March 15, 2013;
Light-activation of the Archaerhodopsin H(+)-pump reverses age-dependent loss of vertebrate regeneration: sparking system-level controls in vivo.
Optogenetics, the regulation of proteins by light, has revolutionized the study of excitable cells, and generated strong interest in the therapeutic potential of this technology for regulating action potentials in neural and muscle cells
. However, it is currently unknown whether light-activated channels and pumps will allow control of resting potential in embryonic or regenerating cells in vivo. Abnormalities in ion currents of non-excitable cells are known to play key roles in the etiology of birth defects and cancer. Moreover, changes in transmembrane resting potential initiate Xenopus tadpole tail
regeneration, including regrowth of a functioning spinal cord
, in tails that have been inhibited by natural inactivity of the endogenous H(+)-V-ATPase pump. However, existing pharmacological and genetic methods allow neither non-invasive control of bioelectric parameters in vivo nor the ability to abrogate signaling at defined time points. Here, we show that light activation of a H(+)-pump can prevent developmental defects and induce regeneration by hyperpolarizing transmembrane potentials. Specifically, light-dependent, Archaerhodopsin-based, H(+)-flux hyperpolarized cells in vivo and thus rescued Xenopus embryos from the craniofacial and patterning abnormalities caused by molecular blockade of endogenous H(+)-flux. Furthermore, light stimulation of Arch for only 2 days after amputation restored regenerative capacity to inhibited tails, inducing cell proliferation, tissue
innervation, and upregulation of notch1
, essential genes in two well-known endogenous regenerative pathways. Electroneutral pH change, induced by expression of the sodium proton exchanger, NHE3
, did not rescue regeneration, implicating the hyperpolarizing activity of Archaerhodopsin as the causal factor. The data reveal that hyperpolarization is required only during the first 48 hours post-injury, and that expression in the spinal cord
is not necessary for the effect to occur. Our study shows that complex, coordinated sets of stable bioelectric events that alter body patterning-prevention of birth defects and induction of regeneration-can be elicited by the temporal modulation of a single ion current. Furthermore, as optogenetic reagents can be used to achieve that manipulation, the potential for this technology to impact clinical approaches for preventive, therapeutic, and regenerative medicine is extraordinary. We expect this first critical step will lead to an unprecedented expansion of optogenetics in biomedical research and in the probing of novel and fundamental biophysical determinants of growth and form.
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Fig. 1. Phenotypes caused by inhibition of the H+-V-ATPase.(A) Dorsal view of normal stage 47 tadpole. White arrows indicate normal eyes, olfactory bulbs and branchial arches. (B–D) Craniofacial phenotypes commonly seen after injection at the one-cell stage with mRNA encoding YCHE78, a dominant negative H+-V-ATPase subunit. B shows abnormally small branchial arches; the arrow in C points to ectopic pigments in the optic nerve; the arrows in D point to a missing eye and an abnormal olfactory bulb. Scale bar in C (for C and D) = 0.5 mm. (E) Profile of a normal stage 42 embryo. Arrows for comparison with F. (F) Stereotypical swellings caused by incubation of tadpoles in 10 nM concanamycin for 24 hours following tail amputation at stage 40. Even if removed from concanamycin at this stage, all tadpoles subsequently die. Anterior is to the left and dorsal is up. (G) A regenerated tail shown approximately 8 days post-amputation. The amputation plane was just anterior to (to the left of) the leftmost white arrow. (H) A non-regenerating tail. After amputation at stage 47, this tail did not grow back, a characteristic of tails cut at this age that defines the refractory stage. The red arrow points to the area referred to as the regeneration bud; the amputation plane is immediately anterior. Except where noted, scale bars = 1 mm.
Fig. 2. Arch is expressed and can be activated in Xenopus cells.(A–C) Expression of Arch-GFP at the stages indicated. mRNA for Arch-GFP was injected at the one cell stage and lasts for at least the 8 days required for the longest experiments. (A) View of animal pole of a Stage 7 embryo, the stage at which electrophysiology was performed. Arch-GFP expression is especially visible at cell–cell boundaries (e.g. white circle) where the cells have less pigment. (B) Arch-GFP is still present in the membranes of hexagonally-packed ectodermal cells at stage 30, well after the developmental stages that are relevant for craniofacial development. (C) Arch-GFP is still strongly expressed at stage 47, the refractory stage, during which, if the tail is cut, it does not grow back due to endogenous inhibition. The black arborized cells are melanophores, the longer aligned cells are muscle cells, and the hexagonally arrayed cells are epidermis. (D) Resting potential (Vmem) of impaled cells in embryos at stage 7. Arch-tomato was injected at 1 cell, then half of these embryos and half of the uninjected (NT = no treatment) controls were placed under blue light while the other halves were kept in the dark until stage 7. The Vmem of untreated embryos was about −29±2 mV and was unaffected by light. Arch-injected embryos kept in the dark were depolarized while Arch-injected embryos kept in the light were hyperpolarized. (E) Explanation of pH measuring experimental protocol. Two dyes were used to compare pH of Arch-tomato expressing cells with pH of cells showing no expression. BCECF is excited by 450 and 500 nm light, which does not activate Arch very strongly, while SNARF-5F is excited by 540 nm, which maximally activates Arch. The prediction is that the pHs measured by BCECF will differ by less than the pHs measured by SNARF because Arch should be maximally activated under the latter conditions, thus causing pH to increase in Arch expressing cells. (F–K) Results of pH measurements using two dyes. (F,I) Brightfield images showing positions of regeneration buds. (G,J) Images of Arch-tomato fluorescence used to choose regions of interest (ROIs) defined by high and low tomato fluorescence. (H,K) ROIs from G and J were transferred to the ratio images generated by the dyes (see Materials and Methods). As predicted, measurements made under low Arch-activation conditions (BCECF, F–H) show only a 0.3±0.2 pH unit difference between Arch-tomato expressing and non-expressing cells. In contrast, under Arch-activating conditions (SNARF-5F, I–K), the pH difference is approximately doubled to 0.7±0.3 pH units. Anterior is to the left in all images except A. Scale bars = 100 µm.
Fig. 3. Arch-Tomato activity promotes regenerative repair. (A) Comparison of regeneration indices, a measure of regeneration success, of the tails of Arch-Tomato expressing tadpoles amputated at the refractory stage. Significantly better regeneration was found in tadpoles that were exposed to Arch-activating light for 48 hours following amputation relative to those kept in the dark (t-test). (B) Comparison of localization of Arch-Tomato in tadpoles exposed to dark versus light for 48 hours post-amputation. Under Arch-activating light, regeneration correlated perfectly with the presence of a group of Arch-Tomato expressing cells at the very tip of the regenerating tail (see C below). In contrast, among the tadpoles kept in the dark, regeneration success did not correlate at all with localization of Arch-tomato. The distributions are highly significantly different (χ2). (C) Arch-Tomato localization in a regenerated tail 8 days after amputation at the non-regenerative stage 47. Arch-Tomato expression is still strong in the uncut part of the tail to the left, and in a small clump of cells at the distal most tip of the regenerating tail (white arrow). Otherwise, as expected, there is very low if any expression of Arch-tomato in the regenerate. Scale bar = 100 µm. (D) Arch-Tomato expression in a tail that did not regenerate. While there is expression all the way to the distal edge of the amputation plane, expression is relatively even, that is, there is no group of particularly bright cells. Scale bar as in C. (E–H) Higher magnification brightfield and fluorescence views of light-stimulated tails and Arch-Tomato expression. Yellow Ns indicate the notochords. White SCs and small arrowheads indicate the spinal cord. Letters were positioned on the brightfield images then transferred to the fluorescence images. At the distal ends (to the right) larger arrowheads indicate the posterior extents of the notochord (yellow) and spinal cord (white). Scale bars = 25 µm. (E,F) Brightfield and fluorescence images of a regenerated tail showing the bright cells to be further distal, and largely ventral, to the spinal cord. The bright cells are indicated by a white arrow in F. (G,H) A refractory tail that did not regenerate showing Arch-tomato expressing neural tissue extending to the distal tip of the non-regenerating bud. For all images, anterior is to the left and dorsal is up.
Fig. 4. Endogenous regenerative pathways follow arch activation by light. (A–D) Whole-mount in situ hybridizations for Notch1 and Msx1, components of regeneration signaling pathways. (A,B) Expression in the regeneration buds of Arch-tomato injected embryos maintained in the dark (white arrows). (C,D) Expression in the 3 days post-amputation (dpa) regeneration buds of Arch-Tomato injected embryos maintained in the light for 48 hours after amputation (red arrows). Expression is much higher in the light-stimulated tails. (E) Comparison of the number of cells positive for phospho-histone H3 (H3P, a marker of mitotic activity) in the regeneration buds of Arch-tomato expressing tails kept in the dark versus the light. There were significantly more in the light-stimulated tails (t-test). (F–I) DIC and fluorescence images of α-tubulin (a neuronal marker) of Arch-Tomato expressing tails at 3 dpa. Tadpoles were kept in the dark versus the light for 48 hpa. (F,G) Non-regenerating tails kept in the dark show the typical arrangement of neurons that have stopped growing or grown in a curve towards the midline. (H,I) Regenerating tails kept in the light show the normal arrangement of neurons in a regenerating tail that is growing parallel to the AP axis of the tail and extending into the growing tail, although not reaching all the way to the distal tip. In all images, anterior is to the left and dorsal is up. Scale bars = 25 µm.
Fig. 5. Summary of the effects of light activation of Arch-tomato on embryos with blocked H+-V-ATPase.(A) Embryos expressing a dominant-negative H+-V-ATPase subunit develop severe craniofacial defects. (B) Illuminating co-expressed Arch-tomato from stage 9 to stage 26 significantly reduces the number of craniofacial abnormalities. (C) Embryos exposed to the highly specific H+-V-ATPase inhibitor concanamycin for 24 hours after amputation at stage 40 develop stereotypical swellings then die, consistent with an effect on osmotic balance regulation. (D) Illuminating Arch-tomato during exposure to concanamycin prevents swelling and subsequent mortality. (E) Tadpole tails amputated at stage 47 normally do not regenerate. (F) Illuminating Arch-tomato for 48 hours after amputation significantly increases the number of regenerating tails if there is a small group of Arch-expressing cells at the distal most tip of the tail. Scale bars = 1 mm.
Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed