Samatha Mathew
E-mail: samatha.mathew@igib.in
The model
Ever put a sea-shell against your ears and heard that noise like that of waves? Well, that sound is of the ‘flow’, or, your blood flow. The network of vessels that carry blood across the body have to be healthy at all times to maintain this flow. However there are conditions where blood vessels malfunction and can manifest as hemorrhages, and even cause strokes, or cause cardio-vascular diseases. Needless to say, understanding the mechanism of how blood vessels function will allow improvement of treatment of a wide range of diseases.
Ever put a sea-shell against your ears and heard that noise like that of waves? Well, that sound is of the ‘flow’, or, your blood flow. The network of vessels that carry blood across the body have to be healthy at all times to maintain this flow. However there are conditions where blood vessels malfunction and can manifest as hemorrhages, and even cause strokes, or cause cardio-vascular diseases. Needless to say, understanding the mechanism of how blood vessels function will allow improvement of treatment of a wide range of diseases.
Biological scientist stereotypes in popular media portray, most often than not, lab-coat adorning people with frowning faces, looking down a microscope or peering at mice in cages. While these portrayals are near accurate, some biologists prefer their striped friends over pearly white mice. Zebrafish are tiny fish that grow to a maximum of 2-3 cm and give rise to about a hundred offspring between a single pair of male and female, every week! Baby zebrafish are transparent, and develop organs such as heart and brain within a day. Blood flow starts soon after. What makes zebrafish dear to scientists is that zebrafish parts can be labeled, and because baby fish are transparent, you could see a beating heart or a forming brain through a microscope, live! What’s more, you could label blood vessels and cells in the blood, and now see how blood flows through blood vessels, and this is what drives the story below.
The question?
Since past two decades, biologists have used high resolution techniques to obtain information hidden in the cells that make up living beings. During this quest, they identified molecules called long non coding RNA, aka lncRNA. RNA stands for ribonucleic acid, a class of molecules which act like messengers, for making building blocks of cells called proteins. What makes lncRNA different from RNA known until recently is that they do not make proteins. Instead they perform regulatory roles in cellular processes. In the past few years thousands of lncRNA have been discovered in different organisms including plants and animals. But even with this explosion of information, we understand very little of what all these lncRNA actually do in a cell.
It has been shown that lncRNA are present in different cells types in humans and other animals. Incidentally, we were interested in finding lncRNA in a particular cell type which forms the inner lining of blood vessels, called endothelial cells. To combine the power of the zebrafish model and to understand the role of lncRNA in blood vessels, our lab identified lncRNA in endothelial cells from one day old zebrafish. This story is about one endothelial lncRNA of the hundred plus identified from this earlier study. Let’s call this endothelial lncRNA ELNC. We asked the following question:
How does ELNC contribute to the function of endothelial cells and affect blood vessel biology?
The story
First, we checked whether ELNC was truly ‘non coding’, that is, does it make a protein? For this we isolated protein making factories of cells, ribosomes. If we could identify an RNA latched to ribosomes, it could make a protein. As expected, ELNC was not found along with ribosomes, suggesting that ELNC is truly ‘non coding’. Now we turned to the aspect of endothelial function of ELNC.
There are synthetic molecules that can suppress the function of RNA. This method is called ‘knockdown’. We introduced such a molecule against ELNC into zebrafish which is still at single cell stage, immediately after the sperm and egg fuse, through a method called microinjection. During microinjection we introduce any material in liquid form at nanolitre volumes. We did this experiment in a double- labeled zebrafish, where both endothelial cells (green) and blood cells (red) could be visualized. Knockdown or suppression of ELNC showed that the vessels were malformed in two day old zebrafish. We also saw hemorrhages in the head region of the same fishes. This showed that disruption of ELNC was disturbing blood vessel functioning.
What if ELNC was present in excess? We artificially synthesized ELNC and introduced it into two day old zebrafish by microinjection. After a day, we could see extra vessels sprouting from a certain kind of vessels with a basket-like structure, called sub-intestinal vessels. When we introduced excess ELNC, we could see extra vessels sprouting off from the basket. This hinted that in normal conditions ELNC is required for routine blood vessel maintenance.
All information in a cell is written in a molecule called deoxyribonucleic acid or DNA. The information or ‘message’ that any RNA holds is ‘copied’ from DNA. Any change in DNA can alter the RNA as well. We used a technique called gene-editing to alter the DNA containing ELNC. Here we aimed to have a complete abolishment of ELNC function or a ‘knockout’, opposed to suppression by knockdown technique. Using microinjection, we introduced artificially synthesized gene-editing molecules into one-cell stage, double-labeled zebrafish. We could see hemorrhages in the head region at ages two days and above in these fishes. This observation backed our results from the knockdown experiment. We further raised these fishes with altered ELNC DNA and generated progeny, which also displayed hemorrhage. This was exciting, because by independent techniques we could show that disruption of ELNC could affect blood vessel architecture or integrity and even lead to hemorrhage!
Now that we knew ELNC was important for endothelial cell function, we wanted to know what exactly the lncRNA did in the cell. A cell is divided in many compartments, each of which perform distinct actions, all hand-in-hand for the survival of the cell. We isolated two fractions of the cell mixture: one that contained the compartment which is like the control center of a cell, called nucleus. The other fraction contained all other compartments, called cytoplasm. The nucleus was further divided in two fractions: One containing DNA called chromatin, and nucleoplasm. We found that ELNC is localized in the chromatin: this told us that ELNC could be involved in processes that happen around DNA or formation of RNA from it.
When we first chose ELNC for our study, we had a hunch that it could be regulating a protein already known to have a function in endothelial cells. This was because in DNA ELNC lies near the DNA stretch that has the information to make this protein, say Protein-E. So in our ELNC knockdown and knockout zebrafish systems showing hemorrhage we started investigating how this Protein-E was affected. While Protein-E itself isn’t affected, we have a hint that ELNC could be regulating the process which generates RNA required to make Protein-E.
Meanwhile, we also wondered if ELNC could be a player in endothelial function in humans. The incredible thing about the zebrafish model is the similarity in DNA between zebrafish and humans, almost 70%! That is, 7 out of 10 times we identify a new protein or RNA in zebrafish we could apply that knowledge to humans. But here’s the tricky part: lncRNA are known to have entirely varying DNA make-ups in different organisms. Yet there is a relatively easy way to find human ELNC. If we could speculate a certain lncRNA in human could be the counterpart of zebrafish lncRNA, it should be able to do the same function. This means that if in the zebrafish ELNC knockdown/knockout systems, we substitute the human counterpart the fishes should be “rescued” from hemorrhages. As described, ELNC is near Protein-E on DNA. And incidentally, Protein-E is one of those proteins which fall in the 70% similar part between zebrafish and human DNA. This made us look for a lncRNA near human Protein-E on DNA. Luckily, there are more than one such lncRNA and we will be testing these for their ability to “rescue” our zebrafish ELNC knockdown/knockout fishes. This is important, because identifying a human ELNC would mean we have identified a regulator than can be targeted in blood vessel diseases in humans, especially the ones involving hemorrhages.
The future
It’s an exciting time in Science; we are leaping from one hypothesis to next using cutting-edge techniques. For our study, to find a human ELNC would mean identifying a potential target for blood vessel diseases. To this end, using both zebrafish and human models (human endothelial cells), we look forward to dissect the mechanism of action of ELNC, and add novel insights to blood vessel biology.
And this time, the flow of information will be from striped friends to stricken humans.