The role of canonical Wnt signalling in embryogenesis of the invertebrate chordate Ciona intestinalis.

Ascidians, as the closest invertebrate sister group of vertebrates, are important to study the development and evolution of our own species. Their gene networks are closely related to those of humans, but without the complexities that were introduced via the whole genome duplications that occurred at the origin of vertebrates. A particular conserved mechanism, canonical Wnt (cWnt) signalling is essential to various development processes, especially in patterning along the anterior-posterior axis. Understanding the role of the cWnt pathway in ascidians would be helpful to elucidate the emergence of chordates and their evolution.

The summer project was complementary to ongoing work in the lab of David Ferrier (in the Scottish Ocean Institute, University of St. Andrews), focused on understanding the mechanisms of cWnt controlling their potential target genes in Ciona, such as the ParaHox genes (Gsx, Xlox and Cdx) that are the evolutionary sisters to the Hox genes, with roles in patterning the anterior-posterior axis in the central nervous system and gut. I had the opportunity to try both experiments with my two lab mates (Dr Nuria Torres-Aguila and Anastasia Ellis, see Figure 3) who use different ways to disrupt the cWnt signalling pathway.

Background

The cWnt pathway features the activation of the β-catenin transcription factor and modulation of specific target gene expression. T-cell factor/lymphoid enhancer (TCF/LEF) is the main transcription factor mediating the cWnt pathway. In the absence of Wnt ligands, responsive genes are generally repressed by TCF/LEF due to the transcriptional cofactor β-catenin constantly being degraded by the proteosome. When Wnt ligands interact with the transmembrane receptor proteins, several proteins (Frizzled, LRP5/6 and Disheveled) are brought to form a multimeric complex attached to the membrane, inhibiting the phosphorylation of β-catenin and its degradation. The stabilised β-catenin then converts the former repressor TCF/LEF into a transcriptional activator of the Wnt-target genes (Gilbert and Barresi, 2022).

Heat shock experiment

Controlling when and where TCF is expressed is useful to help analyse the function of TCF in cWnt signalling. Based on the heat-inducible cis-regulatory element initially characterized by Kawaguchi’s team (Kawaguchi et al., 2014), we tested the efficiency of a DNA construct with heat-inducible gene Ci-HSPA1/2/6-like and mCherry gene in Ciona embryogenesis to adapt this versatile technique to our species and population (i.e. a temperate Scottish population versus a more tropical Japanese population). Heat shock was initiated at different stages of development with different lengths of time and temperatures before being observed under an epifluorescence microscope.

Although the precise heat-shock conditions need to be further refined, it was encouraging to see the induction of mCherry in embryos with this technique (Figure 1), raising the prospects of using the heat-shock approach to over-express genes like TCF in the near future. In this experiment, the microscope was probably the most exciting but also challenging piece of equipment, the microscope was probably the most exciting but also challenging piece of equipment I used. It took me some time to patiently check every detail like light intensity, exposure time, and magnification to ensure comparability between my images. The experience has been valuable for me to be familiar with this essential equipment for studying developmental biology.

Chemical treatment

Chemical treatment investigated whether the TCF-dependent ParaHox gene Cdx is potentially directly controlled by the cWnt pathway. Pharmacological agents, iCRT-14 and azakenpaullone (Akp) were used to downregulate and upregulate the level of the cWnt pathway. Embryos were left to develop to the desired stage for recovery as a post treatment to distinguish rapid (potentially direct) responses from slower (possibly secondary) changes. The samples were processed for in-situ hybridization with a probe against the Cdx gene and images were captured by Nomarski microscopy.

The results showed that the expression of Cdx is possibly regulated by a secondary effect instead of directly disrupted by cWnt cascade, as the samples fixed immediately after treatment showed no obvious changes in expression (data not shown) whereas dramatic anterior extension of Cdx expression was seen with a recovery period in cWnt activator treatment (Figure 2). The lack of apparent response to iCRT14-treated samples may be due to TCF not normally being expressed in tail epidermal cells (Garstang et al., 2016). Further work aiming to check the Cdx expression in deeper cells where TCF is expressed will be conducted in the future.

All of this research helped me to appreciate the importance of time management in the lab. I had the opportunity to repeat the in-situ hybridization several times, and each time I got more skilled in the procedure. My first two times of in-situ hybridization turned out with either the embryos being accidentally lost because of I rushed during wash steps, or they were left in the solution for too long, so I stayed in the lab until very late. It is a relief that eventually they were successfully mounted to be observed, but wisely making use of time during short waits and multitasking largely improved the efficiency in my later experiments.

Personal experience

I appreciate that I have experienced the systematic research process as a whole. From collecting sea squirts in the harbour and manipulating the embryos, to finally assaying their responses to various treatments. I have tried many essential techniques in developmental biology and see how the experiments proceed. I learned how the protocols are designed and improved based on the techniques developed from previous research. Even finding the potential mistakes I made by recalling the steps with my supervisors was a valuable and rewarding experience. Thanks to Dave, Nuria, Anastasia and the friendly people around SOI, all of who created a warm and supportive environment. I am more comfortable working in the laboratory and more determined to pursue further studies now. Thanks to the BSDB and Gurdon scholarship for making this opportunity possible. It has been one of my best memories doing scientific exploration with such a great team, besides the beautiful East Sands beach at St. Andrews.

Reference list:

-Garstang M.G., Osborne P.W. and Ferrier D.E.K. (2016) TCF/Lef regulates the Gsx ParaHox gene in central nervous system development in chordates. BMC Evolutionary Biology 16:57.

-Gilbert S.F. and Baresi M.J (2022) Developmental Biology 12^th edition Chapter 4 Cell to cell communication. Oxford University Press 109-111

-Kawaguchi A, Utsumi N, Morita M, Ohya A, Wada S. (2014) Application of the cis-regulatory region of a heat-shock protein 70 gene to heat-inducible gene expression in the ascidian Ciona intestinalis. Genesis. ;53(1):170-82.

(4 votes)

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Role of PAX6 in human cerebral organoid development

The cerebral cortex contains two major classes of neurons: excitatory and inhibitory. The imbalance between them is postulated to underlie some autism spectrum disorders (ASDs) (Rubenstein, 2010; Uzunova et al., 2016). The transcription factor PAX6 is an important regulator of embryonic forebrain development and mutations in PAX6 have been associated with ASDs (Kikkawa et al., 2019; Manuel et al., 2015). It is therefore possible that PAX6 could be involved in the regulation of the inhibitory-excitatory balance, hence playing a role in the development of ASDs. Supporting this hypothesis, recent findings indicate that PAX6 deletion in mice leads to the appearance of ectopic GABAergic (inhibitory) cells (Manuel et al., 2022). While animal data are useful, the mechanisms of this effect, and whether it is present in humans remains unclear, as human neurodevelopment is difficult to study directly. However, human cerebral organoids offer an exciting, new way to directly investigate early human embryonic neurodevelopment, including the effects of PAX6 mutations (Mason & Price, 2016). Hence, as a first step towards uncovering the mechanisms of the possible PAX6-dependent regulation of the inhibitory-excitatory balance in humans, this project aimed to examine PAX6-/- organoids at early developmental stages to investigate the effects of PAX6 mutations.

Methods

The Mason Lab has previously grown cerebral organoids from PAX6-/- induced pluripotent stem cells and PAX6+/+ controls. At culture day (D)60, the cellular phenotypes of these organoids were tested using scRNA-seq analysis. Indeed, ectopic GABAergic populations were observed in the mutants and not in controls. To examine the earlier stages of PAX6-/- organoid development, I analysed three batches of organoids originating from four cell lines (Controls: Nas2,Cas9; Mutants: A10,B2) at D14 and D30. Organoids were cut into 10μm sections. Inhibitory/excitatory neurons were visualized by tagging Tbr2 (marker of excitatory glutamatergic neurons/progenitors) and Dlx2 (marker of inhibitory GABAergic neurons/progenitors) with fluorescent antibodies. Hence, two distinct cell types could be visualized using fluorescent microscopy (Fig.1A). The number of Tbr2+ and Dlx2+ cells was counted in three randomly selected sections from each organoid using ImageJ software. The cell count was normalized by area of each section and averaged per organoid (Fig.1B-C).

Results

No D14 organoid (apart from an anomalous batch 6 Nas2 organoids) featured Dlx2+ cells. Hence, ectopic GABAergic cells likely arise after D14, which narrows the window of investigation in potential future research. In many cases, the number of Tbr2+ cells was significantly higher in the Nas2 organoids when compared to other cell lines, including the second control line, Cas9 (Fig.1B). This may suggest the presence of unknown variables which affected organoid development, or possibly that PAX6 has an effect on the speed/timing of Tbr2+ neuron emergence.

Both mutant and control organoids at D30 exhibited comparable counts of Tbr2+ and Dlx2+ cells, with significant differences in cell counts present between only a few groups (Fig.1C). This is surprising, as prior literature suggests that ectopic GABAergic cells (in PAX6-/- mice) appear at some point during development due to environmental signals such as Shh found in cerebrospinal fluid (CSF) and/or immigrating interneurons (Manuel et al., 2022). It seems that in human cerebral organoids (in the absence of CSF or immigrating interneurons) GABAergic cells emerge as a normal part of development whether PAX6 is present or not, but disappear by D60 in controls.

PAX6 could possibly be triggering the death of Dlx2+ cells between D30 and D60. PAX6 could also be causing Dlx2+ cells to switch to an excitatory fate by D60. It would be interesting to expand upon these findings by conducting lineage tracing experiments: this would allow for the tracking of the changes in cellular phenotypes to reveal whether the presence of PAX6 affects cell fate decision making.

My experience at Mason Lab

I found this project very exciting. The problem-solving aspect of research following inevitable experimental failures was genuinely great. It was very interesting to brainstorm the next steps when it becomes apparent that the current experimental procedure is not yielding any results. For instance, originally the experiment involved tagging dividing cells with EdU at D13.5, to see whether these cells switch between Tbr2+ and Dlx2+ identities between D14 and D30. However, the experiment had to be modified as the EdU fluorescent signal became too faint at D30 due to dilution during cell division.

The project also showed me the more labour-intensive side of research as I spent many days cutting organoid sections at the cryostat. After the baptism of fire by cutting my finger, I eventually found it a compelling task. As one of the Mason Lab members said, “it builds character”. I was also introduced to fluorescent microscopy and image analysis using ImageJ. The former forced me to become organised and efficient, as one must tame the unruly microscope and take the hundreds of pictures before the booked time runs out. The latter I found challenging as it was my first time using image analysis software, but it will undoubtedly be useful in my future work.

Every part of this project, no matter how menial or monotonous it might have seemed to an observer, I found thrilling. I have obtained experience in numerous laboratory techniques, and again reaffirmed research as my chosen career path. I am very grateful to Mason Lab and BSDB for this opportunity, and I am looking forward to being able to conduct projects of larger scales in the future.

(2 votes)

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A new article on embryos and the beginning of independent human life:

In her January 1 New York Times article, “When does life start? A post-Roe conundrum,” reporter Elizabeth Dias cites some material from a recent paper that I wrote. The article, “Pseudo-embryology and personhood: How embryological pseudoscience helps structure the American abortion debate. Natural Sciences 2022: e20220041″ is open access and can be found at  DOI: 10.1002/ntls.20220041 . The paper reviews scientific opinions as to where independent human life begins, and contends:

• There is no consensus among biologists as to when independent human life begins
• What passes for science is actually a set of outdated myths that are no longer considered valid
• This set of myths denigrates birth and promotes fertilization as the site where personhood begins.

I hope the community of biologists will read and discuss the data and conclusions of this paper.

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Dear Node Colleagues,

In response to the front page article in The New York Times of 2 January 2023, “When Does Life Begin,” I am posting a model for the formation of the earliest stages of the embryo to shed light on the question.

A more complete treatise and graphic presentation is available on www.embryogeometry.com.

I look forward to relevant comments.

Stuart Pivar
___________________________________________________________

The heart of biological science is the search for the way the embryo is formed. The rest is known.

This proposed model is an account of embryogenesis, tantamount to the blueprint for the assembly of the embryo.

Included are the phenomena of morphogenesis, organogenesis, segmentation, limb development, and nerve cord development, all the demonstrable consequence of the singular event of gastrulation. The model is illustrated by hundreds of mechanical animation drawings. 

The model describes the instantaneous catastrophic embryological event called gastrulation as the bursting of the first embryonic structure, the blastula, and its elastic recoil as demonstrably the universal origin of animal form. 

The embryo is the deflated form of the tense, balloon-like blastula membrane upon bursting and elastic recoil. 

Organogenesis is the deformation by elastic recoil of the pattern of self-organized circumferential girdles that encompass the blastula from pole to pole.  

The radial, vermiform, and bilateral body forms result, respectively, from a spherical, cylindrical, or ovoidal blastula. Morphogenesis is the anterior-dorsally directed migration upon bursting failure of the separate layers of the membrane bilayer, governed by the mechanical exigencies of surface wrinkling pattens. Embryogenesis is predictable by the mathematics of topological surface wrinkling patterns.

Taxonomic differentiation is the result of mechanical error in the repetition of the universal anterio-dorsal elastic reaction. Evolution occurs when the modification is recorded and expressed in the genes generationally.  

The genes provide proteins in precisely timed doses that maintain and occasionally change the proportions of the otherwise immutable phyletic form (see S.J. Gould, Ontogeny and Phylogeny, 1977).

The Origin of the Blastula:
Incomplete cell division leaves cells connected by a capillary which itself is divided axially in each cell division. At the eight-cell stage the capillaries intersect and fuse centrally to form the blastocoel, a spherical plenum that enlarges–paved with blastocytes, constituting the blastula, and which eventually bursts and recoils as the embryo. 

The Cause of Segmentation:
The blastula as a water-filled balloon subtends standing oscillatory waves of energy that delineate subdivisions by serial harmonic bisections of the axis that condense chemically at wave intersections and nodes. 

Limb Development:
The separation and dorsal recoil of the pectoral and pelvic girdles at the ventral midline forms the limb buds in vertebrates comparable to the imaginal discs in insects. Development is the reversal of the action.

(1 votes)

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I have become passionate about the field of stem cell biology during my undergraduate degree in Molecular Genetics at the University of Edinburgh. Therefore, I was grateful to be awarded the Gurdon Studentship Award from The British Society for Developmental Biology to conduct a summer research project in Dr Soufi lab in the Institute for Regeneration and Repair (IRR). In this report, I will not only describe the aim of my project and how I proceeded to accomplish it, but also how valuable this experience was for my future studies and career.

In 2006, Yamanaka and Takahashi made a breakthrough in stem cell biology by showing that adult cells can be converted to become induced pluripotent stem cells (iPSCs) using the ectopic expression of four transcription factors (TFs) Oct4, Klf4, Sox2 and c-Myc (Takahashia & Yamanaka, 2006). These iPSCs are very similar to embryonic stem cells (ESCs) and have the unique ability to generate all cell types of the body. Therefore, iPSCs have enormous potential in the area of regenerative medicine. However, reprogramming adult cells into iPSCs is highly inefficient which limits the application of this technology for drug discovery and disease treatment. To improve reprogramming efficiency, further research focusing on fundamental mechanisms by which these TFs facilitate and maintain pluripotency is needed.

In my project I focused on TF Sox2, which is responsible for both somatic reprogramming and the maintenance of pluripotency in ESCs. Previous work, conducted in the Soufi lab, identified critical regions of Sox2 that are responsible for iPSC reprogramming. This was done by carrying out a systematic mutation screen of the Sox2 DNA-binding domain and mapping which regions of this domain are important for reprogramming fibroblasts to iPSCs. This led to the identification of several deletion mutants who were unable to reprogramme. My aim was to test if one of these deletions, called D36, also abolished the ability of ESCs to maintain pluripotency, which would indicate that this region of Sox2 gene is responsible for both reprogramming and pluripotency maintenance (Figure 1).

I used CRISPR-Cas9 technology to generate mouse ESC (mESC) lines expressing D36 Sox2. The first step in this process was to design Sox2-specific guide RNAs (gRNAs) and test their efficiency to target Sox2 gene in mESCs. This was performed by introducing a designed gRNA together with Cas9 nuclease into mESCs by electroporation, followed by alkaline phosphatase (AP) staining of mESCs. AP is one of the best markers of pluripotency (Stefkova et al., 2015) and therefore it was used determine whether these cells differentiated due to Sox2 knock-out (KO). During this phase of my project, I had to design, order, and test multiple gRNAs to find out which one was the most efficient in targeting Sox2. Ultimately, I managed to design a suitable gRNA able to mediate Sox2 KO as was demonstrated by the decreased number of AP-positive colonies after AP-staining.

Next, I performed CRISPR-Cas9 knock-in (KI) to generate mESC cell lines containing D36 deletion mutation by homology-directed repair (HDR). For that, I designed a single stranded DNA (ssDNA) template containing D36 and introduced it into mESCs together with gRNA and Cas9 complex by electroporation. Due to the low efficiency of CRISPR-Cas9 technology, single cell sorting had to be performed to identified mESCs with the mutation of interest. After that, the presence of D36 KI in the picked mESC colonies was evaluated by polymerase chain reaction (PCR) screening using D36-specific primers and confirmed by Sanger sequencing (Figure 2). Out of 38 screened colonies, one colony (colony number 27) was identified as a clear D36 homozygote, together with 6 potential heterozygotes. D36 heterozygosity arose because of low CRISPR-Cas9 efficiency, which was only able to mediate KI of only one Sox2 allele.

Subsequently, the heterozygosity of the potential heterozygous colonies was validated by TopoA cloning. This approach involved the amplification of the Sox2 D36 region by PCR, its ligation into plasmid vectors and their amplification in E.coli. This allowed me to validate the zygosity of D36 mutation by sequencing individual alleles of Sox2 gene. Unfortunately, the homozygosity status of colony 27 with clear evidence of D36 mutation on both Sox2 alleles could not be validated by TopoA cloning due to the lack of time. Interestingly, there were obvious phenotypic differences between this homozygous clone and WT mESC lines (Figure 3). These differences included slow growth, smaller cell size, and greater adherence to the culturing plates. This suggests that D36 deletion most likely does have an impact on maintaining pluripotency of mESCs. Nevertheless, this important conclusion should further be validated, for example by performing embryo contribution assay to assess the ability of D36 mESCs to differentiate and generate tissues and organs.

During my internship, I have not only learnt different laboratory techniques, but also developed various skills and attributes that are essential for a scientific career. Most importantly, this experience helped me enhance my independent critical thinking when designing and planning experiments. Also, I was required to adjust existing protocols to suit my experiments which sometimes took multiple attempts to make it work and is therefore something I have not experienced during my undergraduate studies. Over time, I also became more confident in trusting my own judgments and making independent decisions, but also asking for help and advice when needed. In this respect, I was lucky to be a part of a stimulating scientific environment, surrounded by experienced scientists always willing to help and share their knowledge and skills.

Overall, being a part of Dr Soufi group in IRR was a very useful experience for me which also significantly influenced my future career plans. This internship cemented my aspirations to dedicate my life towards scientific research. Although sometimes difficult if things do not go as planned, it is nevertheless very rewarding and purposeful for me. Therefore, I aim to continue my research in the stem cell field or related areas that I am passionate about by doing a PhD.

References

Takahashi, K. and Yamanaka, S., (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), pp.663-676. Available at: https://doi.org/10.1016/j.cell.2006.07.024

Štefková, K., Procházková, J. and Pacherník, J. (2015). Alkaline phosphatase in stem cells. Stem cells international, 2015:628368. Available at: doi: 10.1155/2015/628368

(3 votes)

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At Development, we know that going on the job market and then setting up a lab is a real crunch time in an academic’s career, and not everyone receives the support they need to manage this transition. As a not-for-profit journal whose aim is to support the research community, we therefore wanted to do something to support individuals through this period. With this aim in mind, we’re excited to launch a new scheme – our Pathway to Independence (PI) programme. This competitive programme will select a small number of researchers (‘PI fellows’) planning to apply for Principal Investigator positions in the coming year, and will provide support, mentorship and networking opportunities.

So if you are a postdoc working in the developmental biology or stem cell field and starting to look for your first independent position, we’d encourage you to apply to this programme. You can find out more information in this Editorial, and also on this webpage. And while we will only be able to select a handful of individuals to become PI fellows, we are also thinking about ways we can support the wider cohort of applicants – we’re hoping to host one or two workshops, and will also give at least some applicants the opportunity to showcase their work through our ‘Development presents…’ webinar series.

This is a pilot scheme, and we’ll be refining the programme with our first cohort of PI fellows. We’re also keen to hear feedback from the wider community, so if you have any thoughts on these plans, do feel free to get in touch.

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It’s musical chairs at The Company of Biologists at the moment (though fortunately without any chairs actually being removed!) – after Seema Grewal left Development last month to become the new Executive Editor of Journal of Cell Science, we’re delighted to have appointed Laura Hankins, currently the Company’s Science Communication Officer, as the new Reviews Editor on Development. Laura started working with us a little over a year ago following a PhD with Jordan Raff in Oxford, and I’m really pleased that she’ll be joining the journal in the near future – having worked with her in her current role, I know she’s going to be a great member of the team. And Helen Zenner – who you’ll know as the Node’s Community Manager – is going to be moving over to our sister site, FocalPlane. Helen’s got a very strong microscopy research background, and has some great ideas for developing FocalPlane.

All this means that we now have two openings for scicomm-type roles! Both these positions are suitable either for current researchers looking to move away from the lab, or for people who already have some scicomm experience. They provide a great opportunity to learn about the scientific publishing world, and to contribute to a not-for-profit organisation that really believes in supporting the biological community. To find out more, take a look at the job adverts for the Node Community Manager and the Science Communications Officer positions, and please feel free to reach out if you want to find out more about either role.

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The European Developmental Biology Congress: a distributed meeting across Oxford, Barcelona and Paris 25th – 28th September 2023

The quadrennial European Developmental Congress usually positions itself neatly at the mid-way point between meetings of the International Society for Developmental Biology, or at least that was the case, until the pandemic-induced postponement of the latest ISDB meeting to October 2021.

This left less than a year between the ISDB and EDBC meetings.  We at the British Society for Developmental Biology, the hosts for #EDBC2023, see this not as a problem but an opportunity – a chance to experiment with some alternatives to the usual international conference format.

For one thing, we have decided to focus EDBC on the ‘rising stars’ of European developmental biology. Most of our invited speakers will be outstanding and engaging speakers who started their own labs within approximately the last five years. We are currently finalising our programme and are always happy to take suggestions of excellent speakers based in European labs from our community, so please do get in touch through our twitter @_BSDB_ or our shiny new mastodon account @BSDB@mstdn.science or by emailing our Meetings Officer sally.lowell@ed.ac.uk. The meeting will also contain all the usual highlights of BSDB meetings, including talks from the winners of the Waddington medal, the Cheryll Tickle medal and the Beddington medal.

Furthermore, in the spirit of experimenting with more accessible conference formats¹ we will distribute the meeting across three different locations in Europe. The main hub of the meeting will be in Oxford, and there will be interlinked one-day ‘spoke’ meetings in Paris, led by Sigolène Meilhac, Nicola Festuccia, Tanya Foley, Tom Cumming & Guillaume Frasca and Barcelona, led by Alejo Fraticelli & colleagues tba.   Talks will stream back and forth between the Oxford hub and the spoke locations, and each of the three sites will also host its own social and scientific events.

We have exciting plans for the scientific and social programmes at all three locations, including lots of opportunities to present your work through short talks chosen from abstracts, flash talks, and lively poster sessions. All will be revealed early next year when the #ECDB2023 website goes live – watch this space! 

We encourage anyone who can’t travel to any of the three locations to consider organising a small scale ‘watch party’, perhaps streaming your favourite sessions to a departmental seminar room and combining this with a local poster session. We particularly hope that PhD students and postdocs might be interested in organising local events of this type – do get in touch (sally.lowell@ed.ac.uk) if you’d like to discuss how BSDB can support you with this.

DATE: Our programme is now complete and registration is open!

We look forward to seeing you in Oxford, Paris or Barcelona!

Sally Lowell, Shankar Srinivas, Ben Steventon & Paul Martin 

British Society for Developmental Biology

¹The future of conferences. Lowell S, Downie A, Shiels H, Storey K. Development. 2022 Jan 1;149(1):dev200438

(4 votes)

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The making of colorful neuromesodermal progenitors

During the embryonic development, while the gastrulation process is taking place, cells within embryos self-organize by creating groups and layers of cells, where each one will develop into different adult tissues. Human and mouse embryos are called triploblastic organisms as they are characterized by the development of three germ layers: ectoderm, which will form epithelial and neural tissues; mesoderm, which will differentiate into muscle, bone, circulatory system and spleen, among other tissues; and endoderm that will produce organs such as gut, lungs, and pancreas.

During gastrulation, when the germ layers are being organized, progenitor cells commit to the generation of one of these three structures and their specific cell types. For example, an ectodermal progenitor will be committed to the generation of either epithelial or neural cell types. However, some studies have described the presence of dual-fated progenitors that could produce both mesodermal and neuroectodermal lineages (Tzouanacou et al., 2009; Wymeersch et al., 2021). These cells were named neuromesodermal progenitors (NMPs) and they are identified by the co-expression of Sox2 and Brachyury (T) genes.

NMPs have risen a lot of interest due to their exceptional behavior and they are being studied with the aim to increase our knowledge about cell fate and cell differentiation, both in developmental biology and biomedical research (Binagui-Casas et al., 2021). Therefore, there has been a lot of effort on the development of in vitro models of these cells, as an easier tool to study their characteristics.

These in vitro NMP-like cells can be risen from Mouse Embryonic Stem Cells (mESC) using protocols such as Gouti et al., 2014, whose outcome has been described to be around an 80% of NMPs, within a mix of other cells in various states of differentiation. In Figure 1 we can see an example of these cultures where in vitro NMP-like cells are identified with Sox2 and T co-expression while there is also the presence of cells committed towards mesodermal lineages, identified with single T expression.

These cultures have been able to then produce both mesodermal and neural lineages at the population level, where mesodermal lineages are identified with single T expression, while neural lineages are characterized by single Sox2 expression. However, there has been a lot of controversy on whether these in vitro progenitors are also dual-fated at a single cell level as described for in vivo NMPs (Tzouanacou et al., 2009). This means that, in a group of in vitro NMP-like cells, some could be more committed to neural lineages while others to mesodermal, and it is not known yet if a single progenitor can generate both lineages.

To better characterize the potency and other characteristics of these in vitro NMP-like cells, Prof Wilson’s lab developed a new double reporter mESC line for Sox2 and T expression. In this case, mCherry protein will report Sox2 expression while GFP protein will report T expression. The knock-in experiments were performed by using gene targeting, to optimize the fidelity of expression, while leaving the endogenous gene’s expression intact. The use of this cell line allows the possibility to obtain a pure population of in vitro NMP-like cells that are double positive for Sox2 and Bra expression by using Fluorescent-activated Cell Sorting (FACS).

During my summer internship in Prof Wilson’s lab and under the supervision of Dr Anahí Binagui-Casas I have been characterizing this new cell line by describing the differentiation outcome of a pure population of in vitro NMP-like cells when cultured in different conditions and plated at different cell densities.

Firstly, in Figure 2 we can see the culture of a pure population of in vitro NMP-like cells in media supplemented with retinoic acid (RA) and smoothened agonist (SAG), as described in Gouti et al. 2014, to induce spinal cord differentiation. We can see the formation of axons and neural rosettes, and they can be identified with Sox2 expression and depleted expression of T, suggesting neural differentiation.

Figure 3 represents another condition where positive cells for Sox2 and Bra were cultured in media supplemented with FGF and CHIR for five days, as described in Tsakiridis & Wilson, 2015 to induce both neural and mesodermal lineages. In this case, there is the presence of cells differentiating towards neural tissues, assembled in rosette-like structures and with single expression of Sox2. Meanwhile, cells differentiating towards mesoderm are identified with single expression of T, or its gene-expression reporter GFP.

These experiments gave us insight about the differentiation outcome of this new cell line towards different lineages by testing multiple protocols. Furthermore, I was able to characterize the culture of in vitro NMP-like cells plated at different cell densities after sorting them using FACS. This knowledge will contribute to future experiments requiring the use of this double reporter mESC line as a tool to characterize in vitro NMP-like cells’ clonal fate and to understand if these cultures are a good model for in vivo NMPs.  

After all this work testing multiple conditions for in vitro NMP-like cells cultures and optimizing their growth, I found out that I really enjoyed working with stem cells and differentiation protocols. Although I spent many hours in the cell culture facility losing track of time, it was very exciting to see how cells grew and changed every day. Specially, I was very excited to see how they were able to organize themselves and create amazing structures, which I then learned how to immunostain and obtain beautiful images under the confocal microscope.

To conclude, this studentship and Prof Wilson’s lab have been a great opportunity for me to learn a lot about how biomedical research is driven and to confirm that I would like to continue developing my career in developmental biology. I would like to encourage students to do these internships because it is an enjoyable way of starting to apply the knowledge acquired during your bachelor’s degree and to investigate if you would enjoy developing your career in this field.

References

Binagui-Casas, A., Dias, A., Guillot, C., Metzis, V., & Saunders, D. (2021). Building consensus in neuromesodermal research: Current advances and future biomedical perspectives. Current Opinion in Cell Biology, 73, 133–140.

Gouti, M., Tsakiridis, A., Wymeersch, F. J., Huang, Y., Kleinjung, J., Wilson, V., & Briscoe, J. (2014). In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity. PLoS Biology, 12(8), e1001937.

Tsakiridis, A., & Wilson, V. (2015). Assessing the bipotency of in vitro-derived neuromesodermal progenitors. F1000Research, 4, 100.

Tzouanacou, E., Wegener, A., Wymeersch, F. J., Wilson, V., & Nicolas, J. F. (2009). Redefining the progression of lineage segregations during mammalian embryogenesis by clonal analysis. Developmental Cell, 17(3), 365–376.

Wymeersch, F. J., Wilson, V., & Tsakiridis, A. (2021). Understanding axial progenitor biology in vivo and in vitro. Development (Cambridge, England), 148(4), dev180612.

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We were lucky enough to have 24 amazing cover images for Development over the past 12 months. Now, it’s your chance to vote for your favourite. To vote, click your choice in the text following the images and then click save. Voting is open until 7 January 2023.

In the battle of the model organisms, mouse comes out on top of the most featured list, followed closely by Drosophila, with honourable mentions for zebrafish, Arabidopsis and Xenopus.

This poll is now closed.

Thanks to everyone that voted and of course, everyone who contributed to such a fantastic year of cover images for Development.

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