Bio Patrika interviews Dr. Das on his thoughts about “how ERK mediated phosphorylation regulate oocyte numbers”

Dr. Debabrata Das’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Das completed his Ph.D. as a DST-INSPIRE-fellow in the Cellular and Molecular Endocrinology Laboratory of Dr. Sudipta Maitra at the Department of Zoology, Visva-Bharati University, Santiniketan, India. His Ph.D. work was focused on the role of insulin-signaling pathway in regulating female reproduction using zebrafish as model organism. After his Ph.D., He joined the Arur Laboratory at the University of Texas MD Anderson Cancer Center in 2017. Currently, he is an Odyssey post-doctoral fellow. Here using C. elegans germline, he is working on the downstream substrates of RAS/ERK signaling pathway in regulating female fertility with special emphasis on meiosis I, as well as their role in cancer progression. Here, Debabrata talks about his PhD work titled “ERK phosphorylates chromosomal axis component HORMA domain protein HTP-1 to regulate oocyte numbers” published as a first author in Science Advances (2020).

How would you explain your paper’s key results to the non-scientific community?

The birth of a healthy baby requires mating of an egg and a sperm. The eggs (or oocytes) in women are generated when she is still in her mother’s womb. This number once formed is fixed and does not increase. If this number is too low, women naturally tend to go through premature menopause and infertility. We wanted to determine the process that regulate egg number in a woman. As the stock of eggs is formed in the woman while she is in her mother’s womb, studying the process in lab mice and humans is difficult and unethical. So, to study this process we use Caenorhabditis elegans as a genetic model system. This is a beautiful system because the genetic circuits that regulate egg production are the same between worms and humans, but worms generate the eggs continuously in the adult ovary. Many years ago our lab discovered a genetic pathway that regulates the number of eggs in worms – the RAS/ERK pathway. This is a signaling pathway, and an increase in RAS through genetic mutations or loss of ERK results in high (Figure 1) or low egg production (Lee et al., 2007). I joined in Swathi’s lab, where I was eager to understand how this signaling circuit controls egg production.

Figure 1. Eggs (oocytes) in the worm ovary of wild-type and RAS (act) mutants. Oocytes are marked according to their development, -1 being the most mature will be fertilized first. Note that the oocytes form much earlier in the mutant (arrowhead).

I discovered a new genetic player which controls egg production but functions together with the RAS/ERK pathway. This player is HTP-1. HTP-1 is modified by ERK, in such a manner that a phosphate molecule is transferred to HTP-1 by ERK. HTP-1 molecule that carries this phosphate molecule then signals to the cell in which it resides that the cell can proceed to go on and form an egg (oocyte) (Figure 2). A cell that does not have the phosphate bound HTP-1 does not proceed to form an egg. This is a mechanism which ensures that the cells are ready to move to the next stage. Thus HTP-1 carrying a phosphate molecule functions like a timer which tells a cell whether it can continue to proceed to form eggs or not. This is a crucial mechanism that regulates female fertility.

Figure 2. Diagram showing the mechanism oocyte number regulation by RAS/ERK signaling. Red region shows where ERK is active.

This work sheds new light on regulators that control female fertility, and importantly we find a link between regulation of female fertility with the nutritional status of the mother. Our lab previously found that maternal nutrition regulates the RAS/ERK pathway in the worms (Lopez et al., 2012). Without food, the RAS/ERK signaling remains off and worms do not make progeny. I added to this knowledge by discovering that ERK transfers a phosphate to HTP-1 and signals the cell to make eggs, which can only happen in a situation where the mother’s nutrition is good. This work reveals a direct connection between female fertility and the nutrition of the mother, and because the genetic circuitry is conserved from worms to humans, I expect that this work will also help address some key questions in human female fertility.

“[….] work reveals a direct connection between female fertility and the nutrition of the mother, and because the genetic circuitry is conserved from worms to humans […].”

What was the exciting moment (eureka moment) during your research?

When Swathi gave me this project, I thought it is a simple problem of increased cell number (oocytes in this case) and it has to be either increased stem cell divisions or decreased cellular death (from my textbook knowledge of cell number regulation!!). So, I first assayed for any changes to the stem cell divisions; and didn’t find any difference. Next, I assayed for cellular death and did not find a correlation with egg numbers. It was an unexpected finding, which was exciting, but at the same time, I remained clueless about the process! I spent hours looking at those images and trying to understand where those extra eggs came from; and one day I noticed that the oocytes in the RAS(act) mutants, actually start to form much earlier in the adult ovary than wild-type animals (Figure. 1, arrowheads). So, I wondered why are these forming early? What is telling them that they can form early, and what stops the eggs in wild-type animals from starting early? It occurred to me maybe what is changing is the timing at which each cell is moving on to form an egg, rather than a new cell being born or cells dying. That whole night I was thinking about this hypothesis and was so excited that I couldn’t sleep! Next morning, I met Swathi, showed her the images and told her my hypothesis. She was very excited too and told me that we are onto something cool here! The rest is history.

What do you hope to do next?

I started working with worms during my post-doc; so, this is the exponential phase of my learning growth curve in this system. I am currently working on a couple of projects. From the work described, we discovered another meiotic regulator protein, HTP-3 as an ERK substrate. I am following up on that. Also, there is another project on the role of a dual-specificity phosphatase (DUSP). How does DUSP regulate the pattern of ERK activation and meiosis in the C. elegans germline. Overall this is an exciting time!

Where do you seek scientific inspiration?

I love to read the stories behind the scientific discoveries and failures, and biographies of scientists, which motivate me greatly. Also, I draw inspirations from my both my mentors, my Ph.D. supervisor, Prof Sudipta Maitra, and my current mentor, Prof. Swathi Arur. I consider myself very fortunate that I am taught and trained by teachers, who are so supportive and have always pushed me that extra bit. I love scientific discussions about ideas with my colleagues and my wife (she is a microbiologist). Good talks also boost me. But not the least, the thrill of seeing something in the microscope or getting the desired Western blot band, which you realize that you are the first person in the world to see at that very moment, is simply awesome! And this thrill is what keeps me going!

How do you intend to help Indian science improve?

I am still in my learning stage, eventually I will return to India and continue my research there. When I think back, I find that I grew my scientific interests in my school days. Thus, alongside with running my own laboratory, I would love to visit nearby schools and talk to the students about science in general and biology in particular. I feel simple biological experiments and projects will help younger generations to be excited about biology and instill curiosity. My efforts will be driven towards motivating young minds to choose to stay in science.

Reference

Lee, M.-H., Ohmachi, M., Arur, S., Nayak, S., Francis, R., Church, D., Lambie, E., Schedl, T. 2007. Multiple functions and dynamic activation of MPK-1 extracellular signal-regulated kinase signaling in Caenorhabditis elegans germline development. Genetics 177, 2039–2062.

Lopez III, A. L., Chen, J., Joo, H.-J., Drake, M., Shidate, M., Kseib, C., Arur, S. 2013. DAF-2 and ERK couple nutrient availability to meiotic progression during Caenorhabditis elegans oogenesis. Dev. Cell 27, 227–240.

Das, D., Chen, S-Y., Arur, S. 2020. ERK phosphorylates chromosomal axis component HORMA domain protein HTP-1 to regulate oocyte numbers. Sci. Adv. 6, eabc5580.

Learn about Swathi Arur’s lab: https://www.mdanderson.org/research/departments-labs-institutes/labs/arur-laboratory.html

Edited by: Govinda Raju Yedida (Volunteer, Bio Patrika)

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