THE GOAL OF THIS PROJECT is to help advance a promising new genetic technology in medicine, sonogenetics, through the discovery of genes that are present in most animals but absent in animals that use echolocation: Bats and toothed whales. We will do this over the next three weeks. Week 1 we will build species trees. Week 2 we will build gene trees. Week 3 we will advance research in sonogenetics (details on what sonogenetics is are below) and hunt for ion channel genes (details on what ion channels are are below) that may have been lost in the evolution of echolocation in bats and toothed whales. In doing this you will gain training in different phylogenetic tools and techniques, including multiple sequence alignment and tree building, how to read and interpret phylogenetic trees, and on mapping traits onto trees to understand their evolution, in addition to learning about vertebrate diversity, mechanosensitive ion channels, and echolocation. Skills learned are highly applicable and can be extended in areas of protein engineering, personalized medicine, genomics, and evolution, including approaches used in characterizing Sars-Cov-2 and its evolution in human populations.
WHY: Sonogenetics is a new emerging technology that could help improve and save lives for millions of people suffering from brain, heart, and other disorders world-wide. The technology holds great promise but is only recently invented. It requires additional development before it can be used in medicine. Advancing sonogenetics is a focus of our research in the Shrek Chalasani laboratory at the Salk Institute in La Jolla, California. The
giganticFISH Channel Hunters project is designed to be a part of our ongoing research to advance sonogenetics, including work that you will do. The project offers you the opportunity to work with research-level phylogenomic tools and data sets to answer questions about species and gene family evolution - and in the process contribute to sonogenetics research and join us in the advance of sonogenetics for science and medicine.
WHAT: Sonogenetics uses pressure waves generated by ultrasound ("sono") to activate ion channels ("genetics" ) and modulate downstream biology. To understand sonogenetics, we need to first understand 1) what these "mechanosensitive" ion channels are and how they work, 2) what ultrasound is and how it works, and 3) how these two things come together to create sonogenetic technology and how it might be used in medicine.
Ion channels are proteins that stretch across the cell membrane and have a hole or pore in their center. This pore is unusual in that it will open or close in response to a stimulus. The pore in a mechanosensitive ion channel is closed at rest but opens in response to a mechanical force, like pressure. The cell membrane is a barrier to the movement of ions into a cell. However, opening of the pore in an ion channel creates a tunnel through the cell membrane that ions can then move through to enter or exit a cell. When the physical force stops, the pore in a mechanosensitive channel closes, and the movement of ions stops. Thus, a mechanosenstive ion channel can provide a signal of when a mechanical force starts and stops through the movement of ions. Cells are highly sensitive to ion concentration, so the movement of ions through a channel can lead to dramatic changes in downstream biology in response to an initial stimulus. For instance, if you sense a poke on your arm and turn your head to see who did it, the origins of your response trace back to mechanosensitive ion channel that opened up and lead to downstream changes.
Mechanosensitive ion channels are present in many types of cells and tissues. For example, as you might have guessed or already know, mechanosensitive ion channels are present in sensory neurons in your skin, where they are part of a pathway that translates a mechanical force on the skin (like a poke on the arm) into a sensation of touch. When you feel touch, it is actually a mechanosensitive ion channel in a sensory neuron in the skin that first sensed and responded to a physical force of pressure. In its response to pressure, the ion channel opens, allowing the movement of ions in and out of the cell. This movement of ions changes the physiology of the cell and leads to downstream changes in biology of the cell and organism. Again, in the case of a sensory neuron in the skin that senses touch, the mechanical force of pressure triggers mechanosensitive ion channels to open. Ions can then enter the sensory neuron through the now open pore of a mechanosensitive ion channel. This movement of ions through an open channel in a sensory neuron leads to production of an action potential by the neuron (action potentials are ion-based electrical signals used for communication between neurons). The actional potential signal travels through the nervous system to the brain. The brain processes the transmitted signal - a signal that again was generated by a mechanical force of pressure that caused opening of a pore in mechanosensitive ion channel in a sensory neuron in the skin. This downstream higher-level processing by the brain leads to you having a sensation of touch on your skin. This entire sequence of events is what happens when a friend pokes you in the arm to get your attention and you feel it. You sense a touch on the arm but it all starts with a mechanical force acting on a mechanosenstiive ion channel that opens its pore and allows movement of ions in a sensory neuron in your skin. It's worth noting that in reality, it is not just one channel or even one neuron but many that are involved in this process.
Ultrasound is a type of mechanical energy in the form of a wave. Waves have frequencies (number of waves over time) and ultrasound waves have frequencies that are distinct from the wave frequencies of sound and infrasound. Mechanical energy waves are physical features of the universe, however, the specific range of frequencies that we call sound is defined by human biology. Sound is the range of mechanical energy wave frequencies that our ears are able to detect and sense as hearing, going from 16 to 20,000 Hertz. Infrasound is defined as mechanical energy waves that occur at frequencies lower than sound waves and what we can hear. Similarly, ultrasound is simply the range of mechanical energy waves that occur at frequencies higher than sound waves and what we can hear. Although we can not hear ultrasounds, many animals can. For instance, mice sing to one another in ultrasound but we hear nothing. Or a dog whistle that your dog can hear but you can not is similarly ultrasound, meaning mechanical energy waves at frequencies that are beyond our ability to hear but are within the range of a dog's ability to hear and sense sound. Of interest for the giganticFISH Channel Hunters project, bats and toothed whales are able to generate high-intensity ultrasound waves for echolocation. In echolocation, ultrasound waves are generated and sent out into the environment by a bat or toothed whale. In the environment, ultrasound waves from a bat or toothed whale are reflected off of objects. The reflected ultrasound waves can then be detected by the bat or toothed whale, allowing them to sense objects in the environment around them. Echolocation is similar to sonar that is used in submarines. Basically, production and detection of ultrasound in echolocation is done by an organism vs. in sonar it is done mechanically by machines.
Ultrasound can generate pressure that acts as a mechanical force on a cell or its immediate environment. Pressures and mechanical forces generated by ultrasound can activate mechanosensitive ion channels in a cell. Ultrasound-based forces that cause opening or closing of mechnosensitive ion channels can lead to downstream changes in activities of the cell and organism. The ability to use ultrasound to generate forces that active mechanosensitive ion channels and alter or modulate downstream biology is the basis of sonogenetics.
Sonogenetics was first created in the Shrek Chalasani lab (where Lola, Jan, and Eric are based) at the Salk Institute as a tool for neuroscience and published in 2015. A mechanosensitive ion channel was expressed in a single neuron in the "brain" of a worm. The neuron is known to cause a worm that is crawling forward to crawl backwards. Prior to expression of the channel in the neuron, applying ultrasound to a worm had no effect on its behavior. After targeting and expressing the mechanosensitive ion channel in the single neuron, application of ultrasound to a worm that was crawling head-first caused the worm to stop and crawl tail-first. Stopping the ultrasound allowed the worm to continue crawling head-first again. You can see an example of this experiment here. The Chalasani lab has a new 2020 paper under review for publication that demonstrates that sonogenetics tools work in human neurons in a dish and in the brain of a mouse to control its behavior, results that move us a step closer towards genetic therapies that could be used in people to help treat diverse illnesses and disease.
An important feature of ultrasound in medicine is that it is safe to use in people and does not require surgery to access tissues and organs deep in the body. You are probably familiar with it its use for visualizing a developing baby during pregnancy. The ultrasound is able to pass harmlessly through tissues and can even transmit through bone to some degree. This means that if we were to genetically target and express a mechanosensitive ion channel in part of the brain or heart that is working incorrectly, we could potentially use ultrasound to activate the ion channel and produce action potentials or other ion-dependent activities that are missing or dysfunctional in a disease. Examples of where a sonogenetic-based therapy might one day be useful in medicine include Parkinson's disease, Post-Traumatic Stress Syndrome (PTSD), or pacemaker irregularities. Current treatments like deep brain stimulation or a medical pacemaker require invasive surgeries, like drilling a hole in skull to insert a probe into the brain. However, the possibility of genetic targeting of a mechanosensitive ion channel to specific cells using safely modified virus-based tools injected into the blood, combined with stimulation of the channel after expression in the cells using non-invasive ultrasound, makes sonogenetics a promising alternative. For example, in the future neurological diseases, like Parkinson's, might be alleviated by sonogenetic treatments, as abilities in the brain that fail to function could be supplemented by stimulation of specific brain regions through expressed channels and ultrasound. However, to achieve this the technology must be developed beyond its use in simple worms or even mice.
HOW: To advance sonogenetic technology, we would like to create ion channels that respond to specific wavelengths and intensities of ultrasound. This will provide more targeted control of the tool, which will be important in developing genetic therapies based on it. To do this, Lola, Jan, and Eric have developed a comparative genomics tool, gigantic, that explores genome-scale genetic diversity across organisms. We started using this tool to look at the evolution of mechanosensitive ion channels to then use in developing sonogenetic tools in the lab.
To identify new mechanosensitive ion channels that may be responsive to ultrasound, we are using a new idea recently developed in the lab. We noticed that the ion channel that responds to ultrasound when expressed in human neurons and in mouse brain in the 2020 manuscript is present in the genomes of nearly all animals that have been sequenced but is absent in toothed whales. Given that the channel is responsive to ultrasound, we had initially thought toothed whales might use it in echolocation. We were very surprised to instead see its unusual loss in toothed whales. However, the channel is expressed in diverse types of cells and neurons in mammals. Given this fact, one way to interpret loss of the channel in toothed whales is that when toothed whales were first evolving high-intensity ultrasound for use in echolocation (vs. low intensity ultrasound that is likely used by mice in singing), the high ultrasound intensities may have caused the channel to suddenly activate in diverse cells and tissues when no activation was wanted. This could have had severe negative consequences and these negative consequences may have lead to growing natural selection pressures against keeping the channel and ultimately loss of the channel in the genome of toothed whales, as echolocation evolved and became increasingly important in their biology.
There are diverse mechanosensitive ion channels but its often unknown which ones are sensitive to ultrasound. It is a big undertaking to test any one channel, so if it's possible to start with channels that are more likely to respond to ultrasound, this can more rapidly, cheaply, and efficiently advance the development of sonogenetic technology. Given this, we realized that understanding the evolution of mechanosensitive ion channels in bats and toothed whales could potentially identify other channels that have been lost in either group and might have been lost due to a sensitivity to high-intensity ultrasound. Thus, we propose as part of the
giganticFISH Channel Hunters project to characterize the evolution of many mechanosensitive ion channels in vertebrates and see if they show unusual patterns of loss in toothed whales or bats. If so, the lab at the Salk will work with the identified channels and test them in human neurons and mice to see if they might be added to the sonogenetic toolkit and help advance the technology.
WHEN: The project is split into three labs over three weeks:
WEEK 1 Species Tree - THIS WEEK
RESEARCH LEVEL 1: Species Tree
Vertebrate species tree are well-resolved. This work will generate a species tree that replicates what is already commonly known. It offers an opportunity to get your
sea legs in phylogenomics research and will verify you have the tools and workflow working correctly.
WEEK 2 Gene Tree - 2nd WEEK
RESEARCH LEVEL 2: TOP SECRET Gene Tree
TOP SECRET gene evolution is well known in many animals but we discovered this summer highly unusual evolutionary patterns of TOP SECRET when looking across vertebrate diversity. Your work here represents critical replication of our gene tree findings to ensure things are what we think they are. TOP SECRET in toothed whales is a super exciting but brand new and still unpublished result - so TOP SECRET is TOP SECRET. The secret will be revealed. Please limit who you tell. Interviews on CNN not advised.
WEEK 3 Gene Tree - 3rd WEEK
RESEARCH LEVEL 3: New Gene Tree
There are over 20 known ion channels that respond to mechanical stimulation in human and may also respond to ultrasound. We have not - and no one in the scientific literature has ever - looked specifically at evolution of these channels in echolocating animals vs all major groups of vertebrates. Your own work in the lab will do produce gene trees for assigned ion channels and break new ground -
we can't wait to see what you DISCOVER!!!
MAJOR STEPS PER LEVEL
A Build alignment
B View alignment
C Build Tree
D View tree
E Annotate tree
F Post tree
