Collective migration of caudal visceral mesoderm (CVM) cells in Drosophila embryos helps form the longitudinal muscles of the larval gut. In their study, Angelike Stathopoulos and colleagues reveal that cell division coordinates two gene expression programmes in migrating CVM cells. To know more about their work, we spoke to the first author, Jingjing Sun, and the corresponding author, Angelike Stathopoulos, Professor in the Division of Biology at the California Institute of Technology, USA.

Angelike Stathopoulos (left) and Jingjing Sun (right)

Angelike, can you give us your scientific biography and the questions your lab is trying to answer?

In my lab, we are interested in the patterning of cells and how this ultimately affects signalling pathway activation. These interests led us to think about the patterning process dynamically, and we work on this problem at many levels: from understanding the dynamics of transcription factor action, studying how receptor and ligand expression domains control the timing of signalling pathway activation, and ultimately then to how these processes control cell migration. Our favourite system of study is the Drosophila embryo. Studying later developmental events can be challenging because one needs to tease apart direct from indirect effects. The early embryo has an advantage since there is less history to deal with. However, we also work in late embryos, larvae and the adult, as we have a newfound interest in neurobiology. We have so many tools and so much of a knowledge base to draw on by working in the Drosophila system. It's a great match for the type of scientific discovery that excites me: questions that I want to ask and the availability of tools to provide answers, with a level of mechanistic detail that satisfies me. Regarding my scientific biography – I was always drawn to math, puzzles and patterns. I considered architecture and medicine as possible career choices but was ultimately drawn to bioengineering/biophysics as it seemed to combine design and biology. In graduate school, I allowed myself to explore new areas and fell in love with genetics (also puzzle-like in nature). While working on calcium signalling in yeast, I stumbled on a transcription factor in a screen, and I have never looked back. I have worked on dorsal-ventral patterning in the fly embryo since my postdoc in the Levine lab. Since 2005, I have been a Professor at the California Institute of Technology (Caltech) where my lab has ventured into several new directions. Caltech is a special place, with access to many resources and extraordinary people. Interacting with so many enthusiastic and creative scientists and engineers makes it a fun place to do research. I also even have some time to do experiments myself – I joke that I am everyone's fly technician and that's just the way I like it.

I joke that I am everyone's fly technician and that's just the way I like it

Jingjin, how did you come to work in the lab and what drives your research today?

I have had a standing interest in understanding the molecular mechanisms underlying collective cell migration since my training in graduate school. I was attracted to the Drosophila model due to the live-imaging technique available to the system and how it allows capturing and quantitative analysis of highly dynamic processes. This was beautifully demonstrated in previous papers published by the Stathopoulos lab and inspired me to continue pursuing the topic. More recently, I have started delving into genomic approaches using single-cell RNA sequencing to study gene expression associated with dynamic developmental processes, which has also inspired our latest work. What still drives my research today is my curiosity about the intricate interactions between molecules and the dynamic cellular behaviours that orchestrate morphogenetic processes in embryonic development. I am most motivated by the opportunities in Angela's lab to leverage cutting-edge genomic and live-imaging tools to uncover the underlying molecular mechanisms governing cellular processes like epithelial-to-mesenchymal transition (EMT), which holds the potential for identifying therapeutic targets and developing treatments to combat human health challenges.

Tell us about the background of the field that inspired your work

AS: I teach a course at Caltech focused on morphogenesis. As part of this course, we discuss anchor cell invasion in C. elegans. We were inspired by a study from 2015 (Matus et al., 2015) that showed anchor cell invasion is blocked when the anchor cell undergoes a precocious cell division. This and other preliminary results we had in the lab led us to investigate the role of cell division in controlling gene expression programs in the caudal visceral mesoderm (CVM) cells we study.

Can you give us the key results of the paper in a paragraph?

JS: In addition to genes that are continuously expressed during migration, there are two groups of genes functioning in temporal order to regulate the behaviours of the longitudinal visceral muscle progenitors: the CVM cells. Those expressed earlier during CVM cell migration (e.g. Doc2 and kontiki) are expressed in a spatially restricted manner, whereas those that control the later phase of migration are expressed uniformly. We found that these two ‘early’ and ‘late’ gene expression programs are largely mutually exclusive and cell cycle progression controlled by Cdc25/string and E2F1 facilitates the progression from the early program to the late one. Furthermore, Doc2 acts to inhibit the expression of CG5080, one of the late-expressing genes associated with muscle cell fate. Interestingly, we found that to initiate the late gene expression program, two conditions need to be met: one, loss of an early gene function; and, second, cell division. In summary, these results support the view that gene expression programs change as cells migrate and that cell division can help to ‘reset’ the cell state.

Jingjing, when doing the research, did you have any particular result or eureka moment that has stuck with you?

JS: Yes. We had always suspected that these two gene programs (early versus late) are co-regulated. It would really strengthen this argument if we could identify a signal or a transcription factor (TF) within the early-expressing gene group that functions to negatively regulate some of the late-expressing genes. When I saw that CG5080 does not turn on in mutants that retain expression of Doc2, I was really excited. This suggested that our hypothesis was right, and that early expressed transcription factors, including Doc2, actively repress the late program.

Lateral view images showing caudal visceral mesoderm (CVM) cell migration in wild type (top panel) and Doc2 overexpression mutant (bottom panel) at stage 11. CVM cells are identified through in situ hybridization with HCR probes to HLH54F (magenta). Doc2 encodes a T-box transcription factor and is transiently expressed in the anterior domain of the migrating CVM cells (green). Overexpression of Doc2 in all CVM cells by 5053-GAL4 results in aberrant CVM cell migration. Scale bar: 50 μm.

Lateral view images showing caudal visceral mesoderm (CVM) cell migration in wild type (top panel) and Doc2 overexpression mutant (bottom panel) at stage 11. CVM cells are identified through in situ hybridization with HCR probes to HLH54F (magenta). Doc2 encodes a T-box transcription factor and is transiently expressed in the anterior domain of the migrating CVM cells (green). Overexpression of Doc2 in all CVM cells by 5053-GAL4 results in aberrant CVM cell migration. Scale bar: 50 μm.

And what about the flipside: any moments of frustration or despair?

AS: We had initially thought that the cis-regulatory logic would become apparent if we isolated 10 or more enhancers that are active in the CVM. However, it did not. This was initially frustrating as similar comparative enhancers studies in dorsal-ventral patterning of embryos had provided key insights into patterning, even when a small set of enhancers was analysed. As described in this study, it was key to understand there are early versus late gene expression programs in the CVM. We found that specific TFs act early in the CVM to control spatial patterning of genes that support invasive cell migration, and then a different set of TFs acts later to control expression of genes that relate to cell differentiation/quality control. We had been lumping all the enhancers together, but now believe that there are at least two programmes at play (early and late) – this better positions us to understand the acting cis-regulatory mechanisms.

Why did you choose to submit this paper to Development?

AS: I personally find papers in Development extremely interesting and important to the field. We wanted to get the work published in a timely manner and in the past the journal has done an excellent job handling our manuscripts in a fair and expeditious manner.

JS: Development is very selective and only publishes quality work. I also felt that we had a good record of publishing in Development in a timely manner.

Jingjing, what is next for you after this paper?

I am interviewing for faculty positions with Angela's support. My training with her, which includes writing this paper, has well-prepared me for establishing an independent research program to continue pursuing my research interests.

Angela, where will this story take your lab next?

We are interested in how the gene expression programmes switch as a result of cell division, and our best guess is that this could be tracked by finding more enhancers – especially those that exhibit temporal differences in expression. In the future, I'd like to learn more about the cis-regulatory logic guiding CVM gene expression changes associated with the transition from early to late programs. Why must cells change their gene expression programmes as they migrate? I think we are in a better position to work on the rules at the cis-regulatory level, as well as to provide insight into this collective cell migration process by understanding what these spatiotemporally regulated genes are doing. There are so many exciting leads because of this work, so I would be happy to recruit new postdocs to help with this project as Jingjing will soon be leaving for a faculty position.

Finally, let's move outside the lab – what do you like to do in your spare time?

AS: I'm married and have two children, with the youngest just having left for college this year. So we are empty nesters (free birds?) and have been enjoying more of what Southern California has to offer. Beach, mountain or desert trips on the weekend. My husband and I are both Greek-American, so we also like to keep up with our traditions, including Greek dancing and cooking Greek food. We love entertaining – so enjoying time with family and friends would be where you find me when I am not in the lab.

JS: I enjoy hiking with my dog on weekends. I am also a frequent moviegoer and I enjoy watching all genres.

California Institute of Technology, Division of Biology and Biological Engineering, 1200 East California Boulevard, Pasadena, CA 91125, USA.

E-mail: [email protected]

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