Friday, February 3, 2012

Ectopic thinking?

Our gene mapping project, which we first blogged about here, is starting to get interesting.  And not necessarily for the reasons that we'd hoped.  You might remember that our project is looking for genes involved in variation in specific craniofacial traits in the F34 generation of descendants of a cross between two inbred mouse lines.  We've measured a handful of traits in over a thousand mouse skulls, and mapped their genetic effects by looking for genetic variation across the genome that might be associated with variation in the traits.

Sparing you the gory details, we'll just say that the chromosomal intervals that one or more of the traits mapped to span 30% of the genome, and 10% of all coding genes. That's 2400 genes or so that could potentially be of interest in affecting head shape in just these particular mice, with the restricted genetic variation they have (because they are descendants of only two inbred parental mouse strains).  That's a lot of genes to wade through to figure out which might be most likely to be involved in the traits we're looking at.

One way to prioritize candidate genes from such a study is to look for the genes in every interval that you know from prior work to be involved in your trait of interest.  Or to identify genes in families that include genes involved in your trait of interest -- these would then be considered guilty by association.

But this means most genes don't have a fighting chance of being considered, because you don't happen to know anything about them, or because nobody knows anything about them, or because what's known about them only partially represents what they do. 

To try to minimize this, many people automate the search, with programs that cull the genes that the literature indicates might be of interest, or that seem to be expressed where you want them to be.  So, this might solve the problem of no one knowing everything about all genes, but it doesn't solve the problem of nothing being known about so many genes, or that there's only partial knowledge.  And it doesn't solve the problem of having to tell the program what to look for, which means you're constraining it in the same way you would if you were doing the search by hand, looking for specific families of genes.  Nor, of course, does it solve the problem of what's happening in all the non-coding DNA that flanks all those genes. 

Thus, we decided that the least biased way to comb the data was to go through all the genes in all the intervals by hand.   We're still making sense of all that, not least because we are hoping not to be constrained by the usual ideas about statistical significance, but we've learned some interesting things along the way.

For example, one of the intervals of interest is loaded with olfactory receptor (OR) genes.  Olfactory receptors reside on the cell surface of olfactory receptor neurons, and are involved in odorant detection.  ORs form the largest family of genes in many genomes -- about 1000 different genes -- and they cluster in sets of genes in various locations on a number of chromosomes.  ORs have a distinctive expression pattern, with only one expressed per neuron in the tissue lining the nose, where they each are sensitive to particular aspects of molecules the animal inhales, and hopes to smell.  How expression of the remaining 999 genes in each cell is blocked is still not known.

ORs are an interesting example of something we've blogged about before, but that continually surprises us.  One of the ways we're evaluating the possible role of all these genes in development of the traits we're looking at is to look at where they are expressed in the developing embryo.  We initially thought this would be helpful for narrowing the search, but it turns out that about 95% or even more of genes (for which there are expression data) are expressed in the head (80% alone in the brain), so it's turning out that expression isn't all that helpful for narrowing the search.  But it does mean we've looked at images of gene expression for around 2000 genes.

Olfr66, GenePaint, E14.5
And ORs are a good example of how what we think we know can inhibit our understanding.  Here, e.g., are the expression results for olfactory receptor 66 (Olfr66) in a developing mouse (at embryonic day 14.5).  Just to orient you if you're not used to looking at such images, it's a single front to back section, the snout halfway down the image and pointing to the left, and the tail at the bottom.  The dark blue is a stain showing cells where the gene is expressed at this particular stage of development.  It's no surprise to see it in the olfactory epithelium in the snout, but notice that it's also in the axial skeleton (vertebral column), probably in cartilage cells that will soon become bone.

What's it doing there?  These are olfactory receptors!  You don't smell with your backbone!  In fact, a lot of ORs are known to be expressed outside the olfactory region, particularly in the testes, but also in the spleen, the thyroid, salivary glands, the uterus, the skin, and other tissues.  A 2006 paper is of interest in this regard, not only because it documents non-olfactory related expression, but because of its title -- "Widespread ectopic expression of olfactory receptor genes".  Ectopic expression, meaning expression where it's not supposed to be. 

But it's only not supposed to be expressed in the axial skeleton because that's not where its name says it will be, not because Nature says so!  People named these genes!  And, there is some discussion in the paper about how ORs might be involved in chemotaxis of sperm as they try to reach and penetrate the egg -- how they direct their movement, based on chemicals in their environment.  Which is equivalent to assuming they are essentially carrying out their olfactory function in the testes, where a different form of molecular reaction than odorant-detection is going on.  But, what about in cartilage, in the image above?  It's hard to imagine chemotaxis has anything to do with OR function here.

Well then, maybe it's an experimental artifact -- maybe the experiment picked up expression of a gene sort of like Olfr66, but not quite, along with Olfr66?  Maybe.  But, then we'd have to explain away all the expression studies showing non olfactory expression of many ORs, and it's rather unlikely that it's all due to experimental artifact.  This is how our own assumptions constrain what we know or even want to know about the function of so many genes.  Maybe Olfr66 has a function we don't yet understand.  As do other ORs.  And, by extension, so many other genes. 

But calling unexpected expression 'ectopic', or naming genes based on only a single role, or in their involvement in disease, when they have other perfectly normal functions, are ways of building in assumptions that, once accepted, can keep us from recognizing that there's a lot we don't yet understand about genes.


Holly Dunsworth said...

So many similarities to the limitations we place on ourselves in paleontology! People named these things.

Anne Buchanan said...

Yes. We too often build entire stories out of incomplete knowledge, and forget that's what we've done.

Ken Weiss said...

Two things are well known about science (and other endeavors, too). Humans seem to want or need to be in groups with shared ethos. That's partly psychological need, perhaps. But in science or the practical world, we also seek or want a framework.

So we invent terms and jargon reflecting--and reinforcing--our framework. We teach the framework. We live within the framework. And we spurn those who challenge it, as heretics.

David Brooks has a column in the NY Times today (Friday Feb 3) on resistance and counter-cultural protests for change--basically a sappy and safe recommendation to seek past precedents and modes of revolt.

Medicine and science have to have their rules of the road and frameworks ('paradigms' in science), and philosophers and historians of science have been pointing this out for much of the past century (Ludwik Fleck, Thomas Kuhn most famously).

You can't get away just by showing, even validly, that current frameworks don't work very well. People resist: they accept only new suggestions for building a new framework. We need our cultural safety-cage, and in many areas (science, religion, education, social status, etc.) it does serve as a cage.

So even validly criticizing business as usual is an uncomfortable role (as we know very well!). The problem is that people don't necessarily want new ideas or good ideas if they challenge the safety zone.

Only occasionally does a transformative idea, with a new framework for action, take hold. Meanwhile, we muddle along even knowing that what we're doing is inaccurate or even wrong, pretending in a sense that our incremental gains are major ones.

Anonymous said...

I've been reading the MT blog for a long time and I finally want to chime in. I do research in evolutionary genetics.

What I appreciate most about the posts here is the championing of wisdom, that is, thinking deeply about why things are being done as they are and pointing out the limitations (i.e., incomplete knowledge) to our various methodologies and paradigms in genetics and evolutionary biology. In my opinion, there is not enough, if any, of this kind of education when it comes to teaching or training undergraduate and graduate students. It's important to educate students about the fact that a single word or phrase can constrain the way they think about the world and how they then make inferences about that world. We train people to become good at information acquisition (an important skill) but don't train them to stop and think how that information was acquired or how possibly incomplete it might be. The result is scientists who go on to maintain the status quo and who don't stop to think there might be another way. After all, isn't every scientific research project an act of irreverence with regards to the research that preceded it (i.e. testing previous hypotheses)? What I find ironic is that the data we collect is always new (that's how we get publications) yet we usually end up shoe-horning that data into a well established paradigm (e.g., OR genes should only be expressed in the olfactory epithelium because they're called OR genes!) without thinking that paradigm may not be even close to complete or accurate. And yet that leap is so simple to think about yet so hard to make in practice. I think Ken's interpretation is correct - it must be some kind of psychological safety net.

I know I'm preaching to the choir here, but I just wanted to express my appreciation for this awesome blog and the great thinking on display here. I don't always agree with everything you write, but hey, that just means you've stimulated reflection.

The theme of today's post reminded me of the mindfulness approach in cognitive psychology, which fosters consideration of alternatives. I want to share a passage from a book on mindfulness as related to health that I think you would appreciate and relevant to many of the discussions on MT:

"Too many of us believe the world is to be discovered, rather than a product of our own construction and thus to be invented. We often respond as if we and/or the world around us are fixed, even when we agree in theory that we are not.... There are many changes we would know how to make to feel better if it only occurred to us to ask. That’s how strong the illusion of stability – mindlessness – is. We imagine the stability of our mindsets to be the stability of the underlying phenomena, and so we don’t think to consider the alternatives. We hold things still in our minds, despite the fact that all the while they are changing. If we open up our minds, a world of possibility presents itself."

-from Counterclockwise by Ellen Langer, 2009, Ballantine Books

Anne Buchanan said...

Thanks so much for your kind words. It's very gratifying to have people resonate with what we try to do here. Of course, much of what we try to do is figure out ourselves what we think! So, if you disagree, do tell us. We'd love to hear why.

One of the things I actually find most uncomfortable about teaching is that students actually believe what I say! Ken and I co-teach a course on biology in society, and our goal is to challenge kids to figure out how they know what they know, and why they believe what they do (about biology). The best students we've had are the ones who challenge us in return.

Absolutely agreed -- "If we open up our minds, a world of possibility presents itself."

Ken Weiss said...

Thanks from me, too, for your kind words! My reply a couple of bumps up on today's Comments, where I mention Kuhn and Fleck, is related to our apparent need structure by which to evaluate the world and our place in it, either because we're social creatures or more likely because our brains have evolved to rely on them because of the relationship between predictability and survival, leads to this phenomenon.

One doesn't have to be a postmodernist in denial of the existence of a real world to see the role of psychology and sociology, as well as the momentum of history, in how we live. The idea of science as our system for getting beyond subjectivity, is something of a self-praising myth. Of course,it's reinforced by the degree to which science really does tell us about the world, and that there are things that, objectively enough, if we don't look out for them, they'll bump us in the head.

I was just reading a chapter by David Hull in the Cambridge Companion to Darwin (2009) in which he has a nice sentence: "Too often it seems that good arguments never convince anyone."

People listen only when a new idea can be seen as giving them something concrete; being illuminating may not be enough for a though to have influence.

Anyhow, of course we write MT to try to communicate our own thoughts, illuminating or otherwise, and we are gratified when we see that some people are thinking about them!

Holly Dunsworth said...

One of the reasons we are kindred spirits, Anne: "One of the things I actually find most uncomfortable about teaching is that students actually believe what I say!"

Anne Buchanan said...

I'm very happy to share this with you, Holly! xoxo

Inti pedroso said...

A quick question and a quick thought ...
Do you only have genotypes and phenotypes for the F34? if you could obtain both, pheno and genotypes, for all (ideally, but some pragmatically) previous generations you could look at the genotype combination at each stage and model the interactions "as the happened" through the pedigree. I guess that could help to account for the genotype inequality problem as you could map different "phenotypic trajectories" on the pedigree and how those are influenced by the genotypes.
I have used some obscure terms in here because I do not really know how to go about the details in practice but I imagine some of the most recent comparative methods could be used for this, e.g., modelling the phenotypes over the pedigree and then you could look at convergence of the phenotypes driven by the the genotypes ... as convergence between species driven by environment or genetic constrains. I think there is potential in treating development and genetic mapping much more alike to comparative analyses on evolutionary biology ... just something that has been occupying my mind lately.

It is just an idea but I would love to know what you think.



Ken Weiss said...

Nice to hear from you. Of course collecting all possible data would in principle provide all relevant answers. In this case, the mice must be CT-scanned for 3D images to evaluate the phenotypes, and it's beyond practicability.

However, if the trait is complex, as it seems to be, and gene expression variable in quantity and location (and developmental age), then we'll end up with more precise estimates of what will be numerous small effects.

And if the effects are context-dependent, and/or non-additive,and context here largely means genomic context; and if there are new mutations and we are using markers rather than wholegenome sequence; and if there are non-sequence-based epigenetic effects; and if many or most genes that are relevant don't happen to vary between the two inbred parental strains and hence to be unmappable in their intercross, then that is like chasing rainbows.

The better way, we think, is to REthink the question about how to deal with inherently polygenic biology.

Young guys like you need to show old guys like us how to think in better ways!