If you were to function as a cell, what type would you be?

Insight from work on Rudhira
Insight from work on Rudhira

Imagine having the supreme ability of transforming yourself to any other functional being of your choice with impeccable precision, just like Mystic from X-men (Marvel fans would get the hint)! Setting aside Sci-Fi, in fact if we take a look inside us, each of us do have such remarkable cells in our body that carries immense potential (although choice is not conscious) to develop into a variety of structurally and functionally diverse cell types, especially in the early stages of life (embryonic stage) as well as during the growth phase. These gems are called Stem Cells; literally living up to their name – being the stem from which several types of cells branch out. Not just that, these cell types even take up the responsibility of replacing damaged cells in certain body parts of adult organisms. Be it understanding how an entire organism develops from a single cell or exploring its regenerative abilities for treating certain chronic diseases, worldwide research on stem cells has progressed appreciably over the last 40 years.

Prof. Maneesha S. Inamdar’s laboratory at the Molecular Biology and Genetics Unit of JNCASR, carries out fundamental research in stem cell and developmental biology using mouse and Drosophila as the model organisms. One of the long-term ongoing research at her lab is on a gene named ‘Rudhira’ that is fundamental to the formation and functioning of new blood vessels during mouse embryonic stage. The gene Rudhira (meaning blood-red) at first, was found to be expressed in the red blood cell lineage of mouse embryonic stem cells and subsequently has been shown to be conserved between Drosophila, mouse and human! The discovery of this novel gene was quite a turning point since the expressed protein has 98% similarity to a protein from the gene overexpressed in human breast cancer cells (human BCAS3). The later has been shown to have expression even in the embryonic stem cells, most importantly detected to have abnormal expression levels in malignant tumors and blood vessels.

Baseline findings
Further in-depth experiments revealed a vital role of Rudhira in directing movement of cells to particular locations required for the process of wound healing. During development in multicellular organisms, errors during the movement of cells to destined locations often result in serious diseases like formation of tumor or vascular defects. On this axis, Prof. Inamdar’s team established that the gene Rudhira codes for a protein that rearranges and promotes cell division control protein (a protein involved in regulation of cell cycle) during the process of wound healing. Lack of this protein was shown to have severe consequences on cell’s cytoskeletal structure (even though cells are microscopic, they have a skeletal structure too that holds them, aids their movement, plays substantial role in cell division) and orientation that ultimately affect the elemental process through which new blood vessels develop.

Current breakthrough
After establishing and functionally characterizing the role of Rudhira in-vitro, it was then time to replicate the results in-vivo. Ronak Shetty and Divyesh Joshi, two of the current graduate students from Prof. Inamdar’s lab involved in this project, continuing work initiated by former graduate student Dr. Mamta Jain, accomplished in generating the first Rudhira knockout mouse (Knockout literally translates to removal; in genetics it is the process through which an existing gene of interest is inactivated or replaced by an artificial piece of DNA with the aim to study what the gene normally function as).

These images depict normal vascular patterning in the control embryo (left) and irregular and discontinuous vasculature in the Rudhira knockout embryo (right) at day 10.5 of embryonic stage.
These images depict normal vascular patterning in the control embryo (left) and irregular and discontinuous vasculature in the Rudhira knockout embryo (right) at day 10.5 of embryonic stage.

In their recently published paper in Scientific Reports, the team details systematic experiments to show major developmental defects in mouse embryos lacking Rudhira. Rudhira knockout mice embryos were unable to survive beyond 9 days of their embryonic stage and were detected by decline in growth and significantly affected patterning in the dorsal aorta of heart. Through immunostaining and subsequent microscopic structure analysis of relevant tissues, the team was able to show that even if the developmental rate was not affected, severe defects in shape and structure of blood vessels in the head and heart of Rudhira knockout embryos were detected (these embryos had shrunken heart chambers and abrupt dorsal aorta among other structural defects in the development). Expression of this gene was further shown to be crucial for normal structuring and functioning in the inner layers of blood vessels.

Dr. Ronak Shetty (left), Prof. Maneesha S. Inamdar and Divyesh Joshi (right) at Vascular biology Laboratory, MBGU, JNCASR.
Dr. Ronak Shetty (left), Prof. Maneesha S. Inamdar (center) and Divyesh Joshi (right) at Vascular Biology Laboratory, MBGU, JNCASR.

This piece of work led by Prof. Inamdar not only reaffirmed the pivotal role of Rudhira in blood vessel development through in-vitro and in-vivo studies, but has also contributed to the field of developmental biology by establishing a mouse model for future studies in stem cell and medical research in cardiovascular development. For more studies from Vascular Biology Laboratory, click here.

Reference
Shetty, R., Joshi, D., Jain, M., Vasudevan, M., Paul, J.C., Bhat, G., Banerjee, P., Abe, T., Kiyonari, H., VijayRaghavan, K. and Inamdar, M.S., 2018. Rudhira/BCAS3 is essential for mouse development and cardiovascular patterning. Scientific reports, 8(1), p.5632.

The article is authored by Manaswini Sarangi, Evolutionary Biology Laboratory, EIBU, JNCASR.

Cover Art by: Manaswini Sarangi.

Ever wondered what keeps one awake when the sun shines!

blog-3_bnl_cover-sketchFruit flies shed light on neurons driving wakefulness during daytime

Like us, some organisms, are active during the day, while some are active at night and some others are active during twilight. It is believed that such patterns are driven by adaptive forces such as the availability of survival resources like food and mates. Be it nocturnal or diurnal, what controls waking up and going to sleep in the living world? The answer is a daily CLOCK! Yes, an organism’s biological clock wakes it up and puts it to sleep by appropriately timing its activities. Scientists have been trying to understand the mechanisms behind sleep and wakefulness behavior and its regulation by the circadian clock.

Sleep is an intriguing phenomenon that has been observed across a variety of species studied, from mammals to insects (1, 2). “About two decades ago, fly researchers woke up to the potential of harnessing the potential of fly genetics to unravel the mysteries of sleep and its underlying cellular and genetic basis. Since then, mutations on several genes have been shown to impact sleep levels, and several distinct brain regions whose electrical activity either induce or reduce sleep have been identified”, says Prof. Sheeba Vasu, an expert in Neurogenetics leading her laboratory at Neuroscience Unit, JNCASR. While human and fly brains are dramatically different in structure and complexity, the states of being awake or sleep share a fair degree of commonality. These parallels between sleep in flies and mammals (3) make fruit flies an excellent choice to study this behavior.

In a report published in August 2018 in eNeuro (4), Prof. Sheeba Vasu and her student Dr. Sheetal Potdar from Neuroscience Unit at JNCASR, showed that a group of dopaminergic neurons under the action of a particular type of neuropeptide called Pigment Dispersing Factor (PDF) keeps the flies awake and active during the daytime. [Quick Fact: Dopaminergic neurons are the brain cells that synthesize a chemical called dopamine. Dopamine serves as a chemical messenger between neurons that is known to promote wakefulness.] “The main motivation behind this study was as part of a bigger question – of whether the two limbs of sleep regulation, sleep homeostat and circadian clocks communicate with each other to regulate sleep and wake”, says Sheetal, lead author of the paper.

Although it is well known that light and dopamine stimulates alertness/wakefulness let us see how the biological clock talks to the neurons to keep the fly awake. In response to the action of light and dopamine, one subset of the Drosophila clock neurons releases a neuropeptide called Pigment Dispersing Factor (PDF). This neuropeptide is expressed only by some specialized clock cells in the fly brain, and is known to have primary functions driving behaviors related to morning and evening times of a given day. The protein PDF has several target spots in the brain depending on the action required, to which Sheetal adds, Since 2008, we know that PDF also functions in promoting wakefulness. So here was a nice opportunity for us to examine if any of the known sleep homeostatic structures responded to signals from the receptor of PDF, in order to tie in with our bigger question of sleep homeostat-circadian clock communication”. [Quick fact: This neuropeptide PDF with similarities to a crustacean hormone was first described by Dick Nassel in 1993 and the gene pdf encoding it was cloned in the laboratory of Nobel Laureate Jeffery Hall, a geneticist and chronobiologist!]

First, Sheetal established that flies carrying dysfunctional receptors for PDF (called PDFR, pdfr being the receptor gene), were unable to remain awake as much as their controls, i.e. these mutant flies slept much more during the day. Second, by manipulating the expression levels of this receptor gene through fly neurogenetic techniques, she conducted an exhaustive screen searching for the cells that are acted upon by the neuropeptide PDF to keep flies awake. Out of the several strains examined, she found a group of dopaminergic neurons that resulted in substantial increase of sleep during the daytime when the receptor gene’s expression levels were turned down and a significant decrease in sleep when this gene was overexpressed in them. When asked about specific challenges during the experiments, Sheetal said, “Absolute levels of sleep vary to a large degree both across flies and assays; trends that you see in one experiment mysteriously disappear in the next one. This was one of the biggest challenges I faced and the only way to overcome this was to repeat all of my experiments multiple times. Most of the data are from experiments repeated anywhere between 2-5 times to ensure that whatever phenotype we report is a true phenotype”.

This figure represents changes in sleep through out the day when expression levels of pdfr were altered (A) lowered expression resulted in increase in day time and nighttime sleep (red line),(B) overexpression resulted in decrease in daytime and increase in nighttime sleep (purple line), compared to their controls.
This figure represents changes in sleep through out the day when expression levels of pdfr were altered (A) lowered expression resulted in increase in day time and nighttime sleep (red line),(B) overexpression resulted in decrease in daytime and increase in nighttime sleep (purple line), compared to their controls.

 

Further detailed examination in the target subsets of the dopaminergic neurons revealed that even though the daytime sleep was significantly enhanced during lowered expression of the receptor gene and significantly reduced during its overexpression, the nighttime sleep remained much higher than their respective controls. Additionally, it was also shown that once lights were turned on during the experiments, the flies which had overexpression of the receptor gene took much longer to fall asleep than their counterparts. The finding was strengthened when they found synaptic connections between the dopaminergic neurons and the PDF-expressing neurons. Further investigations revealed a subtype of neurons (PPM3) that showed significant decrease in intracellular calcium levels under the action of signals from the PDF receptor, particularly during the daytime.

Can you say a little about PPM3/ dopaminergic neurons in general in perspective of sleep research? Was there a ‘eureka’ moment during your experiments? “Previously, the role of dopamine has been reported in promoting wakefulness. Yet, the PPM3 subset that we think at work here could be promoting sleep. So this is a major finding in the sense that it shows that perhaps the broad set of dopamine neurons consist of distinct sleep-promoting and wake-promoting subsets. Furthermore, when I began using the dopamine drivers, I had no a priori reason to believe that it will respond to PDFR signalling. In fact, there was no scientific thought in doing this experiment – it was strictly a serendipitous finding, and I just got lucky! Moreover, at that time in my screen, none of the other drivers had yielded interesting or consistent results, so more than a ‘Eureka!’ moment, it was a ‘thank goodness!’ moment for me!” answers Sheetal.

Fascinating enough, that even though most known dopaminergic neurons are known to improve wakefulness, these researchers at JNCASR were able to show that there are some sub-groups of these neurons that in fact promote sleep!

What is the bigger question of sleep/wake research that you would like to address at some point? “The most mysterious aspect about sleep is what purpose it serves. Realistically, it is possible to address this question. If I am permitted to be more ambitious, I would like to understand why dreams occur – are they a by-product of what happens during sleep or do they serve a specific function?” says, Sheetal.

So, there we go, for remaining awake the circadian neurons send instructions through the neuropeptide PDF receptor to a subgroup of dopaminergic neurons, that otherwise would be promoting sleep. This noteworthy finding is expected to advance our understanding on sleep/wake behavior. For more studies from Prof. Sheeba Vasu’s Behavioral Neurogenetics Laboratory at JNCASR, click here.

References

1. Tobler, Irene. “Phylogeny of sleep regulation.” Principles and Practice of Sleep Medicine (Fifth Edition). 2011. 112-125.
2. Lesku, John A., et al. “Phylogeny and ontogeny of sleep.” The Neuroscience of Sleep (2009): 61-70.
3. Greenspan, Ralph J., et al. “Sleep and the fruit fly.” Trends in neurosciences 24.3 (2001): 142-145.
4. Potdar, Sheetal, and Vasu Sheeba. “Wakefulness is promoted during daytime by PDFR signalling to dopaminergic neurons in Drosophila melanogaster.” eNeuro (2018): ENEURO-0129.

The article is authored by Manaswini Sarangi, Evolutionary Biology Laboratory, EIBU, JNCASR.

Cover Art by:  Manaswini Sarangi.