fabricate the LED holder acrylic right support using a laser cutter. <a href="https://www.jove.com/files/ftp_upload/65733/Supp_File_8_Acrylic_support_right_2023-08-15.DWG" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions at JoVE.com

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

optogenetic-signaling-activation-in-zebrafish-embryos

Research

JoVE Journal

Developmental Biology

2.4K Views.  NIH. Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Video: Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Neuroscience

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Biology

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...

Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses

Source: Palanca A. M. S., et al. <a href="https://www.jove.com/v/50184/" target="_blank">Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato</a>. J. Vis. Exp. (2013).
This video describes optogenetic techniques by activating genetically modified somatosensory neurons and recording elicited behavioral response with a high-speed video camera.

Optogenetyka danio pręgowanego: aktywacja genetycznie zmodyfikowanego neuronu somatosensorycznego w celu zbadania reakcji behawioralnych larw

Source: Palanca A. M. S., et al. Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato. J. Vis. Exp. (2013).
This video describes ...