Slimeballs and eyeballs: hagfish and the evolution of the eye

Charles Darwin famously highlighted the eye, "with all its inimitable contrivances", as one of the hurdles in the acceptance of his theory of natural selection. "[It} seems, I freely confess, absurd in the highest possible degree", that this complex organ arose as the result of natural selection. This confession has in the past been seized upon by the bright lights of the intelligent design movement and their ilk as proof that Darwin himself had doubts about his own theories.

Creationists have gone a bit quiet on this front in recent years as more is known about the evolution of the eye, and they might just be done away with completely if a hypothesis set out in a Nature Reviews Neuroscience paper last December proves to be correct.

"If," Darwin wrote in On the Origin of Species, "numerous gradations from a perfect and complex eye to one very imperfect and simple, each grade being useful to its possessor, can be shown to exist ... and if any variation or modification in the organ be ever useful to the animal under changing conditions of life, then the difficulty of believing that a perfect and complex eye could be formed by natural selection, though insuperable by our imagination, can hardly be considered real."

In the Nature Reviews paper, the team of Professors Trevor Lamb of the John Curtin School of Medical Research at the Australian National University, Shaun Collin of the School of Biomedical Sciences at the University of Queensland and Ed Pugh of the FM Kirby Centre for Molecular Ophthalmology at the University of Pennsylvania have taken up Darwin's challenge and set out a proposed sequence of the evolution of the vertebrate eye which they believe might satisfy Darwin's prescription. And they have even set homework to test the hypothesis.

The team sets out their view of the sequence of events in the evolution of opsins, photoreceptors, the retina and the eye cup in vertebrates. They begin with the separation of primitive bilateral animals into the super-phyla of the protostomes and the deuterostomes approximately 580 million years ago. There have been multiple splits since then, the emergence of the chordates about 550 million years ago being an important one for our purposes.

These little animals, Trevor Lamb says, had developed a notochord and a dorsal nerve chord. They also had developed photosensitive regions at the rostral end. "These were just little spots with a few photoreceptors," Lamb says, "but their primitive receptors were remarkably similar to the cones that we have."

These photoreceptors were ciliary, as opposed to the rhabdomeric photoreceptors primarily found in the protostomes, the invertebrates. And the organs were not eyes as such, having no image forming ability, but presumably were used to distinguish light from dark, to regulate diurnal rhythms and for shadow detection.

"But in order to go to deeper waters, to work in lower light levels, one of these ancestors probably started expanding the photoreceptor region - it just grew and grew, there were more photoreceptors and they could absorb more light so they could work at lower light intensities," Lamb says. "That probably involved a spreading out of this retinal layer sideways."

At the same time, the skull was beginning to develop - the evolution of the craniates. "If you have your photosensitive region on the midline, covered by a skull, that's not going to be much good," he says. "It's going to be advantageous if you expand outwards to the side.

"Then there was probably a contact between this expanding light-sensitive region and the ectoderm [surface]. And what seems likely to have happened is that the proto-eye region induced changes in the surface and kept that surface clear - it stopped the pigmentation there. And subsequently that surface region probably bulged inwards and pushed in as an expansion that ultimately became the lens. Once you've got a thickening of the skin that has any kind of lens-like properties, you start getting some imaging. If you've got a light-sensitive region there, and something that's acting a bit like a lens in front of it, then you've got the rudiments of an imaging eye."

Even with something as rudimentary as this, you are likely to get an advantage. As Shaun Collin puts it, all of a sudden these animals were able to see what was going to eat them. "So they now needed to evolve something to stop being eaten, so you can either camouflage yourself or swim faster, hide or all of the above," he says. "That started what might have been predation and meant that a lot of animals could go in a lot of different directions - all these new groups popped up. Some of them have disappeared since but in this period all these new phyla of invertebrates and of chordates started and we think that the evolution of image-forming eyes was the catalyst to push this forward very quickly."

A perfectly straight-forward hypothesis, but where is the evidence that would have satisfied Mr Darwin? For this team, it lies not just in the fossil record but in the morphological data that can be evaluated in extant species. One of those necessary gradations is found in the hagfish, a rather unattractive species of jawless fish that lurks in deep water and exudes a gooey mess of slime when disturbed. They propose that the hagfish 'eye' is that missing step from primitive photosensitive regions to the development of the modern eye.

---PB--- Slimeballs and eyeballs

The hagfish are commonly grouped together with another extant species, the lampreys, in the cyclostome division (class Agnatha or jawless fish). The jawed fish, or gnathostomes, are a later division in our line. As the researchers write, controversy has long surrounded the interrelationship between hagfish, lampreys and jawed vertebrates. There are two modes of thought: one is that hagfish originated at the same time as the lampreys but diverged and then degenerated, losing their ability to form images. The other school is that hagfish diverged before the lampreys and as such must be considered a sister group.

Shaun Collin knows quite a bit about lampreys and hagfish, their eyes and proto-eyes. One of Australia's few marine neurobiologists, his work involves studying the evolutionary aspects of colour vision and of dim light vision, mostly working on primitive vertebrates.

Trevor Lamb is an expert on photoreceptors and the phenomenon of dark adaptation, the recovery of the visual system after exposure to very intense light. He has been working with psychologist and neuroscientist Ed Pugh for 25 years, collaborating on a unified theory on the photoreceptor's response to light and the cellular and molecular basis of dark adaptation.

Collin and Lamb have known each other for about 10 years and came together on this project when Lamb and Pugh were looking at the similarities and differences between rod and cone receptors within and across different species. Lamb and Pugh were probing the past to discover when rods and cones separated, and began delving even further back. They got to the lampreys, which have very similar eyes to us, but hit a stumbling block. Lamb asked Collin if he knew of something with a simpler retina or simpler photoreceptors and he mentioned hagfish. This is where the collaboration began.

Most people, including the fishermen who despair of catching them in their nets, think hagfish - or slime eels, as they are also known - are right ugly little beasts, Collin says. "They grow up to over one metre in length and we have been studying the New Zealand hag, which is a particularly large species. They've basically got a tough skin and the head region is quite strange.

"It doesn't have a jaw, so they are part of the jawless fishes group, and use a rasping mechanism to scrape flesh off their prey. It has a very strange way of feeding - it can actually attach to dead fish that have drifted down from above and proceeds to tie itself in a knot. The knot starts at the tail end and moves up towards the head, thereby enabling it to have something to hold onto while pulling flesh off."

Hagfish are a deep-water species, some extending down to a thousand metres or more, and they are called slime eels for a good reason, he says. They have slime glands in their skin which, when excited, produce large amounts of a combination of mucins and protein threads that turn the surrounding water into viscous mass of jelly.

"I examined these at the Scripps Institution of Oceanography some years ago and can still remember a colleague of mine putting a hagfish into a bucket, about half or three-quarters full of water, and disturbed the animal just so it swam around in circles," Collin says. "Within a few seconds, he could turn the bucket upside down and no water came out, no animal came out - it was like he'd put a jelly mould into the fridge and it has just gone solid. This is one of the mechanisms this animal uses to immobilise their prey."

While not a particularly pleasant creature, they have provided the perfect model to study the evolution of the eye. Hagfish don't have what we might consider an eye as such, merely photoreceptive tissue embedded in fat underneath a whitish patch of skin, he says.

"They have photoreceptors at the back of the eye which pick up light and dark and we suspect even may respond to different intensities of light, which we haven't proven yet. But there are definitely photoreceptors present, although they look a little bit different to ours."

Lampreys, on the other hand, have an eye that looks quite like ours, he says. "Effectively, it's like a human retina - the lens shape is a bit different because of the optical problems associated with living in water, but the retina looks very much like a rabbit or a squirrel or a human. Therefore, there has been a big jump between early animals and the lampreys. This is the contention of the paper, that the hagfish might be that link between those early eyes that are only able to see light and dark to something that can form an image with some resolution."

---PB--- Hypothesis and homework

The paper traces the development of the vertebrate eye, from the bilateral ancestor of 580 million years ago, in which a primordial opsin - a visual protein that functions as a G-protein-coupled receptor - has evolved into three major classes: rhabdomeric opsins, intermediate photoisomerase-like opsins, and ciliary opsins. Here, protostomes with their rhabdomeric opsins separate from deuterostomes and our ciliary opsins.

Stage two is the development of the protochordates, about 580-550 million years ago, in which the ciliary photoreceptor with a ciliary opsin and a hyperpolarising response continue to evolve. Here, the cephalochordates and the tunicates - represented today by the lancelets and the sea squirt respectively - diverge.

Stage three is the ancestral craniates of 550-530 million years ago, in which a ciliary photoreceptor sends signals to output neurons, a primordial lens develops and the lateral 'eye' invaginates. The researchers say this proto-eye remains present in extant hagfish, which separated from our line 530 million years ago.

Then comes stage four, a period of about 30 million years, in which lamprey-like ancestors evolve and photoreceptors with cone-like features appear, along with an explosion in visual ability. Genome duplications give rise to multiple copies of the phototransduction genes, which allow light to be converted into electrical signals; cell classes diverge into five cone-like receptors; cone bipolar cells and ganglion cells evolve; ganglion-cell axons project into the thalamus and the optic equipment evolves - the lens, the iris and the extra-ocular muscles.

About 500 million years ago the lampreys - containing this modern equipment but lacking intra-ocular muscles - diverge from our line.

Stage five is a long period of about 70 million years in which the jawless fish give rise to the jawed vertebrates and the optic boom continues - rod photoreceptors and rod bipolar cells evolve, along with an iris that can adjust to different light levels. At stage six, 430 million years ago, the last jawless fish separate from the gnathostomes, in which the cornea develops, the lens takes an elliptical shape and eyelids appear.

While some would dispute the divergence of the hagfish and the lampreys, the researchers find evidence in the developmental changes that occur in the larval form of lampreys. This is a period of about five years in which the larval form, or ammocoete, metamorphoses into the adult. The eye of the ammocoete is strikingly similar to that of the hagfish - "they are small, they are buried beneath skin and they possess a relatively undifferentiated retina", they write - but over five years "a slow process of neural differentiation occurs, in the following sequence: ganglion cells, amacrine and horizontal cells, photoreceptors and finally bipolar cells. This is broadly the same order in which the jawed-vertebrate retina differentiates".

To back up their hypothesis, Lamb, Collin and Pugh have set us some homework. They have set out a series of eight predictions they say will test their hypothesis, encouraging other researchers to delve further into the development of cones, rods and bipolar cells, and in particular to study the genes of cyclostomes, to trace definitively when hagfish diverged. They are currently writing a grant application to assist with completing the homework.

---PB--- Shining the light on dark adaptation

If their early careers are anything to go by, Trevor Lamb and Ed Pugh could be considered an odd couple to be researching dark adaptation and the photoreceptor's response to light. Lamb initially studied electrical engineering at the University of Melbourne before moving into physiology, while Pugh's first degree was in mathematics before moving into psychology and later neuroscience.

Lamb became interested in nerves and the brain in his final year of his engineering degree, and did a transfer course into physiology leading to a master's. He then moved to Cambridge University to pursue a PhD with Alan Hodgkin, who had discovered the action-potential impulse of the nerve axon. When Lamb arrived, however, Hodgkin had switched to photoreceptors, and Lamb followed that line.

At Cambridge, where he expected to stay for three years but ended up lasting 31, Lamb became interested in the phenomenon of dark adaptation - the recovery of the visual system after exposure to very intense light. Looking through the literature, he says, it turned out that the best data available was published by one EN Pugh of the University of Philadelphia: Pugh had been working on colour vision and human psychophysical dark adaptation. Lamb wrote to Pugh requesting any further data and was sent a spare copy of Pugh's PhD thesis. They have been collaborating ever since, on and off for 25 years, and were jointly awarded the 2006 Proctor Medal by the US-based Association for Research in Vision and Ophthalmology.

Lamb, who helped develop the suction pipette technique for recording electrically from photoreceptors with King-Wai Yau and Denis Baylor, returned to Australia in 2003 as a Federation Fellow. He runs the Visual Neuroscience Laboratory at the John Curtin School of Medicine at ANU, and is research director of the ARC Centre of Excellence in Vision Science. Here, he is looking at a number of aspects of photoreceptors and early events in the retina.

"We are interested in how it is that the photoreceptor's response to light turns off - the shut-off of the light response - and the molecular mechanisms underlying that," he says. "We are also interested in how this shut-off contributes to recovery after exposure to bright light.

"We are interested in this recovery in two realms: one is the short term, the very rapid shut-off that occurs within a second or so, and then secondly the very slow tail of the shut-off that occurs over many minutes, because that turns out to be involved in dark adaptation recovery. We are going from the level of single cells - using the suction pipette technique on single cells of mice and zebrafish - and on up to humans.

"We record from our own eyes using the electroretinogram to measure signals from the retina to work out what is going on, both in the photoreceptors and also the next layer of cells, the rod bipolar cells. Basically, we are looking at how activity in those cells influences the performance of the overall visual system."

---PB--- Global maps of the retina

As well as researching the strange sight of hagfish and lampreys, Shaun Collin has spent the last three years collecting maps of the distribution of cells in the retina. In association with the Optometrists Association Australia, some research assistants and a lot of hard work, Collin has created an immensely valuable resource of over 770 retinal topography maps covering 160 vertebrate species, all available online.

In what should prove a very powerful research archive, the resource will allow scientists to go online and ask new questions about visual ecology and the visual capabilities of all vertebrate animals, from the lowly lamprey up to humans. "These maps reveal what part of the visual world an animal is most interested in," he says.

"It can give you information about how clearly you see the world or visual acuity. The type of information gleaned from these maps allows predict a lot about the visual capabilities of a particular animal including in what sort of environment it lives and its ability to resolve objects at a particular distance i.e. by the spacing of the neurons across the retina. The archive bridges the gap in our understanding of how animals see and will be invaluable for learning more about animals that are hard to find or live in inhospitable places."

Collin says the resource will now allow scientists to look at the evolution of visual capabilities in relation to the visual field, visual acuity, the type of cells that are involved in processing an image, from lampreys right through to jawed fishes, amphibians, birds, reptiles and mammals."

Many of the maps depict the distribution of a range of retinal neurons such as the photoreceptors, horizontal cells, amacrine cells, bipolar cells and ganglion cells.

"We hope it won't just stop there," he says. "As new maps are completed and published, they will be added to the website. I also hope that scientists who have these maps sitting in a draw and haven't published them will do so online in order to make the archive as up to date and comprehensive as possible. We'd love to get as many maps as possible."

Collin has published a paper on the archive - "A web-based archive for topographic maps of retinal cell distribution in vertebrates" - in the journal Clinical and Experimental Optometry. The archive is at www.optometrists.asn.au/ceo/retinalsearch