“For all the claims that we had finally unlocked the secrets of human biology and were setting off into a new era of gene-driven medicine, there was one rather glaring issue with the first human genome: where were all the genes?”
Presenter Dr Kat Arney
In the latest episode of the Genetics Unzipped podcast, we’re discovering whether size really does matter – when it comes to your genes and genome, that is. Dr Kat Arney gets to grips with why the human genome has so few genes, why some species have more junk DNA than others, and whether you should avoid eating anything with more genes than you.
The Centre for Trophoblast Research (CTR) is looking to recruit a Technician to support cutting edge collaborative research in human biology. To apply for the job please visit: https://www.jobs.cam.ac.uk/job/32716/
The purpose of the role of the CTR Technician is to further support the interface of clinical and basic sciences research within the CTR. Their priorities will be to support the CTR Licencing and Training Coordinator to ensure that research conducted within the CTR is maintained at a high standard and complies with regulatory requirements. The post holder will be responsible for day-to-day maintenance of the CTR Human Uterus in Pregnancy and Disease Biobank and embryos consented and donated for research. The CTR Technician will liaise with clinical collaborators and maintain effective collaborative links.
The CTR Technician will also provide support for the smooth running of the CTR laboratory, ensuring an organized space, stocked core reagents and operational shared equipment to foster further collaborative working. They will be responsible for the management of common CTR equipment and ensure codes of practice and relevant safety regulations are followed within the CTR. They will monitor core stock reagents and ensure restocking. They will supervise lab cleaning and organise rotas for the smooth running of the CTR lab space including any works required.
The post holder will initially gain training in advanced cell and organoid culture and molecular biological techniques under the supervision of the Licencing and Training Coordinator. They will record and interpret data and present the findings. After gaining relevant training in these cutting-edge techniques, the post holder will in turn train others and provide technical advice on the design of experiments. They will coordinate the shipment of reagents or samples to CTR colleagues or external collaborators in consultation with the Licencing and Training Coordinator. They will also provide local guidance and assistance with MTA applications.
The CTR Technician will support the CTR to provide an environment that enables the delivery of research at the highest level and to work in collaborative ways to provide research and training support. Training and mentorship will be provided by the Licencing and Training Coordinator in consultation with the Director. The CTR Technician will need to have a positive approach and be open and willing to engage with diverse groups across the CTR.
Candidates should hold a minimum qualification equitable to HND/HNC, level 4/5 vocational qualifications or an equivalent level of practical experience. Please refer to the further particulars document for a full list of essential skills and qualifications.
We will support visa application through the CTR if assistance is needed. We have a legal responsibility to ensure that you have the right to work in the UK before you can start working for us. Any job application you submit to us will be assessed using criteria based on the knowledge, skills and experience required for the relevant post. You will not be treated less favourably than another applicant on the grounds of national origin.
Appointments will be made on a fixed-term, full-time, basis for a period of 3 years, with the possibility of renewal subject to funding. The salary range is £27,116 – £31,406 (+NI on-costs & Pension).
We would also welcome applications from individuals who wish to be considered for part-time working or other flexible working arrangements.
Instructions for applications: Candidates must submit an application via the Cambridge University Job Opportunities website https://www.jobs.cam.ac.uk/ by the 31st August 2022. References will be requested from candidates after interviews.
We aim to hold interviews shortly after the closing date.
In our recent paper, we uncovered a WIP-mediated embryo-maternal communication in Arabidopsis, which specifically regulates embryonic root development (Fig. 1). WIP2, WIP4 and WIP5 expression in the embryonic root is necessary for the oriented cell division and distal stem cell fates within the root meristem. WIP1, WIP3 and WIP6 are expressed in the maternal tissues surrounding the embryo and suspensor, where they act non-cell autonomously to repress root cell fate specification through SIMILAR TO RADICAL-INDUCED CELL DEATH ONE (SRO) gene family members. The embryonic WIPs functionally oppose those maternal WIPs to orchestrate cell division orientation and cell fate specification in the embryonic root, thereby promoting regular root formation.
It has been shown that loss-of-function wip245 triple mutants are rootless (Crawford et al., 2015). WIP2, WIP4 and WIP5 are expressed in the hypophysis and its derivatives, where they act redundantly to specify distal root stem cell fates (Crawford et al., 2015). We observed that roots were able to form and grow in wip123456 sextuple mutants, this observation prompted us to explore how WIP genes are coordinated to control embryonic root development.
What was already known about the regulation of tissue organization during root development?
Tissue organization during root development requires precise coordination of root cell division, fate specification and differentiation in a spatiotemporal manner. Components in auxin signaling cascade are crucial for root formation in Arabidopsis: the BODENLOS (BDL)/IAA12-MONOPTEROS (MP)/AUXIN RESPONSIVE FACTOR5 (ARF5) and SOLITARY-ROOT (SLR)/IAA14-ARF7/ARF19 modules are required for the initiation of embryonic and lateral roots respectively (Fukaki et al., 2002; Hamann et al., 2002; Okushima et al., 2005; Przemeck et al., 1996). In terms of embryonic roots, BDL and MP expressed in the proembryo cells non-cell autonomously regulate hypophysis specification via two direct target genes of MP, TARGET OF MONOPTEROS5 (TMO5) and TMO7 (Schlereth et al., 2010). It has been shownWIP2, WIP4 and WIP5 act downstream of MP to promote embryonic root formation (Crawford et al., 2015), but hypophysis specification remains normal in wip245 mutants. Auxin and auxin signaling is also considered important in initiating formative cell divisions during both embryonic and lateral root formation (Marhavy et al., 2016; Yoshida et al., 2014). Moreover, PLETHORA (PLT) genes are key effectors for establishment of the root stem cell niche during embryonic pattern formation (Aida et al., 2004; Galinha et al., 2007). Loss-of-function of plt1-/-plt2+/-plt3-/-bbm-2-/- mutants show embryonic root defects that are morphologically similar to the ones displayed in wip245 mutants.
What was the key experiment?
Our results from three experiments made the story outline: the rescued root development in wip123456 sextuple mutants; the identification of SRO gene family members that are responsible for the overexpression of WIP1 induced plant growth arrest from the EMS mutagenesis; the rescued root development in rcd1-4wip245 quadruple mutants.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
When we found that WIP1, WIP3 and WIP6 act maternally in a non-cell autonomous manner to repress root formation. This result points to a WIP-mediated embryo-maternal dialogue.
And what about the flipside: any moments of frustration or despair?
Generation of the multiple mutant combinations was time consuming and demanding.
Where will this story take the lab?
Our lab focuses on the sex determination process in cucurbits. The gynoecious gene WIP1 represses carpel development, causing the formation of male flowers, and its loss-of-function leads to purely female plants (Martin et al., 2009). In Arabidopsis, melon WIP1 and AtWIPs share common functions in inhibiting plant growth (Roldan et al., 2020), suggesting that the molecular network regulated by AtWIPs is likely conserved in plants. Therefore, we aim to transfer the knowledge gained from Arabidopsis back to the sex determination process in melon.
What next for you/your lab after this paper – let us know if you are continuing this research, starting/looking for a new position?
Currently, I am looking for a position and would like to continue the study of maternal-embryo communication in plants. Abdel’s lab will continue to characterize WIP functions in Arabidopsis and in cucurbits.
Christine J Watson, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP
Email: cjw53@cam.ac.uk
The Team (from left to right): Kate Hughes, Jessie Hitchcock, Bethan Lloyd-Lewis, Christine Watson and Sara Pensa
We are excited that our study on immune cells, post-lactational regression of the mammary gland, and tumour growth has been published in a Special Issue of Development on The Immune System in Development and Regeneration. This paper is the culmination of many years of work by a fantastic team of women. For over two decades, my laboratory has been interested in the mechanisms of cell death that are utilised after weaning to remove the milk-producing alveolar cells in the lactating mammary gland as they are no longer required. The mouse mammary gland is a fantastic experimental tool with which to investigate cell death under physiological conditions as the lobuloalveolar structures that make milk during lactation arise de novo, with each and every pregnancy, and are subsequently removed when lactation ceases. The use of milk protein gene promoters to drive expression of Cre recombinase has allowed us to conditionally delete any gene of interest specifically in the alveolar cells and so we can investigate the role of specific genes in lactation and cell death during involution without perturbing normal mouse physiology.
A 3D image of a mammary duct with side branches covered by milk-producing alveoli. Z- stacks are false coloured to show depth, with blue being nearest and red being farthest away.
We showed way back in 1998, using the beta-lactoglobulin promoter to drive Cre expression, that the transcription factor Stat3 was essential for initiating cell death during involution. This was a wonderful collaboration with Rachel Chapman and the late Alan Clarke when our laboratories were based in Edinburgh. We continued our work on the mechanism of Stat3-mediated cell death after my laboratory moved to Cambridge and we were joined by a talented postdoc, Sara Pensa, who had done her PhD with a long-standing collaborator Valeria Poli from the University of Turin in Italy. Valeria’s lab has considerable expertise in Stat3 signalling, cancer and immunology. Among other things, Sara was interested in looking at the effect of age on mammary tumour growth and she initiated work to investigate this in mice. We were joined by a veterinary surgeon and histopathologist, Kate Hughes, who elected to do her PhD in my laboratory and another talented postdoc Bethan Lloyd-Lewis who brought considerable expertise in mammary gland, gained in Trevor Dale’s laboratory in Cardiff. Bethan had an interest in developing lineage tracing and imaging technologies to investigate mammary stem and progenitor cells. Combining these interests and technologies allowed us to investigate multiple aspects of tumour growth utilising a cell culture model of human epidermal growth factor receptor 2 (HER2) overexpression, used in Valeria’s laboratory, and called TUBO cells. HER2 is overexpressed in a subtype of breast cancer that affects about 1 in 5 women with breast cancer usually as a result of the gene encoding HER2 being present in multiple copies. Although this is a more aggressive type of breast cancer, the use of a humanised monoclonal antibody that targets HER2, called Trastuzumab, in combination with other therapies, has proved beneficial.
We decided to use the TUBO cell line as it is a reliable and predicable model of mammary cancer development. We wanted to investigate how the involution process, with its associated extensive cell death and tissue remodelling, would affect the growth of tumours arising from implanted TUBO cells. It is established that the involution process is associated with a transient increase in the risk of developing breast cancer in women, called post-partum breast cancer. However, a full-term pregnancy before the age of 30 reduces the lifetime risk of breast cancer while childbirth after the age of 35 does not provide any protection. It is not well understood how undergoing a full lobuloalveolar development programme during pregnancy can protect from cancer at a young age, why this is abrogated in older mothers, and why the involution process can be pro-tumourigenic. We wished to gain some insights into the molecular and cellular events behind these observations. An interesting aspect of mammary gland involution is that there is an array of immune cell types present in the gland and a dramatic influx of immune cells around day 3 of involution when extensive phagocytosis of dead alveolar cells, milk fat and cellular debris is required along with remodelling of the extracellular matrix and redifferentiation of the white adipocytes in the mammary fat pad. It is remarkable that these processes do not cause overt inflammation. We realised that our team was missing an expert immunologist and we were fortunate to recruit a new postdoc with such expertise. Jessie Hitchcock had just completed her PhD at the University of Birmingham on immunity to infection, focussing on systemic inflammation, and she was keen to move into the cancer field. So, with Jessie on board, we were now well placed to carry out an extensive study on the growth of tumour cells transplanted into involuting mammary glands at various stages.
We were able to analyse immune cells and tumour growth in the mouse mammary gland using a variety of techniques combining our various expertise: histology, deep 3D imaging, flow cytometry, tumour cell implantation and tumour growth measurement. Jessie showed, surprisingly, that leukocytes (marked by CD45 expression) are present not only in the tissue stroma but that a subset intercalate between the myoepithelial and luminal epithelial cells in the ductal epithelial bilayer in virgin mammary gland while during lactation/early involution, these leukocytes co-localise with myoepithelial cells and have a very different shape similar to the star-like morphology of dendritic cells. As observed by 3D immunofluorescent imaging, the density of CD45+ cells in both the epithelium and stroma is greatest at 3 days after forced weaning, and decreases quite dramatically by day 6 of involution, with the leukocytes associating less with the contracting myoepithelium at this stage. Jessie also carried out an extensive analysis of immune cell types present in the mammary gland by flow cytometry at various stages of involution and also in pregnant and non-pregnant mice. Our tumour experiments focussed on injecting TUBO cells into mammary glands of both young and old syngeneic mice at different stages of involution followed by monitoring tumour growth. Syngeneic mice were essential to allow us to investigate whether the fluxes in immune cell types, that we had observed by flow cytometry, had an influence on initial tumour growth.
A single alveolus at 24 hours involution showing contractile myoepithelial cells in turquoise and two individual leukocytes in magenta, closely apposed to single myoepithelial cells.
These were challenging experiments, but we generated some interesting data. Firstly, and surprisingly, we found that the environment in the mammary gland at day 3 involution promoted faster tumour growth compared to nulliparous mice while the environment at day 6 involution suppressed tumour growth considerably compared to day 3 involution and tumours were even slower growing than in nulliparous mice. We were able to correlate these changes in tumour growth rate with the immune cell types present in the gland at these times, and particularly with distinctly elevated CD11b-expressing macrophage populations, that may express inflammatory genes, at day 6 involution compared to day 3 involution. We also found that tumours tend to grow faster at day 3 involution in aged mice (10 months old, equivalent to 38 years of age in women) compared to young mice. Despite differences in growth rate, the immune environment, and the age of mouse, all tumours appeared morphologically similar when assessed both histologically and by 3D imaging of optically cleared tumour tissue.
This work has provided a basis for preclinical studies in women’s breast cancers and for characterisation of weaning-induced breast involution in young women. Furthermore, our study highlights the merits of multidisciplinary work and collaboration between a team of talented and enthusiastic scientists.
Where Science meets Technology – NMMP2022 Summer School in Transcriptomics
Figure 1: Students, speakers, and organizers pose for a group picture.
In Sweden, where the sun doesn’t go down in the summer, the science stays awake!
“57 students, 20 universities and research institutes, 24 nationalities and 1 common interest: learning more about omics approaches.”: tweeted Professor Silvia Remeseiro, which together with Professor Claudio Cantù and Professor Andreas Moor co-organized the first ever NMMP2022 Summer School in Transcriptomics during Development and Cancer in an amazing location of Sandvik Gård on June 27th-30th, 2022.
One of the intriguing features of the Summer School was a relatively low ratio between students and teachers. Ten top-notch scientists coming from all over Europe and beyond helped students dive into a broad range of fascinating topics from newest technologies in single cell transcriptomics to 3D organization of chromatin.
Science unlimited
The four-day program of the Summer School was packed with exciting lectures by keynote and invited speakers, insightful “meet the speaker” sessions providing a glimpse on the life of scientists, and opportunities to discuss students’ projects and questions that are driving them. The Summer School succeeded in bringing together scientists at different career stages from variety of fields inspiring one another.
Figure 2: Picture of the nearby lake taken by Dr. Pierfrancesco Pagella, one of the participants.
Read more about about the speakers and organisers in the “Meet the speakers” paragraph later on this page!
Blurring the lines between dry and wet
Many biological questions require computational approaches to be explored in depth. Experimentalists have to find a common language with bioinformaticians and computational scientists and vice versa, which is not always an easy task. The organizers know it well and designed the program with the hope to generate ample opportunities for “wet” and “dry” scientists to exchange ideas and discuss how to build a bridge between these two domains. Indeed, according to the poll (Fig. 3), the participants had diverse experiences from “dry” and “wet” lab, and almost a fifth was open to transition.
Figure 3: Poll showing percentage of participants coming from dry, wet or mixed labs. Generated with menti.com during the Summer School.
One of the ways to find a common language is to try to explain our projects to researchers outside of our immediate niche. The students tried this during a new poster session format: the “Non-poster” poster session. For two and a half hours, the students divided into small groups presented their posters and provided feedback to each other. The fun part: the projects were as different as it gets. The students left the poster session energized, inspired and willing to try new approaches to their problems. The only complaint they had: “We were enjoying it too much! Two hours and a half were not enough!”. We are sure organizers will take a note for the next edition of the Summer School.
Figure 4: Participants, around the pool of Sandvik Gård, discussing their impressions after the “Non-poster” poster session. Picture taken by Professor Claudio Cantù.
A dive into data
The science at the Summer School was at the top of innovation. For example, students got to hear about a new, fresh from the press, scRNA-seq technology VASAsequencing by Prof. Anna Alemany. Many students presented their projects on developing the technology of the future we are looking forward to putting in practice one day. Perhaps one of the most immersive experiences of the future of transcriptomics data analysis was provided by Dr. Shamit Soneji and his team that developed CellexalVR software. Using virtual reality, students were able to literally dive into single-cell data and visualize the data with a whole new perspective!
Figure 5: Dr. Shamit Soneji (on the right) explaining how CellexalVR works with the help of Oscar Legetth (on the left), software engineer in his research group. Picture taken by Dr. Pierfrancesco Pagella.
Technology has its place in biological research
One could argue that recent fancy technological developments push us paradoxically further away from answering biological questions. Recently, this topic ignited a vivid debate on the Node platform and Twitter. The Summer School was a perfect place to continue discussing this topic as it united biologists experimentalists on one hand and technology fans on the other. Prof. Magda Bienko, avid fan of technological advances and pioneer in understanding the 3D organization of chromatin, added an interesting angle to the debate. We should give enough time for the technology to be developed fully, so all its caveats are addressed before we use it to answer a biological question. If we do it too early, we risk overinterpretation of the results, confusion and mistrust in technology. Technology has a great promise to help us understand the world around us, but we should not rush the process and trust that it will deliver once optimized properly.
Consequently, we witnessed a couple of “conversions” of pure experimentalists willing to give a new technology a try: “Before the Summer School, I saw technology as something that brings me away from the lab work, losing contact with the magic of seeing biology unfolding in front of your eyes. But I realized that technology is just a wonderful mean to get a deeper insight into biological mechanisms. Now I can’t wait to go back and put my hands on one of these amazing tools I learned about here!” stated one of the early-career-stage students.
Such realizations don’t come as a surprise given the keynote and invited speakers are leaders in the leveraging technological advances to gain insights into biology and elicited great enthusiasm in all of the students present at the Summer School with their talks.
Meet the speakers
Figure 6: Organizers, keynote and invited speakers with Universities and Institutes of origin. Picture created with Inkscape by Valeria Ghezzi.
Prof. Sten Linnarsson’s work is shaping the transcriptomics field and transforming our view of the multitude of cells type that emerge during animal development. With his group, he crafted new ways to identify secret messages hidden within transcriptomics datasets.
The work of Prof. Johan Elf is bringing the gene regulation field into a new dimension, as it is producing quantitative physical models and biological observations that, when merged, enable to determinate the real behavior of molecules.
The contributions of Prof. Susan Mango and her group are incredible: from identifying master regulators that shape cell fate, to the compelling and almost counteracting notion that variable environmental conditions play a non-negligible role on developmental trajectories. She is transforming C. elegans into a star of developmental biology.
Dr. Yonatan Stelzer’s group is implementing and developing cutting-edge genome- and epigenomeediting tools together with sophisticated epigenetic and gene expression reporters on embryonic stem cells and developing mice.
Prof. Anna Alemany‘s training and scientific production position her work among the emerging stars in the field of single-cell transcription, and how this can be used to understand cell-fate commitment. She contributed to developing a new scRNA-seq technology – VASAseq – that gives high throughput full gene body read-out with single cell resolution.
Dr. Shamit Soneji’s group is developing analytical pipelines in a new dimension: they recently developed Virtual Reality tools to navigate and analyse most sophisticated datasets. Among these tools, CellexalVR is a platform for visualisation and analysis of single-cell gene expression data.
Prof. Martin Mikl works on generating new ways to identify, in a high-throughput and standardized manner, the rules that govern mRNA function and localization.
Prof. Guillaume Andrey is an emerging leader in the field of the 3D genome, he focuses on the ideological and conceptual innovation of how the genome could be regulated in 3D in addition to the DNA sequence.
Prof. Magda Bienko developed GPseq: a new creative technique that allows mapping the radial organization of the human genome, revealing new patterns of genomic and epigenomic features, gene expression, and activity compartmentalization.
Dr. George Hausmann has an enviable scientific experience and writing dexterity: the entire Department of Molecular Life Science in Zurich competes with his time for help or consultation on manuscript design, writing and perfectionism.
Organizers:
Prof. Claudio Cantù’s Lab, at Linköping University, is focused on discovering the mechanisms of genome regulation that drive specialization during embryonic development using sophisticated tools, from mouse genetics to high-throughput state-of-the-art biochemical approaches. The group is focused on the so-called ‘Wnt signalling pathway’, a molecular cascade important for virtually all aspects of development, and whose deregulation causes human malformations and several forms of aggressive cancers.
Prof. Silvia Remeseiro works as a Wallenberg Molecular Medicine Fellow in Umeå University. Her group is mainly focused on how the reprogramming of regulatory regions and topological changes in 3D chromatin organization determine gene dysregulation in glioblastoma, and how this subsequently contributes to malignancy, heterogeneity and invasiveness.
Prof. Andreas Moor works at the Department of Biosystems Science and Engineer, at ETH Zurich, focusing on exploring the way in which single cells collaborate within tissues to achieve their common functions. His group makes use of quantitative approaches to study cellular and subcellular heterogeneity while preserving information about the spatial tissue context.
Future is in good hands
The Summer School was a dream come true for the organizers Claudio Cantù, Silvia Remeseiro and Andreas Moor, finally taking place after the delay caused by the pandemic. Their hope was to provide similar experiences, that shaped their scientific thinking when they were trainees, to a next generation.
Probably, even their wild imagination was exceeded by the success of this edition of the Summer School: 56 students from all over Europe and one, Vasikar Murugapoopathy, from McGill University in Canada, thanks to a travel grant offered by Antibodies online GmbH.
Figure 7: Countries where the participants are based, with the number of participants stated in each flag. Picture created with Inkscape by Valeria Ghezzi.
High quality of student talks, their engagement and drive to discuss science till late hours impressed the tutors.
When Prof. Susan Mango was asked to describe the Summer School in one word, she answered: “My word would have been STUDENTS. They were great – very interactive, smart questions. A really good lot”.
Prof. Anna Alemany later revealed: “It was really motivating, from the speaker point of view, to discuss the different aspects of science during lunch, coffee breaks, and walks around the lake. The motivation was contagious, I am sure this group of people will achieve anything they want!”.
It looks like the future of life science and molecular medicine is in good hands.
Figure 8: Hanzhao Zhang (on the left) and Alek Gordon Erickson (on the right) talking over Alek’s poster at 11.15 pm. Picture taken by Prof. Claudio Cantù.
Dr. Stefan Pellenz, accomplished scientist and currently product manager at Antibodies online GmbH, shared his insights on how to profile the epigenome with CUT&RUN and CUT&Tag methods.
We hope the Summer School was not a one-time experience and organizers will be able to get support for the next edition.
With an amazing venue, new connections created, excitement shared about the future of science, participant enjoyed the Summer School so much that they unanimously voted for the next edition!
Want to be part of the future? Join the next edition on NMMP2022 Summer School in Transcriptomics!
Doing great science depends on teamwork, whether this is within the lab or in collaboration with other labs. However, sometimes the resources that support our work can be overlooked. Our ‘Featured resource’ series aims to shine a light on these unsung heroes of the science world. In our latest article, we hear from Vitor Trovisco (Curator at FlyBase) and others in the team, who describe the work of FlyBase.
Overview
FlyBase (flybase.org) is the primary knowledgebase and hub for genomic, genetic and functional data on the fruit fly, Drosophila melanogaster. FlyBase was established in 1992, following funding from the National Center for Human Genome Research of the NIH, USA [Ashburner M, 1994; The FlyBase Consortium, 1994], as an online database for information on the fruit fly’s genes and mutations that had previously been collated in the Red Book [Lindsley and Zimm, 1992], and has since accompanied the constant advances in genomics and genetics. Nowadays, FlyBase hosts a comprehensive and ever-growing collection of data curated from large scale projects to primary research publications, which include gene models, expression patterns and function, alleles and transgenic constructs, phenotypes, genetic and physical interactions, disease models, gene groups, large datasets, fly stocks and other reagents. Additionally, FlyBase hosts many linkouts to external resources, particularly those from which it draws data (e.g. UniProt, NCBI, FlyAtlas/2) and several which provide reagents and advanced research tools for fly research (e.g., fly stock centres, DNA clones, Drosophila RNAi Screening Center). Find a comprehensive list of external resources here.
People behind FlyBase
FlyBase is an international consortium of biocurators and IT developers based at Harvard University (USA), Indiana University (USA), the University of New Mexico (USA) and the University of Cambridge (UK). Harvard hosts the IT developers in charge of the database infrastructure, and the team of curators responsible for genomic features, gene models, expression patterns, disease models and physical interactions. Indiana hosts the IT developers entrusted with the website and its query tools. Cambridge hosts the team of curators in charge of genetic entities, phenotypes and genetic interactions, functional data (GO), neuronal gene expression patterns (with VFB), single cell expression data, and ontologies. The team at New Mexico contributes to general curation and physical interactions curation. For the full team, see here.
FlyBase also enjoys great support from its external scientific advisory board, which includes Drosophila researchers and representatives of other genomic databases.
Collaborations
Alliance
FlyBase is part of the Alliance of Genome Resources consortium (the Alliance), together with 5 other model organism genomics databases (Saccharomyces Genome Database, WormBase, Mouse Genome Database, the Zebrafish Information Network, Rat Genome Database) and the Gene Ontology Resource [Alliance of Genome Resources Consortium, 2022]. The Alliance aims to provide better comparative biology data and tools, by bringing together, harmonising and leveraging cross-species genetics and genomics data. As part of the Alliance, FlyBase contributes to and benefits from this improved integration to the advantage of the wider biomedical field.
Virtual Fly Brain
FlyBase is closely intertwined with Virtual Fly Brain (VFB), an interactive web-based tool for neurobiologists. VFB facilitates the study of detailed neuroanatomy, neuron connectivity and expression data of Drosophila melanogaster. VFB aims to make it easier for researchers to find relevant anatomical information and reagents. VFB is a UK-based collaboration between the University of Edinburgh, the University of Cambridge/FlyBase, the MRC Laboratory of Molecular Biology and the EMBL-EBI. FlyBase collaborates in the curation of anatomical entities and transgene expression patterns and provides the transgene expression curation displayed by VFB. In the near future VFB will also provide gene expression summaries derived from single cell data.
Single Cell Expression Atlas
The EMBL-EBI’s Single Cell Expression Atlas initiative re-analyses and standardises publicly-available single cell RNA sequencing studies to make them more comparable and easier to interpret. Through its browser, users can easily visualise clusters of cells, their annotations, and search for gene expression patterns. Our collaboration has expedited the curation of fly datasets and their integration into FlyBase, through dataset report pages and cell type scRNAseq expression summary ribbons on the gene report pages. This work is closely coordinated with Virtual Fly Brain.
Funding
Since inception, FlyBase has had the extraordinary financial support of the National Human Genome Research Institute at the U.S. National Institutes of Health (NHGRI/NIH, currently U41HG000739), in the form of pluri-annual grants that assure FlyBase’s core operations: continual curation of published literature, maintenance and improvement of both the database infrastructure and website. FlyBase has also benefited from grants from other sources to integrate specific new data types. Currently these come from the US’s National Science Foundation (DBI-2035515, 2039324), the UK’s Wellcome Trust (PLM13398) and the UK’s Biotechnology and Biological Sciences Research Council (BBSRC, BB/T014008). Additionally, the UK’s Medical Research Council has provided ongoing funding for gene function annotation since 1996 (currently MR/N030117/1). Despite its continual support, NHGRI/NIH has had to impose significant funding cuts in recent years, putting FlyBase and other model organism genomic databases under some financial strain [Bellen, 2021]. In the face of this and in order to continue providing a high standard of service, FlyBase has had to resort to crowd-funding from the Drosophila research community in the form of annual user fees. Researchers around the world have been extremely generous and their contributions have lessened the impact of the cuts.
Resource overview and highlights
Most data in FlyBase is organised into a series of report pages, corresponding to different data classes (e.g. gene, allele, aberration, dataset), each hosting different types of information. For example, the report page for a given gene displays its associated phenotypes, expression patterns, disease models, and functional data (GO) amongst other data. Each type of data is organised as annotation entries, frequently in table format.
Data are available at different scales to cater to all kinds of users, from the occasional user to the power user – see [Larkin, 2021; Gramates, 2022]. For the most frequent piecemeal use case, the ‘Quick search’ and ‘Jump-to-gene'(J2G) tools allow finding and navigating to individual report pages (see figure). For higher level data-mining there is an array of query tools to explore, such as Batch Download, QueryBuilder, CytoSearch and Feature Mapper (links under ‘Tools’ in the navigation bar). Power users can explore an array of APIs, download precomputed files with the full dataset of several classes of data, and even get hold of the whole database (links under ‘Downloads’ in the navigation bar). Below are a few recent additions.
Interactive HitLists
Most FlyBase tools retrieve their results as Interactive HitLists, or can convert them into HitLists via an “Export to HitList” option, which allow users to view, analyse and export results (see figure). For example, results can be filtered by species or data type. Selecting a single data class allows conversion between associated data types (e.g. genes to alleles) and analysing results by type (e.g. aberrations by mutagen type). Processed results can then be exported as a downloaded file, as a new HitList, or to other tools.
‘Gene groups and pathways’ report pages
These recent additions to FlyBase present sets of related genes, connected by their membership to the same signalling pathway (Pathway reports) or macromolecular complex, or by sharing a common molecular function or biological role (Gene Groups)(see figure). The assembly of these gene sets is based on their underlying GO annotations, which were systematically reviewed from a wide range of sources to ensure accuracy and findability. Gene groups are hierarchical. For example, the “ENZYMES” gene group hosts the “OXIDOREDUCTASES”, “TRANSFERASES”, “HYDROLASES”, “LYASES”, “ISOMERASES”, “LIGASES” and “TRANSLOCASES” child groups, and each of these have their own child groups. Pathway members are organised into “core” members, “positive regulators”, “negative regulators” and “ligand production” members. Gene group and pathway report pages also display GO ribbon stacks, which allow for a quick visual comparison of the group members’ function (see figure).
Experimental tools
‘Experimental tool’ data was introduced to help users find alleles and transgenes with particular characteristics. We define experimental tools as commonly used sequences with useful properties that are exploited to study the biological function of another gene product or a biological process. Examples of different types of experimental tool include those that enable a gene product to be detected (e.g. the FLAG tag, EGFP, mCherry), target a gene product somewhere specific within a cell (e.g. mitochondrial targeting sequence), drive expression in a binary system (e.g. UAS, GAL4) or are used to modify cellular activity (e.g. to inhibit/activate neurons). As new alleles and transgenes are added to the database, they are also linked to any relevant experimental tools, building up a picture of what they are made of. This allows users to easily browse and search for fly stocks with particular properties (e.g. all EGFP-tagged transgenes of their gene of interest).
User support
FlyBase is rooted in the collaborative spirit of the Drosophila research community and good communication is crucial to continue providing a high standard of service. For that, FlyBase sends a couple of surveys a year to the FlyBase Community Advisory Group, which is made up of volunteer users at any career stage, from any biology field, and at any level of expertise on the database resources. Anyone can join by following the link under ‘Community’ in the navigation bar. The surveys try to gauge the level of usage and satisfaction of certain tools and what features could be added or eliminated, and are used to inform the focus of FlyBase resource development.
The query tools and data display are designed to be intuitive, supported by clear help pages. Video tutorials and ‘Tweetorials’ are available for many tools and resources, particularly if new, revamped or heavily used (see full list here).
For more direct interactions with the community, FlyBase tries to be present at major international conferences, such as the US Annual Drosophila Research Conference and the European Drosophila Research Conference. And FlyBase always welcomes suggestions, enquiries and corrections via our
Helpmail (link at the bottom of every page). These messages are read by everyone in the team, so that they can be addressed by the most suitable people.
Help from users
The fly research community has always been extremely supportive and can continue to do so at many levels. In addition to the financial support mentioned above, it is highly important and appreciated if users cite FlyBase whenever possible in articles, presentations and funding applications (citation link at the bottom of every webpage). These acknowledgements make FlyBase’s impact on research more tangible and specifically the article citations provide metrics that can be used for funding applications.
‘Gene snapshot’ summaries
FlyBase welcomes expert researchers to contribute ’Gene Snapshot’ summaries for their favourite genes. These provide a quick overview of the function of a gene’s product, based on key points solicited by FlyBase, and are reviewed by curators.
Help from authors
Authors can also contribute in several ways to simplify the curation of their articles, ultimately allowing their data to be more quickly available on the website.
When you write your paper…
Clear, detailed and accurate descriptions of the experiments and resources minimises the curation effort and reduces the need to contact the authors. Articles should mention official FlyBase identifiers and nomenclature for entities such as genes, alleles, stocks and anatomical structures and should specify the molecular details of newly created alleles.
Once your paper is published…
When a research or review paper is published, authors should get an email from FlyBase asking for their help by filling in the Fast-Track Your Paper (FTYP) form. It requests authors to add the genes their articles focus on, which will become ready to display the next release, and minimal information on the types of experiments performed, which triages and helps prioritise the article for further curation.
Occasionally FlyBase has to send emails with clarification requests. Replying to these queries is greatly appreciated, as it allows for a more complete and accurate capture of the published data and makes it more readily available for display.
Bibliography
Alliance of Genome Resources Consortium. Harmonizing model organism data in the Alliance of Genome Resources. Genetics. 2022 Apr 4;220(4):iyac022.
Ashburner M, Drysdale R. FlyBase–the Drosophila genetic database. Development. 1994 Jul;120(7):2077-9.
Bellen HJ, Hubbard EJA, Lehmann R, Madhani HD, Solnica-Krezel L, Southard-Smith EM. Model organism databases are in jeopardy. Development. 2021 Oct 1;148(19):dev200193.
Gramates LS, Agapite J, Attrill H, Calvi BR, Crosby MA, Dos Santos G, Goodman JL, Goutte-Gattat D, Jenkins VK, Kaufman T, Larkin A, Matthews BB, Millburn G, Strelets VB. FlyBase: a guided tour of highlighted features. Genetics. 2022 Apr 4;220(4):iyac035.
Larkin A, Marygold SJ, Antonazzo G, Attrill H, Dos Santos G, Garapati PV, Goodman JL, Gramates LS, Millburn G, Strelets VB, Tabone CJ, Thurmond J; FlyBase Consortium. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 2021 Jan 8;49(D1):D899-D907.
Lindsley, Zimm. The Genome of Drosophila melanogaster. Academic Press, 1992.
Cell Worlds is an innovative project aiming to bring microscopy images out of the lab to the attention of the general public. It has received a fantastic reaction in the scientific community, and more importantly among their target audience. Cell Worlds takes the viewer on a journey into the microscopic world, while sharing information about the biology behind the beautiful images. To learn more about the background of the project we caught up with the founders of the Cell Worlds, Terence Saulnier and Renaud Pourpre, while on our sister site, FocalPlane, we focus on the microscopists that acquired the image used in Cell Worlds.
Cell Worlds documentary
In addition to the text version of our interview, we have embedded the full audio version below. We did not originally intend to release the recording, but we think that this format gives an even better insight into the enthusiasm and passion of Renaud and Terence. We apologise for some issues with the sound quality and please note that there are a couple of swear words in the recording.
Can you tell us about your background and how you got started with science communication?
Renaud – I have been interested in science since high school and have a PhD in microbiology and epigenetics. When I began my PhD, I became aware of the need to tell our stories and get our messages to people outside of science; there are so many interesting stories! I started doing science communication by myself, alongside my PhD; making some videos and some events. But after I completed my PhD in 2019, I decided that I wanted to build a career in science communication and dedicate myself to working in that space. I started by creating a podcast called ‘The lonely pipette’, with a researcher, ….. Through this, I started to work directly with scientists to help them promote their stories and their research. This was through podcasts, videos and events. At one event, I met Terence and we realised that we had some common goals, and I’ll let him tell that part of our story – how everything really began for Cell Worlds.
Terence – I’m totally in love with science, especially neuroscience. This is why I started as an engineer in neuroscience and neurobiology at the Institut Curie in France. I was spending hours on the microscope looking at fluorescence labelling in cells. I really loved looking at these images and I wanted to share this type of research with the public, with a broader audience. I think it is important to discuss what we are doing in the lab and why we are doing our research. I want to help popularise science with people who would not normally be engaged with it. I do this through conferences and videos, for example those hosted on YouTube. As Renaud mentioned, we met at an event and realised that we had a common goal to work at the interface between research and the public; this was our mission. We want to bring the image of science far from the lab and into the public domain, sharing it on the internet and via digital art.
Photo credit: @youennlerb (fb/ig)
Do you think your background as scientists is important for making you good science communicators?
Renaud – We don’t believe you have to be a scientist to do good science communication. But the good thing about being a former researcher is that we know about the everyday life of the scientists; that they often lack the time and the money to be able to share their research with the public. For Cell Worlds, since we both come from a microscopy background, we knew that scientists had gigabytes of images that were never going to leave the lab. But they have stories and they are beautiful. Because we knew this, we thought that others probably shared our frustration that these images were not being used. Between us, we realised we had a list of inspiring scientists that were already sharing their images on Twitter. When we contacted them, they all just said ‘yes, just do it, you can use our images’.
Terence – I think we are lucky to have this understanding of the lab, but we really think you can speak about science without having a big scientific background. That’s part of the story of Cell Worlds, anyone can understand and participate in science. It is truly open science and this is an important part of our mission.
Aside from the microscopy images, where did the inspiration for Cell Worlds come from?
Renaud – There are many inspirations for Cell Worlds. One thing we were clear about from the start was that we wanted to create a narration that is quite different from classical documentaries. We realised that we share a big passion for music, and we have similar tastes, for example we love electronic music, but also orchestral music, which can bring a different power to the soundtrack. This was the inspiration to pick one musician to become part of our team of three, all with complimentary skills.
Terence – Yes, the music is definitely very cool! We also recognised the role of the internet culture in connecting with the public, especially younger people. The internet has made the way that people get news and access information very different from in the past, especially with social media. For us to have a documentary was very cool, but it was important that we shared it on YouTube, where it is totally free and very easy to share on social media platforms such as Twitter.
Renaud – It brings us back to our open science goals. We produced something that is available to people for free; they can play it on YouTube, or project it somewhere. We think that it is important to make these stories accessible for the public.
Photo credit: @youennlerb (fb/ig)
One thing that struck me about the documentary was that it appealed to such a broad range of people. For example, scientists really love it, perhaps for the images and the music more than the story, whereas children want to hear/see the story as well as the dramatic images and music. Was that broad audience appeal something you were aiming for?
Terence – It’s funny, we found that the documentary took the scientists back to when they started working in science. They told us that it reminded them of why they love science and why they became biologists!
Renaud – We built the documentary to target the same audience as Terence’s YouTube channel, … as that is where we published it. So, we were aiming to connect with teenagers and young adults. But we didn’t really anticipate that younger children would be watching it, but it was amazing, and we realised that our storytelling worked for them as well. We also received messages from teachers asking us about using the documentary to introduce a lesson or showing it to their classes. Another good thing was that scientists became great ambassadors for the documentary, as they were sharing it everywhere! We were so happy to see this collective effort to disseminate it.
Can you tell us more about the exhibition?
Terence – our idea was the exhibition should be an immersive experience. We wanted the audience to be surrounded by the microscopy. The images are projected on all four walls and the floor is a mirror, so you see the reflections from the walls. Of course, it is impossible to be immersed in this world in our reality, so the exhibition creates a dream world for the scientists, the school children, and the public in general.
Renaud – We have a fun story about the exhibition to share with you. We had a small launch, helped by the CNRS, the national institute of research in France. The cool story was seeing some very senior scientists behaving like 5 year olds! Running around searching for their images and even dancing along with them! That was so cool to see, the happiness in that room!
Photo credit: @youennlerb (fb/ig)
Does the exhibition tell the same story as the documentary?
Terence – It is quite different, but the aims are the same. With the exhibition, the idea is to use the entertainment to give the message to the public. But it’s more than just entertainment, we use the beautiful music and images to raise awareness of the science. Only at the end of the exhibition, do you discover what you have been looking at. We write on the wall ‘this is a neuron’ or ‘this is an embryo’, so people now connect that the images they are seeing are actually cells inside them. The museum hosting the exhibition has never hosted any science before, so it was exciting to bring together the art and science worlds.
Can you share the ‘vital statistics’ of the documentary and exhibition?
Renaud – Our nuclear team is three, me, Terence and Youenn, who composed the music. The music is particularly important in the exhibition as there is no voiceover. This means the narrative comes from the music. For the documentary, we also had two actresses helping us with the voiceover. They coached us to use our voices for the audio, as well as adding their own voices. They also tested the script and helped us add an artistic layer to the words. Then we have all the scientists that contributed images. It’s an international team, although initially many of the team were French, with collaborations with some of the top institutes in France. We then had scientists from other institutes join the team; you can see the full list on our website.
In terms of the documentary, we have reached 14,000 views (May 2022). That happened quite quickly! We are continuing to promote the documentary, for example we will share it in a cinema soon. We are also trying to push it to festivals, to really bring it to new audiences, in new places. We really want to get those images to the public, far from the lab.
We think that our exhibition is the first project, at least on this kind of scale (including so many teams, and also in terms of the physical size of the exhibition) to bring biology to the general public in this format. That’s why we called it a world premiere! So, we wanted to know if the people attending the exhibition are seeing a science and art show for the first time. We could collect this information at the end of the exhibitions via our website, which helps connects the exhibition with the documentary. The audience scan a QR code and are taken to a survey asking them if this is their first experience of an art and science show. So far, it is about 85%. We also ask whether people are interested in learning more about the microscopic world. This is about 75%. We think these numbers mean that we are achieving our goal, to get a new audience to engage with science. We chose the art centre because we knew that their main audience are not scientists.
Photo credit: @youennlerb (fb/ig)
How did you select the scientists that became part of the project?
Both – We only had one condition, a willingness to share their images! And all the scientists that we approached were already sharing their research on social media. Often, we only ‘knew’ the researchers because we followed them on Twitter. We just approached people with images that we liked. And we also had recommendations from other scientists either because they had seen some great images, or they were suggesting a new topic for us to cover.
So, which came first the images or the story?
Renaud – It was a little of both! We knew there were some topics that we wanted to cover, and then we looked around to see if there was someone we could connect with on that area. But we also introduced some topics because of the images we had seen. It didn’t matter if we knew that subject, we were interested in sharing the message of the researcher and they know the story, so we just had to look for cool stuff to share!
Terence – So, Cell Worlds is like the theory of evolution; not to have a set plan at the start but the project is made of many different blocks that build the wall.
Where did you get funding?
Terence – The funding came from two sources. Half from the museum that is hosting the exhibition in Bassins des Lumiéres, Bordeaux. And the other half is from le Centre national du cinéma in France, which helped us make the documentary. It was enough to cover our project. We were lucky as getting funding for science popularisation can be complicated.
Renaud – The funding from the le Centre national du cinéma was a competitive process. They were looking for different types of projects in arts and culture. We made an application, trying to be as clear as possible about our vision. They liked the project and decided to support us.
Why both an exhibition and documentary?
Terence – We really think about Cell Worlds as only one project. The images are the same and much of the music is the same. But we can give you a bit of history as to how it came together.
Renaud – When we started thinking about making the documentary, we really wanted it to be an immersive experience. Actually ‘immersive’ is not quite the right word but we wanted the viewer to feel that they were projected into an ecosystem, seeing it like a jungle with all its wildlife. The microscopic world is like another ecosystem, and we help the audience to enter with the voiceover. Whilst working on the project, we were in the museum, and we thought ‘how cool would it be to have a cell that is higher than you’. So, we contacted the museum about using the space. We quickly realised that the exhibition and the documentary would be complimentary – you can enjoy either without seeing the other – but we wanted to build our audiences’ desire to see both. Whilst the images and much of the music are the same, the narrative is different because of the different ways you engage with them. For example, at the exhibition you are more on your own, and if you see if a second time you don’t see it in the same way, because you already have the answers that appear at the end of the show. The voiceover in the documentary gives you more new information. We think that having these different outputs means we can reach a broader audience, and this is one of the main aims of the project. The link between the two is the website, but this is really just to direct people to the output that they have not already seen.
Photo credit: @youennlerb (fb/ig)
What are you working on next, as a team or individuals?
Both – We want to do many things!
Terence – We want to continue to work on open science with our incubator L’Exploratoire. Cell Worlds is a part of this incubator, but we want to continue our mission to explain science to the public in new ways. Not, of course, the academic way of explaining science but allowing the citizens to be actors in science, to participate. This is not really one project but more of an ambition for what we are doing in our other projects. Renaud, maybe you can speak about our new project, ‘Méandres’, ‘Meander’ in English.
Renaud – So, Méandres is about everything being connected or aligned. The project is centred in the notion of open science. We want to connect citizens with science in ways that are not ‘boring’ and allow them to experience science rather than just receiving the information. Méandres has the same team as Cell Worlds. We wanted to keep the same team; we work well together covering science popularisation, culture and music. It just makes sense! We will be working on a completely different project and it is more based on Terence and Youenn’s expertise. It is about how we can talk about neuroscience, emotions and human neurological mechanism through reactions to lost architecture and buildings. In France, we have a lot of heritage buildings, which have fantastic stories and interesting histories. We want to use exploration of abandoned heritage to help talk about neuroscience. Putting people in unknown places raises emotions, so we can see the response and then explain it together. Urban exploration can look spectacular, with many images on the internet, but for us it is really a way to talk about social science and neuroscience. So, the idea is that we will take someone who is not use to those places, we will go with this person to visit an unknown place and observe both the place and the person’s reaction to it, their emotions. There are common emotional reactions to unknown places, especially in terms of fear responses. We would like to demystify the link between the emotions they have experienced, and the neurological mechanisms involved. So, we are talking about neuroscience as part of a shared experience. We will also have the opportunity to talk about these abandoned buildings and their past functions in society, meaning that we can preserve the memories of these places. We already have some locations in mind and we have the people to produce the first episode of the series. Then we will take different people to different places, to cover new topics.
Thanks to Terence and Renaud to talking to us about the fabulous Cell Worlds project as well as giving us a sneak-peek at their new project. We look forward to seeing more of your work in the future.
“A lot of the variation in the big five personality traits is due to genetic differences and there’s suprisingly little effect of the family environment. It seems the way you’re raised doesn’t really affect those particular traits”
Kevin Mitchell, geneticist and neuroscientist
In the latest episode of the Genetics Unzipped podcast, we’re exploring genes, brains and the mind, as we ask how much of our personality is innate, and whether anything we do as adults can change who we fundamentally are. Presenter Dr Sally Le Page sits down with Kevin Mitchell, an Associate Professor of Genetics and Neuroscience at Trinity College Dublin and author of the book Innate: How the wiring of our brains shapes who we are.
If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip
Epithelial to mesenchymal transition (EMT) is an essential process in multiple steps of embryogenic morphogenesis and various pathological conditions. As an example, EMT is involved in gastrulation and neural crest cell (NCC) development during embryogenesis. EMT is also crucial for wound healing, tissue fibrosis and cancer progression. In addition to the cellular and molecular changes facilitating the transformation from epithelial cells into mesenchymal cells, EMT has also been associated with stemness, therapeutic resistance and tumor heterogeneity specifically in the context of malignancy. Due to the inherent differences between different species, cell types and biological contexts, there are variations within phenotypic changes and the underlying molecular mechanisms of different EMT programs.
NCC are an embryonic progenitor cell population that gives rise to numerous cell types and tissues, such as craniofacial bone and cartilage, in vertebrates. Pre-migratory NCCs delaminate from the neuroepithelium via EMT, following which NCCs migrate throughout the embryo and undergo differentiation. Currently, we have limited understanding of the EMT process that gives rise to migratory NCCs in mammals because many NCC EMT related findings in non-mammalian species have not been successfully replicated in mammalian species.
Transcriptional factor Twist1 is one of the major master regulators shown to be involved and to play an important role in EMT throughout both development and carcinoma progression. Previous studies on the role of Twist1 during mammalian NCC development using various mouse models have been thoroughly summarized and reviewed (Zhao and Trainor, 2022). In short, Twist1 null mice exhibit embryonic lethality around E11.5 associated with craniofacial defects such as malformed branchial arches and facial primordia (Chen et al.,2007; Chen and Behringer, 1995). Upon careful experimental testing, these phenotypes were believed to be caused by abnormality of NCC migration and differentiation. Consistent with these in vivo findings, mutations in Twist1 in humans lead to Saethre-Chotzen syndrome, which is characterized by craniosynostosis and cleft palate. In a recent publication “Twist1 Interacts with Beta/Delta-Catenins During Neural Tube Development and Regulates Fate Transition in Cranial Neural Crest Cells”, however, Bertol and her colleagues further depict the neuroectodermal expression profile of Twist1 during early mouse embryogenesis and illustrate potential functions of Twist1 in mouse cranial NCC delamination and EMT.
Key findings
During mouse embryogenesis between E8.5 and E9.5, Twist1 is detected in vesicle-like structures on the apical side of the neuroepithelium/neural plate. Interestingly, such apical expression of Twist1 coincides with the expression pattern of B-catenin and Claudin-1 suggesting an association of Twist1 with adherens and tight junctions in the neuroepithelium. Furthermore, a physical interaction between cytosolic Twist1 and B-catenin is demonstrated by co-immunoprecipitation. When Twist1 is deleted in whole embryos, apical B-catenin in vesicle like structures become diffused and mostly cytosolic in the apical neuroepithelial cells of the neural plate.
Consistent with other studies, Twist1 is also found to be expressed in migratory cranial NCCs at E8.5, E9.5 and E10.5. When Twist1 is conditionally deleted in cranial premigratory NCCs at early E8.5, cranial migratory NCCs are observed throughout the embryos, but there is a fewer number of migratory NCCs in the frontonasal and pharyngeal processes between E9.5 and E11.5. This observation is later confirmed by severe frontonasal prominence defects and neural tube closure abnormalities.
Examinations of remaining post-delamination migratory NCCs in neural tube explants from Twist1 conditional knockout mice reveal that the majority of migratory NCCs exhibits epithelial morphologies, significant cell-cell adhesions and continuous junctional signals of ZO1. Moreover, migratory cranial NCCs in vivo show increased E-cadherin expression, and Specc1 (an actomyosin cytoskeleton regulator) expression is reduced in the hindbrain and first pharyngeal arch. These data indicate disrupted EMT during the delamination of cranial NCCs in an absence of Twist1 expression.
To study the importance of Twist1 phosphorylation in craniofacial tissue development, the researchers have also generated four Twist1 phospho-incompetent mouse lines. Phenotypic characterizations of these mutants demonstrate that S18/20 and S68 phosphorylation sites are critical for craniofacial development.
In summary, the paper contributes a valuable collection of data to fill our knowledge gap of how NCC delamination and EMT are regulated in mammalian species. To my knowledge, this is the first publication that directly studies the role of Twist1 specifically in early NCC development via using Wnt1-Cre and Wnt1-Cre2 driven conditional knockout mouse models. Although I find some parts of the paper slightly confusing regarding the interpretation of certain data and the relevance of IRF6 data to the rest of the paper, the data itself is still very intriguing and thought-provoking. Interestingly, Zeb2 null mutant mouse embryos exhibit similar phenotypes of persistent E-cadherin expression in migratory cranial NCCs (Putte et al., 2003). Similar to Twist1, neither Snail1 or Zeb2 conditional knockout in pre-migratory NCCs completely inhibits NCC delamination and EMT (Murray and Gridley, 2006; Rogers et al., 2013). These previous findings in combination with the proposed function of Twist1 in the completion of mouse cranial NCC EMT could suggest that perhaps EMT master regulators act synergistically in waves to promote the complete transition from neuroepithelial cells to mesenchymal migratory NCCs.
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