Sunday, May 21, 2017

Sponges Are Animals Too

A brief look at some innovations at the bottom of the animal evolutionary tree.

Of the many great innovations in evolution, the development of multicellular animals ranks pretty high. It was based on the many innovations that had previously generated eukaryotes and their ramifications into protists and other complex single cells. It was a big step to realize (unconsciously!) that, however big and complex one could make a lone cell, (think of a paramecium), possibilities of greater scale and specialized organization were being left on the table. Sponges are the most primitive animal that currently exists. To us, sponges are small and simple. But to their protist brethren, they are giants- vacuum cleaners of the sea that suck up protists and other detritus like so many dust mites.

Sponges have several distinct cell types, like outer epidermal cells, pore cells, and interior collar cells that have the flagella that keep water moving through the pores. Sponges also contain wandering cells that secrete collagen in the intersitial areas- the same protein that keeps our bodies together as well. And they have primitive muscle cells / myocytes, which can cause contractions to close pores when needed, and also the larger openings of some species. These myocytes necessarily have some electrical communication, to coordinate their activities around the osculum. So, not exactly Brad Pitt, but they have quite a bit of anatomical complexity

A sponge.

Key innovations of this (relatively) massive and complex organism were not just the novel molecules, such as collagen, and skeletal "spicule" molecules, but the regulatory apparatus that generates different cell types and keeps them at their work, diligently pumping, or waving, or opening/closing, etc. A recent paper looked at this regulatory system and found that sponges already have most of the special mechanisms that other animals use to specify cell fates and body organization.

Those mechanisms revolve around gene control. In the human genome, about 1% of the DNA codes for proteins- the actual stuff of our bodies. Roughly about 7 to 10% of the genome functions in other ways, principally as regulatory regions, RNA-encoding genes, etc. The rest seems to be junk, more or less. The sponge that has been sequenced to date has a genome with roughly the same number of genes as ours (20,000), in 1/20 the genome size. So there is quite a bit less junk and complexity, but still a very large tool chest.

Particularly, it has plenty of regulatory control, and the researchers explore its use of one hallmark of metazoan gene control- histone modification. While the basic role of histones is very simple- to act like tiny wheels around which the DNA wraps, keeping it both secure and compact- its position also allows it to regulate the access to that DNA by other proteins, especially regulators of expression. Higher metazoans have a dizzyingly complex "histone code", composed of chemical modifications to the positively charged tails. These modifications include methylation, di- and tri-methylation, and acetylation, on lysines at positions 4, 5, 9, 14, 20, 27, 79, and 122 of one of three different histones. Each modification and position means something different, and combinations can mean other things again, all affecting the degree and type of gene activity.

A small part of the Amphimedon queenslandica genome, with DNA positions on the X-axis, and annotation tracks in separate rows. Description and codes below.

One example of their data is show above. They tested the locations of five different histone modifications, (H3K4me3 and similar rows), as well as the RNA polymerase (RNAPII) that is the ultimate target of all the regulation. Also laid along the small segment of the genome (X-axis) are rows (tracks) that describe the predicted locations of genes (bottom rows, purple) and the expression of each location of the genome into RNA (dark and light orange, next rows up). At the very top, under the genomic coordinates, are predictions about chromatin states, based on the histone modification analysis, using knowledge from other (higher) animals. The code for this analysis is shown below
Codes for the top annotation tracks above.

The color codes for inferred chromatin states. "Active TSS" means an active transcription start site, or gene being expressed. "Transcr. at gene 5' and 3'" indicates signatures for boundaries of transcription units, though this is clearly not very precise. "Genic activated enhancers" mark upstream regulatory elements that regulate a local transcription start site, sometimes from far away. "Weak enahancers" mark the same, but less active. The "Bivalent/poised" notations indicate non-active transcription units or enhancers, which may have RNA polymerase parked, but not active. Lastly, the gray annotations indicate states of chromative repression of gene activity which are very important, and regulated by histone modification, but not relevant in this genomic location at the (adult) stage sampled.

A few sponge genes with differential expression in larval vs adult stages. Expression in orange and blue,  and the histone marks (H3K4me3) in purple at top. Genes are in purple at the bottom, complete with their complex exon-intron structure.

Another example of their data shows the developmental difference in a small genomic region between larva and adult stages. The triple methylation of Histone H3 at lysine 4 (H3K4me3) turns out to be the most clearly informative chromatin marker, and comes up reliably at active gene start sites/promoters, where it plays a role in activating transcription.

All this goes to show that the tools and knowledge that have been accumulated over the last couple of decades for the study of model animals like mice, fruit flies, and humans, are also totally relevant for the study of sponges at the base of the animal tree. And in turn, that sponges already had and have complex molecular mechanisms that drive their developmental and morphological complexity, such as it is.

If this study revolved around the histone code alone, it would not have been very novel, since yeast cells share much of this apparatus, and are tenaciously unicellular. But the researchers also delve into several other properties that are more diagnostic of animals, such as the the locations of enhancer elements. This sponge genome can have enhancers over 10,000 basepairs away from the sites they regulate, which allows multiple enhancer cassettes can operate on the same gene to provide complex combinatorial and developmentally variable control. Additionally, the large set of gene regulatory proteins is more reminiscent of the set available in animals, many of which themselves have complex enhancer-influenced control.

The large number of introns (interruptions in the blue coding areas in the diagrams above) is another sign of animal-like organization, and in the sponge turn out to be where many of the enhancers live that regulate genes at a distance. Lastly, the researchers mention that about 60 pairs of sponge genes are similarly paired in other animal species, (i.e., they are micro-syntenic), indicating another level of unexpected conservation over about 700 million years. These are some of the ingredients that molecular biologists are learning are important to climb the organizational ladder to multicellularity.