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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On Wednesday 7 December, Development hosted three talks on metabolic and nutritional control of development and regeneration, the topic of our upcoming Special Issue.

Below you’ll find each of the talks and Q&As hosted by our Associate Editor, Irene Miguel-Aliaga (Imperial College London and MRC-LMS, UK)

Natalia López Anguita (PhD student in the Stem Cell Chromatin Group at the Max Planck Institute for Molecular Genetics)
‘Role of hypoxia in pluripotent cells and during differentiation via gastruloid formation’

You can read the Research Article here.

Hannah Brunsdon (Postdoctoral Research Fellow in Liz Patton’s group at the IGC, University of Edinburgh)
‘Aldh2 is a metabolic gatekeeper in melanocyte stem cells’

You can read the full Research Article here.

Benjamin Jackson (MD-PhD Candidate in Lydia Finley‘s group at Memorial Sloan Kettering Cancer Center)
‘A non-canonical tricarboxylic acid cycle underlies cellular identity’

You can read the Research Article here.

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Exploring the role of planar cell polarity in the regulation of the patterning of human axial progenitors

I first became interested in stem cell biology and development while studying Medical Sciences at the University of Edinburgh. During the lockdown, I attended a very inspiring seminar in which Dr Guillaume Blin discussed in-vitro models of developmental patterning. Despite being unable to join the lab in my second year, I explored the topic, and we discussed some ideas from the literature. The BSDB summer studentship provided me with a perfect opportunity to explore these ideas in a lab setting. This summer, I got a chance to work alongside an exceptional team in the Blin lab on my own project at the Centre for Regenerative Medicine (CRM).

I have been interested in the role of planar cell polarity (PCP) in health and disease and decided to investigate its role in developmental patterning. PCP dysregulation can play a significant role in cancers and congenital disorders (Wang, de Marco, Capra and Kibar, 2019). In particular, in congenital malformations of spinal structures, such as neural tube defects (NTDs), where aetiology is still to be elucidated (Chen et al., 2018). A proportion of individuals with NTDs (Chen et al., 2018) and idiopathic scoliosis (Wise et al., 2020) have mutations in some of the core components of the PCP pathway, namely Wnt11, Vangl1 and Vangl2, and Celsr. These components localise asymmetrically within the cell to define cell polarity along the epithelial plane (Butler and Wallingford, 2017).

This pathway is involved in early development, notably when spinal progenitors are established in a structure called the anterior primitive streak (APS) (Andre et al., 2015). As these transient and rapid events occur within a complex 3D environment in-utero, much remains to be understood about cell fate decisions in the APS (Wymeersch et al., 2021). One way to study these processes in a human context is to use human embryonic stem cells to model early development (Blin, 2021). During my project, I worked on a novel in-vitro model that mimics the early stages of human axis elongation. I used this system to test the hypothesis that PCP regulates the patterning and balanced proportion of early spinal progenitors in humans.

My experimental strategy consisted in confining hESC onto custom-made micropatterns and treating the colonies with a spinal fate-inducing medium. In these conditions, hESC self-organise into spatial domains of cell fates and initiate axial growth. I first tested a set of antibodies directed against PCP components. These antibodies did not provide a specific signal, but I obtained images that looked magical, so I was not too disappointed! Next, I decided to use small molecule inhibitors to perturb the PCP pathway. I inhibited either cytoskeleton remodelling downstream of PCP or the secretion of PCP ligands. When I stained for neurectoderm and endodermal cell fate markers, I observed a very severe patterning defect when cytoskeleton remodelling was inhibited (Figure 1).

I was very excited with these clear preliminary results and I wish I had the time to perform additional experiments that would demonstrate the involvement of PCP more specifically, such as PCP components staining and knockdown experiments. I have also assisted my colleagues in testing new micropatterning methods and creating cell lines using transfection methods.

Figure 1. Perturbation of cytoskeletal remodelling downstream of PCP leads to neurectoderm patterning defects. Immunostaining of micropatterned colonies 48h after the induction of differentiation. The endoderm is shown in blue. Neurectodermal cells (green) cluster at the centre in the control condition (a-d) regardless of colony shape, while patterning is perturbed in the treated condition (e-h).

Thinking back to my first days in the lab I was very excited to work alongside experienced scientists but also felt anxious as I needed to use techniques that I was not yet familiar with. During my first week, I worked out how to employ the techniques routinely used in the lab and presented my initial research plan to discuss my approach. Once I became acquainted with the protocols, they became a natural part of my routine. For example, I became proficient in micropatterning, a microfabrication method that makes it possible to standardise the size and geometry of hESC colonies (Blin, 2021). This project also allowed me to try several imaging techniques and perform image analysis using Nessys (Blin, 2019) and PickCells. I really enjoyed organising, sharing, and optimising the protocols in an online lab book. I am so happy I could contribute and that my designs are now used by the lab. We have also collaborated with a computational lab and worked on shared scripts in python.

During my internship, we also attended the Mammalian Synthetic Biology congress taking place in Edinburgh. It was a great opportunity to hear about novel data and techniques from researchers around the world and discuss how we could apply them in our lab. Networking and making friends in a professional environment like the CRM gave me the opportunity to gain unique perspectives from postgraduate students and researchers from various groups. Presenting and discussing my ideas and data allowed me to gain more insight into the dynamics of working in research and academia. These 8 weeks inspired and prepared me to pursue a career in science.

My greatest gratitude goes to my amazing supervisor, Guillaume, for everything that he has done for me. I am so grateful to Miguel for always being there for me, Heather for caring for all of us, and Fatma for the priceless moral support (all below).

Figure 2. The Blin lab (from left): Me, Fatma, Miguel, Guillaume, and Heather.

I want to express my gratitude to the amazing people at the CRM who were always keen to help and explain their experiments with so much passion. Many thanks to the BSDB for giving students like me such a wonderful opportunity to kickstart research careers. I would recommend all students to start looking for a lab they would be interested in doing a BSDB-founded internship in!

References

Andre, P., Song, H., Kim, W., Kispert, A. and Yang, Y., 2015. Wnt5a and Wnt11 regulate mammalian anterior-posterior axis elongation. Development.

Blin, G., 2021. Quantitative developmental biology in vitro using micropatterning. Development, 148(15).

Blin, G., et al., 2019. Nessys: A new set of tools for the automated detection of nuclei within intact tissues and dense 3D cultures. PLOS Biology, 17(8), p.e3000388.

Butler, M. and Wallingford, J., 2017. Planar cell polarity in development and disease. Nature Reviews Molecular Cell Biology, 18(6), pp.375-388.

Chen, Z., Lei, Y., Cao, X., Zheng, Y., Wang, F., Bao, Y., Peng, R., Finnell, R., Zhang, T. and Wang, H., 2018. Genetic analysis of Wnt/PCP genes in neural tube defects. BMC Medical Genomics, 11(1).

Wang, M., de Marco, P., Capra, V. and Kibar, Z., 2019. Update on the Role of the Non-Canonical Wnt/Planar Cell Polarity Pathway in Neural Tube Defects. Cells, 8(10), p.1198.

Wise, C., Sepich, D., Ushiki, A., Khanshour, A., Kidane, Y., Makki, N., Gurnett, C., Gray, R., Rios, J., Ahituv, N. and Solnica-Krezel, L., 2020. The cartilage matrisome in adolescent idiopathic scoliosis. Bone Research, 8(1).

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

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Hannah Cowling, MBiol at Durham University

Introduction

Over summer 2022, I had the opportunity to work with Dr Olena Riabinina in Insect Neuro Lab at Durham University. Her team specialises in neurobiology and neuroecology of insects with established work in mosquito olfaction and bumblebee olfactory neurobiology and ecology. My project was working with PhD student Matthew Quinn, assisting with a chapter of his PhD research project to molecularly characterise the development of the larval visual systems in the Anopheles gambiae mosquito.

Anopheles gambiae are a group of species which include the most significant vectors of deadly disease malaria in sub-Saharan Africa. As malaria poses one of the most significant public health threats worldwide, research surrounding mosquito sensory systems could prove vital in informing vector management techniques. One avenue of Insect Neuro Lab’s previous research has focussed on mosquito olfaction. Significant because research indicates sense of smell is crucial in host seeking behaviour (Wheelwright et al., 2021)(Riabinina et al., 2016). However, as mosquito larvae and pupae are also responsive to visual stimuli, a deeper understanding of their visual development has potential to provide valuable methods of mosquito-driven disease prevention. Visual perception of environmental cues is crucial for insects to, in combination with other senses, avoid predators, source food, mates and ovipositioning.

Compared to the mammalian ‘single-aperture’ eye, mosquitos have poor image resolution, however, can detect comparatively fast movement in a large viewing angle. Each unit is composed of a cornea, lens, and photoreceptor cells which sense light wavelength (colour) and intensity. Opsins are a highly conserved photoreceptor molecules observed in mosquitos and across the animal kingdom. They are membrane-bound proteins which absorb photons and change their conformation, initiating a signalling cascade known as phototransduction (Shichida & Matsuyama, 2009). Previous studies have shown that An. gambiae have 11 opsin genes, 6 of which detect long wave light. This is more than typical for insects which usually have 4. There has been a suggested association of increased long wave opsins being found to accommodate more complex light conditions within aquatic environments (Giraldo-Calderón et al., 2017).

Shown in figure 2, mosquitos have four different stages in their life cycle: Egg, larvae, pupa, and adult. For the larval and pupal stages of development, mosquitos live in an aquatic environment. In contrast, adults are flying insects. It follows that their visual systems will be differently adapted to better assist survival in the contrasting environments. Previous studies involving the larvae of dengue and yellow fever transmitting mosquito Aedes aegypti show that mosquitos develop adult eye cells in late larval and pupal stages. It is predicted that both sets of eyes contribute to visual capabilities but have different photoreceptor cell types (Mysore et al., 2014). This difference in gene expression over time enables genetic targeting and quantification of components of the two visual systems. The aim of this project was to use molecular techniques to characterise expression of opsin genes in larval and pupal An. gambiae and to test the hypothesis that opsin expression profile changes throughout development.

Methods

The project involved synthesizing cDNA from RNA extracted from An. Gambiae at different time stages ranging from egg to adult. CDNA synthesis was necessary because amplification of RNA requires conversion into double stranded form. This was done using a kit containing Moloney murine leukaemia virus reverse transcriptase. The resulting cDNA was used to perform qPCR using ten different opsin and two housekeeping genes as templates. The method utilised real-time fluorescence of a double stranded DNA binding dye to detect amplification at each cycle of PCR. When fluorescent signal is detected above a decided threshold of background fluorescence, a quantification value/ Cq value is determined which calculates relative abundance between samples.

Results

We found that, consistent with previous findings (Jenkins & Muskavitch, 2015), expression of long wavelength-sensing opsin genes was distinct between larval and adult stages. With notably high expression of Opsin 6 in larval stages and high expression of opsins 1,3 and 4 in adult An. gambiae. Opsin 8 which encodes an ultraviolet sensing photoreceptor showed expression in both larval and adult forms which slightly increased in the latter. Lastly, opsin 9 which detects short-wavelength light had very low expression in the early larval stages increasing steadily with time into the adult. This research will be followed up with behavioural assays using knockout mutants to ascertain the roles of the different opsin genes in larvae survival behaviour.

Conclusions and Looking Forward

If I were to have the opportunity to pursue this research further, I would be interested to conduct immunolocalization imaging on larvae and pupae retinas comparative to research seen in figure 3 on adult An. gambiae. To map the different opsin gene expression of the larval and pupal eye structures would shed insight into how the expression levels observed translates structurally.

The project taught me much about experimental design at a PhD level. As I am considering a career in research, it was incredibly useful for me to observe this in action. I understand better how to seek and try to fill a gap in the literature and how to use a range of molecular techniques to test my hypothesis. Furthermore, the project gave me a lot of confidence in the lab and in my own abilities. After studying for the first two years of my bachelors in the climate of covid, our laboratory time had been limited and we spent much of our studies at home watching our lectures on our computers. Hence, experiencing two months of being a part of a friendly and welcoming lab community was so good for me.  Under Matthew’s patient tutelage I added some fundamental molecular skills to my repertoire and deepened my understanding of an area of interest. This knowledge will undoubtably be useful for my degree modules this year and certainly for my level 4 lab-based research year.  Additionally, because of being allowed to do this project I have chosen to do my level 3 literature review module within the subject areas of insect neurology and development. The summer has sparked an interest in new areas for me and shown me the benefits of insects as model organisms.  Many thanks to everyone at Insect Neuro Lab and the BSDB for facilitating this experience.

Giraldo-Calderón, G. I., Zanis, M. J., & Hill, C. A. (2017). Retention of duplicated long-wavelength opsins in mosquito lineages by positive selection and differential expression. BMC Evolutionary Biology, 17(1). https://doi.org/10.1186/s12862-017-0910-6

Hu, X., England, J. H., Lani, A. C., Tung, J. J., Ward, N. J., Adams, S. M., Barber, K. A., Whaley, M. A., & O’Tousa, J. E. (2009). Patterned rhodopsin expression in R7 photoreceptors of mosquito retina: Implications for species-specific behavior. The Journal of Comparative Neurology, 516(4), 334–342. https://doi.org/10.1002/cne.22114

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In 2023, FASEB will host 22 Science Research Conferences (SRCs). SRCs are multiday, in-person meetings featuring discussion of scientific advances and sharing of cutting-edge research through lectures, posters, informal discussions, and social events. The complete SRC schedule is available on our website. You can sort the schedule by month or topic area. 2023 topics include cell biology, neuroscience, clinical and translational medicine, immunology, genetics and genomics, and many other focus areas. Click on the title of the conference you are interested in to see a description, the location, information about registration fees and deadlines, abstract submission instructions, and other key details. The individual conference websites will be updated with additional information over the next few months. For more information, visit www.faseb.org/meetings-and-events/src-events

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