Dissecting embryos from half a billion years ago


There is a treasure trove in China: the well-preserved phosphatized embryos of the Doushantuo formation, a sampling of the developmental events in ancient metazoans between 551 and 635 million years ago. These are splendid specimens that give us a peek at some awesomely fragile organisms, and modern technology helps by giving us new tools, like x-ray computed tomography (CT), scanning electron microscopy (SEM), thin-section petrography, synchrotron X-ray tomographic microscopy (SRXTM), and computer-aided visualization, that allow us to dig into the fine detail inside these delicate specimens and display and manipulate the data. A new paper in Science describes a survey of a large collection of these embryos, probed with these new techniques, and rendered for our viewing pleasure…that is, we've got pretty pictures!

One of the things that can be done is that the embryos can be virtually dissected into their component cells, and even the sub-cellular organelles examined. In the figure below, one animal was scanned and all the boundaries between cells identified, allowing the computer to pull it apart and present each cell separately. This is a 16-cell embryo; about 3/4 of the specimens have 2n cells, an indicator that these are members of a synchronously dividing population, and the roughly equal volumes of the cells also support the idea that these are cells of an early blastula. The remaining quarter of the specimens show deviations that indicate either asynchronous cell divisions or naturally occurring asymmetry.

The techniques allow the interior of the cells to be examined, and in many cases large organelles (Vesicles? Nuclei? Their identity is ambiguous) are preserved, shown as the darker green blobs in the images.

(A) Scanning electron micrograph of a 16-cell embryo. (B) Iso-surface model of the exterior of a 16-cell embryo (specimen DOU-10). (C) Schematic drawing with cells labeled to correspond to their models [(E) to (T)]. (D) An x-ray section showing very faint cell boundaries and subcellular spheroidal structures (arrowheads). Grayscale values in image have been inverted and the image placed on a black background to match (A). (E to T) Models of all 16 cells. Cells with intracellular structures (shaded dark green) are rendered transparent and those without are opaque. Corrugation on cell faces is an isocontouring artifact. (M) shows the internal cell. Scale bar in (A), 200 µm; in (E), 85 µm.

The images below illustrate the level of subcellular detail that can be achieved. The embryos aren't completely preserved—no one is going to pull molecules of a signal transduction pathway out of there—and are subject to all kinds of taphonomic and diagenetic artifacts, some of which I've discussed before. The actual chemical contents have been replaced, so there's no way to tell if, for instance, a large kidney shaped vesicle in the interior once contained massive amounts of DNA (it's a nucleus!) or lysozyme (it's a vesicle!). There are suggestive elements, such as the fact that some of the organelles are consistently aligned relative to the cleavage planes, suggesting that they might be nuclei, which do the same thing in modern embryos. Numerous smaller blobs suggest lipid granules, another common feature of developing cells. In particular, compare the "A" figures to "B"; "A" is a two celled embryo from Doushantuo, while "B" is a two-cell sea urchin embryo. The similarities are striking.

(click for larger image)

(A) A two-cell embryo (specimen MPKxiv). (A1) Surface rendering of the embryo based on tomographic scans, showing the position of the orthoslice (A2), which reveals internal preservation of spheroidal subcellular structures analagous to modern lipid vesicles or yolk granules. (A3) High magnification of an orthoslice at the boundary between the two cells. (B1 and B2) Two-cell embryo of the sea urchin Heliocidaris erythrogamma, including an enlarged view of the lipid vesicles. Such vesicles are spheroidal and can be even larger in large-egged embryos of other modern invertebrates. (C) Orthosliced volume rendering of a possible embryo (specimen SB0604Clea), illustrating intact, deflated, and collapsed spheroidal and ovoidal interior structures, coated by several generations of cement. The left hemisphere is dominated by a clotted fabric. Representative deflated (white arrowhead) and collapsed (black arrow) spheres are indicated. Slices in (A) and (C) are negatives: Darker grayscale values represent lower x-ray attenuation. (D and F) Photomicrographs of four-cell embryos with subcellular structures (arrowheads). (F) Extracted, embedded, and thin-sectioned specimen. (E1) X-ray section through a four-cell embryo (specimen DOU-8), showing three of the cells with paired subcellular structures (one pair is indicated by arrowheads), each with slightly greater attenuation (dark regions in image). (E2 to E4) Isosurface models of the four tetrahedrally arranged cells, with two of the cells extracted. All cells have paired subcellular structures, but only the upper two cells are rendered transparent to show intracellular structures (shaded green). (E4) Embryo from (E2), rotated horizontally 180°;. (G) TEM images of reniform (G1) to spheroidal (G2) structures like those in (D) to (F). Although the exact orientation of the embryo in (G1) is poorly constrained because of difficulties inherent in sample embedding and microtoming, it is hypothesized that this represents a tangential section through a reniform subcellular structure, so that each kidney-shaped structure is represented by two subcircular, more electron-dense areas (dark regions in image). (H) Transmitted-light photomicrograph of much smaller algal or cyanobacterial cells, each with one or more intracellular structures that occupy much of each cell. Relative scale bar size is as follows: for (A) and (B), 170 µm; for (C), 270 µm; for (D), 150 µm; for (E), 150 µm; for (F), 190 µm; for (G1), 8 µm; for (G2), 5 µm; for (H), 20 µm.

The feature that jumps out at us from these fossils are their similarities to modern organisms at early stages of development. However, the authors also not a significant absence, a difference from modern embryos: even in the later stage fossils of a thousand cells or more, there is no sign of epithelialization. This is a major lack. One of the hallmarks of the development of modern metazoans, from sponges on up, is the early formation of sheets of cells—even the terms diploblast and triploblast refer to the number of tissue layers formed, and in derived organisms like the zebrafish what we watch in early development is the formation of ectodermal and mesodermal sheets of tissue, and their movements and interactions. These embryos don't exhibit any sign of doing that! The absence of such a key feature suggests that these animals are very primitive, and that what we're seeing in this collection are the preserved remains of the embryos of stem-group metazoans.

Hagadorn JW, Xiao S, Donoghue PC, Bengtson S, Gostling NJ, Pawlowska M, Raff EC, Raff RA, Turner FR, Chongyu Y, Zhou C, Yuan X, McFeely MB, Stampanoni M, Nealson KH (2006) Cellular and subcellular structure of neoproterozoic animal embryos. Science 314(5797):291-4.

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There are these fossilized embryos from the Ediacaran, approximately 570 million years ago, that have been uncovered in the Doushantuo formation in China. I've mentioned them before, and as you can see below, they are genuinely spectacular. Parapandorina raphospissa But, you know, I work with…

What's a good reference for reading a little on epithelialization? I've googled but I'm just finding stuff on wounds.

By Patrick Caldon (not verified) on 24 Oct 2006 #permalink

Those really are lovely pics. (Though now I'm waiting for Michael Crichton to step forward and holler "Betcha we can pull some fresh DNA outta these suckers!")

Is it possible that those thousand-plus-cell blastula-like items are the actual adult organisms? The resemblance to animal embryos is certainly striking, but is there any indication of further development beyond the hollow-ball-o-cells stage?

Can they tell if they were deuterostomes or protostomes?
I heard somewhere that these were considered to be deuterostome embryos.

Hi, Patrick--

Sorry this is so rudimentary; I'm totally in crunch mode right now, or I'd dig a little more for you.

A PubMed search on:

epithelialization AND invertebrate NOT wound NOT injury NOT trauma NOT burn

turned up the following list:

1: Wang F, Dumstrei K, Haag T, Hartenstein V. The role of DE-cadherin during cellularization, germ layer formation and early neurogenesis in the Drosophila embryo. Dev Biol. 2004 Jun 15;270(2):350-63. PMID: 15183719 [PubMed - indexed for MEDLINE]

2: Mori K, Yang J, Barasch J. Ureteric bud controls multiple steps in the conversion of mesenchyme to epithelia. Semin Cell Dev Biol. 2003 Aug;14(4):209-16. Review. PMID: 14627119 [PubMed - indexed for MEDLINE]

3: Shook D, Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev. 2003 Nov;120(11):1351-83. Review. PMID: 14623443 [PubMed - indexed for MEDLINE]

4: Palacios R, Osorio LE, Grajalew LF, Ochoa MT. Treatment failure in children in a randomized clinical trial with 10 and 20 days of meglumine antimonate for cutaneous leishmaniasis due to Leishmania viannia species. Am J Trop Med Hyg. 2001 Mar-Apr;64(3-4):187-93. PMID: 11442216 [PubMed - indexed for MEDLINE] (this one is almost certainly not what you're looking for)

5: Portereiko MF, Mango SE. Early morphogenesis of the Caenorhabditis elegans pharynx. Dev Biol. 2001 May 15;233(2):482-94. PMID: 11336509 [PubMed - indexed for MEDLINE]

6: Zhu X, Joh K, Hedgecock EM, Hori K. Identification of epi-1 locus as a laminin alpha chain gene in the nematode Caenorhabditis elegans and characterization of epi-1 mutant alleles. DNA Seq. 1999;10(4-5):207-17. PMID: 10727078 [PubMed - indexed for MEDLINE]

7: Holley SA, Nusslein-Volhard C. Somitogenesis in zebrafish. Curr Top Dev Biol. 2000;47:247-77. Review. PMID: 10595307 [PubMed - indexed for MEDLINE]

8: Tamura M, Dan-Sohkawa M, Kaneko H. Coelomic pouch formation in reconstructing embryos of the starfish Asterina pectinifera. Dev Growth Differ. 1998 Oct;40(5):567-75. Erratum in: Dev Growth Differ 1999 Feb;41(1):113-7. PMID: 9783482 [PubMed - indexed for MEDLINE]

9: Miyamoto N, Yoshida M, Kuratani S, Matsuo I, Aizawa S. Defects of urogenital development in mice lacking Emx2. Development. 1997 May;124(9):1653-64. PMID: 9165114 [PubMed - indexed for MEDLINE]

10: Bernacki SH, McClay DR. Embryonic cellular organization: differential restriction of fates as revealed by cell aggregates and lineage markers. J Exp Zool. 1989 Aug;251(2):203-16. PMID: 2671252 [PubMed - indexed for MEDLINE]

Hope some of these may be helpful, even if for nothing more than their Literature Review and References sections; sorry that I only have time for such a superficial search at the moment. Quick PubMed searches are my specialty, but Web of Science and other resources that others here may point you at can also provide a good start.

Aah, I wished you got time to comment on the latest reports, and now you did. Excellent, and a nice post too.

This time I feel a special connection. To way back when I did school practise on a steel mill quality facility, helping crunching statistics from microscopy of inclusions superficially looking like these. Constant nucleation rate while freezing out and then constant growth gave a default log-normal size distribution IIRC, with measurements compensated for cut and small sample effects before extracting quality parameters.

(((Data was collected from the autoscan by computer over HP-IB (IEEE -488) bus, and it was my task to unravel the manufacturers spaghetti control and feature extraction code, fix some common error states, compress it to 1/3, and make place for the full statistics package. (Yes, computers were memory limited way back.) Fun stuff!)))

BTW, it feels funny to me when SEM:s are called new tools. Yes, modern SEM:s are much more capable and ubiquitous. But the technology itself is 70 years; invented 1935, tested in a STEM 1938 and commercialised 1965. I've seen them in labs and diverse production facilities in all my life. History links at http://en.wikipedia.org/wiki/Scanning_electron_microscope which also has some fancy random biological SEM images.

By Torbjörn Larsson (not verified) on 24 Oct 2006 #permalink

Thanks RavenT - in cruch mode myself, but this is heaps and will give me a little weekend reading.

By Patrick Caldon (not verified) on 24 Oct 2006 #permalink

"Can they tell if they were deuterostomes or protostomes?"

As a stem group metazoan probably neither.

By James Orpin (not verified) on 24 Oct 2006 #permalink

Is it possible that those thousand-plus-cell blastula-like items are the actual adult organisms? The resemblance to animal embryos is certainly striking, but is there any indication of further development beyond the hollow-ball-o-cells stage?

I dunno whether this paper says anything, but the Intarweb notes that the Doushantuo organisms tend to be about the same size no matter how many cells they have; that is, their cells were rapidly dividing without growing at all in between divisions. That's characteristic of embryos but, of course, uncharacteristic of adult colonial organisms (since obviously they have to do their feeding and per-cell growth sometime.)

By Anton Mates (not verified) on 24 Oct 2006 #permalink