Genetic Toolkits for Multicellularity and Development

Techne

Registered Senior Member
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).

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Figure 1

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 multicellular body plans (>3 cell types) (Figure 2).*

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Figure 2: Tree of life (Addapted 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 of 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 invertebrates.

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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 however are conserved accross all domains 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.
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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).*​

Note that many of the components of the signaling pathway are present in various bacterial and archaeal lineages. Also note that the origin of multicellular body plans roughly coincide 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 the increase in 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 multicellular body plans (>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].

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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.​

Intriguing questions can arise from these observations.
1) Why does an increase in atmospheric oxygen seem to have the effect of driving eukaryotic multilcellular life but not bacteria and archaea? Is an intrinsic and latent property present in this domain?
2) Gene loss vs innovation: How much gene loss and how much innovation (not just co-option) has occured from the LUCA? (Speculating)
3) Why did all the toolkit parts for the hh-pathway converge on a single sterolation pathway when so many other possibilities are available? Or is it the optimal possibility and random variation and selection processes used by life hit a global optimum?

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.
 
Techne,
are you an instructor?

you would be a fine person to work with.....

would love to go over the "Mechanism of action and signal transduction"

but perhaps another day

thanks for your contributions!

:>:>:>
 
A few interesting observations:

1) Components of the multicellular signaling pathway (sonic-hedgehog) were present in ancestral lineages way before the emergence of multi-cellularity.

2) The origin of multi-cellular body plans roughly coincide with an increase in atmospheric oxygen pressure as well as the first bona fide hedgling.

3) Hedglings are the only examples of post-translational sterolation (addition of cholesterol) of proteins in contempory biology and 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).

4) While large parts of the hedgehog-signaling pathway was present, a little extra oxygen was needed to unlock multicellular signaling capabilities of hedglings.

5) The increase in atmospheric oxygen was bought on about by life itself and this increase in atmospheric oxygen in turn seemed to have unlocked the pathways to multicellular body plans

And now this:
Oxygen Key To 'Cut And Paste' Of Genes
ScienceDaily (July 12, 2009) — An oxygen-sensitive enzyme has been found to play a key role in how genes create the many different proteins that make up our bodies.

The finding shows that the enzyme, termed Jmjd6, directly intervenes in the process in which the DNA of our genes is ‘cut and pasted’ into instructions for the creation of specific proteins.

The discovery, reported in this week’s Science by a team led by scientists from Oxford University and Ludwig-Maximilians-University, Munich, opens up a new area of molecular research into conditions such as heart disease and cancer.

‘Previous work from Oxford has shown that some of these enzymes, called oxygenases, affect which genes are expressed in response to low levels of oxygen. What we have now found is that they also regulate the specific form this expression takes – to give the different proteins that make up everything from heart cells to tumours,’ said Professor Chris Schofield of Oxford University’s Department of Chemistry, one of the authors of the paper.

Genes, stored in the form of DNA, are converted into proteins by a ‘middleman molecule’ called Messenger Ribonucleic Acid – or ‘mRNA’.

Individual genes can often give rise to many different proteins because of a process known as mRNA splicing which enables the cutting and pasting of the mRNA that is produced from DNA. The proteins that the new oxygenase, termed Jmjd6, acts on are involved in regulating the 'cutting and pasting' process.

Angelika Böttger, who led the Munich group, said: ‘The discovery of a role for an oxygenase in mRNA splicing reveals that it is very likely that oxygen levels are involved in regulating almost all steps in the process of gene expression. The challenge now is to determine how the pattern of genes changes in different environments when oxygen is in short supply, enabling us to tackle important questions such as "why do tumour cells respond differently to low oxygen levels than normal cells?"'

A little more about the protein:
This gene encodes a nuclear protein with a JmjC domain. JmjC domain-containing proteins are predicted to function as protein hydroxylases or histone demethylases. This protein was first identified as a putative phosphatidylserine receptor involved in phagocytosis of apoptotic cells; however, subsequent studies have indicated that it does not directly function in the clearance of apoptotic cells, and questioned whether it is a true phosphatidylserine receptor. Multiple transcript variants encoding different isoforms have been found for this gene.
Present where? Well, all over it seems, even proteobacteria (one of the most primitive groups of organisms:
http://www.ebi.ac.uk/interpro/IEntry?ac=IPR003347
 
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