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.

Endocrine disruptors — you’re soaking in them

A human embryo at the 4th week of development is just a tiny bean with a length measured in millimeters, but at this time all kinds of remarkable features are starting to develop. This week in class I talked about urogenital development, which involves forming an array of incredibly delicate, thin tubes from a structure called the urogenital ridge, a thickening of an embryonic membrane which will eventually form a succession of kidneys, the pronephros, mesonephros, and metanephros, only the last persisting into adulthood. The key feature for the story I was telling, though, is that they formed something called the mesonephric duct, and then the paramesonephric duct which parallels it. Another name for the mesonephric duct is the Wolffian duct, and the paramesonephric duct is called the Müllerian duct (personally, I don’t care for the self-serving names given to critical bits of the developing embryo by 19th century men, but it’s what still persists in the embryo. So it goes.)

Both of these ducts are associated with the bipotential or indifferent gonad. There are no sexual differences in embryos this young.

The sex differences emerge later, in response to differential signals. The Müllerian ducts degenerate in males, while the Wolffian ducts persist. In females, the Müllerian ducts persist, while the Wolffian ducts fade away. The bipotential gonad associates with the remaining duct and differentiates into testes or ovaries.

I’ll refrain from delving deeper into the details. My point is that these minuscule ducts and tissues form very early, and are going to expand to form critical, elaborate structures necessary for human fertility. They’re fragile. You really don’t want to perturb the signals and processes going on in a one month old embryo, especially since you may not see the consequences for 15 or 20 years.

In 1941, pharmaceutical companies started to market a synthetic drug with properties similar to estrogen, called diethylstilbesterol, or DES. It wasn’t patented, so anyone could make and sell it, and they pushed it hard to pregnant women. There was weak evidence that it could help sustain pregnancies in women with low progesterone levels, so sure, let’s market it as “routine prophylaxis in ALL pregnancies.” About 4 million pregnant women took this stuff at the suggestion of their doctors, between 1941 and 1971, when it was finally banned.

Think about that. This was an endocrine disruptor, term that wasn’t invented until the 1990s, but everyone knew then that it would have some kind of effect, since it was a functional analog of estrogen. So they gave it to pregnant women, and by that means delivered a potent hormonal signal to their embryos at a time when they were carefully assembling those delicate little tubes. Worse, they knew that high doses given to mice and hamsters caused mammary, cervical, vaginal, and uterine cancers in adult females, and that adult males developed lung cancers, which ought to have set off all kinds of alarm bells. Any tissue that was sensitive to estrogen could be provoked to turn cancerous with DES.

Just for dessert, it was determined in 1953 that DES did nothing to maintain at risk pregnancies. They continued to prescribe the stuff. Just in case, you know.

For additional profit, they also marketed it as a growth hormone for livestock. That continued until it was eventually banned for that purpose in 1979.

Here’s the structure of this potent little molecule.

A is DES; B is estrogen; C is BPA, the common, heavily used plasticizer that we now know is an endocrine disruptor.

You might be wondering what happened to those 4 million women who took the drug. They were fine! Humans and other primates seem to be more resistant to the carcinogenic effects of DES, and they were taking much lower doses than those poor rodents in testing labs who were given massive doses of the drug.

And what about the millions of boomer babies who were doped with it in utero? Again, mostly fine — this is the thing about endocrine disruptors, they tend not to have the gross teratogenic effects we associate with chemicals that cause significant birth defects, like thalidomide. They’re more subtle. They perturb the balance of internal organ systems, and in this case, cause problems in the physiology of reproductive organs, which may lead to fertility issues or some kinds of cancers. I emphasize may because I know DES-exposed people who have had children and are cancer-free; it’s more a matter of letting their gynecologists know to keep an eye on potential warning signs.

But it can go very wrong.

DES is still used in experimental studies because it’s such an interesting molecule. Regular readers probably know about the importance of Hox genes; these are genes expressed along the body axis in pretty much all animals that defined anterior-posterior structures. The same genes also get re-expressed to define the proximal-distal axis of the tetrapod limb. They seem to be a handy-dandy molecular tool for establishing tissue identities along a line.

Here’s another instance of Hox genes defining position on an organ: they’re re-expressed in the Müllerian ducts, which become the fallopian tubes of adult women.

Hoxa9 is expressed throughout the oviduct, Hoxa13 in only the cervix, and Hoxa11 and Hoxa10 in between, forming a kind of positional coding system. This is really neat! I like finding examples of molecular recycling in the evolution of developing systems.

What isn’t so neat is that DES downregulates Hoxa10 by inhibiting an important signaling molecule, Wnt7a, creating coding ambiguities in the structure of that delicate little tube. That leads to poor cell specification and disorganized tissue, erasing what should be clear, sharp boundaries in the organ, which may then be expressed in dysplasias, increasing the odds of cancer.

As if that weren’t enough, we don’t really know what perturbing these signaling pathways does to other developing organs, like the brain. Also, DES affects methylation/demethylation of the genome, so it may have transgenerational effects — pregnant women who took DES may have messed up their children, but there is some evidence (weak, I think) that it also affects their grandchildren.

But wait! It’s banned, so we shouldn’t have to worry about it anymore! That’s partly true, but look at the diagram of the molecules above. Estrogen and DES share similarities to another molecule, bisphenol A, a ubiquitous plasticizer used to make plastic materials less brittle. BPA is found in your food packaging. It lines the interior of aluminum cans. Any plastic you have that is at all flexible has been treated with plasticizers, like BPA or phthalates. It’s leaching into your food and your general environment, and it does not go away. The US has banned its use in baby bottles and baby formula packaging, but not from all your snack food packages and your phones.

If you’re of a certain age, you might recall those commercials for a dishwashing detergent that announced, “you’re soaking in it!“, as if that meant the stuff must be safe. We ought to be aware that capitalist industries have us all soaking in a gentle bath of toxic chemicals right now, and it’s not safe.

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.

Am I creepy? Kooky? Altogether ooky?

There may be something wrong with me. I just spent a happy hour and twenty minutes watching a video about brown recluse spiders, and my only regret was that we don’t have any Loxosceles living anywhere near me. We don’t have any medically significant venomous spiders in this region — it’s one of my only regrets about living in west central Minnesota.

See? Fascinating. Good bit on horizontal gene transfer of the sphingomyelin toxin, lots of practical advice on brown recluse bites, and the spiders are all gentle and generally kind. It tickles my brain in all the right spots. Is that weird?

And then, the best essay I’ve read this week is all about bats and white-nose syndrome. You too can grieve for all the beautiful animals, and you should find them beautiful, that are succumbing to this terrible epidemic.

If you know where and when to look, you can find bats all over the midwest. We’ve got a bunch nesting over our garage, and we put up a bat house near our deck — we’d be thrilled to have even more.

Bats and spiders, and more generally any invertebrate that has a freaky number of legs or eyes — I’m beginning to wonder if maybe I’ve got some kind of exotic disease…a Halloween infection, or Addams syndrome, or something similarly diagnosable.

Of course, one of they symptoms of this syndrome is that I don’t want to be cured. Give me more.

(By the way, I’m teaching a course in science essay writing in the Fall, and am collecting samples. That bat article is going right into the folder. I might be planning to infect impressionable young students with my disease.)

Today is climate change day in the classroom

As I’ve mentioned before, one of the things I’m doing in my Eco Devo class is to throw more of the burden of learning on the students. It would be too easy for me to just get up and lecture, telling them what they should know, and it is often hard for me to just shut up and let the students talk. I’ve split up the course so that Monday is when I start talking and dominate the classroom, Wednesday I ask the students to answer questions about Monday’s lecture and the book chapter, and on Fridays they’re given a paper to analyze.

This week’s paper is Morphological plasticity of the coral skeleton under CO2-driven seawater acidification by Tambutté and others. The context is that we’ve been talking about cellular physiology and development, and responses to environmental stresses, so I figured a primary research article about the effect of rising CO2 levels would be appropriate.

(Answer: more CO2 is not good for corals. Decreasing pH leads to a cnidarian version of osteoporosis.)

(a) Representative longitudinal sections; (b) transverse sections. pH treatment is indicated in the top left corner of each image. Scale bar, 1 mm.

It’s symbiosis week!

Yesterday’s lecture began with a dilemma. The topic this week is all about symbiosis, so of course I had to talk about Lynn Margulis, a very complicated person. I have a lot of respect for her contributions to the field, but also had to mention some of her wrong ideas, like that 9/11 was a false flag operation, and that AIDS was caused by a spirochete. It was also a dilemma back in 2007, when Margulis was a guest on this blog and also on our IRC channel. Whew, that was awkward. There might be a few old timers here who remember that.

Also awkward: most of the students had never heard of Margulis until now (they also had no idea what IRC was). At least I got to expose them to a little significant scientific history, which is my job, even when Margulis expressed the opinion that “I believe at all zoologists are intrinsically poorly educated in biology and that medical people are misinformed.” Ouch. There’s a grain of truth there, but mainly my students got to learn that some famous scientists can be colossal dicks. I did tell my students that if she were alive today she’d be a popular guest on Joe Rogan’s awful show.

Anyway, duty done, I lectured on mycorrhizae and gut microbiomes and a lot on Wolbachia. The paper of the week that the students will be telling me all about on Friday is “Eco-Evo-Devo: developmental symbiosis and developmental plasticity as evolutionary agents” by Gilbert, Bosch, and Ledón-Rettig, which you can read if you want to catch up on the course.

So that’s how things work at Frontiers journals, eh?

That ghastly article with the AI-generated rat testicles has been fully and completely retracted.

Following publication, concerns were raised regarding the nature of its AI-generated figures. The article does not meet the standards of editorial and scientific rigor for Frontiers in Cell and Development Biology; therefore, the article has been retracted.

This retraction was approved by the Chief Executive Editor of Frontiers. Frontiers would like to thank the concerned readers who contacted us regarding the published article.

I would like to know where those “standards of editorial and scientific rigor” were when the article was reviewed and accepted by the editors, because that paper was so blatantly, glaringly, obviously bad that it’s clear that no one actually read the damned thing before stamping it with an “approved” label. I think we’ve just gotten a peek at the process at Frontiers in Cell and Development Biology, and it’s cheap and lazy.

Notice also that no responsibility was taken.

How love can last a lifetime

In today’s eco-devo class, we’re going to be talking about a general phenomenon: the physical reality of your feelings, as witnessed by changes in gene expression. Seems appropriate for Valentine’s Day, right? On Monday I lectured on a few principles of gene regulation, and how environmental factors are transduced into patterns of epigenetic activity. Today, the students are going to answer questions and give explanations on the mechanics of all that, and then on Friday, they’ll discuss this paper: “Maternal care as a model for experience-dependent chromatin plasticity?” by Meaney and Szyf. Here’s part of true love:

The students are going to explain it all to me later this week, so I don’t want to spill all the beans, but in short, these are the results of studies in mice. Happy baby mice are licked and groomed by their mothers, while less happy mice are neglected and stressed. Being groomed increased serotonin levels, which activates adenylate cyclase, which increases cytoplasmic cyclic AMP levels, which activates a serine-threonine kinase called PKA, which activates a DNA binding protein that demethylates specific DNA sequences. Some of these sequences regulate stress responses in the hypothalamic-pituitary-adrenal axis, so those loving mommy-snuggles are changing how baby mice respond to stressful situations, and those responses persist long into adulthood.

So maternal care, or lack of care, is drilling right down to the structure of DNA and making lifelong modifications to your feelings. At least, if you’re a mouse, and humans almost certainly have the same biochemical arrangement. And a scientist can rip out some of your DNA and find a different pattern of epigenetic marks in individuals who had a loving relationship with Mom versus those who were neglected. It’s written in your semi-permanent epigenetic record.

Of course, this is just one pathway, and there are multiple regulatory pathways modulating stress responses, so all is not lost if you have one bad mother. These individual effects are sort of permanent, though, and would require alternative compensatory mechanisms to be overcome. Also, keep in mind that bad mothers could be a product of bad grandmothers, and that these epigenetic modifications can ripple across multiple generations.

Indeed, maternal effects could result in the transmission of adaptive responses across generations. In humans, such effects might contribute to the familial transmission of risk and resilience. Finally, it is interesting to consider the possibility that epigenetic changes could be an intermediate process that imprints dynamic environmental experiences on the fixed genome, resulting in stable alterations in phenotype – a process of environment-dependent chromatin plasticity.

I hope you all have an opportunity to stimulate some environment-dependent chromatin plasticity today. If you don’t have a date, you can at least call your mom, or be kind to a child. Modify someone’s DNA with a hug!