Co-authored by Léa Torcq and Anne Schmidt

Before diving into the science and the path we took to reach our most recent publication in Development¹, let me (Anne Schmidt, senior CNRS scientist, France) take a few lines to introduce the people behind the work.

The first author, Léa Torcq, studied mathematics and developmental biology. Léa began her PhD in October 2019, just a few months before the COVID pandemic. Our second key contributor is Catherine Vivier, our invaluable technician. The third and fourth members of our team are highly dedicated engineers from our platforms, Sandrine Schmutz and Yann Loe-Mie, who brought their excellent expertise in FACS-mediated cell sorting and bioinformatics, respectively. All three of them are staff members of the Pasteur Institute in Paris, France.

Léa and I will take turns sharing our exciting and fruitful collaboration aimed at tracking emerging and newly born hematopoietic stem cells to their implantation sites in developmental niches, using the zebrafish embryo and larva.

Anne: Our project began when — with my background in cellular and bio-membrane dynamics — I decided to investigate fundamental aspects of the cell biology of pre-hematopoietic stem cell emergence. This intriguing and unusual process, first visualized by Karima Kissa and Philippe Herbomel 15 years ago in the zebrafish embryo, is referred to as the Endothelial-to-Hematopoietic Transition or EHT². To our knowledge, this unusual way of emerging from a flat tissue, where the cell bends outwardly from the aortic plane toward the sub-aortic space, appears to be specific to zebrafish (until it is observed in another tissue or species!).

While we began to unveil some fundamental aspects of these intriguing mechanics in 2018³ (see our previous ‘Behind the Paper’ story), we questioned how the luminal membrane of these emerging cells is maintained throughout emergence and how it evolves after completion — with the key feature being the control of apico-basal polarity. Importantly, the evolution of this luminal membrane after release may influence the cell’s behavior, including its migration capacity (for example, if used as a membrane reservoir for cell locomotion), its signaling features (if recycled and/or degraded), and ultimately, its fate.

To tackle these ideas, I developed transgenic lines expressing a well-characterized apical marker, podocalyxin-l2, fused with eGFP (eGFP-podxl). Interestingly, using this line, EHT-undergoing cells imaged with confocal microscopy exhibited obvious asymmetric eGFP-podxl localization, with enrichment at the luminal membrane⁴. Astonishingly, the apical/luminal membrane is extremely dynamic (see Fig. 1) and eventually collapses into a pseudo-endocytic compartment after chasing engulfed intra-aortic fluid, which persists for several hours after release from the aortic floor (a post-EHT signature). This showed that apico-basal polarity is a key feature of EHT cells, maintaining a large apical domain until release, which is unconventional for a cell extruding from a tissue⁵. Perhaps this is the only way a cell can extrude from a flat tissue under strong mechanical tension (the tension exerted on the aortic wall by blood flow and its associated forces⁶), but in fact, it is not! Another outcome from our eGFP-podxl line unambiguously revealed another type of emergence dynamics, which showed no obvious apico-basal polarity. These emerging cells maintain a round shape (not resulting from recent mitosis), with endothelial neighbors crawling on their membrane facing the aortic fluid⁴. These two types of emerging cells, which we called EHT pol+ and EHT pol- cells (for polarized and unpolarized cells, based on podocalyxin localization), suggested that they may have different fates.

Figure 1: Series of z planes showing the dynamics of the EHT cell apical/luminal membrane labelled with eGFP-podocalyxinL2. Spinning disk confocal images obtained with Tg(kdrl:Gal4;UAS:RFP;4xNR:eGPF-podxl2) 50-55 hpf embryos (green: cellular membranes; red: cytosolic RFP). A, single z plane of a longitudinal section of the dorsal aorta (aortic lumen) showing one pre-hematopoietic stem or progenitor cell undergoing EHT (white delimited area on the right). Note the inward bending of the cell, toward the sub-aortic space. B, cropped views of single z planes extracted from a time-lapse sequence starting with the timing point visualized in the field delimited in (A). Images were acquired with 7 minutes intervals, from t=00.00 to t=04.47 hours as indicated in panels 1 and 42, respectively. Numbers 1 to 42 correspond to the progression of the time-lapse sequence throughout time. Note the enrichment of eGFP-podxl2 in the apical/luminal membrane as well as its remarkable dynamics (ex: compare panel 1 with panels 4, 9-12, 14-18). Note also the apparent regression of the apical/luminal membrane in panel 42, indicating that the cell has completed emergence from the aortic floor (we make the interpretation that the cell has detached from the floor in panel 30). Scale bar: 12 µm.

Besides characterizing junctional dynamics at the interface between EHT cells and endothelial neighbors, Léa’s aim was to tackle the question: are EHT pol+ and EHT pol- cells leading to different progenies? The idea was to set up single-cell photoconversion of EHT-undergoing cells, exploring the niches into which progenies establish throughout early larvae (an invaluable advantage of zebrafish, which develops quickly yet remains small) and characterizing their molecular signatures using single-cell RNAseq (sc-RNAseq).

Léa: I joined the lab as a Master’s student to work on this project in January 2019. I had previously studied EHT in Thierry Jaffredo’s lab, conducting work on self-organizing quail embryo explants in vitro. Although such models are fundamental to scientific discovery, I wanted to move toward more physiological, in vivo approaches. When I met Anne and she showed me the movies generated through live imaging of transgenic zebrafish embryos, I was instantly fascinated by this beautiful – both scientifically and aesthetically – approach. I also quickly discovered that Anne was a unique kind of senior researcher. She remains actively involved in performing and analyzing experiments at length with a combination of youthful drive for science with wise meticulousness, which convinced me to follow my initial internship with a PhD, with Anne as my advisor.

I was also drawn to this project because it gave me the opportunity to learn many different techniques, ranging from live imaging to scRNAseq and transgenesis. It came with challenges in optimizing and analyzing these diverse experiments, compounded by the COVID lockdown during most of 2020. Nevertheless, we persevered, and I was fortunate to receive invaluable help from several people, particularly our co-authors. Catherine taught me how to perform in situ hybridization and set up single molecule fluorescence in situ hybridization (smFISH), using RNAScope. Sandrine, from our institute’s cytometry platform, spent around 100 hours sorting cells for subsequent scRNAseq experiments. As for Yann, he originally helped set up the analysis pipeline for MARS-seq and provided guidance when I began training myself in scRNA-seq analysis.

Overall, we used nearly 400 embryos for photoconversion of single cells based on their morphology as they emerged from the aorta. Subsequently, larvae were used to track migration patterns and build precise lineage trees. We also index-sorted the progenies of 2,036 photoconverted cells and generated MARS-seq libraries from them. Separately, we used gata2b and cd41 reporter lines and 10X Chromium to generate a complementary scRNA-seq dataset of more than 30,000 cells, encompassing the whole hematopoietic lineage sorted by their niche of origin. Our main discovery was the differential fate of EHT pol+ and EHT pol- cells, with a bias regarding the lymphoid lineage. We identified different propensities to seed the thymus as well as different abilities to differentiate into T-lymphocytes. Moreover, our work contributes to the characterization of zebrafish hematopoietic cell types with new insights on the origin of some populations, like the ILC2-like and ILC3-like cells, never before observed at such early developmental stages.

Anne: Léa’s hard experimental and bioinformatic work has been extremely fruitful. From our single cell pipelines and their integration, we retrieved informative signatures of Hematopoietic Stem and Progenitor Cell (HSPC) populations. These included the transcription factor gata2b⁷ (which is upstream of runx1, a transcription factor essential for hematopoiesis⁸) and podocalyxin/cd34. Intriguingly, we found that in addition to embryonic HSPCs (eHSPCs) and other multipotent progenitors, gata2b is also expressed in sub-populations of ILC2-like cells enriched in the anterior/trunk region of the larvae, and of young eosinophils. We discovered that eosinophils possess the unique property of differentially expressing genes related to extracellular niche/matrix functions, including serine protease inhibitors of the spink2 family, timp4.2 (an inhibitor of metalloproteases), as well as one specific member of the MFAP4 locus. We then used these markers to investigate the localization of hematopoietic populations through whole-mount in situ hybridization. With Catherine’s expertise, we developed RNAscope applied to zebrafish. While whole-mount RNAscope had rarely been used for zebrafish embryos and larvae at that time – essentially because chromogenic and/or fluorescent in situ hybridization using long antisense nucleotide probes were routinely used and at relatively low cost –, it proved to be a great decision. Because it provided a high signal-to-noise ratio and sensitivity, RNAscope allowed us to investigate cells implanted in niches throughout the entire early larval body, including the pronephros region, which is challenging because it requires deep penetration of probes and low background (see our recent technical paper ⁹).

With the timp4.2 marker highly expressed in eosinophils, we found an intriguing accumulation of cells almost exclusively in the most anterior region of the pronephros, in the 5 dpf larva. These cells, with a maximum of 15 per animal in that region (on average more than in the trunk and the Caudal Hematopoietic Tissue (CHT)), also faintly express eGFP driven by an enhancer of the hematopoietic transcription factor runx1, confirming their hematopoietic origin (see Fig. 2 and Movie 1). This pointed to sub-compartmentation of the pronephros niche. Currently, we do not know if these cells, presumably eosinophils (or progenitors), home there for maturation and/or if they contribute to building a sub-niche hosting specific hematopoietic cell subtypes. Anyhow, these results highlight the functional complexity of the developing pronephros niche and point to the importance of investigating micro-environmental properties supporting the differentiation and/or maintenance of specific hematopoietic populations.

Figure 2: Whole-mount in situ hybridization revealing timp4.2 mRNA expression in hematopoietic and vascular cells using RNAscope. A, C, D, Representative images (Imaris 3D-rendering) of RNAscope ISH for timp4.2 (magenta spots) in 5 dpf Tg(runx1+23:eGFP) larvae. Images show the pronephros region (A, see also Movie 1), the posterior trunk region (C, above the elongated yolk) and the CHT (D). a’, c’, d’ are magnifications of regions outlined with white dashed boxes in (A, C, D), respectively. eGFP positive hematopoietic cells were segmented (green contours). White arrows point at timp4.2 positive hematopoietic cells. The sub-aortic clusters are delimited by yellow dashed lines and the gut by magenta dashed lines. B, Relative position of eGFP positive cells along the antero-posterior axis of the pronephros (n=681 timp4.2– cells, n=46 timp4.2+ cells). E, Percentage of eGFP positive hematopoietic cells expressing timp4.2, n=6 larvae for pronephros, n=3 for trunk and CHT regions. (B, E) Two-sided Wilcoxon tests. NC: notochord. Scale bars: 10 µm.

Movie 1: 3D visualization of RNAscope in situ hybridizations for timp4.2. Timp4.2 (in magenta) in the pronephros region of Tg(runx1+23:eGFP) 5 dpf larvae, 3 representative replicates are shown. Bottom row shows magnifications of the top row. Hematopoietic cells in the pronephros are delineated (green contours). Scale bars: 10 µm.

Finally, the most unexpected results came when using the gata2b probe. We detected strong expression of this transcription factor in endothelial cells of the supra-intestinal artery (SIA), a small vessel located just above the intestinal tract and beneath the posterior cardinal vein (for detailed anatomy, see Isogai et al¹⁰). We obtained these results in March 2023, more than 2 years ago and about a year and a half before submitting our paper to Development. Importantly, our images unambiguously showed that not only do SIA endothelial cells express gata2b, but so do other cells in their direct vicinity, even contacting the SIA wall. These cells also express eGFP driven by the vascular kdrl promoter, and it appeared that many of them express eGFP at levels comparable to SIA endothelial cells.

Léa: When we realized this, we considered that the SIA region might not only be a niche for hematopoietic stem and progenitor cells and more differentiated cells whose ancestors emerged from the dorsal aorta days before (e.g., ILC-like cells with immune functions in the gut¹¹), but that these cells might also derive directly from the SIA wall itself! To reinforce our results, I quantified the number of gata2b-positive cells in the direct surroundings of the SIA as well as near the dorsal aorta, showing that the SIA region is significantly enriched in gata2b cells compared to the dorsal aorta. After 3D segmentation of the cells with Imaris, I quantified their eGFP signals (driven by the kdrl promoter) and found that gata2b-positive cells near the SIA express comparable levels of eGFP to SIA endothelial cells (with no such cells around the dorsal aorta). This suggests they are relatively newly born cells whose eGFPcontent has not been diluted by division cycles, reinforcing the idea that they may originate from the SIA endothelium.

Anne: All this evidence supports the hypothesis that the SIA may be hemogenic. Due to the constant movement of the gut beneath the SIA, we struggled to provide high quality time-lapse sequences for our paper in Development (even acquiring a single complete z-stack was difficult). However, we obtained discontinuous images over relatively short periods of up to 2 hours that strongly suggest emergence from the SIA wall (see Fig. 3). Importantly, cells undergoing apparent emergence remained very ‘sticky’ to the SIA wall, making it difficult to confirm they fully completed EHT. Our results clearly demonstrate that the SIA region is at least a niche hosting HSPCs and suggest that these may be born from this small artery. Hence, the SIA may be hemogenic, a potential novel finding requiring further validation. As discussed in our Development paper¹, this validation will require characterizing the SIA hemogenic endothelium and the fate of the derived EHT cells at the single-cell level.

Figure 3: Evidence of emergence from the SIA endothelium. Top panel: schematic representation of a 5 dpf larva (reproduced with modifications from Schmidt, 2022, doi:10.7554/eLife.64835); red line = aorta, blue line = vein, magenta line = SIA. (a – d’), spinning disk confocal microscopy of a 5 dpf Tg(kdrl:eGFP) zebrafish larva, in the upper part of the trunk region (delimited in the cyan box of the upper cartoon). Panel a (z projection), the upper part of the trunk region encompassing the dorsal aorta, the posterior cardinal vein, and the SIA with the latter passing beneath the swim bladder (SB, on the left side of the image). White and magenta asterisks indicate cells expressing eGFP at apparently comparable level than SIA endothelial cells and that are contacting the SIA wall (note that no such cells are in contact with the ventral floor of the dorsal aorta). (b – d’), single z planes extracted from the z stack projected in (a), with images magnified from the region in (a) delimited by the white rectangle and showing the cell surrounded by the green rectangle in (a) and undergoing emergence between 0 min (b, b’), 10 min (c, c’), and 20 min (d, d’). Panels (b, b’), (c, c’), and (d, d’) are images separated by 1 mm depth in z. Green arrows point at the connection of the emerging cell with the aortic lumen; magenta arrows point at the disappearance of this connection, which suggests completion of the emergence. Scale bar: 20 µm.

This story behind our paper in Development summarizes an exciting research journey that led to several previously undescribed findings. This was made possible by assembling a team of passionate and efficient people and pushing forward the resolution of our analyses, including technically demanding single cell photoconversion, multiple single-cell RNAseq approaches, and powerful smFISH technology using a new generation of small, highly specific probes (we’ve significantly contributed to increasing the number of zebrafish hematopoietic probes in the ACD catalogue!).

Finally, we are convinced that our work opens new avenues for exciting future discoveries in the fields of hematopoietic stem cells and vascular biology.

References

1.   Torcq, L., Vivier, C., Schmutz, S., Loe-Mie, Y., and Schmidt, A.A. (2025). Single-cell and in situ spatial analyses reveal the diversity of newly born hematopoietic stem cells and of their niches. Development 152, dev204454. https://doi.org/10.1242/dev.204454.

2.   Kissa, K., and Herbomel, P. (2010). Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464, 112–115. https://doi.org/10.1038/nature08761.

3.   Lancino, M., Majello, S., Herbert, S., De Chaumont, F., Tinevez, J.-Y., Olivo-Marin, J.-C., Herbomel, P., and Schmidt, A. (2018). Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish. Elife 7, e37355. https://doi.org/10.7554/eLife.37355.

4.   Torcq, L., Majello, S., Vivier, C., and Schmidt, A.A. (2024). Tuning apicobasal polarity and junctional recycling in the hemogenic endothelium orchestrates the morphodynamic complexity of emerging pre-hematopoietic stem cells. Elife 12, RP91429. https://doi.org/10.7554/eLife.91429.

5.   Staneva, R., and Levayer, R. (2023). Cell polarity and extrusion: How to polarize extrusion and extrude misspolarized cells? Curr Top Dev Biol 154, 131–167. https://doi.org/10.1016/bs.ctdb.2023.02.010.

6.   Campinho, P., Vilfan, A., and Vermot, J. (2020). Blood Flow Forces in Shaping the Vascular System: A Focus on Endothelial Cell Behavior. Front Physiol 11, 552. https://doi.org/10.3389/fphys.2020.00552.

7.   Butko, E., Distel, M., Pouget, C., Weijts, B., Kobayashi, I., Ng, K., Mosimann, C., Poulain, F.E., McPherson, A., Ni, C.-W., et al. (2015). Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo. Development 142, 1050–1061. https://doi.org/10.1242/dev.119180.

8.   Gao, L., Tober, J., Gao, P., Chen, C., Tan, K., and Speck, N.A. (2018). RUNX1 and the endothelial origin of blood. Exp. Hematol. 68, 2–9. https://doi.org/10.1016/j.exphem.2018.10.009.

9.   Torcq, L., and Schmidt, A. (2025). Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva. BIO-PROTOCOL 15. https://doi.org/10.21769/BioProtoc.5269.

10. Isogai, S., Horiguchi, M., and Weinstein, B.M. (2001). The Vascular Anatomy of the Developing Zebrafish: An Atlas of Embryonic and Early Larval Development. Developmental Biology 230, 278–301. https://doi.org/10.1006/dbio.2000.9995.

11. Hernández, P.P., Strzelecka, P.M., Athanasiadis, E.I., Hall, D., Robalo, A.F., Collins, C.M., Boudinot, P., Levraud, J.-P., and Cvejic, A. (2018). Single-cell transcriptional analysis reveals ILC-like cells in zebrafish. Sci Immunol 3. https://doi.org/10.1126/sciimmunol.aau5265.

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By Sriivatsan G Rajan and Ankur Saxena

What is this?

A high magnification video of a zebrafish embryo demonstrates sensory neurogenesis in the developing nose (olfactory epithelium), with newly forming neurons labeled in orange and blue cells indicating high Notch signaling activity. The developing eye is also visible nearby. The olfactory epithelium houses remarkable levels of neuroregeneration, including in humans, and is a robust model for investigating the molecular pathways that drive continuous neuronal renewal.

Where can this be found?

Zebrafish are ray-finned fish that are native to freshwater habitats in South Asia and are widely used as vertebrate model systems due to their high degree of genetic similarity to humans. We use a suite of tools to genetically manipulate the organisms, and the optically transparent embryos are amenable to high-resolution microscopy.

How was this taken?

We performed live confocal microscopy of transgenic zebrafish embryos at 2 days post-fertilization (dpf). The embryos expressed red fluorescent protein (orange) in olfactory sensory neurons and destabilized green fluorescent protein (blue) in cells with active Notch signaling. Images were acquired at regularly spaced time intervals for 15 hours using a Zeiss LSM 800 confocal microscope and stitched together to make this timelapse video.

What happens during olfactory sensory neuron (OSN) development?

We discovered that during olfactory sensory neuron (OSN) development, discrete groups of progenitor/stem cells communicate with each other via a unique Notch/Insm1a signaling module to form neighborhoods of cells that act as hot spots of neurogenesis (generation of new neurons). Retinoic acid signaling from the nearby eye influences this intricate process of new OSN formation, and BDNF (brain-derived neurotrophic factor) signaling helps guide new neurons to their final destinations.  

Why should people care about this?

Neurodegenerative disorders are strongly associated with the depletion of neurons across the nervous system. Interestingly, while olfactory sensory neurons (OSNs) are known to be highly regenerative, the loss of smell is often an early indicator of potential neurodegeneration. As a first step to understanding this apparent paradox, we aimed to uncover how new OSNs are generated. Additionally, we hope to discover conserved pathways that might aid neuroregeneration in other organ systems. Finally, our observations of the close coordination and exchange of signals between the nose and the eye shed light on the importance of inter-organ communication for neurogenesis.  

How would you explain this to an 8-year-old?

Our noses have tiny nerve cells that detect different kinds of smells that help you enjoy pizza or not enjoy medicine. While zebrafish don’t eat pizza (as far as we know), they have those types of cells, too. Because you can see through zebrafish pretty well, we can put them under a fancy microscope, watch those nerve cells get made, and learn how that happens. What we learn then allows us to think of ways to make new nerve cells that could help people and keep them healthy.

Where can people find out more about it?

You can read our recent paper in Stem Cell Reports https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(25)00179-1; read a short news story about it https://www.uab.edu/news/research-innovation/sniffing-out-how-neurons-are-made; and check out the fun journal cover image https://www.cell.com/stem-cell-reports/issue?pii=S2213-6711(24)X0010-7#fullCover

To follow other research projects from the Saxena lab, go to www.saxenalab.com

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This year brought the return of our image competition with the MBL Embryology course at Woods Hole. Twenty impressive submissions were received from the 2025 class of students, with images ranging from polychaete worms to butterflies, squids and mice. Here, we interview Arthur Boutillon, the Editor’s Choice winner of the image competition with his submission, ‘Embryonic eye of Anole lizard’. As our Editor’s Choice, Arthur’s image was featured on the cover of a recent issue of Development.  

Can you describe your research career so far?

My research career began during my Bachelor’s studies at the École Normale Supérieure in Paris, France, where I had the opportunity to work with different model organisms through several internships, all focused on morphogenesis. 

I then pursued a PhD in Nicolas David’s team at The Laboratory for Optics and Biosciences, École Polytechnique, Palaiseau, France. Working with zebrafish embryos, my favourite model organism so far, I studied the collective movement of cells during gastrulation using a combination of classical approaches (e.g., grafts) and more advanced techniques (e.g., 3D laser ablations), along with extensive live microscopy. We discovered a novel mechanism by which cells coordinate over long distances, a phenomenon we named “guidance by followers”.  

Can you tell us about your current research?

After defending my PhD at the end of 2021, I moved to Dresden, Germany, to join Otger Campàs’ group in the Cluster of Excellence Physics of Life as a postdoctoral researcher. Here, I study the mechanics of morphogenesis, still using zebrafish as my model system.

By combining physical measurements, quantitative imaging, and genetic perturbations, I investigate the mechanics of somite boundary formation, work that is currently under revision for publication. I also study the link between signaling and the acquisition of tissue mechanical properties.

Attending the Embryology Course at Woods Hole broadened my perspective on the value of different model organisms. I am now developing research projects centered on mechano-evo-devo to apply for PI positions.

What is your favourite imaging technique/microscope?

Anything that allows me to watch development live!

That said, I have a soft spot for the versatility of point-scanning microscopy. 

With our trusted LSM980, I can image both live and fixed samples and perform laser ablation, optogenetics, FRAP, FCS, and more, all on the same instrument! 

What are you most excited about in microscopy currently/in the future? 

I’m excited about all kinds of new developments: every microscopy technique has its strengths and limitations, and it’s ultimately about finding the best fit for each question. What excites me even more are the tools that help with segmentation and image analysis. I’m constantly amazed by what open-source platforms like StarDist, Cellpose, or Mastodon can achieve.

My dream is to see a unified, open-source platform that integrates these tools seamlessly, something that would save long hours of image analysis after acquisition!

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Our September webinar featured three early-career researchers working on gut development. Here, we share the talks from Surojit Sural (Columbia University) and Swarnabh Bhattacharya (Dana-Farber Cancer Institute).

Surojit Sural (Columbia University)

Swarnabh Bhattacharya (Dana-Farber Cancer Institute)

(2 votes)

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Royal Society Publishing has recently published a special issue of Philosophical Transactions A: Biological fluid dynamics: emerging directions compiled and edited by Smitha Maretvadakethope, Marco Polin, David J Smith and Laurence G Wilson and the articles can be accessed directly at  www.bit.ly/TransA2304 

A print version is also available at the special price of £40.00 per issue from sales@royalsociety.org

About this issue
The microscopic world of algae, bacteria, spermatozoa and other swimming microorganisms is fundamental to life on Earth. Here, fluid dynamics follows very different physical laws from those familiar to us. Friction dominates, so cells have to squirm and corkscrew their way through fluid rather than glide. Microorganism have evolved to survive and thrive in the world of biologically active fluids, performing essential functions such as navigating, feeding, cooperating and reproducing. Long-range interactions in microscopic flow can cause beautiful collective effects, such as pattern formation and ‘active turbulence’. Driven by recent advances and touching on topics ranging from new medical technologies to the origins of life itself, this special issue presents contributions at the cutting edge of research in this field.

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Applications are open for 10 ECR funded places at The Company of Biologists Workshop on Novelty, Co-option and Divergence During Gene Network Evolution, organised byJames Hombría and Antónia Monteiro.

The Workshop will bring together researchers working on gene regulation, developmental biology, mathematical modelling and evolution. The invited researchers work on a variety of complex systems and are examining how these systems originated and have evolved over time. By comparing perspectives and experimental approaches to examine the evolution of their specific systems, we hope to draw common threads that may be applicable to most systems, and we aim to highlight these after the meeting in a Review article.

Deadline: Friday 5 December

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Hopeful monsters. Morphospace. Mutation. Natural variation. Mutagenesis screens. Polymorphism. Deformity. Phenotype. Disease. Adaptation. Anomaly. Variant. Error.  

What defines the distinction between defect and difference? Between natural and unnatural? Between right and wrong?

When you are disabled, you are made to feel that you are wrong.

In my evo-devo PhD, my study species was a species of cichlid fish. When people unfamiliar with them ask me what on earth a cichlid is and why on earth I would study it, I launch into explaining what an adaptive radiation is with “you know Darwin’s finches? Like that but fish, and instead of islands, it’s lakes. They’re all sorts of shapes and colours to suit all sorts of ecological niches.” What’s so interesting about them is their diversity. In paper introductions we try to convey the extent of that diversity with adjectives like “remarkable”, “unparalleled”, and “astonishing”. They are words full of wonder. Even with our range of species in the lab, it is something I only really appreciated personally rather than academically when I finally went diving in Lake Malawi/Nyasa.

All of that diversity in form is the product of varying developmental processes, tweaked by genetic differences from an ancestral set of developmental trajectories. So another thing we sometimes say is that cichlids represent a “natural mutagenic screen”¹. Nature has provided a set of morphological differences, so we can investigate the relationship between developmental processes and morphological outcome. Not only does this give us a better understanding of the role of development in evolutionary change, but also it may provide another window into the developmental processes that we still don’t fully understand. The implication is that by studying natural variation, you can better understand “normal” development. If you understand which genetic and developmental differences lead to natural biodiversity, you can gain insights into how genetic and developmental differences lead to pathological deviation from the norm². Take cichlid craniofacial shape diversity, for example. The variation between cichlids reflects adaptations to different feeding strategies. One of the key findings of evo-devo research is that the same developmental pathways and programmes are used across even distantly related organisms. So studying craniofacial development in one organism, such as a fish, can shed light on craniofacial development in another vertebrate, such as humans. By extension, studying how craniofacial development differs between cichlids can shed light on parallel defects in human craniofacial development³.

Who decides what is normal development? Who decides what is natural biodiversity or pathological deviation? Who defines the distinction between defect and difference?

Developmental biology and disability have been two parallel paths in my adult life. Rather, they seemed to be parallel, until more and more ideas from each began to collide. I first stumbled across an article that made me realise I am neurodivergent one evening while undertaking an undergraduate summer research project in a developmental biology lab. Four years later I made it to the top of the waiting list at the NHS clinic and was diagnosed, during my PhD. In the intervening time, I was developing a better understanding of perspectives on evolution and development. In that same intervening time, I read up on neurodiversity theory and disability justice.

Disabled is one of those adjectives that is secretly a verb. Like exhausted, frightened, excited, there is an implicit action that caused this state. I am exhausted – I was exhausted by the swim. I am frightened – I was frightened by the news. I am disabled. The question is: disabled by what? The medical model of disability locates the disability in the person’s body. The social model, on the other hand, locates the disability in the world around me. The world disables me. It disables me by not meeting my access needs. It does not pretend my physiological condition does not exist – but it identifies that the problems related to that condition have external causes. The social model is freeing because it is actionable. If you are shortsighted, you probably don’t consider yourself disabled, and that’s likely because you have prescription glasses or contacts that mean you are not cut off from engaging with parts of the world. The social model is freeing because it means disabled is not a dirty word: you no longer consider it to be an inherent character flaw. Disability is not shameful. Inaccessibility is shameful.

You can repeat those sentences like a mantra as much as you want, but the internalised ableism is hard to shake. It is hard to shake the conviction that you are somehow innately wrong. That it is your fault.

Developmental biology is in the business of understanding how forms are built. With that remit comes the study of how form-building goes awry. Sometimes that’s a tool in the developmental researcher’s toolbox: “Break it, and we understand how a sequence is necessary”⁴. Sometimes understanding how it goes awry by itself is the motivation: so you can suggest how you could fix it. This is often given as justification for the blue-sky discovery science, and is sometimes the central motivation for the work.

About the same time as my diagnosis, I began to lose my hearing – a condition involving excessive bone remodelling that may or may not be inherited, may or may not be exacerbated by oestrogen, but definitely isn’t environmentally induced and that’s about as much as we know. After having covid, I began severely struggling to keep up with my work and life, and about a year later admitted defeat and took long-term sick leave to recover, thankfully returning to finish my PhD despite my fears at the point of intermission. I am accruing disabilities. It is as if I am collecting them like shiny cards. Shiny, shameful, cards.

One of the key perspectives on evolution and development that I like to make clear to people in my non-science life is that there is no such thing as best adaptation. It is a pervasive popular misconception that evolution is a series of advancements and that some species are more evolved than others. The misconception is understandable, given the original scientific thinking on evolution was imbued with the same ideas of advancement and progress. But there’s no such thing as adaptation in a vacuum. Fitness is a concept only in relation to the environment. In my first year undergraduate lectures, we were shown representations of fitness landscapes, and I had fun picturing the landscapes shifting when the environment changed. We stepped through the maths of sickle cell allele fitness in situations with malaria and without malaria. Everything shifts with context, and everything depends on that flimsy, ephemeral balance between organism and environment.

To be glib, a human is no more evolved than a fish, but is better adapted to running long distances to tire out prey, while a fish is better adapted to living underwater. Having lungs instead of gills is a problem if you want to be underwater. To see the cichlids in their natural environment, I donned heavy equipment to take the air down with me. That wouldn’t be something I’d need to consider if I had been looking for finches instead.

The social model of disability is analogous to this argument of adaptation: everything depends on the environment. I’m not arguing that disabilities are an adaptation to worlds that we haven’t yet built. I’m highlighting that the environment determines whether a difference is a disadvantage.

It doesn’t matter if you have insensitive hearing if you are communicating with hand signs. It is not a disadvantage to be sensitive to fluorescent lights if you don’t work in an office rammed full of them. Crucially for the social model of disability, we construct our environment. Humans generally can’t see in UV, so we don’t make road signs with UV markings. If starlings were in charge, things might be different.

Who defines the distinction between defect and difference?

There are many, many aspects of human variation. There are many ways to be disabled. The neurodiversity paradigm makes the argument the most strongly that just as there is bio-diversity between species, within humans there is neuro-diversity. This analogy was arrived at by autistic people on web forums in the 1990s⁵. The differences are physiologically neutral, but disabling because the world is set up to cater by default to the neurotypical neurotype.

Not every neurodivergent person feels the same about these things, of course. And people disabled in other ways feel differently too: people with chronic illnesses, particularly those with chronic pain and energy-limiting conditions, tend to find the social model doesn’t capture the problems they face. In these cases, there is often a dearth of research behind the disabling lack of treatment options. Disabled people are not listened to about which conditions need more research. And in particular, disabled people are not listened to about which aspects of their conditions they would find it useful to have research address. The fact that this research is not done (and sometimes instead unwanted, harmful research is done instead) is an extension of the social model: the lack of appropriate research is itself disabling.

Note my qualifiers: appropriate research, aspects of their conditions. I’ve watched developmental biology presentations where a gene has been identified as of interest through one method or another, and a condition or a whole collection of conditions are listed next to the gene name on the slide. Presumably these have shown up on a human GWAS somewhere. Sometimes it’s physical conditions like spina bifida, or polydactyly. Sometimes it’s neurological conditions like autism, schizophrenia, dementia. It is an off-hand list written to say: ‘look, there could be useful implications to this work! We could be useful and eliminate all these conditions!’ It flattens all these conditions into inherently bad, bad in the same way, with no nuance into what people might actually want to see for improvements to their lives. It is a gut-punch every time. I came to see a seminar about neurons, and I was casually told it would be better if I didn’t exist.

My brilliant friend wrote an article about the futility and danger of the simplifications of identifying genetic associations to complicated human traits in BlueSci, on the search for ‘gay genes’⁶. Not only it is unsurprising that complex, multi-dimensional traits are highly polygenic, but also this pathologising approach encourages the eugenicist perspective: find the cause of difference so you can eradicate it. (And consider: back when homosexuality was in the DSM, would that have shown up on the list of conditions next to the gene of interest in those presentations?) Often, disability-related research similarly enables eugenicist goals. The more I have come to understand this, the more wary I have become of efforts to introduce human embryo gene editing and unwittingly or wittingly make those eugenicist goals possible. With the last of my pre-intermission spare energy, I wrote an article for the Keppel Health Review on autism research, and how autism research funding is often spent on attempts to find the cause of autism, in a way that implies that autism can and should then be eliminated⁷. Rather than listen to criticisms or even make good use of the lived-experience insights from autistic people, high-profile autism research has continued the same research directions while simply pasting inclusive language on top. (Not exactly good practice Public and Patient Involvement!) I was excitedly gearing up to write about the use of language about disability in developmental biology and the intertwined history of eugenics when, ironically, I had to pause working due to my long-term illness.

I’m not arguing that disabled people don’t want research, or new treatments or interventions. I’m highlighting that disability can’t be flattened into something that is uniformly and inherently wrong, for the scientist to swoop in to save us all from.  

Who defines the distinction between defect and difference?

Medical research can transform lives. We live in the age of miracles – I believe this genuinely and viscerally. Transformative medical advances can rest on developmental biology research. And more broadly, developmental and evolutionary biology concepts shape the way we see ourselves as human.

So, it is possible to do better. It is possible to engage with disabled people when you realise your development research is related to disability. Too often, attempts at engagement with marginalised groups relevant to research boils down to pasting inclusive language over the same narrow negative perspectives. Genuine engagement is as simple as listening. Listen to how disabled people feel about their conditions and their circumstances. Listen to what they would have change if they could. Listen to how they relate to their conditions and how they prefer to describe them. Listen to how they explain their experience of their conditions, because it might give you ideas and insights into what to research next. Listen to what kind of research they would find useful and what kind of research they would find harmful. Listen to people with the relevant conditions, because disability isn’t a singular experience. Listen to a range of people with those conditions because, again, disability isn’t a singular experience. And crucially, change your approach to be consistent with what you hear. Change your language, yes, but also interrogate what views underlie the language you were previously using and challenge those views. I think the field could be so much richer for it.

Who defines the distinction between defect and difference? Surely it should be those who live with those differences.

My favourite part of my PhD was any time I had the chance to look at my embryos under the microscope. They were strange, gorgeous things. Developmental biology is fundamentally beautiful. We are no less beautiful for our variation. Instead, perhaps we are more so. Perhaps we are remarkable. Perhaps we are full of wonder.

1. Kocher. (2004). Adaptive evolution and explosive speciation: the cichlid fish model. Nature Reviews Genetics 5, 288–298 https://doi.org/10.1038/nrg1316
2. Diogo, Guinard, Diaz Jr (2017). Dinosaurs, chameleons, humans, and evo-devo path: Linking Étienne Geoffroy’s teratology, Waddington’s homeorhesis, Alberch’s logic of “monsters,” and Goldschmidt hopeful “monsters”. J. Exp. Zool. (Mol. Dev. Evol.) 328B: 207–229 https://doi.org/10.1002/jez.b.22709
3. Powder, Albertson (2016). Cichlid fishes as a model to understand normal and clinical craniofacial variation. Developmental Biology 415, 338-346 https://doi.org/10.1016/j.ydbio.2015.12.018
4. Cooper (2024). The case against simplistic genetic explanations of evolution. Development 151 https://doi.org/10.1242/dev.203077
5. Martijn Dekker (2023). A correction on the origin of the term ‘neurodiversity’ https://www.inlv.org/2023/07/13/neurodiversity-origin.html
6. Chay Graham (2021). What have we learnt by searching for gay genes? BlueSci 52 https://www.bluesci.co.uk/posts/what-have-we-learnt-by-searching-for-gay-genes
7. Clark (2023). Beyond box-ticking: redressing communication power imbalances in autism research. Keppel Health Review. No longer available online; author copy at https://1drv.ms/b/c/de730b21dd0b2d0d/EX6XCAnx–hMo-_vuM-yF-4B4qjWEE5tZbKHYpgeVvjP5A?e=3Pqnix

(10 votes)

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Doctoral programs say they want the brightest, most creative, and motivated students. But once you enter, creativity often gets replaced by execution, independence by subordination, and discovery by survival. Why does this paradox exist, and what does it mean for the future of science?

The academic path looks simple on paper: PhD, to postdoc, to PI. In practice, most doctoral students never become principal investigators, and many who do spend more time chasing funding cycles than pursuing the questions that first drew them to science. Yet, in this high-pressure system, the bottleneck begins far earlier – during the PhD itself.

Doctoral training should be about developing the ability to ask new questions, test risky ideas, and learn through failure. Instead, many students are trained mainly as “hands”. They join ongoing projects, collect and analyze data, write papers, and keep the lab productive. That technical training is valuable, but it does not cultivate the creative independence required of a scientist.

This is a structural trap. A PhD student may be talented, motivated, and full of ideas, but the funding architecture rarely treats them as independent scientists. Instead, they are seen as extensions of their PI: useful hands within someone else’s grant, not originators of their own research. Their ability to explore depends entirely on whether a PI has the time, money, and open-mindedness to support side projects. Exploration in science rarely follows a straight line: when you begin working within a PI’s broader framework, small and unexpected findings often emerge. These fragments, seemingly minor at first, can combine later to sharpen or even overturn an initial hypothesis. But following up on them usually demands extra experiments, financial investment, and time – luxuries a student cannot access independently. Awarding small research grants directly to students could support such exploratory work, giving them the chance to refine an idea, craft a proposal, and navigate submission guidelines. This process itself is vital training in independence – not only in how to build a project, but also in how to cope with the inevitability of rejection and try again.

Funding systems reinforce the trap. In the US, there are prestigious opportunities such as the NIH F31 predoctoral fellowship or the NSF Graduate Research Fellowship Program; funding programs that allow students to pursue independently led scientific projects. These awards are fiercely competitive, but applying is itself a form of training: students must learn the system, engage with program officers, and craft proposals that stand a chance of success. Even without funding, the experience prepares them for future large-scale NIH or NSF applications. By contrast, in Europe such funding opportunities for student-led projects are scarce. Large initiatives like Marie Skłodowska-Curie Actions fund doctoral networks, but the money is formally awarded to the PI, not the student. Seed grants for doctoral candidates are rare, and existing options, such as EMBO or Company of Biologists travel grants, support mobility and training, but not the independent pursuit of a research project.

The result is a predictable cycle: new cohorts of doctoral students become experts in executing tools, presenting data, and meeting deadlines, but not necessarily in generating ideas that push science forward.

Lab culture compounds this problem. When the lab leader values only hierarchy, the PI’s ideas reign supreme. When junior researchers don’t feel safe or encouraged to voice critique, propose hypotheses, or share their own ideas – creativity is stifled. But in labs where every opinion is listened to, where mistakes are not punished but discussed, where funding applications from students are encouraged regardless of seniority – that is where scientific innovation grows.

This is not a problem of talent. PhD students are often able to push the frontiers of science, but only if given the resources and freedom to pursue new ideas. If doctoral training is to form scientists rather than technicians, then structures should be in place to make this possible: funding lines that support student-led discovery, PIs who act as co-mentors rather than gatekeepers, and programs that reward exploration as much as publication.

The paradox is clear. Doctoral programs attract creative minds, but the system too often suppresses the very qualities it claims to seek. And the consequence is equally clear: creative people leave academia for start-ups, biotech, and other environments where risky ideas are supported and failure is treated as progress. If this trend continues, academia risks not only losing its brightest people but also its role as the primary driver of scientific discovery.

(6 votes)

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A recent paper by Otsuki and colleagues investigates the molecular mechanisms driving limb regeneration in axolotl

The question

You may know the axolotl (Ambystoma mexicanum), its funny face and gills floating around its head.

What you may not know is that it is also a model organism for organ regeneration thanks to its ability to regenerate many body parts, including its limbs. This is possible because cells know and remember where they are and can use this knowledge to inform the regeneration process. Cells found at the front of the limb possess so-called anterior identity, while those at the back hold posterior identity information. After amputation, cells from the anterior and posterior parts of the stub meet and trigger correct limb regeneration.

But how do cells know to produce a new limb after limb amputation, and not a tail or head instead?

The molecular bit

A recent study by Otsuki and colleagues¹, highlighted in a News & Views article², investigates the process of limb regeneration in axolotl through transgenic lines, transcriptomics and grafting experiments.

Otsuki and colleagues found that posterior identity in axolotl is established and maintained by a positive feedback loop that involves Hand2, a protein that controls the expression of other genes, and Shh (Sonic hedgehog), a signalling protein involved in limb growth. During development, Hand2 is expressed in posterior cells, and it is present at a steady state in adults. During regeneration, Hand2 is necessary and sufficient to induce the expression of Shh, which in turn activates Hand2 expression in nearby cells, sustaining the establishment of posterior identity in the new limb. After regeneration, Shh expression stops but residual Hand2 ensures lasting positional memory.

The unexpected discovery and why it matters

Interestingly, Otsuki and colleagues were able to rewire anterior-posterior memory, but only during regeneration and in one direction: anterior cells can stably acquire posterior identity when placed in posterior zones (or upon transient Shh signalling), but the opposite leads to defective limb regeneration.

The results presented by Otsuki and colleagues represent an important step forward in the understanding and manipulation of organ regeneration, and future studies into therapeutic applications in humans will benefit from this important work.

References

1. Otsuki, L., Plattner, S. A., Taniguchi-Sugiura, Y., Falcon, F. & Tanaka, E. M. Molecular basis of positional memory in limb regeneration. Nature 1–9 (2025) doi:10.1038/s41586-025-09036-5.

2. Wu, S. Y. C. & Whited, J. L. How axolotl cells ‘remember’ development to rebuild a lost limb. Nature d41586-025-01447–8 (2025) doi:10.1038/d41586-025-01447-8.

(6 votes)

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The Kahneman Chronicles #1: When a Nobel Laureate Fixed Our Lab’s Scheduling Disasters

Daniel Kahneman (1934-2024) was a legendary psychologist who revolutionized our understanding of human decision-making and became known as the “grandfather of behavioral economics.” Awarded the 2002 Nobel Prize in Economics, Kahneman’s groundbreaking research with Amos Tversky revealed systematic biases and mental shortcuts leading people to make irrational choices. 

This article series imagines what would transpire when Daniel Kahneman took a sabbatical and worked in a fly lab. 
Part of “The Kahneman Chronicles: Lessons from a Fly Lab” – A report from our imaginary interdisciplinary fellowship program

On the day Nobel Laureate Prof. Daniel Kahneman arrived for his sabbatical, our Drosophila lab buzzed with nervous excitement. Here was the legend himself—extraordinary psychologist who’d won economics’ highest prize, revolutionizing our understanding of errors in decision-making.

The ghost of Thomas Morgan urged us to do our best. We’d prepared our most impressive experiments, polished our presentations, and practiced our pitch for explaining fly development.

What we hadn’t prepared for was Kahneman spending his entire first morning silently observing us work. Often he scribbled notes in a small black notebook with the focused intensity of Jane Goodall studying chimps.

Why do we spend so much time in the lab?

“I’ll just quickly mount these embryos—twenty minutes, tops,” announced postdoc Shweta. This became a two-hour odyssey involving broken coverslips and dried glue. Followed by an existential crisis, wondering if the fluorescent blob she saw was signal or autofluorescence from a properly developing embryo.

“Quick PCR setup, maybe thirty minutes,” declared grad student Fillip, before vanishing into an afternoon-long quest. Missing primers. Buffer math. Finding the thermal cycler waited on “infinite hold” since previous Tuesday. You know the drill.

“Fascinating,” Kahneman murmured after each wildly inaccurate prediction.

By day three, a pattern was undeniable. Every time estimate in our lab was spectacularly yet consistently wrong. “Simple” tasks morphed into epic quests.

The Intervention

Kahneman approached the whiteboard where we’d sketched our weekly schedule – optimistically planning seventeen different experiments into forty work hours.

“Let’s implement realistic time budgeting,” he announced with the calm authority of someone who’d spent decades studying how humans delude themselves. Our simple thirty-minute embryo injections were now allocated one-hour blocks.

The room erupted in protests. “But we’ve done these injections hundreds of times!” “We know exactly how long they take!”

Kahneman smiled. “You’re all victims of the planning fallacy. Your System 1 is wildly optimistic about everything. Your mind accounts only for quick needle preparations while forgetting inevitable moments someone drops the cover slip itself”

“Your intuitive mind,” he explained, “only remembers the core task—actual injection. It conveniently forgets the setup, troubleshooting, inevitable equipment malfunction, and time spent staring at embryos wondering if they are worth injecting at all.”

The Planning Fallacy: The tendency to underestimate time, costs, and risks of future actions while overestimating their benefits. Even when people know similar tasks have taken longer than expected in past, they still predict future tasks will take less time.

System 1 vs. System 2 : Kahneman’s framework for two modes of thought. System 1 is fast, automatic, and intuitive (like quickly estimating “this should take twenty minutes”). System 2 is slow, deliberate, and logical (like carefully calculating each step: needle prep, embryo collection, injection setup, actual injection, cleanup, and imaging).

The Kahneman Method in Action

His solution was deceptively simple: multiply every time estimate by two, then add buffer time for “unknown unknowns.” “There are things you know will probably go wrong—known unknowns, like occasional broken needle or contaminated sample,” he explained.

“But then there are unknown unknowns—the completely unexpected problems you can’t even anticipate. The incubator that dies on a weekend, the new batch of reagent that behaves differently, or the day your hands just won’t stop shaking. You can’t plan for specific unknown unknowns, but you can acknowledge they exist.”

He made us track everything for two weeks: actual injection times, PCR setup duration…and the data was humbling. Our “standard” twenty-minute procedure had a median time of 40 minutes, with some taking over 1.5 hours when equipment misbehaved.

We tried his interventions skeptically. To our disbelief, the results were miraculous and maddening in equal measure.

For the first time in lab history, experiments actually finished when scheduled. Postdocs stopped working until midnight to complete “quick afternoon experiments.” Stress level plummeted as people stopped running late for their next commitment.

“Your emotional attachment to each experiment makes you treat it as special,” Kahneman explained. “You think ‘this time will be different’ or ‘I’m more prepared now.’ But from a statistical perspective, today’s PCR is just another data point in the distribution of ‘times PCR has taken in this lab.’
The planning fallacy tricks you into believing you can beat the historical average through wishful thinking.”

The lesson was profound: scientists are ultimately human and prone to same cognitive biases that affect everyone else. We bring these same mental shortcuts to our labs, our experimental designs, and our data interpretations. The first step toward better science maybe a more nuanced use of an important equipment—our own minds.

Have you experienced similar planning fallacies and overcome them? Do share in the comments.

What else did the Prof. Kahneman advise us on? Stay tuned for the next article in the series.

Practical Applications: The Kahneman Time Revolution

1. Track Reality First: Record actual times for routine procedures for couple of weeks.

2. Use the 1.5x Rule: Multiply routine task estimates by 1.5.

3. Use the 3x Rule: Triple your estimate for novel experiments.

4. Build Break Points: Schedule natural stopping points in long experiments, to allow buffers for unknown unknowns.

5. Try the Three-Point Method: For familiar tasks, estimate your best-case time (everything goes perfectly) and worst-case time (multiple things go wrong). Then calculate the geometric mean (root of the product) for a realistic schedule estimate.

Example: Embryo injection times (Best case 20 minutes, worst case 1 hours), geometric mean√(20 × 60) = √1200 ≈ 34 minutes.

Sameer Thukral is a post doc in the lab of Yu-Chiun Wang at RIKEN-BDR, Kobe, Japan, where he loves discussing science in the healthy and respectful lab environment. He is a developmental biologist with a focus on mechanics of yolk-blastoderm interactions. He is also the co-founder of BDR-Launchpad, a post-doc network for supporting ECRs with the hidden curriculum of science.

The observations made here are his own and do not reflect the opinions of the employer. This article was written by Sameer Thukral, with formatting, structuring and framing support of Claude AI.

(8 votes)

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