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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Building a light box &#38; Light exposure protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>#8 x 1&#34; 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 
			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&#34; 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&#34; thick black silicone</td>
			<td>McMaster-Carr</td>
			<td>5812T12</td>
			<td>1.4.3 
			1.4.4</td>
		</tr>
		<tr>
			<td>LED microplate illuminator</td>
			<td>Prizmatix</td>
			<td>NA</td>
			<td>1.1 
			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 
			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 
			4</td>
		</tr>
		<tr>
			<td>Red gel filter paper #E106</td>
			<td>Rosco / B&#38;H Foto &#38; Electronics</td>
			<td>110084014805-E106</td>
			<td>4.2.1</td>
		</tr>
		<tr>
			<td>Side Brackets (1/4&#34; 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&#34; 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&#34; wd 1/4&#34; tk</td>
			<td>McMaster-Carr</td>
			<td>8694K12</td>
			<td>1.8</td>
		</tr>
		<tr>
			<td>Generating mRNA</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>Injecting mRNA</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&#34; 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.

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

Optogenetic Signaling Activation in Zebrafish Embryos

To establish the embryo's body plan during development cells are thought to decode signaling intensities dynamics and combinations. We are using optogenetics to manipulate these signaling features in zebrafish embryos. This approach will help us understand how embryonic cells decode signaling to make some of the earliest sulfate decisions The optogenetic approaches discussed here use light as a remote control to activate BMP or nodal signaling rapidly and reversibly.Because light exposure is easy to direct, these approaches can offer improved experimental control over signaling. Our protocol introduces using these light sensitive tools in zebrafish embryos. With this improved temporal and spatial control, it should now be possible to perform precise signaling modifications during zebrafish developments.We expect this to deepen our understanding of how cells decode signaling to make faint decisions, which will help reveal how the embryonic body plan develops.

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

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.

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

optogenetic-signaling-activation-in-zebrafish-embryos

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JoVE Journal

Developmental Biology

Injecting bOpto-BMP&#47;Nodal Constructs into Zebrafish Embryos 

Injecting bOpto-BMP/Nodal Constructs into Zebrafish Embryos   

To begin, use an incubator and an LED Microplate Illuminator to create a light box for controlling light exposure and temperature. Then, position a six well plate on the upper shelf of the incubator, and check whether the light beam evenly encompasses it. Use a sheet of paper to visualize the distribution of light coverage.Use a light meter to measure irradiance and spatial uniformity. At least one day before injections, make injection dishes and agarose-coated six well plates. Rinse an injection dish mold with embryo medium, and place gently onto molt and agarose.Once the agarose has solidified, delicately use the tab to remove the mold. Next, prepare flamed glass pipettes for immunofluorescence experiments on dechlorinated embryos. Place the end of a glass pipette into a bun and burner flame, and rotate continuously until the edges become smooth.Prepare an injection mix using the mRNAs shown here. For the phenotyping assay, introduce the needle through the corion directly into the center of the cell at the one cell stage, and inject one nanoliter of the injection mix. If conducting an immunofluorescence assay, target the center of the cell of coated embryos at the one cell stage.For both assays, prepare enough embryos for the unexposed non-injected, unexposed injected, exposed non-injected, and exposed injected embryos. Select at least 30 embryos per condition. For the immunofluorescence assay, prepare an additional dish containing non-injected embryos as a proxy to assess the progression of developmental stages.After injection, incubate the embryos in Petri dishes at 28 degrees Celsius.

Light Exposure Experiment to Activate BMP or Nodal Signaling in Zebrafish

To begin, incubate the injected zebrafish embryos at 28 degrees Celsius. Around 1.5 HPF at the four to 16 cell stage, remove unfertilized and unhealthy embryos. For the phenotyping assay, wrap the unexposed control plate with noninjected and injected embryos in aluminum foil.Position the foil covered plate on the lower shelf, and the exposed plate on the upper shelf of the 28 degrees Celsius light box. After confirming that the dish cover is in place, activate the blue light, and shut the light box door to prevent unintended room light exposure. For the immunofluorescence assay around 1.5 HPF, individually wrap the exposed and unexposed dishes in aluminum foil while keeping the proxy dish unwrapped.Put all the dishes in the 28 degrees Celsius light box without activating the LED. Close the light box door to prevent inadvertent exposure to room light. At approximately five HPF, use a dissecting scope to evaluate the developmental stage of the proxy embryos.Also remove the wrapped dishes to ensure uniform temperature exposure across all dishes. Once the proxy embryos reach 40%epiboly, uncover the exposed dish and place it on the upper shelf of the light box. Keep the unexposed dish covered on the lower shelf.Immediately activate the blue light, close the door, and set a timer for 20 minutes. To prepare for fixation, eliminate as much room light as possible. Just before fixation, take tubes containing 4%formaldehyde from four degrees Celsius and position them beside the light box.After 20 minutes, open the door of the light box and remove the dish that wasn't exposed to light. Use a flamed glass pipette tip to remove the light sensitive embryos from the dish. Then to immerse the embryos in 4%formaldehyde, submerge the flamed glass pipette tip in the formaldehyde, and allow the embryos to sink into the liquid.Store fixed embryos at four degrees Celsius overnight.

Evaluation of bOpto-BMP&#47;Nodal Expressing Embryos After the Light Exposure Experiment

Evaluation of bOpto-BMP/Nodal Expressing Embryos After the Light Exposure Experiment  

For the phenotyping assay, perform the light exposure experiment on bOpto-BMP bOpto-Nodal expressing zebrafish embryos, and remove them from the light box at one day post-fertilization between 24 to 32 HPF. Using a dissecting scope, evaluate phenotypes and establish a scoring rubric. Assess embryos while they remain within the chorion for convenience.Utilize a pipette to probe to reposition embryos for viewing from different angles. For the immunofluorescence assay, after incubating embryos in 4%formaldehyde at four degrees Celsius overnight, remove the formaldehyde and rinse them three to five times with PBST. Discard the PBST and add 100%methanol.Seal the tubes and gently invert them to mix residual PBST and methanol. After washing the embryos two more times with methanol, store them at minus 20 degrees Celsius for at least two hours up to several years. Using a microscope equipped for optical sectioning, capture images of immunostained embryos.Immunofluorescent staining experiments with appropriate mRNA amounts and light exposure conditions showed that the pSmad levels were similar and non-injected in unexposed embryos, whereas injected light exposed embryos exhibited higher levels of Smad phosphorylation.

1.5K 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.

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Summary