Pharyngula

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What are the key innovations that led to the evolution of multicellularity, and what were their precursors in the single-celled microbial life that existed before the metazoa? We can hypothesize at least two distinct kinds of features that had to have preceded true multicellularity.

  • The obvious feature is that cells must stick together; specific adhesion molecules must be present that link cells together, that aren’t generically sticky and bind the organism to everything. So we need molecules that link cell to cell. Another feature of multicellular animals is that they secrete extracellular matrix, a feltwork of molecules outside the cells to which they can also adhere.

  • A feature that distinguishes true multicellular animals from colonial organisms is division of labor — cells within the organism specialize and follow different functional roles. This requires cell signaling, in which information beyond simple stickiness is communicated to cells, and signal transduction mechanisms which translate the signals into different patterns of gene activity.

These are features that evolved over 600 million years ago, and we need to use a comparative approach to figure out how they arose. One strategy is to pursue breadth, cast the net wide, and examine divergent forms, for instance by
comparing multicellular plants and animals. This approach leads to an understanding of universal properties, of how general programs of multicellular development work. Another is to go deep and examine closer relatives to find the step by step details of our specific lineage, and that’s exactly what is being done in a new analysis of the choanoflagellate genome.

So what is a choanoflagellate? They are members of a diverse and common group of single-celled eukaryotes that possess a flagellum for motility and a collar of slender processes called microvilli that it uses to capture bacterial prey. It’s a very successful lifestyle that has allowed them to flourish in both marine and freshwater environments.

i-2a1d4626ae8081fa01395bf7823a7174-choanoflagellate.jpg
Choanoflagellate cells bear a single apical flagellum (arrow, b) and an apical collar of actin-filled microvilli (bracket, c). d, An overlay of β-tubulin (green), polymerized actin (red) and DNA localization (blue) reveals the position of the flagellum within the collar of microvilli. Scale bar, 2 µm.

Here’s the connection to multicellular animals, the metazoa: choanoflagellates are markedly similar to the choanocytes of sponges. These are cells lining the interior channels of the sponge, which beat their flagella to propel water through the animal, and use the microvilli to filter out food particles. Right away, we can see an adaptive reason for the evolution of multicellularity: ancestral choanoflagellate-like organisms that teamed up could more efficiently filter water to extract food. The question here is the identity of the specific molecules they used to form early colonies.

King and others have sequence the entire genome of Monosiga brevicollis and compared what they found there to similar genes in other organisms. The genome itself is about 41.6 megabases, and contains approximately 9,200 genes (about half of what is present in humans). These genes were then compared to those of a suite of organisms to sort out what was held in common with the multicellular animals, the metazoa, and what was different from our distant cousins, the fungi and plants.

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The close phylogenetic affinity between choanoflagellates and metazoans highlights the value of the M. brevicollis genome for investigations into metazoan origins, the biology of the last common ancestor of metazoans (filled circle) and the biology of the last common ancestor of choanoflagellates and metazoans (open circle). Genomes from species shown with their abbreviation were used for protein domain comparisons in this study: human (Homo sapiens, Hsap), ascidian (Ciona intestinalis, Cint), Drosophila (Drosophila melanogaster, Dmel), cnidarian (N. vectensis, Nvec), M. brevicollis (Mbre), zygomycete (Rhizopus oryzae, Rory), basidiomycete (Coprinus cinereus, Ccin), ascomycete (Neurospora crassa, Ncra), hemiascomycete (Saccharomyces cerevisiae, Scer), slime mould (Dictyosteliumdiscoideum, Ddis) and Arabidopsis (Arabidopsisthaliana, Atha).

So what did they find? Choanoflagellates have a surprisingly rich repertoire of cell adhesion molecules, with many members of families of genes that the metazoa also use. They have at least 23 cadherin genes; cadherins are calcium-dependent cell adhesion molecules that are not found in other multicellular organisms like fungi and plants, and are present in animals where they are used for essential processes in development like cell sorting and polarization, and in regulation of the morphogenetic movements of sheets of tissues … and there they are in the choanoflagellates as well. While some species of choanoflagellates will form clusters and at least transient colonies, M. brevicollis is not known to make such associations, so the function of these molecules in these particular organisms is a bit mysterious. They also contain integrin-α genes and genes with immunoglobulin domains — while you may be familiar with immunoglobulins as key proteins of the vertebrate immune system, the immunoglobulin motif is also a more general cell adhesion domain that is also found in many cells of the nervous system.

While these proteins that metazoans use to mediate interactions between cells are exciting to find in a choanoflagellate, and while their presence opens up new questions about their function, there’s another class of genes that are even more peculiar to find in a single-celled organism: genes for proteins that bind to the extracellular matrix. These are important in animals like us; we construct layers of extracellular matrix proteins during our development that are contained within our bodies, and cells bind to them and take advantage of them in embryogenesis. What do choanoflagellates use them for? They may be important in substrate attachment, or possibly these organisms secrete a more complex suite of molecules into their environment than is known.

So we see some remarkable homologies between choanoflagellates and metazoans in the genes that mediate cell adhesion and adhesions between cells and a matrix of molecules in the environment — our single-celled ancestors first built up a collection of tools to make them sticky, a cookbook of glue molecules that would later enable more sophisticated patterns of attachment to one another. The table below also shows that the choanoflagellates and their last common ancestor with the metazoa also evolved some common transcription factors, or gene regulators.

Note: sticky proteins and transcription factors are not unique to choanoflagellates and metazoans — bacteria, plants, fungi, etc. all also have them. What this work is showing is that the choanoflagellates and metazoa share an idiosyncratic, special set of sticky molecules and transcription factors.

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M. brevicollis possesses diverse adhesion and ECM domains previously thought to be unique to metazoans (magenta). In contrast, many metazoan sequence-specific transcription factors are absent from the M. brevicollis gene catalogue. For adhesion and ECM domains, a filled box indicates a domain identified by both SMART and Pfam, a half-filled box indicates a domain identified by either SMART or Pfam, and an open box indicates a domain that is not encoded by the current set of gene models. The presence (filled box) or absence (empty box) of transcription factor families was determined by reciprocal BLAST and SMART/Pfam domain annotations. EC, extracellular domain; cyto, cytoplasmic domain; asterisk, collagen triple-helix-domains occur in the extended tandem arrays diagnostic of collagen proteins found only in metazoans and choanoflagellates.

At the beginning of this article I said that there were two properties essential to multicellularity: adhesion and signaling. Choanoflagellates have the molecular precursors needed for metazoan-style adhesivity, but they lack metazoan-specific signaling pathways. This makes sense. A general property like adhesion may well have utility to a single celled organism, but the specific pathways that would trigger region- and tissue-specific differentiation would be a later innovation.

Even in the case of these unrepresented elements of the metazoan genome, though, we see premonitions in the choanoflagellate. While complete, recognizable homologs of important signaling genes like hedgehog and
Notch are not found, fragments of them are found scattered about. They didn’t appear out of nowhere — rather, there was a process of domain shuffling during metazoan evolution that built new signaling molecules by recombining elements present in the ancestral genome. So, while choanoflagellates reveal that the ancestor almost certainly did not have a true Notch gene, we can find 3 genes that contain pieces of Notch: one has the EGF domain, another the NL domain, and another the set of ankyrin repeats … and it’s easy to see that the Notch gene was not generated ex nihilo, but was assembled by splicing bits of pieces of extant genes into a novel protein.

i-3e88f6157235d74963693cf244bf43d7-choano_shuffling.jpg
Analysis of the draft gene set reveals that M. brevicollis possesses proteins containing domains characteristic of metazoan Notch (a, N1?N3) and hedgehog (b, H1 and H2). Some of these protein domains were previously thought to be unique to metazoans. The presence of these domains in separate M. brevicollis proteins implicates domain shuffling in the evolution of Notch and Hedgehog. Hh, hedgehog; N-hh, hedgehog N-terminal signalling domain; Hint, hedgehog/intein domain; TM, transmembrane domain; VWA, von Willebrand A domain.

Transitional fossils always get all the attention — and you’ve got to admit, a new collection of old bones is a sexy thing, an arresting attention grabber that has a lot of visual appeal. I think the more powerful modern evidence for evolution, though, are these examples of molecular transitions in which we can reconstruct the details of ancient changes. Not to belittle the fossil evidence, but changes within a single narrow lineage within a single phylum aren’t quite as dramatic or impressive as the kind of radical evolutionary event we see here — not just the reshaping of a femur, for instance, but the acquisition of whole new capabilities, the novel potential to build a femur in the first place. This is big stuff, a peek into core innovations that led to insects and jellyfish and grasshoppers and snails and cows, and that are held in common among all of us.

Yet despite the magnitude of the potential evolutionary consequences, we can also see revealed the mechanisms underlying them, and that they are small and simple changes, an expansion of capabilities present in miniscule, single-celled creatures. When evolving such fundamental and revolutionary features as multicellularity is such a patently feasible and explainable event, it does seem absurd that some people can still question relatively minor transformations, such as between varieties of ape.


King N, Westbrook MJ, Young SL, Kuo A, Abedin M, Chapman J, Fairclough S, Hellsten U, Isogai Y, Letunic I, Marr M, Pincus D, Putnam N, Rokas A, Wright KJ, Zuzow R, Dirks W, Good M, Goodstein D, Lemons D, Li W, Lyons JB, Morris A, Nichols S, Richter DJ, Salamov A, Sequencing JG, Bork P, Lim WA, Manning G, Miller WT, McGinnis W, Shapiro H, Tjian R, Grigoriev IV, Rokhsar D. (2008) The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans. Nature 451(7180):783-788.

Comments

  1. #1 David Marjanovi?, OM
    March 3, 2008

    Scale bar, 2 µm.

    Wow, that’s small for a eukaryote. Escherichia coli is 1 x 1 x 3 µm.

    Creationists declare frontloading in 5…4…3…

    Would be stupid of them — Monosiga, existing today, is not one of our ancestors. But I suppose that’s your point… :-)

  2. #2 David Marjanovi?, OM
    March 3, 2008

    No, it’s a different kind of stupid. See

    Oh. Yeah.

    Did I miss something that rules out the hypothesis that these animals, rather than being a sister clade to sponges, are descended from sponges, such that they could have degraded adhesion and signaling rather than precursor?

    Yes, a few molecular phylogenetic analyses for example.

  3. #3 David Marjanovi?, OM
    March 3, 2008

    Oops — they aren’t the sister-group of the sponges, we are. Choanoflagellates and animals are sister-groups or nearly so.

  4. #4 David Marjanovi?, OM
    March 4, 2008

    …or “we” would be, if the “sponges” weren’t turning out to be so damn paraphyletic. We can be the sister-group to calcareous sponges, how’s that?

    Yes, or even to a part of the calcareous sponges, the Homo…basalomorpha they are called, I think. We’re living in interesting times!

    I’ve never heard of a nucleated eukaryotic cell that was much less than 10 microns in approximate diameter.

    There are plenty. The record is a spherical marine green alga 0.8 µm in diameter — contains not only a nucleus (with a single pore), but also a chloroplast and a (single) mitochondrion. I’ll post a link later today. Or google “picoplankton”. Brown tides consist of tiny eukaryotes, for example…

  5. #5 David Marjanovi?, OM
    March 4, 2008

    Are there any known examples of a unicellular descendent from a multicellular ancestor [...]?

    Yeast: secondarily unicellular ascomycetes. Some yeasts, like Candida, retain the linear (hyphal) organization, though.

    —————–

    Contrary to my assertion, Ostreococcus tauri has several mitochondria. The website I was thinking of, which has nice pictures, isn’t among the first 50 Google results anymore.

    Brown tides are composed of pelagophytes that are apparently up to 3 µm in diameter.

  6. #6 David Marjanovi?, OM
    March 4, 2008

    BTW, I have made the rather stunning experience that there are molecular biologists who sometimes talk of “higher eukaryotes” and mean “everything except yeast”/”plants and animals”. They don’t know what they are talking about. The term is positively misleading.

  7. #7 Alma
    November 24, 2008

    As to size variations in eukaryotes: 20 malarial merozoites will fit into one red blood cell, which is a relatively small cell, itself.