Wednesday, October 15, 2014

A Random Walk into the Genetics of Axon Guidance



The man claimed he was a wizard and had cast the “spell of attraction” on the target.  Now the target would guide his arrow to the mark.  So we gave the man a broken arrow and watched to see if this arrow could hit the target.  The man took the arrow and flung it at the target.  Indeed, this arrow too could hit the target. 


Credit: Nina Matthews Photography
Perhaps we had asked the wrong question.  Instead of looking at whether the arrow made it to the target, we needed to look at how the arrow went to the target. 



Genetics

One of the goals of genetics is to use mutations to reveal the normal function of a gene.  My laboratory uses genetics.  We study mutations in the nematode model organism Caenorhabditis elegans.  By analyzing mutations in genes that control axon guidance, we hope to gain a better understand of how axon guidance works.

In the past, we simply looked at a mutant that had a loss of gene function and asked, “Did the axon reach its target?”   If not, then our interpretation was that the gene is needed for axon guidance.  If the axon did reach its target, than our interpretation was that the gene is not needed for axon guidance (or that the gene is redundant with a second gene whose function can compensate of the loss of the first gene). 

The attraction and repulsion model

This is an illustration from our 2002 review article.  It shows two different neurons. One axon extends toward the ventral source of the extracellular UNC-6 guidance cue and expresses the UNC-40 receptor.  The other axon extends away from the ventral source of UNC-6 and expresses the UNC-40 and UNC-5 receptor.  


The thought was that the UNC-40 receptor alone allows an attractive response towards the UNC-6 source, whereas the UNC-5 receptor allows a repulsive response away from the UNC-6 source. 

When we made mutations in the  unc-5 gene, we found that axons could still migrate towards the UNC-6 source, which was consistent with the idea that UNC-5 is the repulsive receptor and is not needed for attraction.  

The random walk response model

In previous posts I presented the hypothesis that the direction of axon guidance is not determined by directional responses to the guidance cues, but rather by the succession of randomly directed movement.  See my post  "Random Walks, the Brain Initiative, and the Genius of Einstein's Brain". 

Because of the random walk response model,  we wondered if we should look at guidance in a different way.  Instead of looking at whether the axon makes it to its target, we should instead look at how the axon is moving towards the target.  Could UNC-5 affect how the axon moves towards the UNC-6 source?

To do this we looked at the probability of outgrowth in each direction in an unc-5 mutant.  See my post "Axon Guidance Meets Statistical Physics".

We found that the probability of axon outgrowth from each side of the neuron was:


direction probability
ventral .8
anterior .2
posterior  0
dorsal  0


We could now use this probability distribution to simulate a simple random walk.  The results give us a picture of how all the guidance cues acted together when the axon extended from the neurons's cell body to set the probabilities of outgrowth for each direction.  Although in this case, without the function of UNC-5.  The simulation shows the directional bias for the movement of the axon as it first extended.  Of course the random walk of the axon in reality is not a simple walk.  For one thing, the probability distribution would not remain identical as the axons moves through its environment and encounters new cues and different concentrations of the cues.  Therefore, the simulations provide us with a relative directional bias.  How did getting rid of the function of UNC-5 alter the directional bias relative to the directional bias created in a normal, wild-type animal? 


10 simulations each of simple random walks based on the 
probabilities of axon outgrowth in each direction. 
As this illustration shows, when UNC-5 function is missing the outgrowth activity changes.  The relative directional bias shifts.  Although in unc-5 mutants this axon still reaches its target, it does so with a broken guidance system.  

Our results contradicted the notion that UNC-5 is only a "repulsive" receptor, since in this case it is involved in the guidance of an axon towards the source of the guidance cue.   


And this leads to new directions

Instead of observing whether the axon makes it to the target in a mutant, we can now observe the relative directional bias in a mutant.  A new phenotype!  For geneticists, this means looking at more mutants to see how other genes affect the phenotype.  And making combinations of mutations to see genetic interactions.  

And that's we did.  One of the most interesting results was the genetic interaction between unc-5 and unc-53, a gene that encodes a protein that is found within the neuron.  Whereas the loss of function of either gene alone causes only a slight shift in the relative directional bias, the loss of both together causes a dramatic shift.  What's more, we found that in the double mutants the UNC-40 receptor doesn't work properly.   

So by looking at how the axon went to its target, we have discovered more about the molecular mechanisms by which an axon finds its target.


References:

Wadsworth, W. (2002). Moving around in a worm: netrin UNC-6 and circumferential axon guidance in C. elegans Trends in Neurosciences, 25 (8), 423-429 DOI: 10.1016/S0166-2236(02)02206-3


Kulkarni, G., Xu, Z., Mohamed, A., Li, H., Tang, X., Limerick, G., & Wadsworth, W. (2013). Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity Biology Open, 2 (12), 1300-1312 DOI: 10.1242/bio.20136346


Yang, Y., Lee, W., Tang, X., & Wadsworth, W. (2014). Extracellular Matrix Regulates UNC-6 (Netrin) Axon Guidance by Controlling the Direction of Intracellular UNC-40 (DCC) Outgrowth Activity PLoS ONE, 9 (5) DOI: 10.1371/journal.pone.0097258


Xu, Z., Li, H., & Wadsworth, W. (2009). The Roles of Multiple UNC-40 (DCC) Receptor-Mediated Signals in Determining Neuronal Asymmetry Induced by the UNC-6 (Netrin) Ligand Genetics, 183 (3), 941-949 DOI: 10.1534/genetics.109.108654

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