We teach developmental biology at UMM

I’m back to teaching developmental biology this term, and one of the things I do in my upper level classes is have students write blog entries on the themes of the course. In the past, I’ve given them space right here to do that, which I’ve found to be a parlous course of action — the commentariat here is savage and brutal, and infested with trollish nitwits who can derail threads spectacularly, so I’m doing it a little differently this time around. I’ve had them create their very own blogs on their own spaces, which has the additional benefit that maybe they can keep them going after they graduate.

So here’s the list of student blogs from my course. Feel free to visit, criticize, comment, etc., but do remember that they’re just now learning, so constructive discussion is far better than our usual ravaging ferocity. They’ve also been warned to be thick-skinned, though, so you don’t have to be too gentle.

Developmental Biology blogs

There isn’t much there yet in most cases, since they’ve just set them up, but I’ll be making weekly links to relevant articles in the future.

While I’m at it, I’ll mention that a former student, Levi Simonson, has a blog from his perspective as an ex-UMM student, now a graduate student at the wonderful University of Oregon. People do leave here to go on to interesting work!

Beehive development

A beekeeper did something simple: he put a bell jar over an opening in his beehive. The bees obliged by building a hive within it, where you could see it, and captured the process in a series of photos.

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I’m fascinated by the fact that the first struts in the framework are regularly spaced vertical bars of wax — how did they do that? I want to see videos of those early stages when the bees are working to initiate the strips. I suspect there are some interesting bee:bee interactions going on as they set them up.

Ray Kurzweil does not understand the brain

There he goes again, making up nonsense and making ridiculous claims that have no relationship to reality. Ray Kurzweil must be able to spin out a good line of bafflegab, because he seems to have the tech media convinced that he’s a genius, when he’s actually just another Deepak Chopra for the computer science cognoscenti.

His latest claim is that we’ll be able to reverse engineer the human brain within a decade. By reverse engineer, he means that we’ll be able to write software that simulates all the functions of the human brain. He’s not just speculating optimistically, though: he’s building his case on such awfully bad logic that I’m surprised anyone still pays attention to that kook.

Sejnowski says he agrees with Kurzweil’s assessment that about a million lines of code may be enough to simulate the human brain.

Here’s how that math works, Kurzweil explains: The design of the brain is in the genome. The human genome has three billion base pairs or six billion bits, which is about 800 million bytes before compression, he says. Eliminating redundancies and applying loss-less compression, that information can be compressed into about 50 million bytes, according to Kurzweil.

About half of that is the brain, which comes down to 25 million bytes, or a million lines of code.

I’m very disappointed in Terence Sejnowski for going along with that nonsense.

See that sentence I put in red up there? That’s his fundamental premise, and it is utterly false. Kurzweil knows nothing about how the brain works. It’s design is not encoded in the genome: what’s in the genome is a collection of molecular tools wrapped up in bits of conditional logic, the regulatory part of the genome, that makes cells responsive to interactions with a complex environment. The brain unfolds during development, by means of essential cell:cell interactions, of which we understand only a tiny fraction. The end result is a brain that is much, much more than simply the sum of the nucleotides that encode a few thousand proteins. He has to simulate all of development from his codebase in order to generate a brain simulator, and he isn’t even aware of the magnitude of that problem.

We cannot derive the brain from the protein sequences underlying it; the sequences are insufficient, as well, because the nature of their expression is dependent on the environment and the history of a few hundred billion cells, each plugging along interdependently. We haven’t even solved the sequence-to-protein-folding problem, which is an essential first step to executing Kurzweil’s clueless algorithm. And we have absolutely no way to calculate in principle all the possible interactions and functions of a single protein with the tens of thousands of other proteins in the cell!

Let me give you a few specific examples of just how wrong Kurzweil’s calculations are. Here are a few proteins that I plucked at random from the NIH database; all play a role in the human brain.

First up is RHEB (Ras Homolog Enriched in Brain). It’s a small protein, only 184 amino acids, which Kurzweil pretends can be reduced to about 12 bytes of code in his simulation. Here’s the short description.

MTOR (FRAP1; 601231) integrates protein translation with cellular nutrient status and growth signals through its participation in 2 biochemically and functionally distinct protein complexes, MTORC1 and MTORC2. MTORC1 is sensitive to rapamycin and signals downstream to activate protein translation, whereas MTORC2 is resistant to rapamycin and signals upstream to activate AKT (see 164730). The GTPase RHEB is a proximal activator of MTORC1 and translation initiation. It has the opposite effect on MTORC2, producing inhibition of the upstream AKT pathway (Mavrakis et al., 2008).

Got that? You can’t understand RHEB until you understand how it interacts with three other proteins, and how it fits into a complex regulatory pathway. Is that trivially deducible from the structure of the protein? No. It had to be worked out operationally, by doing experiments to modulate one protein and measure what happened to others. If you read deeper into the description, you discover that the overall effect of RHEB is to modulate cell proliferation in a tightly controlled quantitative way. You aren’t going to be able to simulate a whole brain until you know precisely and in complete detail exactly how this one protein works.

And it’s not just the one. It’s all of the proteins. Here’s another: FABP7 (Fatty Acid Binding Protein 7). This one is only 132 amino acids long, so Kurzweil would compress it to 8 bytes. What does it do?

Anthony et al. (2005) identified a Cbf1 (147183)-binding site in the promoter of the mouse Blbp gene. They found that this binding site was essential for all Blbp transcription in radial glial cells during central nervous system (CNS) development. Blbp expression was also significantly reduced in the forebrains of mice lacking the Notch1 (190198) and Notch3 (600276) receptors. Anthony et al. (2005) concluded that Blbp is a CNS-specific Notch target gene and suggested that Blbp mediates some aspects of Notch signaling in radial glial cells during development.

Again, what we know of its function is experimentally determined, not calculated from the sequence. It would be wonderful to be able to take a sequence, plug it into a computer, and have it spit back a quantitative assessment of all of its interactions with other proteins, but we can’t do that, and even if we could, it wouldn’t answer all the questions we’d have about its function, because we’d also need to know the state of all of the proteins in the cell, and the state of all of the proteins in adjacent cells, and the state of global and local signaling proteins in the environment. It’s an insanely complicated situation, and Kurzweil thinks he can reduce it to a triviality.

To simplify it so a computer science guy can get it, Kurzweil has everything completely wrong. The genome is not the program; it’s the data. The program is the ontogeny of the organism, which is an emergent property of interactions between the regulatory components of the genome and the environment, which uses that data to build species-specific properties of the organism. He doesn’t even comprehend the nature of the problem, and here he is pontificating on magic solutions completely free of facts and reason.

I’ll make a prediction, too. We will not be able to plug a single unknown protein sequence into a computer and have it derive a complete description of all of its functions by 2020. Conceivably, we could replace this step with a complete, experimentally derived quantitative summary of all of the functions and interactions of every protein involved in brain development and function, but I guarantee you that won’t happen either. And that’s just the first step in building a simulation of the human brain derived from genomic data. It gets harder from there.

I’ll make one more prediction. The media will not end their infatuation with this pseudo-scientific dingbat, Kurzweil, no matter how uninformed and ridiculous his claims get.

(via Mo Constandi)

I’ve noticed an odd thing. Criticizing Ray Kurzweil brings out swarms of defenders, very few of whom demonstrate much ability to engage in critical thinking.

If you are complaining that I’ve claimed it will be impossible to build a computer with all the capabilities of the human brain, or that I’m arguing for dualism, look again. The brain is a computer of sorts, and I’m in the camp that says there is no problem in principle with replicating it artificially.

What I am saying is this:

Reverse engineering the human brain has complexities that are hugely underestimated by Kurzweil, because he demonstrates little understanding of how the brain works.

His timeline is absurd. I’m a developmental neuroscientist; I have a very good idea of the immensity of what we don’t understand about how the brain works. No one with any knowledge of the field is claiming that we’ll understand how the brain works within 10 years. And if we don’t understand all but a fraction of the functionality of the brain, that makes reverse engineering extremely difficult.

Kurzweil makes extravagant claims from an obviously extremely impoverished understanding of biology. His claim that “The design of the brain is in the genome”? That’s completely wrong. That makes him a walking talking demo of the Dunning-Kruger effect.

Most of the functions of the genome, which Kurzweil himself uses as the starting point for his analysis, are not understood. I don’t expect a brain simulator to slavishly imitate every protein, but you will need to understand how the molecules work if you’re going to reverse engineer the whole.

If you’re an acolyte of Kurzweil, you’ve been bamboozled. He’s a kook.

By the way, this story was picked up by Slashdot and Gizmodo.

Blaschko’s Lines

One of the subjects developmental biologists are interested in is the development of pattern. There are the obvious externally visible patterns — the stripes of a zebra, leopard spots, the ordered ranks of your teeth, etc., etc., etc. — and in fact, just about everything about most multicellular organisms is about pattern. Without it, you’d be an amorphous blob.

But there are also invisible patterns that you don’t normally see that are aspects of the process of assembly, the little seams and welds where disparate pieces of the organism are stitched together during development. The best known ones are compartment boundaries in insects. A fly’s wing, for instance, has a normally undetectable line running across the middle of it, a line that cells respect. A cell born on the front half of the wing will multiply and expand its progeny to cover a patch on the surface, but none of its offspring cells will cross over the invisible line into the back half. Similarly, cells born on the back half will never wander into the front.

We can see these invisible lines by taking advantage of mosaicism: generate a fly wing with two genetically distinct cell types, for instance by making one type express a pigment marker and the other not, and the boundaries become apparent. There are many ways we can generate mosaics, but in Drosophila we can use somatic recombination — with low frequency, chromosomes in the fly can undergo crossing over in mitosis, not just meiosis, so sometimes the swapping of chromosome segments will turn a daughter cell that should have been heterozygous for an allele into one that is homozygous, allowing a marker allele to express itself.

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(Click for larger image)
(A) The shapes of marked clones in the Drosophila wing reveal the existence of a compartment boundary. The border of each marked clone is straight where it abuts the boundary. Even when a marked clone has been genetically altered so that it grows more rapidly than the rest of the wing and is therefore very large, it respects the boundary in the same way (drawing on right). Note that the compartment boundary does not coincide with the central wing vein. (B) The pattern of expression of the engrailed gene in the wing. The compartment boundary coincides with the boundary of engrailed gene expression.

It’s like a secret code written in molecules hidden to the eye until you illuminate it in just the right way to expose it. And these lines aren’t just arbitrary, they’re significant. The wing boundary defines the expression of important molecules that define the identity of specific structures. The posterior half of the wing is the domain of expression of a molecule called engrailed, which is part of the machinery that makes the back half a back half. We can also stain a wing for just that gene product, and also expose the hidden lines.

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We can also mutate the pathway of which engrailed is part, and do interesting things to the fly wing, like turn the back half into a mirror image of the front half. So these lines actually matter for the proper development of a fly.

So you might be wondering if we have anything similar in humans…and no, we don’t have strict compartment boundaries like a fly. However, we do have normally invisible lines and stripes of subtle molecular differences running across our bodies, which are occasionally exposed by human mosaicism. These are marks called the lines of Blaschko, after the investigator who first reported a common set of patterns in patients with dermatological disorders in 1901.

Don’t rip off your shirt and start looking for the Blaschko lines — they’re almost always invisible, remember! What happens is that sometimes people with visible dermatological problems — rashes, peculiar pigmentation, swathes of moles, that sort of thing — express the problems in a stereotypically patterned way. On the back, there are V-shaped patterns; on the abdomen and chest, S-shaped swirls; and on the limbs, longitudinal streaks.

Here is the standard arrangement:

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And here are a few examples:

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Note that usually there isn’t a whole-body arrangement of tiger stripes everywhere — there may be a single band of peculiar skin that represents one part of the whole.

Where do these come from? The current hypothesis is that a patch of tissue that follows a Blaschko line represents a clone of cells derived from a single cell in the early embryo. These clones follow stereotypical expansion and migration patterns depending on their position in the embryo; this would suggest that a cell in the middle of the back of a tiny embryo, as it grows larger with the growing embryo, would tend to expand first upwards towards the head and then sweep backwards and around to the front. One way to think of it: imagine taking a piece of yellow clay and sandwiching it between two pieces of green clay into a block, and then pushing and stretching the clay block to make a human figurine. The yellow would make a band somewhere in the middle, all right, but it wouldn’t be a simple rectilinear slice anymore — it would express a more complex border that reflected the overall flow of the medium.

What makes the lines visible in some people? The likeliest example is mosaicism, a difference between two adjacent cells in the early embryo that then appears as a genetic difference in the expanded tissues. There are a couple of ways human beings can be mosaic.

The most common example is X-chromosome inactivation in women. Women have two X-chromosomes, but men only have one; to maintain parity in the regulation of expression of X-linked genes, women completely shut down one X. Which one is shut down is entirely random. That means, of course, that all women are mosaic, with different X-chromosomes shut down in different cells. This normally makes no difference, since equivalent alleles are present on each, but occasionally an X-linked skin disorder can manifest itself in a splotchy pattern. Another familiar example is the calico fur color in female cats, caused by the random expression of a pigment gene on the feline X chromosome.

A more spectacular example is tetragametic chimerism. This rare event is the result of the fusion of two non-identical twins at an early stage of development, producing an embryo that is a kind of salt-and-pepper mix of two individuals. After the fusion, the embryo develops normally as a single individual, but genetic or molecular tests can detect the patches of different genotypes. (No scientific tests can tell whether the individual has two souls, however.)

Another way differences can arise is by somatic mutation. Mutations occur all the time, not just in the germ line; we’re all a mixture of cells with slightly different mitotic histories and some of them contain novel mutations, usually not of a malign sort, or you wouldn’t be reading this right now. But what can happen is that you acquire a mutation in one cell that may predispose its clone of progeny to form moles, or acquire a skin disease, or even tilt it towards going cancerous. It’s a fine thing to undergo genetic screening to find that you may not carry certain alleles associated with cancer, but you aren’t entirely off the hook: you may have patches of tissue in your body that are perfectly normal and functional except that they carry an enabling mutation that occurred when you were an embryo.

One final likely mechanism is epigenetic. Throughout development, genes are switched on and off by epigenetic modification of the DNA. This process can vary: epigenetic silencing doesn’t have to be 0 or 100% absolute, but can differ in degree from cell to cell. It can also vary by chromosome — you’re all diploid, and epigenetic modification may affect one chromosome of a pair to a different degree than the other. Since epigenetic modifications are inherited by the progeny of a cell, that means these differences can be propagated into a clonal patch…that on the skin, will likely follow the lines of Blaschko.

Don’t fret over these lines; they aren’t a disease or a problem or even, in most cases, at all visible. The cool thing about them is that there is a hidden map of your secret history as an individual embedded in silent patterns in your skin — you were not defined as a single, simple, discrete genetic entity at fertilization, but are the product of complicated, subtle changes and errors and shufflings and sortings of cells. We’re all beautiful pointillist masterpieces.

Engineering lungs

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This past weekend, I attended my 35th high school reunion. It’s a strange phenomenon to be meeting people you haven’t seen since you were 18, and further weirdness ensues when we discover that most of them are already grandparents. There have been a lot of life changes in 35 years. Saddest of all, though, is the ritual listing of the deceased…and I learned that one of my former classmates died in her late 40s of emphysema, a progressive and irreversible lung disease that leads to the near complete loss of lung function. The only cure right now is a lung transplant, and patients who’ve been suffering with emphysema for long typically have so many other problems that they’re poor candidates for surgery, not to mention that the waiting list for organs is long, and a transplant means a life-long commitment to anti-rejection drug treatments.

There will come a day, though, when new lungs can be built and replacement surgeries more common. That day is definitely not yet here, though, but there are promising signs on the horizon. One such sign is recent work in tissue engineering to grow lungs in a dish.

We can grow functional rat lungs in a tissue culture chamber now, which show some limited ability to support respiration when transplanted back into another rat. Here’s an overview of the procedure; I’ll go through it step by step.

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Schema for lung tissue engineering. (A) Native adult rat lung is cannulated in the pulmonary artery and trachea for infusion of decellularization solutions. (B) Acellular lung matrix is devoid of cells after 2 to 3 hours of treatment. (C) Acellular matrix is mounted inside a biomimetic bioreactor that allows seeding of vascular endothelium into the pulmonary artery and pulmonary epithelium into the trachea. (D) After 4 to 8 days of culture, the engineered lung is removed from the bioreactor and is suitable for implantation into (E) the syngeneic rat recipient.

The first step is to collect a donor rat lung. The organ is cut out of a living rat, who will quickly become a dead rat; this part of the procedure probably can’t be scaled up to human transplantations, for obvious reasons. However, lungs from dead people or perhaps even lungs from appropriately sized animals will be adequate.

This donor lung is then stripped of all of its living cells. This is done by perfusing it with a detergent solution. Membranes dissolve and rupture in the presence of detergent, so all of the cells in the lung are basically destroyed, their contents, including cytoplasm, nucleus and DNA, and membranes and organelles are washed away.

What’s left then? Connective tissue and extracellular proteins. All that’s left is a frothy, fragile matrix of fibers like collagen and elastin, and imbedded signaling proteins that will be useful in supporting the growth of new cells. It leaves behind a kind of porous, pale ghost lung, a collection of microscopic struts that just needs to be draped with new cells.

This is the key step. Lungs are complicated, delicate membranous structures, and it would be hard to regrow it entirely from scratch. Starting with an acellular scaffold, though, a kind of skeleton of a lung that sketches out the arrangement of alveoli and blood vessels gives the tissue engineering a head start. It really is amazing how much of the organization of the lung is still left when all of the cells are removed: the photos below show the air spaces left behind (on the left) and the major blood vessels (on the right). There are no cells here! This is just an outline of the structure left by the still extant connective tissue!

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What’s promising about this so far is that almost all of the antigenic components of the lung are removed; if this lacework of proteins were transplanted into another animal, it probably wouldn’t provoke an immune response. Of course, it also wouldn’t work as a lung, because there are no gas exchange surfaces present, and everything is leaky. It’s only a skeleton of a lung, remember…so let’s fix that.

The next step is to suspend the lung framework in a chamber, with tubes attached to the airways and to the blood vessels. Living, isolated lung epithelial cells are then introduced into the airways, and lung endothelial cells (the cells that line blood vessels) are introduced into the pulmonary arteries and veins. These cells stick to the scaffolds and grow. They actually grow better in the lung matrix than they do in a petri dish, probably because of the presence of growth factor proteins lurking in the acellular scaffold.

The new cells use the matrix as a guide and repopulate and rebuild the cellular structure of the lung. During this whole process, the tissue culture medium is pumped through the arteries to mimic the effects of blood pressure, and air is pumped into the airways. It works beautifully. New columnar epithelial cells grow to line alveoli, and endothelial cells line the blood vessels appropriately, and the cells produce the right secreted proteins, like surfactants that reduce surface tension and are important for allow lungs to inflate. It takes about a week for the new lung tissues to form, and they are robust, producing sheets of cells that can tolerate the same pressures as intact, normal lungs.

Now for the real test. The regrown lungs were transplanted into living rats for 45 minutes to two hours. They worked! Everything stitched together nicely, and the engineered tissues pinked up nicely as blood flowed into them. Blood was visibly oxygenated as it passed through the new lung, and measurements of the partial pressure of oxygen and carbon dioxide in the blood showed that the former went up and the latter went down, exactly as it is supposed to do.

This is all very promising, but the transplantation times were very short, and you might be wondering why. These lungs aren’t perfect. There was bleeding from the blood vessels into the airways, and also some blood clotting — for this to work, the barrier membranes have to be essentially perfect, and they aren’t, yet. The poor rats’ lungs would have spluttered and fallen apart with prolonged use, and the blood clots would have led to thrombosis eventually.

Another problem, and one that has to be solved eventually if this is ever to work for human transplantations, is the source of the cellular components. The connective tissue stuff is only weakly antigenic, so isn’t going to provoke a strong immune response, but repopulating it with foreign cells brings the rejection problem roaring right back. What we need next for long term success is to find a source of lung adult stem cells, or a way to induce pluripotent stem cells to make the cell types needed.

That’s right, we need more human stem cell research. If you need a new lung, and we’re going to have to reengineer one for you in a dish, we’ll need a population of autologous cells — cells from you — to reseed an acellular matrix taken from a cadaver or a pig with lung tissue that won’t trigger an immune response.

This is a long way from being useful for humans, with two big problems still facing us, refinement of the tissue-engineering technique to produce more reliable and complete membranes, and the all-important stem cell research to allow us to create sources of immunologically-compatible cells, but the cool thing is that the questions that need to be answered are in crystal-clear focus, and there are strategies to get us the answers. Time and money and a less restrictive regulatory environment for stem cell work is all that is needed.

Petersen TH, Calle EA, Zhao L, Lee EJ, Gui L, Raredon MB, Gavrilov K, Yi T, Zhuang ZW, Breuer C, Herzog E, Niklason LE (2010) Tissue-engineered lungs for in vivo implantation. Science 329(5991):538-41.

No metazoan is an island

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I’m one of those dreadful animal-centric zoologically inclined biologists. Plants? What are those? Fungi? They’re related to metazoans somehow. Lichens? Not even on the radar. The first step in fixing a problem, though, is recognizing that you have one. So I confess to you, O Readers, that my name is PZ, and I am a metazoaphile. But I can get better.

My path to opening up to wider horizons is to focus on what I find most interesting about animals, and that is that they are networks of cells driven by networks of genes that generate patterned responses of expression by cell signaling, or communication. See? I’m already a little weird. Show me a baby bunny, and I don’t just see a cute little furry pal with an adorable twitchy nose, I see an organized and coherent array of differentiated tissues that arose by a temporal sequence of cell-cell interactions, and I just wanna open him up and play with his widdle epithelial sheets and dismantle his pwetty ducts and struts and fibers and fluids, oochy coo. And ultimately, I want to take apart each cell and ask why it has its particular assortment of genes switched off and on, and how its state affects its neighbors and the whole of the organism.

Which means, lately, that I’ve acquired a growing interest in bacteria. If I were 30 years younger, I could probably be seduced into a career in microbiology.

There are a couple of reasons why an animal-centric biologist would be interested in bacteria. One is the principle of it; the mechanisms that animal cells use to build complex arrangements of tissues were all first pioneered in single-celled organisms. We have elaborated and added details to gene- and cell-level phenomena, but it’s a collection of significant quantitative differences, with nothing known that is essentially new in metazoan cells. All the cool stuff was worked out by evolution in the 3-4billion years before the Cambrian, a potential that simply blossomed in the past half-billion years into big conglomerations of cells. Understanding how the building blocks of multicellularity work individually ought to be a prerequisite to understanding how the assemblages work.

But there’s another reason, too, a difference in perspective. It is our conceit to regard ourselves as individuals of Homo sapiens, a body of cells clonally derived from a single human cell. It’s not true. It turns out that each one of us is actually a whole population of species, linked by our evolutionary history and lumbering through the world as a team. Genus Homo is also genera Escherichi and Bacteroidetes and Firmicutes and many others.

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Physiology

Let’s begin with the most widely known factor: we’re mostly bacterial in cell numbers, with about ten times as many bacterial cells as human cells. Most of these are nestled deep in our guts, where they are indispensible. In mammals, they help break down complex polysaccharides which we can then absorb through the wall of the digestive tract — these are compounds that would be simply lost without bacterial assistance. Even more dramatically, termite guts contain colonies of bacteria that produce enzymes to break down cellulose. Another insect, aphids, live in plant saps which have negligible protein components, and they rely on gut bacteria that can synthesize nine essential amino acids. One cool feature is that the bacteria can’t complete the synthesis of leucine; the last step is carried out by aphid enzymes. The synthetic pathway is split acros two different species!

Another weird twist is that gut bacteria can affect morphology (or vice versa; physiology influences which gut bacteria thrive). Mice with a genetic predisposition to obesity were found to have a different distribution of gut bacteria; fat mice are full of Firmicutes, while lean mice are loaded with Bacteroidetes. Something in the genetics of the obese mice seems to favor the proliferation of that one species. Cause and effect is not so easily separated, though, since doing a fecal transplant and inoculating the guts of germ free mice with the bacteria from obese mice vs. lean mice has a surprising effect: the mice given obese mouse fecal enemas subsequently increased their body fat by 60%. The bacteria promoted more fat storage in the host animal.

So what, you may be thinking, it’s mice. However, it turns out that obese humans tend to have reduced amounts of Bacteroidetes species in their guts than lean people, and weight loss is accompanied by an increase in Bacteroidetes. Fecal transplants are not recommended as a weight loss technique…at least not yet.

They have worked for some other problems. Crohn’s disease and ulcerative colitis are diseases that involve intestinal inflammation, and they’re also associated with imbalances in the species distribution of gut bacteria. Some promising treatments have involved collecting feces from healthy individuals, and using a nasogastric tube to inoculate the guts of Crohn’s patients with the stuff. Ick, I know, but it seems to have worked surprisingly well in a small number of patients.

Development

Bacteria are present in the gut from a very early age, and populate the digestive epithelia. There must be interactions going on, and it appears that the bacteria are actually regulating the growth of the gut lining.

Germ-free zebrafish lines have no gut bacteria, and they also have problems. The intestinal lining arrests its development and fails to fully differentiate; the lining also grows much more slowly. They also have difficulty absorbing some nutrients. Add bacteria, though, and growth and differentiation resume. This is a case where the developmental program and the bacterial influences are interdependent, and it makes sense — they’ve co-evolved.

It’s not just fish, either — these are conserved interactions across the vertebrates. Mice exhibit the same dependence on gut flora for development of the intestinal lining.

The very best example of a developmental dependence on bacteria, though, is in squid. The bobtail squid has a light-emitting organ that relies on colonization by a luminescent bacterium, Vibrio fischeri. The animal gleans the bacteria from the water with a special ciliated epithelium and secreted mucus that seems to be just the right flavor for Vibrio, and the bacteria migrate deep into the light-emitting organ. Once colonized, the squid dismantles the harvesting cilia and downregulates the secretion of mucus. If no bacteria of the right species are present, it maintains the cilia. If the bacteria in the organ die, resumes mucus production.

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Bacterial symbionts induce light-organ morphogenesis in squid. A Adult squid (E scolopes). SEM images of epithelial fields before B and after C regression of ciliated appendage. Scale bar, 50 mm. Ciliated appendages are marked by an orange dashed line.

Evolution

If something affects development and physiology, it affects evolution, so evolutionary importance is simply rather unavoidable. However, there’s also one somewhat surprising observation (to me, at least — microbiologists probably expect it): different species of related organisms can have different microbial populations, even when raised in identical conditions. Different Hydra species in the lab under controlled conditions have recognizably different populations of bacteria living on their epithelia, and Hydra of the same species collected in the wild have similar distributions of species. The properties of each Hydra species uniquely favor different distributions of bacteria, and the bacteria are also preferentially colonizing particular species of Hydra.

Hydra are wonderful experimental animals in that one can ablate stem cells for a particular tissue type, and still get an animal that develops and lives; do the same thing to a vertebrate, for instance knocking out the mesodermal lineage in the embryo, and you get an aborted blob. In Hydra, you get a tissue that survives and is colonized by bacteria…but the kinds of bacteria populating it is different from the populations in the intact animal. The animal and the bacteria are swapping molecular signals that specify favored relationships. Again, these are coevolved populations that recognize molecular properties of the host and symbiont.

This is all getting very complicated. I’m used to thinking in terms of networks of genes: there are regulatory interactions between genes in a single cell that establish cell-type specific patterns of gene activity; all express a common core of genes, but different cell types, such as a neuron vs. a cell of the digestive epithelia, will also have their own unique special-purpose genes switched on. I’m also comfortable thinking of networks of cells: cells are in constant negotiations with their neighbors, mainting a common pattern of expression within a tissue, and defining interacting edges with other tissues. Cells are continually sending out messages about their state into the system and responding to local and global signals. All this is part of the normal process of thinking developmentally.

Now, though, there’s another layer: we have to think in terms of networks of species that cooperate in the development and physiology of individual multi-cellular organisms. Purity is compromised. My precious animalia — they’re inconceivable without bringing bacteria into the picture.

Fraune S, Bosch TCG (2010) Why bacteria matter in animal development and evolution. Bioessays 32:571-580.

Attention, perversely assertive women! You are abnormal!

Important clarification: CAH is a real and serious disease. There are no objections to pediatricians treating the physiological disorders in utero. However, lesbianism, traditionally masculine career choices, and disinterest in having children are not diseases…and the problem in this work is that the doctors involved clearly think they are, and are interested in using the drug dexamethasone to modify behavioral choices. That’s the scary part.

Ladies, are you independent, stubborn, or mildly aggressive in your social interactions? Are you perhaps less interested in having sex with men than your neighborhood nymphmaniac? Are you possibly even lesbian or bisexual? Worst of all, are you pursuing a career in a masculine profession, and possibly deferring pregnancy and child-rearing to a later date, or even indefinitely?

*Researchers couldn’t possibly have suggested this, could they? Yes, they did.

In a paper published just this year in the Annals of the New York Academy of Sciences, New and her colleague, pediatric endocrinologist Saroj Nimkarn of Weill Cornell Medical College, go further, constructing low interest in babies and men – and even interest in what they consider to be men’s occupations and games – as “abnormal,” and potentially preventable with prenatal dex:

“Gender-related behaviors, namely childhood play, peer association, career and leisure time preferences in adolescence and adulthood, maternalism, aggression, and sexual orientation become masculinized in 46,XX girls and women with 21OHD deficiency [CAH]. These abnormalities have been attributed to the effects of excessive prenatal androgen levels on the sexual differentiation of the brain and later on behavior.” Nimkarn and New continue: “We anticipate that prenatal dexamethasone therapy will reduce the well-documented behavioral masculinization…”

See? It’s abnormal to have career interests that are not aligned with your gender!

By the way, “46,XX” just means chromosomally normal.

Your diseased state may be due to a congenital abnormality, prenatal exposure to excess androgens. You’ve been mildly masculinized. There are specific heritable traits such as congenital adrenal hyperplasia that can, in extreme cases, lead to ambiguous genitalia, but even at low levels of effect may contribute to such horrors as female homosexuality, childhood playing with trucks, and the ambition to pursue careers in physics or medicine or computer science, where you don’t really belong.*

I fear that if you’re reading Pharyngula, you’re probably one of those more assertive, man-like women, and you’re rapidly reviewing your personal history and realizing it’s true: you didn’t make Scarlett O’Hara your role model, you aren’t submissive to your husband (if you even have one!), and the prospect of churning out a baby a year does not appeal to you. And you’re wondering what went wrong with your life, and what can you do to change your personality to something more demure, more delicate, more passive.

I’m sorry, it’s too late. There’s nothing that can help you now. I told you it’s caused by congenital exposure to androgens. Weren’t you listening, woman?

But wait, don’t despair. You might be condemned to a life of misery trying to compete with men by your aberrant brain, but your children don’t have to. What you need to do is use your ovaries and get pregnant right now — don’t complain, it’s your natural destiny — and if it’s a boy, be happy and relieved, but if it’s a girl, there is a drug you can take that might make her a girly girl girl, one who is joyously heterosexual (unlike you), happy to have sex with men (unlike you), a perfect fit to traditional gender roles (unlike you), and enthusiastic about having babies (unlike you, who is only doing this out of a sense of duty and a diminished self-esteem).

Now the evidence that prenatal androgenization is the cause of your weird unladylike attitude is a little shaky, and the use of this drug is experimental and hasn’t really been tested that well to see if it works as claimed, but never mind all that — its use has been endorsed by the American Academy of Pediatrics, the Lawson Wilkins Pediatric Endocrine Society, the European Society for Paediatric Endocrinology, the European Society of Endocrinology, the Society of Pediatric Urology, the Androgen Excess and PCOS Society, and the CARES Foundation. The rather tenuous chain of evidence for this claim, and the peculiarity of diagnosing as a problem something that most of the poor, afflicted, purblind women do not recognize as a problem, is being disregarded because it is so very important that we reduce the incidence of lesbianism by an entirely hypothetical and unmeasured percentage. Lesbianism is just that bad.

Ask for it by name. It’s a glucosteroid called dexamethasone, and tell your doctor you need it because you want to make sure your baby likes pink frilly dresses when she grows up and doesn’t try to compete with men. It’s a convenient anti-uppitiness pill for your baby!

Meanwhile, just sit around and feel miserable about your congenital failures.

Oh, by the way — dexamethosone is a potent little steroid, and there may be a few trivial side-effects of the chronic exposure to the hormone to you while pregnant, including weight gain, diabetes, immunosuppression, hypertension, catabolic muscle atrophy, osteoporosis, and psychiatric disturbances, like mania, depression, and mood swings. It’s just one of those little sacrifices I’m sure you’ll be happy (remember—destiny, natural order, womanly role, etc.) to make in order that your baby grows up knowing her place as an appropriately obedient little receptacle of manly desires, and liking it.

Before you less-than-hyper-macho men get all smug, though, let me warn you: prenatal hormone effects is a hot, hot topic in the heteronormative world of pediatrics. You’re going to be diagnosed as suffering from a prenatal androgen deficiency and shamed if you’re anything less than a man’s man with stereotypic masculine interests. Look for intrauterine testosterone treatments for women carrying boy children, just to make sure they grow up to like football (American, not that pansy soccer stuff) and follow macho careers!

I, for one, look forward to our brave new world of drug-enhanced sexual dimorphism and the extermination of all sexual ambiguity and androgyneity. Aren’t you?

If you aren’t, give us time: we’ll come up with a pill for that, too.

How insects and crustaceans molt

I was mildly surprised at the reaction to this cool timelapse video of a molting crab — some people didn’t understand how arthropods work. The only thing to do, of course, is to explain the molting process of insects and crustaceans, called ecdysis.

Let’s go back to the basics first. In the beginning was the epithelium, a continuous sheet of linked cells that envelops multicellular organisms. These are living, dividing, dynamic cells that are flexible, can repair damage to themselves, and represent the boundary between the carefully maintained internal environment of the organism, and the more variable and often hostile external environment. And that’s where the problem lies: living cells are relatively fragile and sensitive, and in particular don’t cope well with drying out. Cells like it wet, yet if you look at insects and people, we live under horrible conditions for living cells, surrounded by dryness and heat and cold.

Our external epithelia have evolved different solutions to this problem of the basic inhospitability of terrestrial life. In us, our bounding epithelia divide frequently, pushing new cells outward. As these cells move, they commit suicide, producing a fibrous protein called keratin which forms dense, matted tangles inside the cells; these cells also build tight protein connections between their neighbors. It is these dead, protein-packed cells that face the outside world, protecting the delicate interior. These cells are steadily worn away and cast off — dandruff flakes, for instance, are sheets of these dead epithelial cells — and new protective cells produced by cell division and pushed up from the inside out to replace them. It’s a good solution that allows for constant growth and flexibility.

Arthropods, on the other hand, start with a similar sheet of living epithelial cells, but do something completely different. Instead of pushing out a continuous column of dying cells, they secrete dense layers of complex chemical compounds that harden into a tough cuticle. The exoskeleton of an insect or crustacean is acellular — the living cells have protected themselves by secreting an initially fluid set of chemicals that harden like epoxy to form a tough protective armor around themselves. We protect ourselves with sheets of leather; arthropods make plates like fiberglass on their outsides.

And there’s the rub. The cuticles of insects do not gradually slough away, replaced steadily by the addition of new material from the inside. They’re mostly fixed and rigid and static. This does have the advantage of providing a solid protective armor and a rigid framework for muscles, but isn’t so great for accommodating growth. Fiberglass isn’t stretchy and flexible!

Here’s a closer look at the structure of the arthropod cuticle.

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In the diagram on the left, the living epithelium is at the bottom, labeled “epidermis”. Above it are multiple acellular layers called the cuticle made up of substances like chitin and waxes (notice that it is also perforated by pores containing ducts of the glands that secrete the chemical substances, and also places where hairs called setae can dangle into the exterior.

In order to grow, the animal must discard the old cuticle and build a new one from the inside out. In (b), this process begins by peeling away the living epidermal cells from the dead cuticle, creating a gap called the exuvial space, which is filled with a fluid called molting fluid. The cells then begin secreting a new cuticle from underneath, which is initially flexible.

What is poorly shown in these diagrams is that the new cuticle can be larger than the old. What that means is that epithelium inside the old cuticle is wrinkled and convoluted to have a larger surface area. Again, it is soft, not hard, so it can wrinkle up freely to fit. Also, to make room, the molting fluid in (c) is busily digesting the old cuticle from underneath, and the protein components are absorbed and reused to build the new cuticle.

In (d), the new cuticle is nearly fully formed, the old cuticle has been reduced to a thinner rind, and the two are separated by a thin fluid-filled space. Ecdysis, the actual molt, then occurs, and the old cuticle is discarded. Free of its confining shell, the animal inflates itself to extend the wrinkled new cuticle into larger smoothness, and the process of sclerotization, or hardening of the cuticle, begins from the outside in. Tanning agents, like polyphenols are secreted through ducts onto the surface, where they are oxidized into quinones, which trigger chemical reactions that cross-link the various substances of the cuticle into a rigid structure.

If you’ve ever eaten soft-shell crabs, you’ve caught the poor creature just after a molt and before its cuticle has hardened — in large arthropods, it can take several days for the post-molt cuticle to be fully cured. The hardening is also regional. Next time you’re eating a crab leg, notice that the shaft of the limb is rigid and strong and a bit brittle, but it grades into softer, less thickly sclerotized material at the joints called arthrodial membranes, which retains the flexibility of the pre-molt cuticle.

Now go watch the video again, and it should make more sense. What you’re seeing near the end is the crab pulling soft and rubbery limbs out of the shell of its old legs, and then resting as the new cuticle slowly hardens.

The secret life of babies

Years ago, when the Trophy Wife™ was a psychology grad student, she participated in research on what babies think. It was interesting stuff because it was methodologically tricky — they can’t talk, they barely respond in comprehensible way to the world, but as it turns out you can get surprisingly consistent, robust results from techniques like tracking their gaze, observing how long they stare at something, or even the rate at which they suck on a pacifier (Maggie, on The Simpsons, is known to communicate quite a bit with simple pauses in sucking.)

There is a fascinating article in the NY Time magazine on infant morality. Set babies to watching puppet shows with nonverbal moral messages acted out, and their responses afterward indicate a preference for helpful agents and an avoidance of hindering agents, and they can express surprise and puzzlement when puppet actors make bad or unexpected choices. There are rudiments of moral foundations churning about in infant brains, things like empathy and likes and dislikes, and they acquire these abilities untaught.

This, of course, plays into a common argument from morality for religion. It’s unfortunate that the article cites deranged dullard Dinesh D’Souza as a source — is there no more credible proponent of this idea? That would say volumes right there — but at least the author is tearing him down.

A few years ago, in his book “What’s So Great About Christianity,” the social and cultural critic Dinesh D’Souza revived this argument [that a godly force must intervene to create morality]. He conceded that evolution can explain our niceness in instances like kindness to kin, where the niceness has a clear genetic payoff, but he drew the line at “high altruism,” acts of entirely disinterested kindness. For D’Souza, “there is no Darwinian rationale” for why you would give up your seat for an old lady on a bus, an act of nice-guyness that does nothing for your genes. And what about those who donate blood to strangers or sacrifice their lives for a worthy cause? D’Souza reasoned that these stirrings of conscience are best explained not by evolution or psychology but by “the voice of God within our souls.”

The evolutionary psychologist has a quick response to this: To say that a biological trait evolves for a purpose doesn’t mean that it always functions, in the here and now, for that purpose. Sexual arousal, for instance, presumably evolved because of its connection to making babies; but of course we can get aroused in all sorts of situations in which baby-making just isn’t an option — for instance, while looking at pornography. Similarly, our impulse to help others has likely evolved because of the reproductive benefit that it gives us in certain contexts — and it’s not a problem for this argument that some acts of niceness that people perform don’t provide this sort of benefit. (And for what it’s worth, giving up a bus seat for an old lady, although the motives might be psychologically pure, turns out to be a coldbloodedly smart move from a Darwinian standpoint, an easy way to show off yourself as an attractively good person.)

So far, so good. I think this next bit gives far too much credit to Alfred Russel Wallace and D’Souza, though, but don’t worry — he’ll eventually get around to showing how they’re wrong again.

The general argument that critics like Wallace and D’Souza put forward, however, still needs to be taken seriously. The morality of contemporary humans really does outstrip what evolution could possibly have endowed us with; moral actions are often of a sort that have no plausible relation to our reproductive success and don’t appear to be accidental byproducts of evolved adaptations. Many of us care about strangers in faraway lands, sometimes to the extent that we give up resources that could be used for our friends and family; many of us care about the fates of nonhuman animals, so much so that we deprive ourselves of pleasures like rib-eye steak and veal scaloppine. We possess abstract moral notions of equality and freedom for all; we see racism and sexism as evil; we reject slavery and genocide; we try to love our enemies. Of course, our actions typically fall short, often far short, of our moral principles, but these principles do shape, in a substantial way, the world that we live in. It makes sense then to marvel at the extent of our moral insight and to reject the notion that it can be explained in the language of natural selection. If this higher morality or higher altruism were found in babies, the case for divine creation would get just a bit stronger.

No, I disagree with the rationale here. It is not a problem for evolution at all to find that humans exhibit an excessive altruism. Chance plays a role; our ancestors did not necessarily get a choice of a fine-tuned altruism that works exclusively to the benefit of our kin — we may well have acquired a sloppy and indiscriminate innate tendency towards altruism because that’s all chance variation in a protein or two can give us. There’s no reason to suppose that a mutation could even exist that would enable us to feel empathy for cousins but completely abolish empathy by Americans for Lithuanians, for instance, or that is neatly coupled to kin recognition modules in the brain. It could be that a broad genetic predisposition to be nice to fellow human beings could have been good enough to favored by selection, even if its execution caused benefits to splash onto other individuals who did not contribute to the well-being of the possessor.

But that idea may be entirely moot, because there is some evidence that babies are born (or soon become) bigoted little bastards who do quickly cobble up a kind of biased preferential morality. Evolution has granted us a general “Be nice!” brain, and also that we acquire capacities that put up boundaries and foster a kind of primitive tribalism.

But it is not present in babies. In fact, our initial moral sense appears to be biased toward our own kind. There’s plenty of research showing that babies have within-group preferences: 3-month-olds prefer the faces of the race that is most familiar to them to those of other races; 11-month-olds prefer individuals who share their own taste in food and expect these individuals to be nicer than those with different tastes; 12-month-olds prefer to learn from someone who speaks their own language over someone who speaks a foreign language. And studies with young children have found that once they are segregated into different groups — even under the most arbitrary of schemes, like wearing different colored T-shirts — they eagerly favor their own groups in their attitudes and their actions.

That’s kind of cool, if horrifying. It also, though, points out that you can’t separate culture from biological predispositions. Babies can’t learn who their own kind is without some kind of socialization first, so part of this is all about learned identity. And also, we can understand why people become vegetarians as adults, or join the Peace Corps to help strangers in far away lands — it’s because human beings have a capacity for rational thought that they can use to override the more selfish, piggy biases of our infancy.

Again, no gods or spirits or souls are required to understand how any of this works.

Although, if they did a study in which babies were given crackers and the little Catholic babies all made the sign of the cross before eating them, while all the little Lutheran babies would crawl off to make coffee and babble about the weather, then I might reconsider whether we’re born religious. I don’t expect that result, though.

Everything old is new again

If you’ve ever invited me out to give a science talk, you know that what I generally talk about is this concept of deep homology: the discovery that features that we often consider the hallmarks of complex metazoan life often have at their core a network of genetic circuitry that was first pioneered in bacteria. What life has done is taken useful functional elements that were worked out in the teeming, diverse gene pools of the dominant single-celled forms of life on earth and repurposed it in novel ways. The really interesting big bang of life occurred long before the Cambrian, as organisms evolved useful tools for signaling, adhesion, regulation, and so forth — all stuff that was incredibly useful for a single cell negotiating through space and time in a complex external environment, and which could be coopted for building multicellular organisms.

But if you don’t feel like flying me out to tell you all about it, Carl Zimmer has an excellent article on deep homology in the NYT, and he uses a new example I’ll have to steal: a genetic module that we use to regulate blood vessel growth that can also be found in yeast cells, where it is used to maintain cell walls.