XB-ART-50582Int J Dev Biol January 1, 2014; 58 (10-12): 851-61.
Optogenetics in Developmental Biology: using light to control ion flux-dependent signals in Xenopus embryos.
Developmental bioelectricity, electrical signaling among non-excitable cells, is now known to regulate proliferation, apoptosis, gene expression, and patterning during development. The extraordinary temporal and spatial resolution offered by optogenetics could revolutionize the study of bioelectricity the same way it has revolutionized neuroscience. There is, however, no guide to adapting optogenetics to patterning systems. To fill this gap, we used optogenetic reagents, both proteins and photochemical switches, to vary steady-state bioelectrical properties of non-spiking embryonic cells in Xenopus laevis. We injected mRNA for various proteins, including Channelrhodopsins and Archaerhodopsin, into 1-8 cell embryos, or soaked embryos in media containing photochemical switches, then examined the effect of light and dark on membrane voltage (Vmem) using both electrodes and fluorescent membrane voltage reporters. We also scored tadpoles for known effects of varying Vmem, including left-right asymmetry disruption, hyperpigmentation, and craniofacial phenotypes. The majority of reagents we tested caused a significant increase in the percentage of light-exposed tadpoles showing relevant phenotypes; however, the majority of reagents also induced phenotypes in controls kept in the dark. Experiments on this "dark phenotype" yielded evidence that the direction of ion flux via common optogenetic reagents may be reversed, or unpredictable in non-neural cells. When used in combination with rigorous controls, optogenetics can be a powerful tool for investigating ion-flux based signaling in non-excitable systems. Nonetheless, it is crucial that new reagents be designed with these non-neural cell types in mind.
PubMed ID: 25896279
Article link: Int J Dev Biol
Species referenced: Xenopus
Genes referenced: gopc
Article Images: [+] show captions
|Fig. 1. Expression of optogenetic reagents in Xenopus embryos at different ages. Embryos in A to H are 1.2 mm in diameter. (A-F) mRNA for constructs was injected at the one or two cell stage. A to F are taken from a time lapse movie of an embryo that had been injected with LiGluR-tom. By monitoring tomato fluorescence we can see that the protein begins to accumulate at the cell surface by 85 minutes post-injection (B) and within another 10 to 15 minutes (C,D), it’s expression is quite strong. It is also clear that the bolus does not diffuse far from the injection site, thus in this embryo, only one cell is positive for the protein (E) until almost four hours post injection (F). Embryos continue to express the proteins through late gastrula stages (G,H), here illustrated by the expression of Vcomet-HRDAtom in all the cells that also contain lineage tracer to follow those cells that received the mRNA. Protein is also still clearly visible in cells, over 7 days after injection (I) illustrated here by expression of ChR2-C128T-GFP in skin cells of a tadpole.|
|Fig. 2. Power spectra of illuminators used to activate or de-activate channels. Two types of illumination systems were used, LED arrays and fiberoptically delivered LED lights inside light-tight cubes. Arrays comprised six LEDs arranged in a circle and aimed towards a central point. Irradiance inside the cubes was measured by the manufacturer at lmax. Irradiance of the LED arrays was measured using a USB650 spectrometer, an LS1-CAL lamp for calibration, and SpectraSuite software. As much as possible given the difference in thicknesses, the sensor was placed at the same distance from the light as the embryos, as indicated in the table. To calculate irradiance, each curve was integrated from l max – 10 nm through l max + 10 nm.|
|Fig. 3. Vmem of early Xenopus embryo cells (blastomeres) treated with AAQ. Resting potential of individual cells was measured using microelectrodes. Uncertainty bars are standard deviations. The measurement of untreated embryos match published values, and addition of a depolarizing solution caused the measurement to change appropriately (data not shown). In contrast, embryos that had been exposed to AAQ for 20 minutes prior to measurement were hyperpolarized relative to the untreated controls at some stages. These results are opposite our prediction of depolarization.|
|Fig. 4. Relative Vmem of Xenopus embryonic cells expressing Vcomets. We measured relative Vmem of cells in embryos transfected with VcometHRDA and Vcomet-HRFY. These reagents differ at two amino acids, and both were predicted to cause depolarization. (A-C) A ±128-cell embryo expressing Vcomet-HRDA in a subset of cells. Those cells expressing higher levels (example marked with up arrow), are hyperpolarized (labeled with -) relative to those expressing less (marked with down arrow). (D-F) A stage 20 embryo; cells expressing Vcomet-HRFY (marked with up arrow) and not expressing (labeled with down arrow). In this embryo, the cells expressing the construct are depolarized (+) relative to those expressing less or none (-). The embryo in A-C is 1.2 mm in diameter. The embryo in D-F is approximately 1.4 mm in length (left to right) and 1.2 mm tall (top to bottom).|
|Fig. 5. Phenotypes caused by treatment with optogenetic reagents. These phenotypes are commonly found when bioelectrical signaling is disrupted during Xenopus development. Images are dorsal views unless otherwise indicated; V = ventral, p = profile. Tadpoles are anesthetized for imaging. (A-C) Views of normal tadpole with relevant structures outlined. E = eye; j = jaw; ob = olfactory bulb; b = brain; oc = otocyst; ba = branchial arches. Three categories of phenotype were counted, hyperpigmentation, heterotaxia, and craniofacial abnormalities. (D) A tadpole with hyperpigmentation, which is characterized by the presence of more pigment cells, cells in normally clear regions, and greater area covered by pigment due to spreading of the cells in the plane of the skin. Spreading cannot be seen because anesthesia causes the pigment to collect in the center of the cell; however, pigmented cells can be seen covering the entire surface, including abnormal locations such as between the brain and the eye. (E) A tadpole that is heterotaxic. Heterotaxia is defined here as a reversal in the left-right patterning of one, two, or three organs, specifically the heart, gall bladder and/or gut. This example has a reversal of the loop of the heart. (F-L). Examples of the most common abnormalities of the face. (F) This profile shows a malformed anterior dorsal region. (G) This tadpole has black pigment associated with the optic nerves; the pigment appears to join the two eyes across the midline. (H) This tadpole has developed extra brain tissue. (I) The olfactory pits of this tadpole appear as one large organ. (J) The left eye of the tadpole is missing. (K) The jaw and branchial arches of the left side of this tadpole are small and malformed. (L) The head skeleton of this tadpole is much smaller than normal.|
|Fig. 6. Titration of ChR2-C128T. Transfection of Xenopus blastomeres by microinjection is normally accomplished by injecting nanograms of mRNA. To find a dose of ChR2-C128T mRNA that did not lead to phenotypes in the absence of light, serial dilution was used to lower the amount of mRNA injected. Black indicates no significant difference between treated and controls; red indicates a significant difference. Even as little as 0.5 picograms of mRNA led to significant numbers of tadpoles with phenotypes|