Awesomely horrible

We have a long history in developmental biology of studying the most amazing freaks of nature — damage to developing organisms can produce astonishingly ghastly results as the embryo tries to regulate and recover, yielding results that are almost normal. There’s even a whole subdiscipline of the field, teratology, dedicated to studying aberrations of embryology. The word is perfect, since it is derived from a Greek root that means both “wonders” and “monsters”.

An unfortunate child in Colorado was the recipient of one of these wonders/monsters. Diagnosed with a brain tumor, when surgeons opened up his skull, they found fragments of a fetus inside: two tiny feet, part of a hand, coils of intestine. The surgery was successful and the child is doing fine now, but this was the most well-organized ‘tumor’ I’ve ever heard of. It’s not clear exactly what it was; there are things called teratomas, where a particular kind of cancer recapitulates a developmental program and builds tissues, things like skin with hair or teeth or chunks of muscle and bone and gland, but those aren’t this well organized. They tend not to produce complete organs, but partially differentiated sheets and lumps. Another possibility is fetus in fetu, where a fragment of the very early embryo is isolated and begins its own independent pattern of normal development, and then is engulfed by the larger and faster growing sibling embryo. Sometimes people late in life will be surprised to learn that there is a partially developed twin imbedded deep in their body. There is no question in any of these cases, however, that the tissue is not an autonomous individual. It is a piece of human-derived tissue that has executed part of the program of cell:cell interactions and induction that these kinds of cells are capable of doing.

Something struck me when I saw the photograph of this particular surgery. Here it is, a photo of a fetal foot flopping out of a bloody baby’s brain (don’t click if you’re squeamish). As I’m sure you’ve noticed, anti-choice people love to parade about with gory photos of aborted fetuses, and they love to dwell on little details like a recognizable hand or face. This picture is exactly like those, yet realize this: there was no human being behind those little baby toes. The existence of these fragments of non-sentient tissue endangered the life of a child, and there was no question that they needed to be extracted.

This is also how we should view abortion. It’s ugly and messy, and there’s something disquietingly resonant of humanity in the pieces of the embryo or fetus, but we shouldn’t be fooled. Those are beautifully patterned collections of differentiated cells, but there is no person there.

A Natural History of Seeing: The Art and Science of Vision

Simon Ings has written a wonderful survey of the eye, called A Natural History of Seeing: The Art and Science of Vision(amzn/b&n/abe/pwll), and it’s another of those books you ought to be sticking on your Christmas lists right now. The title give you an idea of its content. It’s a “natural history”, so don’t expect some dry exposition on deep details, but instead look forward to a light and readable exploration of the many facets of vision.

There is a discussion of the evolution of eyes, of course, but the topics are wide-ranging — Ings covers optics, chemistry, physiology, optical illusions, decapitated heads, Edgar Rice Burroughs’ many-legged, compound-eyed apts, pointillisme, cephalopods (how could he not?), scurvy, phacopids, Purkinje shifts…you get the idea. It’s a hodge-podge, a little bit of everything, a fascinating cabinet of curiousities where every door opened reveals some peculiar variant of an eye.

Don’t think it’s lacking in science, though, or is entirely superficial. This is a book that asks the good questions: how do we know what we know? Each topic is addressed by digging deep to see how scientists came to their conclusion, and often that means we get an entertaining story from history or philosophy or the lab. Explaining the evolution of our theories of vision, for example, leads to the story of Abu’Ali al-Hasan ibn al-Hasan ibn al-Haythem, who pretended to be mad to avoid the cruelty of a despotic Caliph, and who spent 12 years in a darkened house doing experiments in optics (perhaps calling him “mad” really wasn’t much of a stretch), and emerged at the death of the tyrant with an understanding of refraction and a good theory of optics that involved light, instead of mysterious vision rays emerging from an eye. Ings is also a novelist, and it shows — these are stories that inform and lead to a deeper understanding.

If the book has any shortcoming, though, it is that some subjects are barely touched upon. Signal transduction and molecular evolution are given short shrift, for example, but then, if every sub-discipline were given the depth given to basic optics, this book would be unmanageably immense. Enjoy it for what it is: a literate exploration of the major questions people have asked about eyes and vision for the last few thousand years.

The heartbreaking beauty of development

This is a spectacular video of the development of Clypeaster subdepressus, also called a sand dollar or sea biscuit. These are stunningly beautiful creatures (as are we all, of course), and it is so cool to see them changing here. The video starts with a little echinoderm porn — these animals are profligate with their gametes — and then we see early divisions, gastrulation, the formation of the pluteus larva, metamorphosis into Aristotle’s lantern (one of the more charming names for a developmental stage), and into an ungainly spiky juvenile.

This is why some of us are developmental biologists: it’s all about the exotic weirdness and delicate loveliness of transformation.


A Sea Biscuit’s Life from Bruno Vellutini on Vimeo.

Why can’t we have this shown in the US?

Surely there can’t be anything objectionable to the Religious Right in a documentary called Animals in the Womb, can there? It sounds like it could be fun, with videos shot using tiny little cameras (and some simulations) of developing embryos in vivo. The only thing I object to is the silly title, since they do have invertebrates and non-mammalian vertebrates on the show, so “womb” is a major misnomer.

It’s going to be on Channel 4 in the UK — I suppose I’ll have to wait for it to be released on DVD.

Watching every cell of the developing zebrafish

Blogging on Peer-Reviewed Research

How can I respond to a story about zebrafish, development, and new imaging and visualization techniques? Total incoherent nerdgasm is how.

Keller et al. are using a technique called digital scanned laser light sheet fluorescence microscopy (DSLM) to do fast, high-resolution, 3-D scans through developing embryos over time; using a GFP-histone fusion protein marker, they localize the nucleus of every single cell in the embryo. Some of the geeky specs:

  • 1500×1500 pixel 2-D resolution

  • 12 bits per pixel dynamic range

  • Imaging speed of 10 million voxels per second

  • Complete scan of a 1 cubic millimeter volume in 3µm steps in 90 seconds

  • Efficient excitation (5600 times less energy than a confocal, one million times less than a two-photon scope) to minimize bleaching and photodamage

Trust me, this is great stuff — as someone who was trying to do crude imaging of fluorescently labeled cells in the 1980s using a standard fluorescence scope and storing stills on VHS tape, this is all very Buck Rogers. Just load your embryo into the machine, start up the scanner, and it sits there collecting gigabytes of data for you for hours and hours.

But wait! That’s not all! They’ve also got sophisticated analysis tools that go through the collected images and put together data projections for you. For instance, it will color code cells by how fast they are migrating, or will count cell divisions. Similar tools have been available for C. elegans for a while now, but they have an advantage: they’re tiny animals where you might have to follow a thousand cells to get the full story. In zebrafish, you need to track tens of thousands of cells to capture all the details of a developmental event. This gadget can do it.

Here, for instance, are a couple of images to show what it looks like. The right half is the raw embryo, where each bright spot is a single cell nucleus; the left is one where the pattern of cell movement is color-coded, making it easier to spot exactly what domains of cells are doing.

i-a993b2c6d92e3964a73f6707b3cee9ca-dslm.jpg
Cell tracking and detection of cell divisions in the
digital embryo. (A) Microscopy data (right half of embryo:
animal view maximum-projection) and digital embryo (left
half of embryo) with color-encoded migration directions (see
movie S9). Color-code: dorsal migration (green), ventral
migration (cyan), towards/away from body axis (red/yellow),
toward yolk (pink).

I grabbed one of their movies and threw it on YouTube for the bandwidth-challenged. It’s not very pretty, but that’s the fault of reducing it and compressing it with YouTube’s standard tools. This is an example with color-coded migration (blue cells are relatively motionless, orange ones are moving fast), and you can at least get the gist of what you can detect. You can see the early scrambling of cells in the blastula, migration during epiboly and blastopore closure, and convergence in the formation of the body axis fairly easily. Well, you can if you’re familiar with fish embryology, anyway.

This crappy little video doesn’t do it justice, however. Take a look at the Zebrafish Digital Embryo movie repository for much higher resolution images that are crisp and sharp and unmarred by compression artifacts. It contains DivX and Quicktime movies that are somewhat large, 10-40M typically, that represent visualizations of databases that are several hundred megabytes in size.

What can you do with it? They describe observations of early symmetry breaking events; patterns of synchrony and symmetry in cell divisions; direct observations of the formation of specific tissues; and comparisons with mutant embryos that reveal differences in cell assortment. It’s fabulous work, and I think I’m going to be wishing for a bank of big computers and lasers and scopes for Christmas—only about $100,000 cheap! Until then, get a fast internet connection and browse through the movies.

Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EHK (2008) Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy. Science 2008 Oct 9. [Epub ahead of print].

Reprogramming the pancreas

Blogging on Peer-Reviewed Research

Wow…so have you heard about this result?

One goal of regenerative medicine is to instructively convert adult cells into other cell types for tissue repair and regeneration. Although isolated examples of adult cell reprogramming are known, there is no general understanding of how to turn one cell type into another in a controlled manner. Here, using a strategy of re-expressing key developmental regulators in vivo, we identify a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble β-cells. The induced β-cells are indistinguishable from endogenous islet β-cells in size, shape and ultrastructure. They express genes essential for β-cell function and can ameliorate hyperglycaemia by remodelling local vasculature and secreting insulin. This study provides an example of cellular reprogramming using defined factors in an adult organ and suggests a general paradigm for directing cell reprogramming without reversion to a pluripotent stem cell state.

This is a big deal, I think, so allow me to translate.

First, a little caveat: this is a recent result published in Nature, and it is basic science, not clinical work. Before you start thinking it’s a new treatment for diabetes, I have to dash a little cold water on you and warn you that this has a long, long way to go before it can be applied to humans…but it does open the door to some future strategies that might be applied.

The pancreas is a fairly complicated organ. It’s made up of a variety of different cells that we can toss into a couple of different classes. There are garden variety support cells — mesenchyme, connective tissue, components of the circulatory system, and the ductwork of the organ — that provide building services for the other cell types. Then there are exocrine cells, cells that produce quantities of important substances that are piped directly into the digestive tract via ducts. Among the most important materials exported by this route are bicarbonate buffers to neutralize stomach acids and enzymes like amylase to digest sugars. Finally, the class of cells that most people are familiar with, because they are the subject of a common disease, are the endocrine cells. These are cells that generate hormonal signals that are secreted into the blood stream, and the most familiar of these are the beta (β) cells, which are organized into clumps called islets and which secrete insulin…and if something goes awry with the β cells, the resulting disease is called diabetes.

What the researchers did was identify a small subset of transcription factors, the genes Ngn3, Pdx1 and Mafa, that are sufficient to switch on the insulin production genes in non-insulin-producing cells of the pancreas. They can turn exocrine cells into β cells, which produce insulin, and these cells reduce the effects of diabetes.

The way they did this was to insert the transcription factors (and a gene that makes a glowing protein, GFP, as a marker) into adenoviruses, and then inject the virus directly into the pancreases of genetically immunodeficient (to reduce immune response complications) adult mice. The viruses infected a subset of the pancreatic cells, preferentially the exocrine cells, and started pumping out the transcription factors. As is common in these kinds of genetic engineering experiments, the use of viral transfection is perhaps the scariest part of the story; viruses aren’t trivial to keep in check. However, they report that they also did later PCR tests of adjacent tissues and found no evidence that the virus spread beyond the target organ; they also found that inducing the expression of the 3 transcription factors in other kinds of cells, like muscle, seems to do nothing. These genes are only potent in pancreatic cells that are already primed to be competent to respond to the signals generated by the transcription factors.

The virus is also not needed for long term maintenance of these cells. The virus in the pancreas, as determined by PCR, is cleared away after about 2 months. It seems that all it takes is a brief jolt of expression of Ngn3, Pdx1 and Mafa to switch susceptible cells into the β cell state, and that the developmental program is then self-sustaining.

The authors also made diabetic mice by injecting them with streptozotocin, which kills islet β cells, and then gave them the viral cocktail injection. It did not cure their diabetes, but it did give them significantly greater glucose tolerance, and they did measure increased blood insulin levels. One reason the treatment may not be as effective as it could be is that it simply converts random, scattered exocrine cells into single β cells that are not organized into the islets of the normal pancreas.

A lot of attention has been paid to embryonic stem cell and adult stem cell technologies, and those are both important and provide research and treatment opportunities that must not be neglected, but this is a third way: mastering the developmental control genes of the cell so that we can reprogram mature cells into any cell type we need. While injecting a person’s pancreas with a collection of viruses to rebuild missing cell types might be a little hazardous and crude, there may come a day when we can collect a few cells from an individual by a scraping or biopsy, grow them in a dish to get enough, tickle their transcription factors to cause them to differentiate into the cell, tissue, or organ type we want, and transplant the final, immunocompatible product right back into the patient.

This is the direction developmental medicine can take us — I hope you’re all ready to support it.

Zhou Q, Brown J, Kanarek A, Rajagopa J, Melton DA (2008) In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature Aug 27. [Epub ahead of print].

Blurring the distinction between contraception and abortion

Monday morning, PST: time for some science with a side of controversy, Danio-style

There’s a Department of Health and Human Services document circulating that’s got the pro-choice lobby up in arms. Afarensis and The Questionable Authority weighed in on the sociopolitical impact of such a policy last week, but in addition to the significant threat to reproductive rights that it presents, this proposal is yet another example of the complete lack of scientific expertise informing decisions about public health.
At issue is the determination of a time point that marks the beginning of pregnancy. The consensus of the medical community is that an established pregnancy occurs at the point when the blastocyst successfully implants into the uterine wall. This time point makes a lot of sense in considering early events in the reproductive process. Pre-implantation embryos have a vast distance to travel, complex chemical cues to navigate, and a ticking biological clock to contend with within the bounds of the female reproductive cycle. Roughly 40% of all embryos don’t survive the ordeal. These odds are one good reason to hold off on crying ‘pregnant’ until a successful implantation is achieved; another is that implantation signifies the beginning of the physiological impact of a pregnancy on a woman’s body. Developmental events prior to implantation have essentially no impact on maternal tissues, which are just marking time until the beginning of the next menstrual cycle. The massive signaling between embryonic and uterine tissues that occur during implantation, the establishment of maternal and embryonic connections and boundaries, delineating the difference between ‘self’ and ‘not self’, are all medically relevant occurrences in terms of the physiology of the female patient, hence the general accord within the medical community in marking this time point, and none before it, as the point at which a pregnancy is established.
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Basics: Sonic Hedgehog

Every time I mention this developmentally significant molecule, Sonic hedgehog, I get a volley of questions about whether it is really called that, what it does, and why it keeps cropping up in articles about everything from snake fangs to mouse penises to whale fins to worm brains. The time seems appropriate to give a brief introduction to the hedgehog family of signaling molecules.

First, a brief overview of what Sonic hedgehog, or shh, is, which will also give you an idea about why it keeps coming up in these development papers. We often compare the genome to a toolbox — a collection of tools that play various roles in the construction of an organism. If I had to say what tool Sonic hedgehog is most like (keeping in mind that metaphors should not be overstretched), it would be like a tape measure. It’s going to have multiple uses: as a straightedge, as a paperweight to hold down your blueprints, as something to fence with your coworkers on a break, and even to measure distances. It will be pulled out at multiple times during a construction job, and it’s generically useful — you don’t need one tape measure to measure windows, another to measure doors, and yet another to measure countertops. Sonic hedgehog is just like that, getting whipped out multiple times for multiple uses during development, often being used where structures need to be patterned.

Let’s dig into some of the details. I’m using the 2006 review by Ingham and Placzek for most of this summary, so if you really want to get deeper into the literature, I recommend that paper as a starting point.

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Evolving snake fangs

Blogging on Peer-Reviewed Research
i-3c3ae12d88b9d206fc84149b28a6cf52-fang_embryo.jpg
Ontogenetic allometry in the fang in the front-fanged Causus rhombeatus (Viperidae) displaces the fang along the upper jaw. Scale bars, 1 mm. We note the change in relative size of the upper jaw subregions: i, anterior; ii, fang; iii, posterior. d.a.o., days after oviposition.

I keep saying this to everyone: if you want to understand the origin of novel morphological features in multicellular organisms, you have to look at their development. “Everything is the way it is because of how it got that way,” as D’Arcy Thompson said, so comprehending the ontogeny of form is absolutely critical to understanding what processes were sculpted by evolution. Now here’s a lovely piece of work that uses snake embryology to come to some interesting conclusions about how venomous fangs evolved.

Basal snakes, animals like boas, lack venom and specialized fangs altogether; they have relatively simple rows of small sharp teeth. Elapid snakes, like cobras and mambas and coral snakes, are at the other extreme, with prominent fangs at the front of their jaws that act like injection needles to deliver poisons. Then there are the Viperidae, rattlesnakes and pit vipers and copperheads, that also have front fangs, but phylogenetically belong to a distinct lineage from the elapids. And finally there are other snakes like the grass snake that have enlarged fangs at the back of their jaws. It’s a bit confusing: did all of these lineages independently evolve fangs and venom glands, or are there common underpinnings to all of these arrangements?

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Snake segmentation

Blogging on Peer-Reviewed Research

Life has two contradictory properties that any theory explaining its origin must encompass: similarities everywhere, and differences separating species. So far, the only theory that covers both beautifully and explains how one is the consequence of the other is evolution. Common descent unites all life on earth, while evolution itself is about constant change; similarities are rooted in our shared ancestry, while differences arise as lineages diverge.

Now here’s a new example of both phenomena: the development of segmentation in snakes. We humans have 33 vertebrae, zebrafish have 30-33, chickens have 55, mice have 65, and snakes have up to 300 — there’s about a ten-fold range right there. There are big obvious morphological and functional differences, too: snakes are sinuous slitherers notable for their flexibility, fish use their spines as springs for side-to-side motion, chickens fuse the skeleton into a bony box, and humans are upright bipeds with backaches. Yet underlying all that diversity is a common thread, that segmented vertebral column.

i-44c0b44c78c71955b53b0a7f004b7ea4-snakeseg.jpg
(Click for larger image)
Vertebral formula and somitogenesis in the corn snake.
a, Alizarin staining of a corn snake showing 296 vertebrae, including 3
cervical, 219 thoracic, 4 cloacal (distinguishable by their forked
lymphapophyses) and 70 caudal. b, Time course of corn snake development
after egg laying (118-somite embryo on the far left) until the end of
somitogenesis (~315 somites).

The similarities are a result of common descent. The differences, it turns out, arise from subtle changes in developmental timing.

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