In some ways, it’s difficult to imagine a better model of regeneration than the planarian flatworm. These comical, googly-eyed creatures have captivated scientists for centuries with their remarkable regenerative potential (Dalyell., 1814; Abeloos, 1930). Famously, planarians can be cut into small fragments, each of which is capable of regenerating the full body, (including the brain, eye-spots and intestine), an ability owed to the distribution of potent adult stem cells, known as neoblasts, which are distributed throughout the body and ‘hyperproliferate’ following amputation (Ivankovic et al., 2019; Lee et a., 2022). In this first instalment, I discuss the highlights from the following two articles:

Size of Fragment and Rate of Regeneration in Planarians
Agnes Brøndsted and H. V. Brøndsted

https://doi.org/10.1242/dev.2.1.49

Danish couple, Agnes and Holger Brøndsted, married in 1919 and had the culmination of their work studying planarian fragment size regeneration published in March 1954, in the first issue of the second volume of JEEM/Development (Brøndsted and Brøndsted, 1954). At this time, the regenerative ability of planarian fragments was well established and the presence of neoblasts was accepted. However, Agnes and Holger’s article came at a time of confusion in the field, when conflicting evidence made it unclear whether the number of cells in each fragment contributed to the speed at which the fragment regenerated. They set out to address this question with the minimalistic approach of cutting animals into fragments of different sizes and measuring the length of time eye-spots took to regenerate. Their work began by circling the shores of Lake Fures near Copenhagen, Denmark, lifting rocks to collect planarians from the wild. They ended up with a collection of a few hundred animals from four different species: Dendrocoelum lacteum, Bdellocephala punctata, Euplanaria lugubris and Polycelis nigra. The authors highlight that these four species do not reproduce asexually by fission, removing a confounding variable from their studies. For reasons, I admit, aren’t so clear to me, they dissect each of the species in different ways to produce fragments of different sizes (Fig. 1). However, regardless of the size or shape of the fragment, the rate of regeneration was the same, showing conclusively that the number of cells (and thus the number of neoblasts) in the fragment does not contribute to regeneration speed, suggesting that neoblasts are unlike to migrate within the fragment.

Around 70 years later, planarians are still a potent and popular organism for studying regeneration; however, the variety of species used as models has largely converged on two species: Schmidtea mediterranea and Dugesia japonica. Significant technical advances, not least of which the culture of planaria in the lab, have allowed researchers to understand the cellular and molecular mechanisms of stem cell behaviour during regeneration and mean that hunting for critters under rocks is now a pastime, rather than a scientific necessity.

Loss of plac8 expression rapidly leads pluripotent stem cells to enter active state during planarian regeneration
Hayoung Lee, Kanon Hikasa, Yoshihiko Umesono, Tetsutaro Hayashi, Kiyokazu Agata and Norito Shibata

https://doi.org/10.1242/dev.199449

Last year in Development, Hayoung Lee (Kyoto University) and colleagues capitalised on modern innovations, such as markers for neoblasts and genetic knockdown experiments, to understand the role of a specific gene, plac8-A, during regeneration in D. japonica (Lee et al., 2022). Unlike the species used by the Brøndsteds, D. japonica can reproduce asexually via fission. The authors of the present study employ this fission phenomenon as a measure of cell proliferation and show that knockdown of plac8-A increases the number of fission events. Using imaging and expression analyses, Lee and colleagues demonstrate that plac8-A is expressed by neoblasts and is downregulated before the neoblasts proliferate (Fig. 2). Finally, through chemical treatments, Lee and colleagues show that ERK signalling acts upstream of plac8-A and inhibits plac8-A expression during regeneration via the JNK signalling pathway. Together, these findings demonstrate that plac8-A acts as a molecular switch that regulates neoblasts’ regenerative behaviour.

Together, these two articles address a similar question: What influences neoblast proliferation during planarian regeneration? The Brøndsteds show that, following amputation, not all cells hyperproliferate and that the quantity of cells doesn’t regulate this process, while Lee and colleagues determine that plac8-A must be downregulated in order for neoblast hyperproliferation to occur and loss of plac8-A can induce neoblast proliferation across the whole organism. I hope you enjoyed the post and join me again tomorrow when we will be revisiting the archives to look back on skeletal muscle regeneration.

References

M. Abeloos. Recherches expérimentales sur la croissance et la régénération chez les Planaires. Bull. biol. 1930; 64, 1–140.

Agnes Brøndsted, H. V. Brøndsted; Size of Fragment and Rate of Regeneration in Planarians. Development 1 March 1954; 2 (1): 49–54. doi: https://doi.org/10.1242/dev.2.1.49

J. G. Dalyell. Observations on Some Interesting Phenomena in Animal Physiology, Exhibited by Several Species of Planariae: Illustrated by Coloured Figures Of Living Animals. 1814. Edinburgh: Archibald Constable & Co.

Mario Ivankovic, Radmila Haneckova, Albert Thommen, Markus A. Grohme, Miquel Vila-Farré, Steffen Werner, Jochen C. Rink; Model systems for regeneration: planarians. Development 1 September 2019; 146 (17): dev167684. doi: https://doi.org/10.1242/dev.167684

Hayoung Lee, Kanon Hikasa, Yoshihiko Umesono, Tetsutaro Hayashi, Kiyokazu Agata, Norito Shibata; Loss of plac8 expression rapidly leads pluripotent stem cells to enter active state during planarian regeneration. Development 1 February 2022; 149 (3): dev199449. doi: https://doi.org/10.1242/dev.199449

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The field of regenerative biology has grown considerably since the millennium and, with the creation of the International Society of Regeneration Biology a couple of years ago (Poss and Tanaka, 2021), you’d be forgiven for assuming that it’s a relatively modern field. However, a quick peak through the archives of Development, or the Journal of Embryology and Experimental Morphology (JEEM) as it used to be known, demonstrates that regeneration was – and is – a key focus in the journal since its conception in 1953.

In honour of the inaugural ISRB meeting starting today, this one-week series will take a retrospective look back through some of the earliest regeneration articles published in Development, comparing the research questions, approaches and technologies to more recent publications.

Here are the posts in the series:

Planarians aplenty
Learn about a Danish couple that enjoyed long walks on the beach collecting flatworms and the work of Hayoung Lee, Kiyokazu Agata, Norito Shibata and colleagues.

Muscle memory lane
We meet East Africa-based chiropteran-crusher, J.C.T. Church, and take a whistle-stop tour through the work of Corey Flynn, Deneen Wellik and colleagues.

Time heals all wounds
In this collagen-centric third instalment, we discuss the work of amateur guinea pig tattoo artists, together with Filipa Simões, Paul Riley and colleagues’ study of cardiac regeneration.

A budding tale
Introducing zoologist, engineer, Lieutenant and author David Newth, and his work on epimorphic tail regeneration, complemented by recent studies by Momoko Deguchi, Taro Fukazawa and Takeo Kubo.

Hands-on hard graft
We revisit Dr. D.R. Newth’s newts and their mysterious limb regeneration abilities, compared with Takashi Takeuchi, Haruka Matsubara and colleagues’ modern perspective.

Go fish
The last post says “goodbye and thanks for all the fish”, featuring work from a Nobel Prize winner and Lili Zhou, Ken Poss, Massya Mollaked and colleagues.

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Is it time to re-define what a human embryo is?

With the recent advances in human stem cell-derived embryo models, a team of researchers have suggested that perhaps it is time to redefine what a human embryo is.

Read the Perspective “An ethical framework for human embryology with embryo models” and this News article: What is an embryo? Scientists say definition needs to change.

If you’d like to read more about the technical challenges of studying early human development, check out this Spotlight on Development.

preLights on dev bio

Check out the preLights in #devbio published from March to July this year: preLights in the field of developmental biology (March – July 2023) | Development | The Company of Biologists

A featured preLight is on the preprints ‘Cell polarity linked to gravity sensing is generated by protein translocation from statoliths to the plasma membrane’ by Takeshi Nishimura et al. and ‘Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants’ by Jiayue Chen et al.

News from the community

Learning from our friends over at ZebrafishRock, the Node would like to be more intentional in celebrating the various achievements of people in the developmental and stem cell biology community. We have trawled through social media (which is a bit all over the place nowadays) to look for any relevant news in the past month, but fill in this form if you know someone who deserves a mention, and we’ll consider sharing the piece of news in the next installment of ‘Developing news’.

Newly minted PhDs:

Promotions

Awards:

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Learning from our friends over at ZebrafishRock, the Node would like to be more intentional in celebrating the various achievements of the wonderful people in the developmental and stem cell biology community.

In the latest ‘Developing news‘, we have trawled through social media (which is a bit all over the place nowadays) to look for any relevant news in the past month — newly minted PhDs, promotions, awards — but we know we’ve definitely missed some. That’s why we want to hear directly from you!

If you know someone who deserves a mention, fill in this form, and we’ll consider sharing the piece of news in the next installment of ‘Developing news’.

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A recent paper in Science Advances titled ‘Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly wing patterns’ explores the expression and function of Wnt signaling pathway members in setting up butterfly wing patterns. We caught up with first author Tirtha Das Banerjee and corresponding author Antόnia Monteiro from the National University of Singapore to learn about the behind the paper story.

What was known about Wnt signalling and the butterfly wing patterning before your work?

Tirtha: Wnt signalling is a fundamental signalling pathway that regulates cell communication, cell growth, and cell proliferation in metazoans. A lot is known about this pathway in classical model systems such as Drosophila, but little is known in other systems, such as in butterfly wings. In butterfly systems a few Wnt ligands, primarily WntA and Wnt1, had been associated with the development of bands and eyespots, but most of the other ligands in the pathway, and their receptors, had not been examined in any species.

Antónia: Back in 2006, in my lab at Buffalo, we had visualized Wg/Wnt1 (using an antibody against the Wnt1 protein in humans) at the center of butterfly eyespots, but it wasn’t until we were able to produce a transgenic line, expressing two copies of wg back to back, that folded upon each other when transcribed, that we were able to knock-down this gene to observe its effects on eyespots. A graduate student in my lab in Singapore, Nesibe Özsu, worked on this, and she was able to see smaller eyespots developing when the double stranded RNA was transcribed inside cells via a heat-shock. Tirtha, however, used CRISPR to try and get stronger phenotypes on the wing.

How did this project get started? And Tirtha, what brought you to Antónia’s lab?

Tirtha: This work was partially inspired by my previous work on venation patterning published in Development in 2020 where I observed a very dynamic pattern of Armadillo (Arm) in the larval wings of butterflies. Since Arm is an important component of Wnt signaling, I hypothesized that the ligands (the Wnts) and their receptors (the Frizzleds) might also show dynamic patterns of expression as the wing develops. I started examining their expression, one by one, and uncovered that these other components of Wnt signaling are also extremely dynamic across both larval and pupal wings.

I visited Antónia’s lab back in 2014 during a summer internship program from my graduate studies at NIT-Durgapur. Back then, I visualized the expression of two transcription factors, Engrailed and Spalt, using antibodies that produced some interesting patterns. Even though these early stains were extremely blurry and saturated with colors, they were super cool to me. I proposed an hypothesis for butterfly venation patterning based on the data but due to the limited time of my internship (of 2 months), I was unable to continue the work. Later after graduation I continued this work, which later got published. I was hooked into the evo-devo of wing vein (and color) patterning ever since.

Why did you choose the butterfly wing as a model to undercover the complexities of Wnt signalling?

Tirtha: Developing butterfly wings are simple 2D sheets of cells where numerous ligands, receptors, signal-transducers, and transcription factors orchestrate the specification of extremely complex patterns (of colour) that will be visible in the adult wing. Cells send and receive cues, and interact with each other during development, to specify these colour patterns. Since larval and pupal wings are miniature versions of adult wings, it is easy to map the molecules involved in these signalling processes to the final colour patterns they are likely affecting. 

Antónia: I started working on butterfly wing patterns as an honours student in 1990. I thought butterflies would make great genetic and developmental models because they can produce a ton of eggs and the wings are large and flat, like Tirtha said. In addition, they were much prettier than Drosophila, and their intricate colour patterns were the real hook.

Can you summarise your key findings?

Tirtha: In our recent work we found that the expression and function of different Wnt signaling members varies quite a bit during wing development. For example, Frizzled4, one of the Wnt receptors, is expressed quite uniformly during larval development, but is missing from the future eyespot centers, where canonical Wnt signaling, mediated by Armadillo, is taking place. During the pupal stage, however, the expression of Frizzled4 completely changes and now it is co-expressed in the eyespot centers together with Armadillo. During these two stages Frizzled4 is also likely playing different roles: it is involved in the localization of the eyespot centers during the larval stage because its removal leads to eyespot center duplications, and it is involved in wing scale orientation during the pupal stage. We observed similar dynamics for many other genes we tested in the study and proposed mechanisms of how these genes are likely interacting to specify the multiple cell fates on the wings of butterflies.

Antónia: A key realization for me was observing that a patchwork of different Frizzled ligands and receptors is expressed across the whole wing, which means that every cell of the wing is either producing or receiving Wnt signals, but processing them in different ways. How this patchwork of different Wnt ligands and receptors work together will be interesting to investigate in future.

Did you have any particular result or eureka moment that has stuck with you?

Tirtha: Well one of the exciting moments was when I observed the armadillo CRISPR phenotype showing a double eyespot on the wing. I was also very excited the day I observed the different frizzled patterns on the pupal wings. It made me wonder how nature, over the course of evolution, has been intricately patterning wings by activating certain genes at some location while repressing them in other locations. It’s really incredible, and makes you sit down and think how things which we consider simple are so complex and elegantly tuned at the molecular level.

And the flipside: were there any moments of frustration or despair?

Tirtha: Ohh there were many. As a scientist I believe we all have accepted that there will be more moments of frustration than excitement. For example, for one of the Wnt ligands called Wnt1, I tried to knock it out with  over ten different CRISPR guides, and injected over 5000 embryos. I got no results. Doing stainings with the traditional enzymatic in-situ hybridization was also a painstaking job. I am glad at least those days are over with the new HCR technique we have adapted in the lab.

A recent Development paper also used CRISPR to look at WntA and Frizzled receptors in the butterfly wing patterning. How does the two papers complement each other?

Tirtha: The study from Arnaud Martin’s lab is extremely impressive. Their lab has consistently produced papers that have advanced the field of biological colour pattern evolution.  The authors have generated a massive amount of information in different species of butterflies on how WntA is likely being transduced via the Frizzled2 receptors. They have also gathered functional  data on other receptors such as frizzled, frizzled3, and frizzled4 patterning different aspects of the adult wing scales and venation which are not present in our work. The use of RNAi as a gene knockdown methodology they used would also be extremely useful for other labs working on similar lepidopteran tissues. The gene expression data from their lab and our lab confirms the presence of the different receptors during larval and pupal stages in similar conserved domains strengthening our hypothesis that these receptors are involved in patterning adult wings across butterflies.  

Where will this story take the lab?

Antónia: Well, we have only touched the tip of iceberg with the present study. This work has opened up many new avenues for new students to work on. For example, a student in the lab is now testing a hypothesis we proposed on the interaction of Frizzled4 and Arm in larval wings and the function of frizzled2 and frizzled9 in the development of scales and colour patterns. Another student is testing where the rest of the Wnt signalling members are expressed and what function they play. A postdoc in the lab is trying to visualize whether there is a Wnt1 morphogen gradient around the eyespot centers, as hypothesized nearly 45 years ago.

Tirtha, what’s next for you?

Tirtha: Well, I am currently involved in the development of more advanced technologies for spatial transcriptomics that will allow us to multiplex the number of genes we tested in these studies. Basically, we would be able to understand how perturbations to individual members of Wnt signaling affect the expression of other Wnt pathway members or downstream targets. I hope the upcoming work will have broad applicability across different model systems.

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Our Congenital Anomalies Cluster invites you to join them for an exciting hybrid event at the Advance Training Centre at MRC Harwell on the 23^rd and 24^th of November.

The meeting title ‘From Cardiac Gene Variant to Mouse Model’ is a good summary of the aims of this informal meeting which are to:

• Create a framework for accurately modelling and phenotyping human congenital heart defects in mouse.
• Link human and mouse phenotyping.

The target audience is varied and expected to comprise clinicians, clinical geneticists and developmental biologists interested in finding new genes for congenital heart defects and validating them in mouse models.

For more information and to register for the meeting please download our programme

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Lessons from developmental biology in how to navigate the scientific career-scape

Between the fifth and tenth days the lump of stem cells differentiates into the overall building plan of the mouse embryo and its organs. It is a bit like a lump of iron turning into the space shuttle.

~Miroslav Holub, Czech poet and developmental biologist

My email pings less than 20 minutes after I work up the courage to send a follow-up message to the biology PhD program. It’s April 14, 2020 — one month after Florida shut down for the COVID-19 pandemic.

As I read the message — I would say there is virtually no chance you would hear positively from us — the proverbial rug is pulled out from under me, the vision of my scientific career destabilizing as fast as ocean waves disintegrating a child’s sandcastle. I am set adrift, yet at the same time frustratingly stuck, forced to face the seemingly infinite possibilities in response to the question: What do I do now?

See the landscape for what it is

It’s been three years, and that question still nags at me as I try to answer it. My conclusion? Maybe the path of a scientific career isn’t linear. Maybe it’s more like Waddington’s epigenetic landscape — a metaphorical description of development that begins with a single egg, with its myriad potential like a marble poised at the peak of a complex and shifting geography. This marble can roll down any number of paths into valleys representing cell divisions and their subsequent differentiations into terminal cell fates.

At first glance, this landscape appears rather straightforward. We don’t see the system of “guy ropes” attached to “pegs” that shape the surface area at the whims of genetic and environmental inputs.

I always assumed that a true science career began with graduate school. But the odds of getting into a PhD program, at least in the US, is only about 20%. That means for every applicant who shouts in excitement after receiving an acceptance notification, four others are staring blankly at their computer screens, hearts thumping as they wonder: What do I do now?

In 2020, I was part of the 80% majority. But I felt very much alone in the quiet lockdown of COVID-19. I didn’t even feel like I had a right to be sad — after all, I had my health, still had my job as a laboratory technician. Surely there were other opportunities for a fledgling scientist like me.

I looked for them, too. In my mind, I needed something that was still applicable to my interest in biology. With the pandemic, I wanted something that would allow me to work from home, if need be. Bioinformatics was appealing, although given my lack of computer savvy I knew there would be a learning curve. I just hoped I could crawl up that steep slope of my personal landscape.

In the fall of 2020, I took bioinformatic classes through the online Harvard Extension biotechnology program. I was enamored with the name and the prestige — so much so that I lost sight of the “wondrousness of this wonder” of biology as I got bogged down in computations and repeated syntax errors flashing on my computer screen, stuck in the rut of wanting a degree for a degree’s sake. It didn’t take me long to realize that this online program was not the right fit for me.

Here, once again, my personal landscape shifted and the possibility of a career in science seemed beyond my reach. I struggled to sleep at nights. I even tried distracting myself with daily 7 mile runs, every pounding step the question echoing: What do you do when you don’t know what to do?

Lean into conflicting signals

Much of early development is determined by antagonistic relationships of gene expression — a wrestle between conflicting signals.

One well known example is the role of sonic hedgehog (Shh) and Bmp/Wnt in forming the vertebrate spinal cord. High concentrations of Shh, secreted from the floorplate, instruct nearby cells to be “ventralized.” But the signal gradually fades and eventually meets the opposing Bmp/Wnt signals coming from the roof plate promoting more dorsal cell identities. It’s in this conflict of gradient signals that a unique cell identity code emerges, specifying the neural progenitor cell subtypes along the axis of the future spinal cord. It’s fascinating to me how seeming conflict — an identity crisis, if you’ll allow the anthropomorphism — eventually resolves itself into a whole organism, given the proper signaling gradient across time and space.

In the months and years after my rejected grad school applications, I went through my own identity crisis. On an intellectual level, I knew the notifications may be more a reflection of a lack of funding, space, or resources and not necessarily of my abilities as a future scientist. On an emotional level, I couldn’t shake my feelings of worthlessness. But sharing my loss felt taboo while a pandemic raged, killing thousands of people or leaving them hooked up to ventilators. So I tried to ignore my feelings and push through, to climb up the cliff of my career’s landscape and remake myself.

One night at a socially distant social activity, I struck up a conversation with local news editor Brian McMillan. We talked about our interests in science and writing and the need for better science communication. He encouraged me to begin writing clips, starting with our local Palm Coast Observer. His invitation took root in my brain, a planted seed that just needed some nourishment and gentle coaxing to grow into something larger than what I saw at the time.

Since then, I’ve begun exploring the world of science communication. I was invited to attend the Creative Science Writing Workshop hosted by The Company of Biologists where I met others who wanted to tell the stories of science in ways that could move people. In October of last year, I was granted a New Horizons Fellowship to attend the SciWri22 conference in Memphis, Tennessee. There, I not only discovered a community devoted to telling the stories of science — I found the words to express the grief I didn’t know I was feeling and could finally begin the slow process of healing.

During one of the scientific sessions, St. Jude social worker Erica Sirrine introduced me to the phrase “disenfranchised grief,” or “grief experienced by those who incur a loss that is not, or cannot be, openly acknowledged, publicly mourned, or socially supported.” I saw myself — or rather, my experience — reflected in those words. I felt seen and acknowledged by a community, finally empowered to begin making sense of my hidden grief.

Find your niche community and join in

The growth of any science career, like the proliferating cells of a growing organism, requires collaborative interactions between individuals. No scientist, no matter their passion, can be formed in isolation. Mentors, both formal and informal, help us see ourselves — our strengths and weaknesses — and offer organizing principles that shape our career’s landscape. We do ourselves a disservice when we ignore these outside influences.

As a research community, we must crack the myth of a single career trajectory from masters to PhDs to post-docs to tenured faculty. That’s merely one path in the maze of the scientific landscape, a snapshot that misses the full dynamics of what a science career can be, where boundaries blur and overlap, molded into something no less miraculous than a space shuttle forming from a lump of iron ore while in outer space.

Focusing on tenure professorships at the exclusion of any other career path is myopic, even problematic. The number of PhD applications in the US swelled to 770,000 in 2021, growing nearly 10% since my applications were rejected. Of the 651,000 doctoral students in Europe that same year, nearly 40% were in the STEM field. In Asia, there are over 285,000 doctoral students. These statistics, incomplete as they are, hint that the supply of doctoral degrees is fast outpacing the demand for limited tenured track positions. It’s not the role of mentors to shape their pupils after their own image, but rather to help their pupils find a career path that is suitable for them.

That may be as practical as encouraging science trainees to engage in professional workshops like the one The Company of Biologists offered me last year. We need to hear the stories of scientists who have walked those paths less traveled and see the potential of exploring and even creating new spaces in science. So-called “alternative careers” are as real as academia and need to be de-stigmatized just as much as the grief that comes when doors seem so firmly shut.

A science career is not, nor has it ever truly been, a terminally differentiated state. It’s more like a stem cell niche, maintained in perpetuity until the right signal comes along. Then that marble can start to roll through its personal landscape. If a lab isn’t a right fit, well — there’s always another path, even if it’s unclear where it might lead.

I’m one of those still stuck in the in-between of an undifferentiated career state. But I’ve found a supportive community to encourage me along the way. And that has made all the difference.

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Krista Gert, a recent doctoral graduate from Andrea (Andi) Pauli’s lab at the Research Institute of Molecular Pathology (IMP) in Vienna, Austria, recently published a study on how Bouncer, a small egg membrane protein required for sperm binding, governs compatibility between the sperm and eggs of different fish species. Her study, published in Nature Communications in June, juxtaposes two distantly related, reproductively isolated fish species—zebrafish and medaka. Using a structure-function approach guided by evolutionary analysis, her work uncovers what makes a Bouncer protein compatible with one species’ sperm but not another’s, providing an intriguing molecular explanation for fish hybridization. We caught up with Krista to find out more about the story behind the paper. 

What brought you to Andi’s lab and how did the project start?

I first heard about Andi’s lab through an advertisement for the Vienna BioCenter Summer School program back in 2016 while I was doing my master’s degree at Linköping University in Sweden. I had previously participated in a summer REU (Research Experience for Undergraduates) program while a bachelor’s student and was keen on doing a summer research project again. Developmental biology fascinated me, so I applied to Andi’s lab because of a project on a small protein called Toddler and its role in regulating cell migration during gastrulation. I enjoyed being in Andi’s lab and working with zebrafish that summer so much that I came back for my master’s thesis project in 2017. For that project, I changed gears from working on Toddler to Bouncer and its role in fertilization with a then Ph.D. student, Sarah Herberg. The work I did during that time set the stage for my own Ph.D. project and led to my discovery that Bouncer keeps fertilization species-specific between medaka and zebrafish—a major finding in our first publication on Bouncer in Science. These exciting results provided the momentum for my own Ph.D. project. I dove into exploring how Bouncer mediates specificity in sperm-egg interaction between species, the main goal of my paper in Nature Communications.   

What was known about cross-fertilisation between different species before your work? 

In general, scientists had observed a high frequency of hybridization among fish species compared to other animal groups, but no one had really sought to understand the molecular basis for it given the fact that we didn’t even know what proteins are generally required for sperm-egg membrane interaction in fish. That’s part of why it was so exciting to work on Bouncer—it was the first egg molecule shown to be required for fertilization in any fish species, opening the door to not only studying its function in sperm binding, but also probing how it might limit binding to only sperm from closely related species or allow hybridization across greater phylogenetic distances. 

Can you summarise your key findings? 

When I started working on Bouncer, I initially observed that Bouncer enabled fertilization in a species-specific manner: eggs expressing zebrafish Bouncer could be fertilized by zebrafish sperm, but not by medaka sperm. At the beginning of my current study, however, I soon found that there was more to it than that—zebrafish sperm turned out to be compatible with several different species’ Bouncer proteins, and remarkably, some Bouncer proteins could even work with both medaka and zebrafish sperm. 

However, taking advantage of Bouncer’s medaka/zebrafish specificity, I defined features within Bouncer orthologs, both on the amino acid and post-translational modification levels, that are required for interaction with zebrafish or medaka sperm. In terms of evolution, we found that Bouncer remains largely similar from fish species to fish species, yet we identified a medaka-specific change in the amino acid sequence (site 63) which contributes to the medaka/zebrafish cross-fertility block. In zebrafish, this site contains arginine (R), whereas medaka Bouncer contains a leucine (L). We furthermore found that the N-glycosylation pattern of Bouncer really matters for medaka sperm compatibility but does not influence zebrafish sperm compatibility. In this way, I elucidated some of the molecular details as to why zebrafish and medaka cannot cross-fertilize and furthermore demonstrated that there are different levels of stringency for sperm-Bouncer interaction depending on the species, likely impacting their ability to hybridize with more distantly related species vs. only close relatives. 

Were you surprised to find that seahorse and fugu Bouncer are compatible with both zebrafish and medaka sperm?

Yes, completely! This result was particularly surprising when considering that both seahorse and fugu Bouncer are ~40% identical to zebrafish Bouncer, whereas medaka Bouncer shares just under 39% identity with zebrafish Bouncer. As I mentioned before, it was also quite a surprise since we originally expected that Bouncer would show strict species specificity beyond just the medaka/zebrafish combination. Nonetheless, it was an informative result because I could then focus on sequence elements that were different between medaka and zebrafish Bouncer and ignore any changes that were also present in fugu and seahorse Bouncer, allowing me to narrow the field in determining what underlies Bouncer’s medaka/zebrafish specificity.  

What was it like working with two different species of fish?

For me, working with animals is one of the most enjoyable parts of being a biologist, so I gladly accepted the challenge of introducing a new model species into the lab. Before my project, we had only zebrafish, and I needed to set up all the protocols and tools for working with medaka. It was not easy, but I found it very rewarding once I got everything up and running! Having the side-by-side comparison of medaka and zebrafish was also very informative in terms of their intrinsic species differences, particularly in the process of fertilization itself. 

Did you have any particular result or eureka moment that has stuck with you?

My biggest “eureka moment” would have to be when I saw zebrafish eggs fertilized by medaka sperm for the first time. During my master’s project, I had made several bouncer mutant zebrafish lines expressing other species’ bouncer genes to test whether they could rescue fertilization with zebrafish sperm. I tested the Bouncer proteins from human, mouse, Xenopus, and medaka, but none of them were compatible with zebrafish sperm. But what about the other way around? Could I fertilize these zebrafish eggs with sperm that was species-matched with the expressed Bouncer protein? The scenario most likely to work was medaka sperm with medaka Bouncer-expressing zebrafish eggs, so I procured several medaka males from a neighboring lab and gave IVF a try. After a few attempts and to my great surprise, I caught sight of cleavage-stage embryos in the petri dish as I peered through the dissection scope a few hours later—the very first zebrafish-medaka hybrids to ever exist!

And the flipside: were there any moments of frustration or despair?

When I started this project, I believed that I would be able to pinpoint the exact changes needed to make zebrafish Bouncer compatible with medaka sperm. Each time I made a new line, I thought for sure that this would be the one that would finally work. There is a significant time lag between making a new line and getting the results since you need to wait for two generations, and it takes about 3 months for zebrafish to reach sexual maturity. After making and testing all my amino acid substitution mutants without much luck, I was convinced that combining the R63L change with the medaka N-glycosylation pattern in zebrafish Bouncer would be sufficient for medaka sperm to work. It was disappointing when it didn’t, and even more so when I then tried the entire medaka finger 3 sequence plus the N-glycosylation pattern in zebrafish Bouncer, and this still failed to work with medaka sperm. Looking back, though, I am glad I kept trying even though I didn’t quite solve the entire puzzle of species specificity. 

My initial discovery of Bouncer as the main block to cross-fertilization between medaka and zebrafish provided a versatile system for uncovering Bouncer’s interaction partner on sperm. On top of this, my current paper highlights the importance of site 63 in determining specificity, and it may therefore also be critical for interaction with the sperm binding partner. Current work in the lab by my colleagues Andreas Blaha and Victoria Deneke uncovered that Bouncer may interact with a heterotrimeric complex on sperm consisting of Izumo1, Spaca6, and a new factor, Tmem81, as they report in their recent bioRxiv preprint (Deneke, Blaha, et al., 2023). This is groundbreaking for the fertilization field since the only sperm-egg protein interaction pair of which we know currently is IZUMO1 and JUNO in mammals. What’s especially striking to me is that when you look at the AlphaFold-predicted model of zebrafish Bouncer and the sperm trimer, the tip of finger 3 where R63 sits in Bouncer is right at the interface between Spaca6 and Izumo1. It’s exciting to see how my work was able to pinpoint a potential key interaction site even though I had only half of the picture. Now that we know Bouncer’s interaction partner on sperm, we can work toward defining the Bouncer-trimer interaction interface and understand why zebrafish sperm are able to bind many Bouncer orthologs, whereas medaka sperm maintain higher specificity.

And personally, what is next for you after this paper?

Discovering how to make hybrids between medaka and zebrafish spawned another whole project on which I embarked during my Ph.D. I used them to address another question of species specificity: how is the timing of zygotic genome activation (ZGA) determined? Medaka and zebrafish have proved to be a very interesting comparative system for this study as well, given their different times of ZGA onset during embryogenesis. Currently, I’m finishing up work in the lab for a paper on how we used these hybrids to explore this question. In case you are curious, there’s already a preprint on bioRxiv (Gert et al., 2021).  

I defended my Ph.D. earlier this summer and am currently looking for a post-doc position to continue working on fertilization or reproductive biology. I am especially excited to work on understudied species and to establish new animal models, as my work with Bouncer in two distantly related fish species has really convinced me of the value of a comparative system. In the future, I hope to work with endangered species and develop new reproductive technologies to aid their conservation. 

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Where is the lab?

We are based in the MRC Human Genetics Unit in the Institute for Genetics and Cancer at the University of Edinburgh. It is close to the Stockbridge neighbourhood with lovely views over the city and surrounding countryside.

Lab website: Jenny Nichols Research Group | The University of Edinburgh

Research summary

We are interested in early mammalian development, using mouse and human as model systems. We have a particular focus on preimplantation cell lineage acquisition and gastrulation, also specialising in derivation and use of stem cell lines from both species.

Can you give us a lab roll call, with a sentence including what each person works on and career stage

Jenny: PI.

Elena: Postdoctoral research associate, in collaboration with Kevin Chalut; investigating the role of the mechanical microenvironment and signalling mechanisms during cell lineage specification in pre-implantation mammalian embryos

Takuya: Postdoctoral research associate; investigating the role of Stat3 signalling in the gastrulation stage of mouse development using embryos and stem cell derived ‘gastruloids’.

Lawrence: Postdoctoral research associate, studying events in gastrulation, integrating signalling and transcriptional networks.

Kasia: Edinburgh Clinical Academic Track PhD student, co-supervised by Jenny and Hannah Long. I’m a plastic surgery trainee interested in developmental biology and genetics. I am particularly focused on the role of neural crest in cranio-facial and appendage development using stem cell derived models. I’m using SOX9 gene as a model locus to understand the impact of coding and non-coding mutations in human disease.

Favourite technique, and why?

Jenny: Blastocyst injection is my favourite technique. Apart from being a useful tool for testing stem cell potency and generating mutant mouse lines, it allows us to address interesting questions, such as cell competition in the primary lineages and early post-implantation embryo. In collaboration with Bertie Gottgens’ lab in Cambridge we have been using single cell chimera-seq as a powerful technique to study the function of genes that would result in early embryonic lethality in complete knock-outs.

Apart from your own research, what are you most excited about in developmental and stem cell biology?

Jenny: I am fascinated by the potential to replicate specific organ units from stem cells using quite basic culture regimes and no obvious directional cues. These are invaluable for scrutinising developmental mechanisms in a 3Rs compliant manner and have obvious translatable value.

How do you approach managing your group and all the different tasks required in your job?

Jenny: I see myself more as a facilitator than manager. I am fortunate to have a small lab of highly motivated young scientists inspired by their particular interests. In between all the admin tasks, I like to work in the lab beside my team. I provide them with a coffee machine and unlimited refills, and the door is always open for discussions.

What is the best thing about where you work? 

Jenny: It is a very friendly and welcoming environment, and there are lots of opportunities to attend seminars to hear about the work of our colleagues. Edinburgh University as a whole also has a great developmental biology community with plenty of seminars.

Elena: Microscopes and machines to fabricate our own glass tools! We moved to the HGU recently and our lab neighbours – the Mill lab – have been incredibly helpful and welcoming to us, not only scientifically but also with baked goods! 

Takuya: Technical staff, especially FACS, microscopy and image analysis.

Lawrence: There’s a great social community in our institute.

Kasia: I enjoy the collaborative and interdisciplinary environment in the institute. It’s interesting to interact and learn from scientists coming from a variety of different backgrounds. Core facilities are approachable and helpful.

What’s there to do outside of the lab?

Jenny: It is lovely to walk in the local hills, highlands or along the coast. There is also plenty of culture to enjoy in Edinburgh throughout the year, especially music.

Elena: Take a bus to the south of the city and enjoy a hike in the Pentland Hills! Closer to the HGU are the old railway paths with no cars and surrounded by trees, they take you to Haymarket, Wardie Bay and Leith, I normally use them to run home after work.

Takuya: I enjoy visiting the Highland area of Scotland and the distilleries.

Lawrence: I was thrilled to find places that serve a really good coffee!

Kasia: Edinburgh is a great place to live with access to amazing restaurants, famous theatre and comedy festival and many tourist attractions.  I particularly enjoy yoga and doing sports with my kids. We’ve recently discovered a junior parkrun for kids and parents in our local park.

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“Vaccines don’t just stop short-term disease. They can shape your whole health future”

Dr John Tregoning, Imperial College London

In the latest episode of the Genetics Unzipped podcast, we’re going “Vax to the Future” to look at the science behind DNA and RNA vaccines. How do they work? What can they do, and how can they be made at scale so that more people around the world can benefit from them?

Genetics Unzipped is the podcast from The Genetics Society. Full transcript, links and references available online at GeneticsUnzipped.com.

Subscribe from Apple podcasts, Spotify, or wherever you get your podcasts.

Head over to GeneticsUnzipped.com to catch up on our extensive back catalogue.

If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip

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