Toolkit Parts for Multicellularity

The article about the evolutionary history of body size on Earth has raised some interest. Words like "latent evolutionary potential was realized", "realize preexisting evolutionary potential" and" a major innovation in organismal complexity—first the eukaryotic cell and later eukaryotic multicellularity" seem to have raised a few eye brows. Are "latent", "pre-existing","innovation" and "potential" the appropriate words? From the following figure, the earliest multicellular (Grypania spiralis) eukaryotic fossil dates back ±1.6 billion years ago (bya) and at present the earliest evidence for eukaryotic cells is posited to have existed 1.68-1.78 bya , (perhaps 1.8 bya or possibly even 2.1 bya) (Figure 1).

Figure 1: From the article.

The following tree is adapted from discoverlife.org with the tentative dates for the origins of archaea and bacteria, eukaryotes, as well as the origins of multicellularity and body plans (Figure 2).*

Figure 2: Tree of life (Adapted from discoverlife.org)*

As suggested by the paper, increases in cellular sizes roughly coincide with the alleviation of at least one environmental constraint, namely low atmospheric oxygen pressure. The origin of body plans (±0.6 bya) also seem to coincide with an increase in atmospheric pressure. Why could that be?

A look at hedgehogs

With about 6, 000 spines on their back, an excellent sense of smell, a running speed of 4.5 mph a normal heart rate of up to 19o bps and 10 bps during hibernation, hedgehogs are interesting little animals. Hedgehog (hh) genes are equally fascinating. The reason for the name of this gene is that a malfunctioning hh gene often results in the formation of small pointy projections on embryos, similar to that of a hedgehog. So what does it do?

Functions

The hh signaling pathway plays a fundamental role in cell pattrerning, cell proliferation and participates in the development of tissues and organs during the stages of animal development. It exerts its effect by influencing the transcription of many target genes in a concentration dependent manner.

Mechanism of action and signal transduction: Hints from hedgelings and hoglets

The hh protein comprises of two domains, namely the hedge domain (hedgling) and the hog domain (hoglet). The hedge domain acts as a ligand after processing and binds to a set of conserved receptors to activate downstream signal transduction pathways [1]. After transcription, the hh-gene undergoes a post-translational autocatalyzing editing process initiated by the hoglet resulting in the formation of the hedgling protein. Further processing of the hedgling occur and include the palmitoylation and sterolation (addition of cholesterol) of the ligand (Figure 3). Interestingly, hh proteins are the only examples of sterolation in contempory biology (more on that later) [2]. After processing, the hedgling ligand is transported through the Dispatched receptor where it binds to a specific lipd transport molecule (different in invertebrates and vertabrates) and is transported and binds to the 12-transmembrane protein called Patched. Internalisation od Patched alleviates the inhibitory effect of Patched on the 7-transmembrane protein Smoothened. This in turn activates the hedghog-related transcription factors (Gli in vertebrates and Ci in invertabrates) (Figure 3) [2]. This relatively simple pathway plays a crucial role in the unfolding of the developmental program in vertebrates and invertabrates.

Figure 3: Hedgehog signal transduction. The hedgehog protein is post-translationally modified through autocatalyzation and palmitate and cholesterol addition. Processed hedgelings are transported to the extracellular matrix through dispatched receptors and in turn transported by lipid transport molecules to bind to patched receptors. Binding of hedgling molecules to Patched receptor results in the subsequent activation of hedgehog mediated transcrition factors e.g. Gli in vertebrates and Ci in invertebrates.

With the knowledge of some of the proteins that play a part in hh-signal control, let's look at the evolution and origin of some of the components. The following proteins can be used for BLAST.
Hedgling (Amphimedon Queenslanica)
Hoglet (Monosiga Ovata)
Patched (Ciona Intestinalis)
Dispatched (Ciona Intestinalis)
Suppresor of Fused (Sufu) (Ciona Intestinalis)
Smoothened (Ciona Intestinalis)
Fused (Drosophila)
Gli1 (Human) or Ci (Drosophila)
Kif27 (vertebrate) or Cos2 (Drosophila)

Using the InterProScan Tool with these sequences, the following results were obtained:
Hedgling: The oldest (phylogenetically) bona fide hedgeling found so far is in the genome of the sponge, Amphimedon Queenslanica. However, the structure of this domain is structurally homologous to the zinc-binding motif in bacterial D-alanyl-D-alanine carboxypeptidases (the same motif found in beta-lactamases and the various nylonase genes).
Hoglets: Hoglets are typical Intein (internal protein) proteins also known as HINTs (hedgehog inteins) [3]. Inteins are selfish DNA elements that are distributed accross all the domains of life [4].
Patched: Patched is a transmembrane protein with a sterol sensing domain (SSD) and is also distributed in all the domains of life.
Dispatched: Dispathed is also a transmembrane protein with a SSD and forms a subfamily of the sterol sensing receptors. Also present in all the domains of life.
Fused: Fused is kinase conserved in all the domains of life.
Suppresor of Fused (Sufu): Sufu yielded an interesting result. Acting as a suppressor of the hh-signaling pathway, it is limited to the bilaterians and cnidaria and bacteria. it seems to have been lost in other linages.
Smoothened (Frizzled domain, G-protein-coupled receptor (GPCR) domain): Smoothened contains a frizzled domain and a GPCR domain. The frizzled domain is limited to eukaryotes, while the GPCR domain is conserved in all the domains of life.
Gli1: This protein (and cos2) is transcription factor and hh-signaling converges to control the activity of this protein. It is a zinc-finger protein. While zinc-finger proteins are conserved in all domains of life, this particular protein seems to be limited to eukaryotes.
Kif27: Kif27 (and Cos2) is a kinesin-related protein (KRP). Kif27 appears to be functional molecular motor while Cos2 seems to have lost the ability to function as a motor protein. KRPs are conserved accross all eukaryotic forms of life [5]. A conserved function of KRPs is to facilitate movement of vesicle along microtubules and one of the functions of Cos2 seems to be just that [6].

From the above, the following picture of the components of the hh-signaling toolkit can be drawn.

Figure 4: Origins of the parts in the hedgehog signaling pathway. (Red = absent, Orange = reasonable sequence and/or structural simlarity, Green = present, Graded green = part of the same family, Brown = unsure .

Note that many of the components of the signaling pathway are present in various bacterial and archaeal lineages. Also note that the origin of multicellul body plans roughly coincides with an increase in atmospheric oxygen pressure as well as the first bona fide hedgling. Remember, hedglings are the only examples of post-translational sterolation (addition of cholesterol) of proteins in contempory biology. Why is this interesting? Well, oxygen is needed for cholesterol synthesis, more importantly, oxygen is needed for placing the hydroxyl group in the 3-position of cholesterol which plays a crucial role in subsequent transformations (including sterolation). Thus, while large parts of the hh-signaling pathway was present, a little extra oxygen was needed to unlock multicellular signaling capabilities of hedglings.

Therefore, words like "pre-existing", "latent" and "potential" seem apt in describing the hedghog signaling pathway and the unfolding of multicellular body plans in relation to atmospheric oxygen pressure. "Innovation" perhaps not so much, seeing that only real innovation was bought on about by life itself namely the increase in atmospheric oxygen. This increase in atmospheric oxygen in turn seemed to have unlocked the pathways to multicellularity (more than 3 cell types).

Gene loss vs Innovation
Looking at the hh-signaling pathway, there seem to be very little innovation, and a lot of co-option of pre-existing information into new functions. Sufu was an interesting example of gene loss only to be co-opted later into a role in the hh-signaling pathway. With this in mind, what can one expect to find in the Last Universal Common Ancestor (LUCA)? Also consider the following. The Tetrahymena thermophila (alveolate) genome has been sequenced, and a number of genes that are absent in yeast (fungi), are found in amoeba, vertebrates, invertebrates as well as in the Tetrahymena genome. It paints the following picture (Figure 5) [7].

Figure 5: Genes present in Tetrahymena thermophila but absent in yeast indicate either convergnece in higher organisms or that the genes were present in the eukaryote common ancestor.

References
1. Matus DQ, Magie CR, Pang K, Martindale MQ, Thomsen GH. The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution. Dev Biol 2008; 313: 501-518.
2. Bijlsma MF, Spek CA, Peppelenbosch MP. Hedgehog: an unusual signal transducer. Bioessays 2004; 26: 387-394.
3. Perler FB. Protein splicing of inteins and hedgehog autoproteolysis: structure, function, and evolution. Cell 1998; 92: 1-4.
4. Pietrokovski S. Intein spread and extinction in evolution. Trends Genet 2001; 17 465-472.
5. Varjosalo M, Taipale J. Hedgehog: functions and mechanisms. Genes Dev 2008; 22: 2454-2472.
6. Ogden SK, Ascano M Jr, Stegman MA, Robbins DJ. Regulation of Hedgehog signaling: a complex story. Biochem Pharmacol 2004; 67: 805-814.
7. Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan M, Wortman JR. et al. Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol 2006; 4: e286.

J. L. Payne, A. G. Boyer, J. H. Brown, S. Finnegan, M. Kowalewski, R. A. Krause, S. K. Lyons, C. R. McClain, D. W. McShea, P. M. Novack-Gottshall, F. A. Smith, J. A. Stempien, S. C. Wang (2009). Two-phase increase in the maximum size of life over 3.5 billion years reflects biological innovation and environmental opportunity Proceedings of the National Academy of Sciences, 106 (1), 24-27 DOI: 10.1073/pnas.0806314106

*-corrections are welcomed.

Update 2009-08-17.
Following has been changed:
KRPs however are conserved accross all domains of life [5].
This should read:
KRPs are conserved accross all eukaryotic forms of life [5].

It is unlikely that KRPs are found in bacteria and archaea. The taxonomic figure here related to KRPs is a bit misleading as it might refer to just the ATP-binding site of KRPs and that might well be conserved accross all domains of life.

Thanks Steve