evaluation of bopto-bmp&#47;nodal expressing embryos after the light exposure experiment

Investigando o desenvolvimento com manipulação de sinalização optogenética em peixe-zebra

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.

Autor em destaque: Manipulando a sinalização em embriões de peixe-zebra para decodificar decisões de destino celular 

Ativação da Sinalização Optogenética em Embriões de Zebrafish

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.

Watch this Scientific Journal Video about Evaluation of bOpto-BMP/Nodal Expressing Embryos After the Light Exposure Experiment at JoVE.com

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

Avaliação de embriões com bOpto-BMP/nodal após experimento de exposição à luz

Para o ensaio de fenotipagem, realizar o experimento de exposição à luz em embriões de peixe-zebra bOpto-BMP expressando embriões de zebrafish e retirá-los da caixa de luz em um dia pós-fertilização entre 24 e 32 HPF. Usando um escopo dissecante, avalie fenótipos e estabeleça uma rubrica de pontuação. Avalie os embriões enquanto eles permanecem dentro do córion por conveniência.Utilize uma pipeta para sondar para reposicionar embriões para visualização de diferentes ângulos. Para o ensaio de imunofluorescência, depois de incubar os embriões em formaldeído a 4% a quatro graus Celsius durante a noite, remova o formaldeído e lave-os três a cinco vezes com PBST. Descarte o PBST e adicione 100% de metanol.Sele os tubos e inverta-os suavemente para misturar PBST residual e metanol. Depois de lavar os embriões mais duas vezes com metanol, armazene-os a menos 20 graus Celsius por pelo menos duas horas até vários anos. Usando um microscópio equipado para corte óptico, capture imagens de embriões imunomarcados.Experimentos de coloração por imunofluorescência com quantidades adequadas de RNAm e condições de exposição à luz mostraram que os níveis de pSmad foram semelhantes e não injetados em embriões não expostos, enquanto embriões expostos à luz injetados exibiram níveis mais altos de fosforilação de Smad.

evaluation-bopto-bmpnodal-expressing-embryos-after-light-exposure

Research

JoVE Journal

Developmental Biology

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

371 visualizações.  NIH. Para o ensaio de fenotipagem, realizar o experimento de exposição à luz em embriões de peixe-zebra bOpto-BMP expressando embriões de zebrafish e retirá-los da caixa de luz em um dia pós-fertilização entre 24 e 32 HPF. Usando um escopo dissecante, avalie fenótipos e estabeleça uma rubrica de pontuação. Avalie os embriões enquanto eles permanecem dentro do córion por conveniência.Utilize uma pipeta para sondar para reposicionar embriões para visualização de diferentes ...

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

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.

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 ...

Biologia do Desenvolvimento

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.

Injetando bOpto-BMP/Nodal Constructs em Embriões de Zebrafish 

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.

Experimento de Exposição à Luz para Ativar BMP ou Sinalização Nodal em Zebrafish

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.