Can Voltage Gradients Be Altered to Control Organ Development in Tadpoles?

[Pythagorean]

prepare to have your mind blown.

they changed the voltage gradient of cells in the tadpoles' back and tail to match that of normal eye cells. The eye-specific gradient drove the cells in the back and tail -- which would normally develop into other organs -- to develop into eyes.

http://www.sciencedaily.com/releases/2011/12/111207175740.htm

peer-reviewed article:

Vaibhav P. Pai, Sherry Aw, Tal Shormat, Joan M. Lemire. Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis. Development, December 20, 2011 DOI: 10.1242/dev.073759

 

[Ygggdrasil]

they changed the voltage gradient of cells in the tadpoles' back and tail to match that of normal eye cells. The eye-specific gradient drove the cells in the back and tail -- which would normally develop into other organs -- to develop into eyes.

The press release on Science Daily seems to exaggerate greatly some of the claims made in the paper. For example, the quote above that you used is misleading; it suggest that the authors had control over the location where they changed the voltage gradient and could control the strength of the voltage gradient. In reality, the authors could do neither. In order to induce ectopic eye development, the authors injected the mRNA for various ion channels into a frog embryo at the four cell stage. As the cells divide, the injected mRNAs get randomly segregated into different cells of the developing embryo, and the ion channels transcribed from the mRNA then cause some of the cells to have a higher membrane potential than normal. This intervention causes only 7.5% of the injected embryos to show complete eye structures on different parts of the tadpole's body (although the Science Daily press release claims that these structures are working eyes, I see no data in the paper showing that the eyes are functional). Because of the limitations of their mRNA injection method, the authors cannot control where the eyes develop. Furthermore, the claim that changing the membrane voltage to match that of eye cells is not investigated; the authors have no control over how much the voltage channels affect the membrane potential during development nor do they attempt to quantify the effect.

Now, a really cool experiment would be to use some of the recent developments in optogenetics to design a system where the experimenters could control the precise location of the voltage perturbation as well as the strength of the voltage perturbation. To do this, you would engineer the tadpoles to express channelrhodopsin, an ion channel whose activity is controlled by blue light. Such a system would allow the experimenter to selectively depolarize a subset of cells in the embryo by simply shining a blue laser on those cells. By including some sort of voltage-sensing mechanism (i.e. a calcium-sensitive fluorescent dye or a voltage-sensitive fluorescent protein) that allows the experimenter to monitor the voltage change to the cell, one could automate a feedback loop to control the laser intensity in order to set the strength of the voltage perturbation (as demonstrated by http://dx.doi.org/10.1038/nmeth.1700 ). An experimental system like this would allow the researchers to look at the role of voltage in eye development much more quantitatively.

Now lest I sound overly negative about the study, I do want to point out that the experiment is a really nice demonstration that changing membrane potential is sufficient to induce eye development. Is the result so mind blowing? Perhaps not. As the authors point out in their paper, cell contain numerous voltage-gated calcium channels that will open when cells become depolarized. Opening these channels let's calcium into the cell and calcium ions are very well known to have a number of roles in cellular signaling and in regulating gene expression. Indeed, the authors show that they can prevent their mRNA injection procedure from creating ectopic eyes by adding drugs that block voltage-gated calcium channels, providing some nice data that demonstrate the role of calcium signalling in eye development.

 

[Evo]

Science Daily isn't exactly a proper source.

 

[atyy]

Now lest I sound overly negative about the study, I do want to point out that the experiment is a really nice demonstration that changing membrane potential is sufficient to induce eye development. Is the result so mind blowing? Perhaps not. As the authors point out in their paper, cell contain numerous voltage-gated calcium channels that will open when cells become depolarized. Opening these channels let's calcium into the cell and calcium ions are very well known to have a number of roles in cellular signaling and in regulating gene expression. Indeed, the authors show that they can prevent their mRNA injection procedure from creating ectopic eyes by adding drugs that block voltage-gated calcium channels, providing some nice data that demonstrate the role of calcium signalling in eye development.

But why so specifically the eye? Isn't calcium involved in lots of things? Or is it that only in eye development calcium has to enter through voltage gated channels?

 

[Pythagorean]

The press release on Science Daily seems to exaggerate greatly some of the claims made in the paper. For example, the quote above that you used is misleading; it suggest that the authors had control over the location where they changed the voltage gradient and could control the strength of the voltage gradient. In reality, the authors could do neither. In order to induce ectopic eye development, the authors injected the mRNA for various ion channels into a frog embryo at the four cell stage. As the cells divide, the injected mRNAs get randomly segregated into different cells of the developing embryo, and the ion channels transcribed from the mRNA then cause some of the cells to have a higher membrane potential than normal. This intervention causes only 7.5% of the injected embryos to show complete eye structures on different parts of the tadpole's body (although the Science Daily press release claims that these structures are working eyes, I see no data in the paper showing that the eyes are functional). Because of the limitations of their mRNA injection method, the authors cannot control where the eyes develop. Furthermore, the claim that changing the membrane voltage to match that of eye cells is not investigated; the authors have no control over how much the voltage channels affect the membrane potential during development nor do they attempt to quantify the effect.

Now, a really cool experiment would be to use some of the recent developments in optogenetics to design a system where the experimenters could control the precise location of the voltage perturbation as well as the strength of the voltage perturbation. To do this, you would engineer the tadpoles to express channelrhodopsin, an ion channel whose activity is controlled by blue light. Such a system would allow the experimenter to selectively depolarize a subset of cells in the embryo by simply shining a blue laser on those cells. By including some sort of voltage-sensing mechanism (i.e. a calcium-sensitive fluorescent dye or a voltage-sensitive fluorescent protein) that allows the experimenter to monitor the voltage change to the cell, one could automate a feedback loop to control the laser intensity in order to set the strength of the voltage perturbation (as demonstrated by http://dx.doi.org/10.1038/nmeth.1700 ). An experimental system like this would allow the researchers to look at the role of voltage in eye development much more quantitatively.

Now lest I sound overly negative about the study, I do want to point out that the experiment is a really nice demonstration that changing membrane potential is sufficient to induce eye development. Is the result so mind blowing? Perhaps not. As the authors point out in their paper, cell contain numerous voltage-gated calcium channels that will open when cells become depolarized. Opening these channels let's calcium into the cell and calcium ions are very well known to have a number of roles in cellular signaling and in regulating gene expression. Indeed, the authors show that they can prevent their mRNA injection procedure from creating ectopic eyes by adding drugs that block voltage-gated calcium channels, providing some nice data that demonstrate the role of calcium signalling in eye development.

Hrm, thank you for reporting the paper's content, I haven't been able to get access to it. I liked science daily a lot when I first found it, but it's gradually knicking away at what little faith I had left in scientific journalism. Have you found a free copy anywhere? My research library doesn't seem to carry development.

The mind-blowing part to me is that electrical activity can guide organesesis (regardless of whether the PI's had the technique/technology to directly control it directly). I would be excited to see what a precise control system (such as I was led to believe they used) could produce. I've always wanted to get into optogenetics. It seems like the perfect technique for testing hodgkin-huxley ion-conductance network models.

 

[atyy]

I've always wanted to get into optogenetics. It seems like the perfect technique for testing hodgkin-huxley ion-conductance network models.

With the DIY molecular biology lab and iphone

 

[Pythagorean]

I wonder if you could just buy a 7$ C. elegans strain from the CGC and a 7$ laser pointer and go to town.

 

[Pythagorean]

no ZX299, AQ2334, or ZX460 in the CGC. Share your strains, optogenetic peoples!

 

[Ygggdrasil]

But why so specifically the eye? Isn't calcium involved in lots of things? Or is it that only in eye development calcium has to enter through voltage gated channels?

That's a good question. I'm sure there are other factors involved as well. I'm neither a developmental biologist nor a neurobiologist, but I remember hearing about research showing that waves of spontaneous activity across the retina are important for the proper development of certain structures in the eye, so the specific spatial and temporal patterns of electrical activity may also play some important role. These are things that an optogenetics experiment could probably investigate in greater detail.

Hrm, thank you for reporting the paper's content, I haven't been able to get access to it. I liked science daily a lot when I first found it, but it's gradually knicking away at what little faith I had left in scientific journalism.

Science Daily for the most part just copies university press releases and republishes them on its site. Look at the note at the bottom of the article regarding the story source. So, the inaccuracies aren't really Science Daily's fault, but the fault of the university PR office.

Have you found a free copy anywhere? My research library doesn't seem to carry development.

Sorry, I don't know if there is a free copy anywhere. Sometimes professors will post a copy on their website. If the research was funded by the NIH, the article will appear in PubMed Central six months after publication.

 

[atyy]

That's a good question. I'm sure there are other factors involved as well. I'm neither a developmental biologist nor a neurobiologist, but I remember hearing about research showing that waves of spontaneous activity across the retina are important for the proper development of certain structures in the eye, so the specific spatial and temporal patterns of electrical activity may also play some important role.

That's an interesting hypothesis. Any pointers for reading? A quick search turned up this review, but the waves there seem to start late, being "first detected a few days before birth" in mice. Wrong species too, I should probably look for frogs.

Actually, now reading the http://dev.biologists.org/content/early/2011/12/07/dev.073759.abstract, they say "During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes." So I'd guess the eye specificity is from the "dorsal lineages" (whatever that means), and the reason one can get weird eyes outside the anterior neural field is that there are these dorsal lineages outside the anterior neural field. Just a guess to check when this becomes free in 6 months' time:)

I looked for free versions of other articles by the same authors. Maybe that'll at least give us some background.
http://www.ncbi.nlm.nih.gov/pubmed/19406249
http://www.ncbi.nlm.nih.gov/pubmed/19823012 (Figure 3 seems to have a preliminary version of the OP paper)
http://www.ncbi.nlm.nih.gov/pubmed/20959630

 

[Pythagorean]

Good call atyy, the first one you linked has a lot of background on the subject and they talk a bit about the technique Yggg mentioned with mRNA injection in that figure from link 2.

Science Daily for the most part just copies university press releases and republishes them on its site. Look at the note at the bottom of the article regarding the story source. So, the inaccuracies aren't really Science Daily's fault, but the fault of the university PR office.

That's even more disturbing... it worked though, here we are. Go PR.

 

[Ygggdrasil]

That's an interesting hypothesis. Any pointers for reading? A quick search turned up this review, but the waves there seem to start late, being "first detected a few days before birth" in mice. Wrong species too, I should probably look for frogs.

The review likely covers the phenomenon I mentioned earlier. The particular study I had in mind was done in ferrets (http://www.ncbi.nlm.nih.gov.ezp-prod1.hul.harvard.edu/pubmed/17046688 , disclaimer: one of the authors is a colleague of mine). These studies are not necessarily relevant to what's happening in the frog embryos, but rather they point to the fact that temporal patterns of electrical activity are important in regulating development. Indeed, the role of oscillations in calcium concentrations in cell signalling is a problem that has been extensively studied (this review is a bit old, but see http://dx.doi.org/10.1146/annurev.cb.06.110190.003435).

Actually, now reading the http://dev.biologists.org/content/early/2011/12/07/dev.073759.abstract, they say "During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes." So I'd guess the eye specificity is from the "dorsal lineages" (whatever that means), and the reason one can get weird eyes outside the anterior neural field is that there are these dorsal lineages outside the anterior neural field. Just a guess to check when this becomes free in 6 months' time:)

Here's what the authors have to say on the matter (copied from the discussion section of the paper):

"Neural tissue and Otx2 expression are thought to be crucial for eye induction (Zuber et al., 2003). The ability to form ectopic eyes without induction of additional primary axes or central nervous system tissue (lack of ectopic Otx2) is very interesting and suggests a more direct (downstream) linkage of Vmem to eye-specific cascades (Fig. 5A). Indeed, bioelectrically induced eyes can be formed on the gut or within lateral mesoderm – non-neural tissues outside the known competence lineage for eyes. Similarly, patches of lens/retina readily form in the tail. By contrast, Pax6 alone is incapable of initiating an eye field in regions outside the competent neurectoderm, suggesting that additional factors might also be involved in positive feedback interactions with Vmem. However, Vmem is able to initiate not only Pax6 expression but also the conditions that make tissues competent to form eyes upon Pax6 transcription.

Temporally and spatially restricted hyperpolarization has previously been shown to drive cellular fate (Hinard et al., 2008; Konig et al., 2004; Konig et al., 2006; Sundelacruz et al., 2009). Moreover, specific cellular responses to windows of Vmem have also been documented (Gruler and Nuccitelli, 1991; Jurkat-Rott et al., 2009; McDonald et al., 1972). Our data suggest the presence of a narrow hyperpolarization range that specifies the Vmem signal for eye induction (Fig. 5C)."​

So the author's argument is that a specific value of the membrane potential is what specifically induces the cells to differentiate into eye cells. While their experiments messing with eye development seem to support this hypothesis, their experiments with ectopic eye development are still very messy and as noted before, do not allow the researchers to control or monitor the strength of the voltage perturbation and therefore provide no evidence whether the strength of the Vmem perturbation is key. Although the hypothesis is plausible, a much more careful analysis of the ectopic eye development experiments is required to isolate all of the important factors involved. In fact, although I previously posted "the experiment is a really nice demonstration that changing membrane potential is sufficient to induce eye development", I want to amend that to say "the experiment is a really nice demonstration that changing membrane potential is sufficient to induce eye development in a subset of cells." To demonstrate the former claim requires showing that any arbitrary cell can have its cell fate changed to that of an eye cell, something that this paper has not demonstrated.

 

[Pythagorean]

So I first got interested in this question because I was curious what effects membrane potential has on normal processes in the cell.

For instance, if we ignore voltage-gated calcium signaling, does a change in membrane potential change the energy landscape enough to effect the on/off times of ligand-receptor kinetics or other underlying processes or is this pretty much solely a case of Ca influx (which then goes on to molecular pathways)?

I'm not asking for published proof, so much as an estimate of the reasonableness that membrane potentials would have enough energy to effect binding kinetics.

 

[atyy]

So I first got interested in this question because I was curious what effects membrane potential has on normal processes in the cell.

For instance, if we ignore voltage-gated calcium signaling, does a change in membrane potential change the energy landscape enough to effect the on/off times of ligand-receptor kinetics or other underlying processes or is this pretty much solely a case of Ca influx (which then goes on to molecular pathways)?

I'm not asking for published proof, so much as an estimate of the reasonableness that membrane potentials would have enough energy to effect binding kinetics.

Like these?
http://www.ncbi.nlm.nih.gov/pubmed/6325946
http://www.ncbi.nlm.nih.gov/pubmed/1645414
http://www.ncbi.nlm.nih.gov/pubmed/16921022

This review of L-type calcium channels says "Inhibition by dihydropyridines is state-dependent: enhanced at depolarized membrane potentials that open the channel, but reduced at hyperpolarized membrane potentials (Bean 1984; Berjukow et al. 2000)."

 

[Pythagorean]

I was thinking more of metabolic processes and gene expression. Mg coincidence detection is fairly well known in neuroscience and has some implications for conditioning behavior such as observed in Pavlov's dog; any ion/current gating (such as with sodium, potassium, and calcium channel varieties) is well known to operate on membrane potential (hodgkin huxley type models actually model exactly this dependency).

In this case, I was more thinking of passive electrical processes. Most of the above are more active voltage responses, so it's not surprising they're voltage-sensitive. But do metabolic and expression processes get affected by either passive currents (through gap junctions from neighboring cells that have active currents) or from the active processes inside the active cell themselves?

 

[atyy]

I assumed you were thinking of ligand binding to a transmembrane receptor, since that's where the potential difference is. You are thinking of a potential difference within the cell?

BTW, are there known cases of calcium entry from "hyperpolarization"? Most examples seem to be due to "depolarization". (Probably ok, but this is a physics site isn't it, so an action potential really isn't a "depolarization";)

 

[Pythagorean]

I assumed you were thinking of ligand binding to a transmembrane receptor, since that's where the potential difference is. You are thinking of a potential difference within the cell?

BTW, are there known cases of calcium entry from "hyperpolarization"? Most examples seem to be due to "depolarization". (Probably ok, but this is a physics site isn't it, so an action potential really isn't a "depolarization";)

No, I still mean across the membrane, but it's not active. I was just thinking of passive/diffusive travel of electrical currents through, for instance, non-neural/muscle cells. The target would be processes inside the cell. Of course, through gap junctions, the ultimate source of the perturbation could have been an active current, but the point is that the perturbations travel a particular spatiotemporal profile given by the capacitance and resistance of the model compartment so there will be an inherent range of frequencies that the perturbations oscillate with.

Is it such that the intensity and frequency of these waves, washing over a tissue of cells (through gap junctions) drives metabolic and/or genetic/expression processes?

To your question, I remember something about that in my molecular neuro class. Here's a paper I see whose title fits (haven't read it though)
http://www.pnas.org/content/101/4/1051.short

 

[atyy]

http://www.pnas.org/content/101/4/1051.short

Thanks!