A spider with two hoo-has

Nice.

Almafuerte peripampasica, female. A, prosoma and opisthosoma (dorsal view). B, opisthosoma (ventral view, arrow pointing to the duplicated epigyne). Note the difference in size and the slightly asymmetry. C, normal epigyne (arrow pointing to larva). D, duplicated epigyne. Specimen locality: Argentina: Córdoba: Parque Nacional Quebrada del Condorito, “sitio 2”, -31.63481, -64.71087, alt. 1846m, M. Izquierdo, D. Abregú, C. Mattoni, col. Sep. 16, 2019, under stones. LABRE-Ar 498, voucher MAI-4754.

Developmental variations always catch my eye, and this one is interesting from the standpoint of looking for spatial specifications. The normal blueprint for the spider body plan puts the epigyne at the anterior end of the abdomen, but here’s one way back near the spinnerets. What triggered formation at that position? It’s also non-functional, they think — I’d want to know more about the internal organs, what’s connected to what.

In general, malformations in spiders involve chelicerae, pedipalps, walking legs, and eyes, but those affecting female reproductive structures are not so frequent. A teratological case of a spider with a duplicated reproductive structure is described. The female specimen has the typical epigyne and a second one near the spinnerets. The second epigyne is less developed and seems to be non-functional. Similar malformations have been reported for Amaurobiidae and Salticidae, and here is presented for Gnaphosidae. Although it is widely known that temperature and humidity may induce abnormalities under experimental conditions, the causes behind teratological genitalia in wild females are unknown. This case opens the question of the origin of such a malformation and the ontogeny of female reproductive organs in spiders.

Also, that’s an adult of respectable size, so the ectopic organ doesn’t seem to have affected its viability. I’m going to have to spend some time looking up spiders’ skirts this summer.

Sex combs!

I mentioned sex combs a while back, so I thought I’d clarify a bit — I hope none of you rushed out to buy one for yourself (I don’t think human sex combs exist, but if they do, I don’t need to know.) Sex combs are secondary sexual characteristics found in on the forelegs of only male Drosophilidae. They are small dark patches of bristles on the tarsus of the first leg, and they are not something you’d notice if you saw a fly buzzing in your kitchen — you have to knock them out and carefully scrutinize the limbs with a hand lens or microscope to see them, but they’re important for recognizing the sex of a fly definitively. I tell my genetics students that you can tell the sexes apart by the shape of the abdomen or the pigment patterns, but to be really sure you should check for the presence or absence of the sex combs.

Sex comb in Drosophila melanogaster male: a) front leg with sex comb marked with black arrow; b) sex comb bristles

They don’t look like much, but they also matter to female flies. Mutants or surgically modified male flies with the sex combs reduced suffer with lower reproductive success. The flies use them as gentle grasping tools to separate her wings, grasp her abdomen, and tease open the genitals, so of course they’re subject to selection. Different species of fruit flies exhibit different patterns of sex combs, and we observe natural variation within a species.

Sex comb (SC) diversity. A phylogeny of eight comb-bearing species, assuming that the montium subgroup is a sister-taxon to the
Oriental lineage, as in Kopp (2006). Branch lengths are not to scale. Note the variation in the length, size and orientation of the SCs.

Variation within and between species makes sex combs of great interest to evolutionary biologists. Here’s an illustration of the variation we can see.

Variation in sex comb tooth number and development. (A–C) Exam-
ples of Drosophilidae forelegs with long combs. (D and E) D. melanogaster
forelegs. (F–J) Schematics of foreleg development of the top Drosophila legs.
(K–O) Examples of D. melanogaster perturbations in sex comb tooth number.
(A–C and F–H) Drosophila species with long sex combs achieve vertical orien-
tation by different mechanisms: (i) Teeth initially form in a vertical orientation
(F) (e.g., D. ficusphila t1–t2); (ii) rotation of a long row (G) (e.g., D. guanche t1–t2
and D. rhopaloa t1); (iii) rotation of multiple small rows and posterior fusion
into a long sex comb (H) (e.g., D. rhopaloa t2). (D, E, I, and J) In D. melanogaster, the male sex comb rotates from a horizontal to a vertical position
(diagrammed in D), while TRs remain horizontal. The only exception is the
most distal transverse row (red dotted box in D and I), which bends proximally
close to the top part of the sex comb. In contrast, the female rows of bristles
homologous to the sex comb remain static during development (brackets in E
and J). In order to study the phenotypic and developmental effect of changing
the number of sex comb teeth, this trait was perturbed using artificial selection
(K and L), mutants (M–O), and UAS-Scr RNAi transgenic lines . Gray circles represent sex comb teeth in the initial position and
black circles represent sex comb teeth in the final position. Empty circles represent the TR bristles. Gray arrows indicate movement of individual tooth or
sex comb rows. Red brackets indicate sex combs or homologous female bristles. Red numbers represent the range of sex comb teeth in each line. babPR72, bric à bracPR72; scd, sex combs distal; Scr, sex combs reduced; t1 and t2, first and
second tarsal segment, respectively. Distal is down and posterior is to the right.
Scale bar: 20 μm.

Focus for now on the top panel on the right, which shows a male and a female foreleg. They both have hairy legs, and in the default pattern seen in the females is a series of bristles in transverse rows (TRs) in arrays marching down the leg. The flies specifically use these bristles to groom their eyes — if you look closely, flies are remarkably tidy and neat.

The TRs are illustrated diagrammatically as small open circles in rows, the base pattern. These bristles are also developmentally interesting, because the way you make a sex comb is you express a set of specific genes in the distal two rows, and the whole structure rotates 90° to form a longitudinal comb. This opens up a whole set of informative interactions — the rotation is essential for function, and is subject to constraints imposed by adjacent tissues. I’ve been reading papers for the past week focused on the developmental and evolutionary significance of this tiny, odd, little known structure in flies. You should read some of these papers, too! I’m a sucker for anything evo-devo, and that’s what this little patch of hairs illustrates.

The most complex and diverse secondary sexual character in Drosophila is the sex comb (SC), an arrangement of modified bristles on the forelegs of a subclade of male fruit flies. We examined SC formation in six representative nonmodel fruit fly species, in an effort to understand how the variation in comb patterning arises. We first compared SC development in two species with relatively small combs, Drosophila takahashii, where the SCs remain approximately transverse, and Drosophila biarmipes, where two rows of SC teeth rotate and move in an anterior direction relative to other bristle landmarks. We then analyzed comb ontogeny in species with prominent extended SCs parallel to the proximodistal axis, including Drosophila ficusphila and species of the montium subgroup. Our study allowed us to identify two general methods of generating longitudinal combs on the tarsus, and we showed that a montium subgroup species (Drosophila nikananu) with a comb convergently similar in size, orientation and position to the model organism Drosophila melanogaster, forms its SC through a different developmental mechanism. We also found that the protein product of the leg patterning gene, dachshund (dac), is strongly reduced in the SC in all species, but not in other bristles. Our results suggest that an apparent constraint on SC position in the adult may be attributable to at least two different lineage-specific developmental processes, although external forces could also play a role.

Atallah J, Liu NH, Dennis P, Hon A, and Larsen EW (2009) Developmental constraints and convergent evolution in Drosophila sex comb formation. Evolution & Development 11(2): 205-218.

Malagón JN, Ahujab A, Sivapatham G, Hung J, Leea J, Muñoz-Gómez SA, Atallah J, Singh RS, and Larsena E (2013) Evolution of Drosophila sex comb length illustrates the inextricable interplay between selection and variation www.pnas.org/cgi/doi/10.1073/pnas.1322342111

Another step in the evolution of multicellularity

I’m not a fan of phys.org — they summarize interesting articles, but it’s too often clear that their writers don’t have a particularly deep understanding of biology. I wonder sometimes if they’re just as bad with physics articles, and I just don’t notice because I’m not a physicist.

Anyway, here’s a summary that raised my hackles.

Chromosphaera perkinsii is a single-celled species discovered in 2017 in marine sediments around Hawaii. The first signs of its presence on Earth have been dated at over a billion years, well before the appearance of the first animals.

A team from the University of Geneva (UNIGE) has observed that this species forms multicellular structures that bear striking similarities to animal embryos. These observations suggest that the genetic programs responsible for embryonic development were already present before the emergence of animal life, or that C. perkinsii evolved independently to develop similar processes. In other words, nature would therefore have possessed the genetic tools to “create eggs” long before it “invented chickens.”

First two words annoyed me: Chromosphaera perkinsii ought to be italicized. Are they incapable of basic typographical formatting? But that’s a minor issue. More annoying is the naive claim that a specific species discovered in 2017 has been around for a billion years. Nope. They later mention that it might have “evolved independently to develop similar processes”, which seems more likely to me, given that they don’t provide any evidence that the pattern of cell division is primitive. It’s still an interesting study, though, you’re just far better off reading the original source than the dumbed down version on phys.org.

All animals develop from a single-celled zygote into a complex multicellular organism through a series of precisely orchestrated processes. Despite the remarkable conservation of early embryogenesis across animals, the evolutionary origins of how and when this process first emerged remain elusive. Here, by combining time-resolved imaging and transcriptomic profiling, we show that single cells of the ichthyosporean Chromosphaera perkinsii—a close relative that diverged from animals about 1 billion years ago—undergo symmetry breaking and develop through cleavage divisions to produce a prolonged multicellular colony with distinct co-existing cell types. Our findings about the autonomous and palintomic developmental program of C. perkinsii hint that such multicellular development either is much older than previously thought or evolved convergently in ichthyosporeans.

Much better. The key points are:

  • C. perkinsii is a member of a lineage that diverged from the line that led to animals about a billion years ago. It’s ancient, but it exhibits certain patterns of cell division that resemble those of modern animals.
  • Symmetry breaking is a simple but essential precursor to the formation of different cell types. The alternative is equipotential cell division, one that produces two identical cells with equivalent cellular destinies. Making the two daughter cells different from each other other opens the door to greater specialization.
  • Palintomic division is another element of that specialization. Many single-celled organisms split in two, and each individual begins independent growth. Palintomic division involves the parent cell undergoing a series of divisions without increasing the total cell volume. They divide to produce a pool of much smaller cells. This is the pattern we see in animal (and plant!) blastulas: big cell dividing multiple times to make a pile of small cells that can differentiate into different tissues.
  • Autonomy is also a big deal. They looked at transcriptional activity to see that daughter cells had different patterns of gene activity — some cells adopt an immobile, proliferative state, while others develop flagella and are mobile. This is a step beyond forming a simply colonial organism, is a step on the path to true multicellularity.

Cool. The idea is that this organism suggests that single-celled organisms could have acquired a toolkit to enable the evolution of multicellularity long before their descendants became multicellular.

I have a few reservations. C. perkinsii hasn’t been sitting still — it’s had a billion years to evolve these characteristics. We don’t know if they’re ancestral or not. We don’t get any detailed breakdown of molecular homologies in this paper, so we also don’t know if the mechanisms driving the patterns are shared.

I was also struck by this illustration of the palintomic divisions the organism goes through.

a, Plasma membrane-stained (PM) live colonies at distinct cell stages, highlighting the patterned cleavage divisions, tetrahedral four-cell stage and formation of spatially organized multicellular colonies (Supplementary Video 5). b, Actin- (magenta) and DNA-stained (blue) colonies at distinct cell stages showcasing nuclear cortical positioning, asymmetrical cell division (in volume and in time) and the formation of a multicellular colony. This result has been reproduced at least three independent times.

Hang on there : that’s familiar. D’Arcy Wentworth Thompson wrote about the passive formation of cell-like cleavage patterns in simple substrates, like oil drops and soap bubbles, in his book On Growth and Form, over a century ago. You might notice that these non-biological things create patterns just like C. perkinsii.

Aggregations of oil-drops. (After Roux.) Figs. 4–6 represent successive changes in a single system.

Aggregations of four soap-bubbles, to shew various arrangements of the intermediate partition and polar furrows.

An “artificial tissue,” formed by coloured drops of sodium chloride solution diffusing in a less dense solution of the same salt.

That does not undermine the paper’s point, though. Multicellularity evolved from natural processes that long preceded the appearance of animals. No miracles required!

Wilson’s Principles of Teratology

It’s another busy week of EcoDevo, and even though the campus was closed I still had to give a lecture on endocrine disruptors. I started by laying out Wilson’s Principles of Teratology…wait, what? You don’t know them? I guess I’d better explain them to the internet at large.

These principles are a bit like Koch’s Principles, only for teratology — you better know them if you want to figure out the causes of various problems at birth, and you do: about 3% of all human births express a defect serious enough for concern. Here’s the list:

  1. Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with adverse environmental factors.
  2. Susceptibility to teratogenesis varies with the developmental stage at the time of exposure to an adverse influence. There are critical periods of susceptibility to agents and organ systems affected by these agents.
  3. Teratogenic agents act in specific ways on developing cells and tissues to initiate sequences of abnormal developmental events.
  4. The access of adverse influences to developing tissues depends on the nature of the influence. Several factors affect the ability of a teratogen to contact a developing conceptus, such as the nature of the agent itself, route and degree of maternal exposure, rate of placental transfer and systemic absorption, and composition of the maternal and embryonic/fetal genotypes.
  5. There are four manifestations of deviant development (death, malformation, growth retardation and functional defect).
  6. Manifestations of deviant development increase in frequency and degree as dosage increases from the No Observable Adverse Effect Level (NOAEL) to a dose producing 100% lethality (LD100).

The first two tell you what is tricky about teratology. There are multiple variables that affect the response: genetic variability in the conceptus (and, I would suggest, maternal variations), and also timing is critical. A drug might do terrible things to an embryo at 4 weeks, but at 3 months the fetus shrugs it off.

Ultimately, though, the teratogen is having some specific effect (3) on a developing tissue. We just have to figure out what it is, while keeping in mind that that effect might be hiding in a maze of genetics (1) and time (2).

Another complication is that in us mammals the embryo is sheltered deep inside the mother, who has defense mechanisms. The agent has to somehow get in (4). A complication within a complication: sometimes the teratogenic agent is harmless until Mom chemically modifies it as part of her defense, and instead creates a more potent poison.

#5 is just listing the terrible outcomes of screwing with development.

#6 I do not trust. It’s saying the effect is going to follow a common sense increase with increasing dosage, but even that isn’t always true. There is a phenomenon called the inverted-U response where the effect increases with dosage, then plateaus, and then drops off at high concentrations. We’re dealing with complex regulatory phenomena with multiple molecular actors that may have unpredictable interactions. There are teratogens that do terrible things to embryos at low concentrations, but do nothing at ridiculously high concentrations — as if the high dose triggers effective defense mechanisms that the low dose sidesteps.

I had to review these principles in class yesterday, because although I’d also discussed them earlier in the semester, we are currently dealing with teratogens of monstrous subtlety, these compounds that mimic our own normal developmental signals, the same signals our bodies use to assemble critical organ systems. It’s as if some joker were placing inappropriate traffic signals along a busy highway — most would do no harm, but some may totally confuse travelers who then end up detouring up into the kidneys rather than down the genitals, as they preferred, or they end up crashing into the thyroid.

Unfortunately, in this case the responsible jokers are mainly gigantic megacorporations who are spewing these dangerous signals all over the countryside…and then we get to wait until the people swimming in them try to have children, and then the teratologists get to say “death, malformation, growth retardation and functional defect”.

In case you were wondering, Wilson didn’t come up with his list first — a 19th century scientist named Gabriel Madeleine Camille Dareste did it first. No, not first. Lots of people have been documenting these developmental problems as long as there’s been writing, like on this Chaldean tablet:

When a woman gives birth to an infant:
With the ears of a lion There will be a powerful king
That wants the right ear The days of the king will be prolonged
That wants both ears There will be mourning in the country
Whose ears are both deformed The country will perish and the enemy rejoice
That has no mouth The mistress of the house will die
Whose nostrils are absent The country will be in affliction and the house of the man will be ruined
That has no tongue The house of the man will be ruined
That has no right hand The country will be convulsed by an earthquake
That has no fingers The town will have no births
That has the heart open with no skin The country will suffer from calamities
That has no penis The master of the house shall be enriched by the harvest of his field
Whose anus is closed The country shall suffer from want of nourishment
Whose right foot is absent His house will be ruined and there will be abundance in that of the neighbor
That has no feet The canals of the country will be cut and the house ruined
If a queen gives birth to:
An infant with teeth already cut The days of the king will be prolonged
A son and a daughter at the same time The land will be enlarged
An infant with the face of a lion The king will not have a rival
An infant with 6 toes on both feet The king shall rule the enemies’ country

Nowadays we’re more interested in causes than imagined consequences, I hope.

You know, alcohol is not good for children and other growing things

A few weeks ago, I had an absolutely delicious stout at a brew pub in Alexandria. I’m going to have to remember it, because it may have been the last time I let alcohol pass these lips. Why? Because I’m slowly turning into one of those snooty teetotalers who tut-tut over every tiny sin. It started with vegetarianism, now it’s giving up alcohol, where will it end? Refusing caffeine, turning down the enticements of naked women, refusing to dance? The bluenose in me is emerging as I get older. I shall become a withered, juiceless old Puritan with no joy left in me.

It didn’t help that last week I was lecturing on alcohol teratogenesis in my eco devo course, and it was reminding me of what a pernicious, sneaky molecule it is. I’ve known a lot of this stuff for years, but there’s a kind of blindness brought on by familiarity that led me to dismiss many of the problems. You know the phenomenon: “it won’t affect me, I only drink in moderation” and other excuses. Yeah, no. There are known mechanisms for how alcohol affects you, besides the obvious ones of inebriation.

  1. It induces cell death.
  2. It affects neural crest cell migration.
  3. It downregulates sonic hedgehog, essential for midline differentiation.
  4. It downregulates Sox5 and Ngn1, genes responsible for neuron growth and maturation.
  5. It weakens L1-modulated cell adhesion.

I already knew all about those first four — I’ve done experiments in zebrafish like these done in mice.

Take a normal, healthy embryo like the one in A, expose it to alcohol, and stain the brain for cell death with any of a number of indicator dyes, like Nile Blue sulfate in this example B (I’ve used acridine orange, it works the same way). That brain is speckled with dead cells, killed by alcohol. If you do it just right, you can also see selective cell death in neural crest cell populations, so you’re specifically killing cells involved in the formation of the face and the neurons that innervate it. In C, you can see the rescuing effects of superoxide dismutase, a free radical scavenger, and that tells you that one of the mechanisms behind the cell death is the cell-killing consequences of free radicals. I could get a similar reduction in the effects with megadoses of vitamin C, but that doesn’t mean a big glass of orange juice will save you from your whisky bender.

I was routinely generating one-eyed jawless fish, a consequence of the double-whammy of knocking out sonic hedgehog and cell death in the cells that make branchial arches.

You can wave away these results by pointing out those huge concentrations of alcohol we use to get those observable effects, but we only do that because we don’t have the proper sensitivity to detect subtle variations in the faces of mice or fish. So we crank up the dosage to get a big, undeniable effect.

I only just learned about the L1 effects, and that’s a case where we have a sensitive assay for alcohol’s effects. L1 is a cell surface adhesion molecule — it helps appropriate populations of cells stick together in the nervous system. It also facilitates neurite growth. It’s good for happy growing brains.

It also makes for a relatively easy and quantitative assay. Put neuronal progenitors that express L1 in a dish, and they clump together, as they should in normal development. Add a little alcohol to the medium, and they become less sticky, and the clumps disperse.

What’s troubling about this is the dosage. Adhesion is significantly reduced at concentration of 7mM, which is what the human blood alcohol level reaches after a single drink. The fetal brain may not be forming as robustly when Mom does a little social drinking that doesn’t leave her impaired at all, not even a slight buzz.

Maybe you console yourself by telling yourself a little bit does no harm, your liver soaks up most of the damage (and livers are self-repairing!), that it’s only binge drinkers who have to worry about fetal alcohol syndrome, etc., etc., etc. We have lots of excuses handy. Humans are actually surprisingly sensitive to environmental insults, we have mechanisms to compensate, but there’s no denying that we’re modifying our biochemistry and physiology in subtle ways by exposure to simple molecules.

Now maybe you also tell yourself that you’re a grown-up, I’m talking about fetal tissues, and you also don’t intend to get pregnant in the near future or ever. I’m also a great big fully adult person who is definitely not ever going to get pregnant, but development is a life-long process, and we’re all fragile creatures who nonetheless soak up all kinds of interesting and dangerous chemicals during our existence. We know alcohol will kill adult brain cells, but what else does it do? Do you want to be a guinea pig? I think that, as I age, I am becoming increasingly aware of all the bad stuff I did to myself in my heedless youth, and am starting to think that maybe I need to be a little more careful, belatedly.

Oh, you want some reassuring information? Next week we’re discussing endocrine disruptors in my class — DDT, DES, BPA, PCB, etc. — all these wonderful products of plastics and petrochemical technology. You’re soaking in them right now. They never go away. How’s your sperm count looking? Any weird glandular dysplasias? Ethanol looks pretty good compared to chlorinated and brominated biphenyls.

Grow a segmented spine!

You want to see something awesome? This is a video of human embryonic cells developing in a dish and activating the segmentation clock to build a series of somites.

That embryos do this is pretty neat but not surprising. It’s easy to see that process going on in zebrafish embryos, where a new segmental blob pinches off every 20 minutes. What’s cool is that they’ve pared down the system to just what looks like presomitic mesoderm — no gut endoderm, no nervous system or ectoderm, just those mesodermal cells grouping and organizing to make pre-muscle tissue. At first, I thought, no big deal. We’ve observed vertebrate segmentation in fish and frogs and chicks for many decades, why would we need to look at it in human embryos? They almost certainly use the same molecules and interactions as mice or any other vertebrate. It’s got to be using Notch signaling, after all.

What’s special here, though, is that they’ve reduced the complexity of the system to a remarkable degree. You don’t need a nervous system or a gut or even, it seems, a notochord to assemble segmental organization. They’re even getting colinear expression of HOX genes! That’s the surprise to me, that so little is required to trigger oscillatory expression and patterning. And that’s the point the authors emphasize.

Our bottom-up experimental approach demonstrates that complex developmental events such as somitogenesis, can be deconstructed and dissected into discrete “building blocks” of developmental principles which are usually intricately connected and cannot be easily uncoupled in vivo. Axioloids, a self-organizing in vitro model of human axial development allowed us to individually assess and manipulate such building blocks at the molecular, cellular and morphogenetic level. Axioloids -a surrogate model of the human embryonic tail and forming axis- are capable of recapitulating core features of human somitogenesis, and represent an exciting new platform to investigate axial development and disease in a human context.

Don’t read the YouTube comments. There are people complaining that it’s a human life with a unique genetic code (no, it’s not a person, and these embryonic cultures cannot develop into a person — while scraps of mesoderm might be enough to make somites, it’s not sufficient to build a whole human being) and Stop trying to PLAY GOD!!! (this isn’t god-like, it’s biology).

Bad science tries to drip its way into everything

You want to read a really good take-down of a bad science paper? Here you go. It’s a plea to Elsevier to retract a paper published in Personality and Individual Differences because…well, it’s racist garbage, frequently cited by racists who don’t understand the science but love the garbage interpretation. It really is a sign that we need better reviewers to catch this crap.

The paper is by Rushton, who polluted the scientific literature for decades, and Templer, published in 2012. It’s titled “Do pigmentation and the melanocortin system modulate aggression and sexuality in humans as they do in other animals?”, and you can tell what it’s trying to do: it’s trying to claim there is a genetic linkage between skin color and sexual behavior and violence, justifying it with an appeal to biology. It fails, because the authors don’t understand biology or genetics.

They’re advocating something called the pleiotropy hypothesis, which is the idea that every gene has multiple effects (this is true!), and that therefore every phenotype has effects that ripple across to every other phenotype (partially, probably mostly true), so that seeing one aspect of a phenotype means you can make valid predictions about other aspects of the phenotype (mostly not at all true). This allows them to abuse a study in other mammals to claim that human outcomes are identical. Here’s the key graf:

The basis of the pleiotropy hypothesis presented by Rushton and Templer hinges on a citation from Ducrest et al. (2008), which posits ‘pleiotropic effects of the melanocortins might account for the widespread covariance between melanin-based coloration and other phenotypic traits in vertebrates.’ However, Rushton and Templer misrepresent this work by extending it to humans, even though Ducrest et al. (2008) explicitly state, ‘these predictions hold only when variation in melanin-based coloration is mediated by variation in the level of the agonists at MC1R… [conversely] there should be no consistent association between melanin-based coloration and other phenotypic traits when variation in coloration is due to mutations at effectors of melanogenesis such as MC1R [as is the case in humans].’ Ducrest et al. continue, ‘variation in melanin-based coloration between human populations is primarily due to mutations at, for example, MC1R, TYR, MATP and SLC24A5 [29,30] and that human populations are therefore not expected to consistently exhibit the associations between melanin-based coloration and the physiological and behavioural traits reported in our study’ [emphasis mine]. Rushton and Templer ignore this critical passage, saying only ‘Ducrest et al. (2008) [caution that], because of genetic mutations, melanin-based coloration may not exhibit these traits consistently across human populations.’ This is misleading. The issue is not that genetic mutations will make melanin-based pleiotropy inconsistent across human populations, but that the genes responsible for skin pigmentation in humans are completely different to the genes Ducrest et al. describe.

To translate…developmental biologists and geneticists are familiar with the concept of an epistatic pathway, that is, of genes affecting the expression of other genes. So, for instance, Gene A might switch on Gene B which switches on Gene C, in an oversimplified pattern of regulation.

Nothing is ever that simple, we know. Gene A might also switch on Gene Delta and Gene Gamma — this is called pleiotropy, where one gene has multiple effects. And Gene Gamma might also activate Gene B, and Gene B might feed back on Gene A, and B might have pleiotropic effects on Gene Beta and Gene E and Gene C.

This stuff gets delightfully tangled, and is one of the reasons I love developmental biology. Everything is one big complex network of interactions.

What does this have to do with Rushton & Templer’s faulty interpretation? They looked at a study that identified mutations in a highly pleiotropic component of the pigmentation pathway — basically, they’re discussing Gene A in my cartoon — and equating that to a terminal gene in humans, equivalent to Gene C in my diagram. Human variations in skin color are mostly due to mutations in effector genes at the end of the pathway, like MC1R. It will have limited pleiotropic effects compared to genes higher up in the epistatic hierarchy, like the ones Ducrest et al. described. Worst of all, Ducrest et al. explicitly discussed how the kind of comparison Rushton & Templer would make is invalid! They had to willfully edit the conclusions to make their argument, which is more than a little dishonest.

It reminds me of another recent disclosure of a creationist paper that also misrepresented its results. This paper, published in the International Journal of Neuroscience, openly declared that it had evidence for creationism.

In the paper, Kuznetsov reportedly identified an mRNA from one vole species that blocked protein synthesis in a related vole species. That same mRNA, however, did not block translation in the original vole species or another species that was more distantly related. The finding, Kuznetsov wrote in his report, supported “the general creationist concept on the problems of the origin of boundless multitudes of different and harmonically functioning forms of life.”

I vaguely remember reading that paper and rolling my eyes at how weak and sloppy the data was — it was never taken seriously by anyone but creationists. I don’t recall the details, though, because it was published 30 years ago, and is only now being retracted, after decades of the author fabricating data and being so obvious about it that he was fired as editor of two journals in 2013. The guy had a reputation, shall we say. Yet he managed to maintain this academic facade for years.

Phillipe Rushton had similarly managed to keep up the pretense of being a serious academic for an awfully long time, right up until his death in 2012. He used his reputation to spray all kinds of fecal nonsense into the scientific literature, and that’s why you have to maintain a skeptical perspective even when reading prestigious journals.

Tissue Organization Field Theory

It’s been a while since I brought everyone up to date on the progress of my Ecological Development course, because I’ve been busy. So have the students. After our spring break I subjected them to the dreaded oral exam, which actually isn’t so bad. I tried to engage them less in an adversarial role and more as a quiet conversation between two people on science. Some students took to it easily — the more outgoing ones — others were noticeably nervous, which was OK, and I hope they learned that it isn’t that terrifying to have a discussion with a mentor.

Then the next few weeks were a mad whirl of horrible things done to babies: teratogens, endocrine disruptors, multi-generational epigenetic inheritance, all that fun stuff. We wrapped up with the depressing stuff for me, although the young’uns were more sanguine, I think. We talked about adult onset developmental diseases (it’s good to look at heart disease through the lens of developmental biology), aging, and cancer. Next week they get some time off, because I’m being drawn away to a conference on the east coast, but they’re supposed to spend it preparing for their final presentations, which will consume the last two weeks of class. And then we’re all done. School’s out for summer!

I’m going to say a bit about our last class discussion, because it got into some interesting territory and reflects the theme of the course well. We talked about that theory mentioned in the title of this article, and the origins of cancer, and to do that I have to give you all a little background.

Tissue Organization Field Theory (TOFT) is an alternative to what is sort of the dominant paradigm in cancer biology, the Somatic Mutation Theory (SMT). I have to say “sort of” because what I get from the literature is that SMT is more of a working assumption, and that cancer biologists are open to new ideas. The SMT is a useful molecular perspective on carcinogenesis. It postulates that cancer is a cellular disorder in which the genetic material has been perturbed to produce a lineage of cells with aberrant characteristics, that if we want to figure out what the primary cause of a cancer was, we can trace it back to a somatic mutation, or a change in a critical gene or, more likely, multiple genes, that lead to uncontrolled proliferation. So we pursue oncogenes, genes that have the potential to acquire mutations that trigger cell division or bypass control points, and tumor suppressor genes, protective genes that, when damaged, remove essential regulators of growth.

So, under the SMT, cancer is a disease caused by the progressive accumulation of mutations in cells of the body, as they divide. These mutations gradually strip away the normal restraints on cell division, and on immune system recognition, and on cell death activation, etc., etc., etc. until you have a rogue cell that can seed the growth of a massively disruptive tumor.

And it’s not wrong! Cancer biology has been immensely productive in identifying the enabling mutations, and even developing treatments that specifically target molecular agents of cancer. We know that somatic mutations are a routine part of the progression of cancer, and we also know that there heritable alleles that can affect the likelihood of the disease. The SMT is a tool to explain many of the phenomena of cancer, and it’s not going to just go away. It’s also a tool that is amenable to a reductionist approach to cancer biology, and is well-adapted to the utility of molecular biology.

Tissue Organization Field Theory is an alternative explanation for the origins of cancer.

TOFT argues that the focus of the SMT on single cell events is inappropriate and misses a whole range of effects at the level of tissue organization, effects which are more important in creating a pathological environment in which those mutations can accumulate. Further, it gets into field theory, which is important in developmental biology but isn’t exactly the subject of common conversation. Here’s one standard definition of a field: “a morphogenetic (or developmental) field is a region or a part of the embryo which responds as a coordinated unit to embryonic induction and results in complex or multiple anatomic structures.” If that’s not helpful — and it probably isn’t, we’d have to go over a textbook if we wanted to explain developmental field theory — here’s a diagramatic metaphor. Do you see the field in this picture?

There’s something special about part of that image, but it’s not that the individual subunits are intrinsically different — it’s tied up in the relationships between the central set of blocks and the blocks outside of it. There’s something different going on with a subset of the blocks, but it’s not necessarily best described by explaining the details of single blocks, but is more easily explained at a higher level, as properties of a tissue within a tissue. Of course, what will eventually happen in a developing organism is that those central blocks will express a unique pattern of genes, so eventually it’s identifiable by molecular markers, but the field first arises in a sea of genetically and epigenetically uniform cells.

Another important property of a field is that it is not itself uniform. It’s going to acquire complex spatial properties over time. Insect limbs, for instance, arise from a disc-shaped field with extensive patterning information within them, so the central region will become the distal tip of the limb, and there is information that is interpreted as polar coordinates that specifies what portion of the limb is anterior, posterior, medial, and lateral (the limb is not a uniform cylinder). Similarly, vertebrates have a limb field represented in the limb bud, with gradients of morphogens specifying the orientation of the limb, and with re-expression of Hox genes used to specify longitudinal positions. Hox genes in a limb field are interpreted in different ways than Hox genes along the body axis, obviously.

The key factor here is that in field theory cells are not simply independent units — they are part of a larger assemblage, a tissue, that has complex fates that are not easily summarized by individual gene expression. They have to be understood as a network.

That’s the first thing to remember: TOFT is treating a cancer as a field, with field properties, which are not adequately described if you only look at cancer as a collection of autonomous cells all doing their own thing at the command of their broken genes. Aberrant disruption of the field can produce aberrant structures without requiring any genetic changes.

This is the difference between a mutagen and a teratogen. The effects of a mutagen are caused directly by damage to the structure or sequence of DNA; they produce heritable changes to the cells of an organism. Teratogens, on the other hand, are not necessarily mutagenic at all — they disrupt the normal pattern of development without changing genes at all. Thalidomide babies, for example, had some extreme morphological changes, like phocomelia or truncated limb development, but those are not heritable, and the people affected by thalidomide can grow up to have normal, healthy children.

TOFT argues something similar, that there is a disruption of a tissue that initializes aberrant growth, that may then be an enabling precondition for the accumulation of mutations. One piece of evidence for this is a set of experiments on tissues, illustrated below.

Most cancers arise in epithelial tissues, like the sheets of cells that line glands or your organs, in large part because those are the cells that divide most frequently. These epithelial cells, also called parenchyma, do not typically grow in isolation, but on a substrated of connective tissue, extracellular matrix, and other cell types, called stroma. The stroma supports and signals the overlying epithelium, and vice versa, and together they make a coherent functional tissue.

The theory suggests that cancers can arise in epithelia by way of disruptions in signaling in the stroma. A carcinogen could distort the interactions between stroma and epithelium at the level of the stroma, and the epithelium then goes nuts and proliferates to produce a pre-cancerous mass.

One test of the theory would be to separate stroma and epithelium, expose the stroma to a short-lived teratogen, and then after the teratogen was washed out, re-associate the two and determine whether there was an increase in the frequency of cancers in the epithelium, which has not been exposed to teratogens.

The experiment has been done. Here are the results for rat mammary gland tissue in which the epithelium was exposed to the solvent vehicle but no N-methyl nitorsourea (a potent mutagen), while the stroma was soaked in NMu, labeled VEH/NMu. The numerator describes the epithelial condition, and the denominator is the stroma condition, so NMu/NMu means both were hit with the mutagen, VEH/VEH means both were exposed only to the vehicle, and NMu/VEH means the epithelium was poisoned with NMu, while the stroma was not.

There’s an awfully strong positive correlation between exposing the stroma to mutagens and getting tumors, and a negative correlation with exposing epithelia to mutagens and tumors.

You want more evidence? Here’s a very interesting experiment. Start with aggressively metastatic melanoma cells from a human patient (labeled in green, below). Inject them into a completely different environment, the neural crest pathway in a developing chick embryo. Surprisingly, if you accept the SMT, the cancer cells calm right down and are conditioned by their environment to participate in normal development in the chick and get incorporated into the facial cartilages and sympathetic ganglia.

I suspect those melanoma cells do carry somatic mutations, and are not actually “cured” of a predisposition to cancer. What the experiment says, though, is that environmental influences are extremely important in regulating the behavior of these cells, and that modifying the cells communicating with the cancerous cells can have a profound effect on how they act.

Note that this is not a pathway to a cure. It’s all well and good to say that if we could break up a tumor, separate the individual cells and put them in a more nurturing, embryo-like environment, they’ll stop acting up and resume normal, regulated growth, but if we could do that, slicing out the tumor and tossing it in an incinerator would also be effective. The problem is that in a human patient we do not and cannot have such precise control of the micro-environment of the cancer, and in fact, the tumor itself is a kind of bubble of micro-environment that actively reinforces cancer growth.

My students and I read a paper from Carlos Sonnenschein, who is a major proponent of TOFT, as well as our textbook summary. The paper was titled The tissue organization field theory of cancer: a testable replacement for the somatic mutation theory. They’re a smart bunch, and they see the promise of the idea, in part because this whole course is about thinking a level above reductionist cell biology, but they also found the word “replacement” off-putting. It doesn’t invalidate everything about the SMT, but it does support an important alternative route for carcinogenesis. They also weren’t impressed by the rather aggressive insistence by some TOFT proponents that they have the One True Explanation, and that their observations are sufficent to explain cancer — we came up with a few alternative interpretations of their own favorite experiments that they haven’t nailed down completely just yet.

One thing that amused me is that the class consensus actually converged on the views of another paper by Bedessem and Ruphy, which I did not assign them to read, largely because of its more philosophical argument (I’ve focused on empirical/experimental papers in the class). This is how I feel about it, too.

The building of a global model of carcinogenesis is one of modern biology’s greatest challenges. The traditional somatic mutation theory (SMT) is now supplemented by a new approach, called the Tissue Organization Field Theory (TOFT). According to TOFT, the original source of cancer is loss of tissue organization rather than genetic mutations. In this paper, we study the argumentative strategy used by the advocates of TOFT to impose their view. In particular, we criticize their claim of incompatibility used to justify the necessity to definitively reject SMT. First, we note that since it is difficult to build a non-ambiguous experimental demonstration of the superiority of TOFT, its partisans add epistemological and metaphysical arguments to the debate. This argumentative strategy allows them to defend the necessity of a paradigm shift, with TOFT superseding SMT. To do so, they introduce a notion of incompatibility, which they actually use as the Kuhnian notion of incommensurability. To justify this so-called incompatibility between the two theories of cancer, they move the debate to a metaphysical ground by assimilating the controversy to a fundamental opposition between reductionism and organicism. We show here that this argumentative strategy is specious, because it does not demonstrate clearly that TOFT is an organicist theory. Since it shares with SMT its vocabulary, its ontology and its methodology, it appears that a claim of incompatibility based on this metaphysical plan is not fully justified in the present state of the debate. We conclude that it is more cogent to argue that the two theories are compatible, both biologically and metaphysically. We propose to consider that TOFT and SMT describe two distinct and compatible causal pathways to carcinogenesis. This view is coherent with the existence of integrative approaches, and suggests that they have a higher epistemic value than the two theories taken separately.

Anyway, keep an eye open for more on the tissue organization field theory — there seems to be a fair bit of ongoing debate in the scientific literature about it. I’ll keep telling everyone cancer is a developmental disease, so you need more developmental biologists to study it. Or, alternatively, every cancer biologist is already a developmental biologist.

Bedessem B, Ruphy S. (2015) SMT or TOFT? How the two main theories of carcinogenesis are made (artificially) incompatible. Acta Biotheor. 63(3):257-67.

Soto AM, Sonnenschein C (2011) The tissue organization field theory of cancer: a testable replacement for the somatic mutation theory. Bioessays. 33(5):332-40.

Steve Pinker’s hair and the muscles of worms

I’ve been guilty of teaching bean-bag genetics this semester. Bean-bag genetics treats individuals as a bag of irrelevant shape containing a collection of alleles (the “beans”) that are sorted and disseminated by the rules of Mendel, and at its worst, assigns one trait to one allele; it’s highly unrealistic. In my defense, it was necessary — first-year students struggle enough with the basic logic of elementary transmission genetics without adding great complications — and of course, in some contexts, such as population genetics, it is a useful simplification. It’s just anathema to anyone more interested in the physiological and developmental side of genetics.

The heart of the problem is that it ignores the issue of translating genotype into phenotype. If you’ve ever had a basic genetics course, it’s quite common to have been taught only one concept about the phenotype problem: that an allele is either dominant, in which case it is expressed as the phenotype, or it’s recessive, in which case it is completely ignored unless it’s the only allele present. This idea is so 19th century — it’s an approximation made in the complete absence of any knowledge of the nature of genes.

And the “one gene, one trait” model violates everything we do know about the phenotype and genotype. Every gene is pleiotropic — it influences multiple traits to varying degrees. Every trait is multigenic — multiple genes contribute to the expression of every phenotypic detail. The bean-bag model is totally inadequate for describing the relationship of genes to physiology and morphology. Instead of a bean-bag, I prefer to think of the genome as comparable to a power spectrum, an expression of the organism in a completely different domain. But I wrote about that previously, and I’ll make this explanation a little simpler.

Here’s the problem: you can’t always reliably predict the phenotype from the genotype. We have a skewed perspective on the problem, because historically, genetics has first searched for strong phenotypes, and then gone looking for the genetic cause. We’ve been effectively blind to many subtle phenotypic effects, simply because we don’t know how to find them. When we go the other way, and start by mutating known genes and then looking for changes in the phenotype, we’re often surprised to discover no detectable change. One of the classic examples is the work of Elkins (1990), who found that mutating a neural cell adhesion gene, Fasciclin I, did not generate any gross defects. Mutating another gene, a signal transduction gene called Abelson tyrosine kinase, similarly had no visible effects. Mutating the two together, though — and this is a major clue to how these strange absences of effect could work — did produce gross and obvious effects on nervous system development.

Providing another great example, Steve Pinker examined his own genome, and discovered that his genes said he was predisposed to be red-haired and at high risk for baldness. If you’ve seen Steve Pinker, you know he’s neither.

How can this be? As any geneticist will tell you, the background — the other alleles present in the organism — are important in defining the pattern of expression of a specific gene of interest. One simple possibility is that the genome contains redundancy: that a trait such as adhesion of axons in the nervous system or the amount of hair on the head can be the product of multiple genes, each doing pretty much the same thing, so knocking out one doesn’t have a strong effect, because there is a backup present.

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Genetic interactions provide a general model for incomplete penetrance. Representation of a negative (synergistic) genetic interaction between two genes A and B.

So Steve Pinker could have seen that he has a defective Gene A, which is important in regulating hair, but maybe there’s another Gene B lurking in the system that we haven’t characterized yet, and which can compensate for a missing Gene A, and he has a particularly strong form of it. One explanation for a variable association between an allele and the phenotype, then, is that we simply don’t have all the information about the multigenic cause of the phenotype, and there are other genes that can contribute.

This doesn’t explain all of the observed phenomena, however. Identical twins who share the same complement of alleles also exhibit variability in the phenotype; we also have isogenic animal lines, where every individual has the same genetic complement, and they also show variability in phenotype. This is the problem of penetrance; penetrance is a genetics term that refers to the likelihood that an individual carrying an allele will actually express the phenotype associated with that allele…and it’s not always 100%.

Again, the explanation lies in the other genes present in the organism. No gene functions all by itself; its expression is dependent on a cloud of other proteins — transcription factors, enhancers, chaperones — all of which modulate the gene of interest. We also have to deal with statistical variation in the degree of expression of all those modulatory factors, which vary by chance from cell to cell, and so the actual degree of activation of a gene may follow a kind of bell curve distribution. In the cartoon below, the little diamonds represent these partners; sometimes, just by chance, they’ll be present in sufficiently high numbers to boost Gene B’s output enough to fully compensate for a defective Gene A; in other cases, just by chance, they’re too low in concentration to adequately compensate for the absence.

i-4e599384fc1ad3453f1e71306459fde4-penetrance.jpeg
Genetic interactions provide a general model for incomplete penetrance. A model for incomplete penetrance based on variation in the activity of genetic interaction partners.

What the above cartoon illustrates is the concept of developmental noise, the idea that the cumulative total of statistical variation in gene expression during development can produce significant phenotypic variation in the absence of any differences in the genotype. Developmental noise is a phrase bruited about quite a bit, and there’s good reason to think it’s valid: we can see quantitative variation in gene expression with molecular techniques, for instance. But at the same time we have other concepts, like redundancy and canalization, that work to buffer variation and produce reliable outputs from developmental processes, so we don’t have many good examples where we can directly correlate subtle variation at the molecular level with clear morphological differences.

To test that, we have to go to simple animal models (it turns out that Steve Pinker is a rather intractable experimental animal). And here we have a very nice example in the nematode worm, C. elegans. In these experiments, the investigators were dealing with an isogenic strain — the genetic background was identical in all of the animals — raised in a uniform environment. They were looking at a mutant in the gene tbf9, which causes defects in muscle formation, but only 50% penetrance; that is, half the time, the mutants appeared completely normal, and the other half of the time they had grossly abnormal muscle development.

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Genetic interactions provide a general model for incomplete penetrance. Inactivation of the gene tbx-9 in C. elegans results in an incompletely penetrant defect, with approximately half of embryos hatching with abnormal morphology (small arrow).

See the big red question mark? That’s the big question: can we trace the abnormal phenotype all the way back to random fluctuations in the expression of other genes in the animal? Yes, they can, otherwise it would never have been published in Nature and I wouldn’t be writing about it now.

In this case, they have a situation analogous to the Gene A/Gene B cartoons above. Gene B is tbx-9; Gene B is a related gene, a duplicate called tbx-8 which acts as a redundant copy. In the experiments below, they knock out tbx-9 with a mutation, and then measure the quantity of other genes in the system using a very precise technique of quantitative fluorescence. Below, I’ve reproduced the entirety of their summary figure, because it is awesome — I just love the idea of being able to count the number of molecules expressed in a developing system. In order to avoid overwhelming everyone, though, I’ll just describe a couple of the panels to give you the gist of the work.

First, just look at the top left panel, a. It’s a plot of the level of expression of the tbx-8 gene over time, where each line in the plot is a different animal. The lines in black are in the wild type animal, with fully functional copies of bothe tbx-8 and tbx-9, and you should be able to see that there’s a fair amount of variation in expression, about two-fold, in different individuals. The lines in green are from animals mutant for tbx-9; it’s messy, but statistically what happens when tbx-9 is knocked out, more tbx-8 gene product is produced.

Panel e, just below it, shows the complementary experiment: the expression of tbx-9 is shown for both wild type (black) and animals with tbx-8 knocked out. Here, the difference is very clear: tbx-9 levels are greatly elevated in the absence of tbx-8. This shows that tbx-8 and tbx-9 are actually tied together in a regulatory relationship where levels of one rise in response to reduced levels of the other, and vice versa.

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(Click for larger image)
Early inter-individual variation in the induction of ancestral gene duplicates predicts the outcome of inherited mutations. a, Quantification of total green fluorescent protein (GFP) expression from a tbx-8 reporter during embryonic development in WT (black) and tbx-9(ok2473) (green) individuals. Each individual is a separate line. a.u., Arbitrary units. b, Boxplot of tbx-8 reporter expression (a) showing 1.2-fold upregulation in a tbx-9 mutant at comma stage (~290 min, P=1.6×3 10-3, Wilcoxon rank test). c, Expression of tbx-8 reporter in a tbx-9(ok2473) background for embryos that hatch with (red) or without (blue, WT) a morphological defect. d, Boxplot of c showing tbx-8 expression is higher in tbx-9 embryos that develop a WT phenotype (blue) compared with those that develop an abnormal (red) phenotype at comma stage (P= 6.1×10-3). e, Expression of a ptbx-9::GFP reporter in WT (black) and tbx-8(ok656) mutant (green). f, Boxplot of tbx-9 reporter showing 4.3-fold upregulation at comma stage (~375 min, P=3.6×10-16). g, Expression of tbx-9 reporter in a tbx-8(ok656) mutant background, colour code as in
c. h, Boxplot of g showing tbx-9 expression is higher in tbx-8 embryos that develop a WT phenotype (P=0.033). i, Expression of a pflh-2::GFP reporter in WT (black) and flh-1(bc374) mutant (green). j, Boxplot of flh-2 reporter expression (i) showing 1.8-fold upregulation in a flh-1 mutant at comma stage (~180 min, P=2.2×10-16). k, Bright-field and fluorescence image of an approximate 100-cell flh-1; pflh-2::GFP embryo. Red arrow indicates the local expression of flh-2 reporter quantified for flh-1 phenotypic prediction.
l, Boxplot showing higher flh-2 reporter expression at approximate 100 cells for WT (blue) compared with abnormal (red) phenotypes (P=0.014). Boxplots show the median, quartiles, maximum and minimum expression in each data set.

Now skip over to the right, to panel c. All of the lines in this plot are of tbx-8 expression in tbx-9 mutants, and again you see a wide variation in levels of gene expression. In addition, the lines are color-coded by whether the worm developed normally (blue), or had the mutant phenotype (red). The answer: worms with low tbx-8 levels were more likely to have the abnormal phenotype than those with high levels.

Panel g, just below it, is the complementary analysis of tbx-9 levels in tbx-8 mutants, and it gives the same answer.

Obviously, though, there is still a lot of variability unaccounted for; having relatively high levels of one or the other of the tbx genes didn’t automatically mean the worm developed a wild-type phenotype. There’s got to be something more that is varying. Look way back to the second cartoon I showed, with the little diamonds representing the cloud of transcription factors and chaperone proteins that modulate gene expression. Could there also be correlated variation there? And yes, there is. The authors looked at a chaperone protein called daf-21 that is associated with the tbx system, and found, in mutants for tbx-9, that elevated levels of daf-21 were associated with wildtype morphology (in blue), while lowered levels of daf-21 were associated with the mutant phenotype.

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(Click for larger image)
Expression of daf-21 reporter in a tbx-9(ok2473) mutant background. Embryos that hatch into phenotypically WT worms (blue) have higher expression than those hatching with a morphological defect (red) at the comma stage (P=1.9×10-3).

I know what you’re thinking: there isn’t a perfect correlation between high daf-21 levels and wildtype morphology either. But when they do double-label experiments, and take into account both daf-21 and tbx-8 levels in tbx-9 mutants, they found that 92% of the animals with greater than median levels of expression of both daf-21 and tbx-8 had wildtype morphology. It’s still not perfect, but it’s pretty darned good, and besides, it’s no surprise that there are probably other modulatory factors with statistical variation lurking in the system.

What should you learn from this? Developmental noise is real, and is a product of statistical variation in the degree of expression of multiple genetic components that contribute to a phenotype. We can measure that molecular variation in living, developing systems and correlate it phenotypic outcomes. None of this is surprising; we expect that the process of gene expression is going to be a bit noisy, especially in these transcriptional regulators that are present in low concentration in the cell, anyway. But the other cool thing we can observe here is that having multiple noisy systems that interact with each other can produce a more reliable, robust signal and contribute to the fidelity of developmental outcomes.

Burga A, Casanueva MO, Lehner B (2011)
Predicting mutation outcome from early stochastic variation in genetic interaction partners. Nature 480(7376):250-3.

Elkins T, Zinn K, McAllister L, Hoffmann FM, Goodman CS (1990)
Genetic analysis of a Drosophila neural cell adhesion molecule: interaction of fasciclin I and Abelson tyrosine kinase mutations. Cell 60(4):565-75.

(Also on FtB)