Understanding how cells differentiate during embryonic development is invaluable for the in vitro derivation of functional cells from stem cells. However, mapping the human embryo, including characterization of all the cell types that make up the developing and mature human body, and of all embryonic progenitor cell types that appear in intermediate developmental stages, is an overwhelming challenge.

The LifeMap Discovery database has taken a lead role in this effort, providing the research community with an easy to use data portal describing embryonic development, along with substantial information about stem and progenitor cells, relevant differentiation protocols and cell therapy applications.

The database has been designed to integrate data derived from in vivo and in vitro experimental setups, including gene expression and signaling information in developing cells. This knowledge is essential for identification and classification of laboratory-derived stem cells and can be used to match such with in vivo cells sharing an identical or similar gene expression profile.

The database is divided into the following modules:

1. In Vivo Development

LifeMap Discovery provides easy access to a wealth of information on mammalian development, integrated with stem cell biology and regenerative medicine information.

Highlights:

– A detailed description of the developmental ontology of organs/tissues, anatomical compartments and cells

– Manually curated gene expression relating to all developmental stages, as well as data extracted from high throughput experiments and large scale in situ databases

– Detailed information on signals that regulate cells during development

2. Stem Cell Differentiation

LifeMap Discovery describes cultured stem, progenitor and primary cells, along with related differentiation protocols. Integration of stem cell biology and embryonic development knowledge provided in the database can be harnessed by stem cell researchers to better characterize experimentally obtained cell derivatives and to develop or improve stem cell differentiation protocols.

Highlights:

– Provides valuable information regarding stem cell types, such as gene expression profile and cellular markers

– Contains a large collection of stem cell differentiation protocols

– Enables accurate characterization of cultured stem and progenitor cells during differentiation processes

– Promotes an in-depth understanding of how stem cells differentiate, and of the key signals governing the process

3. Regenerative Medicine

LifeMap Discovery summarizes cell-based therapies that aim to apply stem, progenitor or primary cells towards treatment of degenerative diseases.

Highlights:

– A concentrated source of information on cell therapies spanning the different stages of development

– The provided information has been manually curated from multiple literature sources, such as: scientific publications, press releases, patent applications and clinical trials registries.

– The comprehensive information presented for each cell therapy includes: an overview of the therapy, a list of therapeutic cells utilized in the specific treatment, mode and regimen of cell delivery, mechanism of action, formulation, in vitro data, animal models, preclinical data and related clinical trials.

 4. Gene Expression

LifeMap Discovery also functions as a gene expression database, aiming to sketch a systematic map of gene expression profiles within cells and tissues.

Highlights:

– Gene expression profiling of developing and adult mammalian organs, tissues, anatomical compartments and cells, as well as for cultured stem, progenitor and primary cells, or cells derived using differentiation protocols; this genetic information enables characterization of annotated cells by their gene expression patterns.

– GeneAnalytics^TM, a powerful gene expression analysis tool, has recently been added to the LifeMap Discovery tool box. The platform integrates and clusters data extracted from multiple and variable resources and supports simultaneous analysis of multiple genes, applying a novel algorithm to match gene sets to tissues, anatomical compartments and cells within the database.  The GeneAnalytics application is the most comprehensive analysis tool currently available for modeling gene expression data in the embryo and the adult body.

LifeMap Discovery is a free tool for academics. We welcome you to watch our short introductory movie to learn more about the database and hope you will find LifeMap Discovery a useful tool for your ongoing and future research!

Please do not hesitate to contact me, by commenting on this post, for more information about LifeMap Discovery.

Dr. Ariel Rinon

LifeMap Discovery Team

References:

1. LifeMap Discovery is available at: http://discovery.lifemapsc.com/
2. Edgar R, Mazor Y, Rinon A, Blumenthal J, Golan Y, Buzhor E, Livnat I, Ben-Ari S, Lieder I, Shitrit A, Gilboa Y, Ben-Yehudah A, Edri O, Shraga N, Bogoch Y, Leshansky L, Aharoni S, West MD, Warshawsky D, Shtrichman R (2013).  LifeMap Discovery™: the embryonic development, stem cells, and regenerative medicine research portal. PLoS One. 2013; 8(7): e66629.

(4 votes)

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Last year we interviewed Kara Nordin, who won the SDB poster prize at the ISDB meeting in Cancun. Kara’s prize was to travel to Warwick to attend this year’s BSCB/BSDB Spring meeting. Continuing the interview chain, Kara interviewed Zarah Löf-Öhlin, who won the BSDB poster prize there. As a prize, Zarah will be attending the coming SDB meeting in July, which will take place in Seattle, USA.

KN: Congratulations, your hard work paid off!

ZLÖ: Thank you!

KN: How are you feeling?

ZLÖ: Really good! It is amazing to win this prize.

KN: What does your lab work on?

ZLÖ: I work in Henrik Semb’s lab, at the Danish Stem Cell Centre in Copenhagen. Our lab is divided into two different branches. Half of the lab works on the developmental biology of the pancreas, trying to understand what it takes for β-cells to develop. The other half works on human embryonic stem cells, trying to differentiate them towards β-cells, making use of the information we get from the developmental side.

KN: How long have you been there?

ZLÖ: I started out in Henrik’s lab in 2008, in Lund. However, two years ago we moved to Copenhagen. I have been around for a while!

KN: Can you tell me more about your recent findings, and what you presented in your poster?

ZLÖ: My project tries to link polarity and differentiation. I am working on a small RhoGTPase called Rac1. My work investigates how this protein controls apical polarity in cells, and how apical polarity inhibits differentiation of β-cells.

KN: And what is next for you?

ZLÖ: There are still some details that I want to investigate in my project, before we tie it all together. We are looking into the mechanism behind our observations, and we have identified Pl3 kinase and EGF receptor signalling as being involved in this process. I am also in the final year of my PhD, so I definitely want to finish my thesis by the end of the year.

KN: Have you won poster prizes before at any other meeting?

ZLÖ: No. I came first runner up on the Stem Cell Niche meeting that took place in Copenhagen two years ago, but that was the closest I have ever got to winning a prize!

KN: And will you be attending the SDB meeting?

ZLÖ: It has been a great experience to attend this meeting, so yes, I hope so!

Zarah Löf-Öhlin (left) and Kara Nordin (right)

(5 votes)
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Below are links to  two positions available at Department of Anatomy and Developmental Biology, within the School of Biomedical Sciences, Monash University, Clayton campus, Australia

Lecturer/Senior Lecturer (Anatomy – Education Focussed)

http://jobs.monash.edu.au/jobDetails.asp?sJobIDs=522377&lWorkTypeID=&lLocationID=&lCategoryID=641, 640, 636&lBrandID=&stp=AW&sLanguage=en

Lecturer / Senior Lecturer or Associate Professor – Developmental Biology

http://jobs.monash.edu.au/jobDetails.asp?sJobIDs=522375&lWorkTypeID=&lLocationID=&lCategoryID=641, 640, 636&lBrandID=&stp=AW&sLanguage=en

— Dr Megan Wilson, ANZSCDB

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This is the first of several Node Posts that the Developmental Neurobiology Seminar Class at Reed College in Portland, Oregon (USA) will be posting. Each week, 12 advanced undergraduate students and one professor get together to discuss a concept and paper related to developmental of the visual system. Some of the papers spark a lot of discussion and interest and small groups of students work together to write posts that are summative, insightful, and hopefully thought-provoking. Hope you enjoy sharing in our “conversation”.

Cell regionalization versus cell fate in zebrafish anterior neural plate development

Is there anything quite like hearing a great song for the first time? Or smelling the perfume of someone you haven’t seen in too long? Or living without the taste of sandwiches? Maybe not—but anyone who has been in the dark without a flashlight knows that sight may be the most important sense we possess.

Recently, we read and discussed two papers (Cavodeassi, et al., 2013 Development 140, 4193-4202 and Bielen, H. and Houart, C., 2012 Developmental Cell 23:4, 812-822.) that investigated the early development of the tissues that allow us to see. Regionalization of the forebrain development is one of the first events of vertebrate eye morphogenesis. Eye-fated cells must segregate from the surrounding telencephalic-fated cells and then evaginate to form optic vesicles, which eventually give rise to eyes. Specific molecular cues (like BMPs from the non-neural ectoderm and Wnts from the caudal region of the neural plate) seem to be important for specifying eye and telencephalic fate, but many questions about how these two groups of cells segregate and coalesce remain.

How do cells know where to go? Recent work from Cavodeassi and co-workers (2013) suggests that one of the master transcriptional regulators of eye fate, Rx3, helps keep eye-fated cells in the right place by regulating Eph/Ephrin expression.

Eph/Ephrin signaling is like a molecular handshake: both the Ephrin ligands and the Eph receptors are membrane bound proteins, making signaling possible only with cell-cell contact. Unique among receptor-tyrosine kinases, both cells receive a message from the binding event, just like when you shake. Unlike the normal attraction that comes with hand-shaking, Eph/Ephrin signaling is usually repulsive, causing Eph-expressing cells to congregate together, away from Ephrin-expressing cells, and visa-versa.

Cavodeassi and colleagues show that Ephrins are expressed in the eye field and Ephs in the developing telencephelon. They also provide convincing evidence that Rx3 inhibits Ephrin expression in eye-fated cells. The signals upstream of Eph expression in the eye-field remain mysterious. The authors next performed a series of transplantation experiments to examine how specific Ephs and Ephrins influence cell sorting and eye morphogenesis. When cells expressing specific Ephs or Ephrins are transplanted into the ANP (anterior portion of the neural plate), most cells expressing the Eph ephb4a segregate to the telencephalon, whereas most transplants expressing the Ephrin efnb2a segregate to the eye field (see reprinted Figure 5 from their paper below).

Figure 5 from Cavodeassi et al., 2013. Transplants of cells expressing ephb4a or efnb2a segregate to the telencephalon or the eye field, respectively. Forebrain (frontal view) cell transplants at 1-2 somite stage expressing GFP (A,B) and ephb4a (C,E,F) or efnb2a (D,G,H). Rx3 expression is shown in the eyefield (A,B,E-H) and F-actin is seen along the eye/telencephalic boundary (C,D). Dashed lines mark the eye field, arrows indicate the eyefield boundary, and asterisks identify transplanted cells. F and H are details from E and G, respectively.

Regionalization versus cell fate: Interestingly, Cavodevassi and colleagues go on to show that this segregation is independent of cell fate. To understand this distinction, imagine a room full of red-shirted carpenters on one side and green-shirted electricians on the other. If some of the carpenters were forced to don green shirts and then ended up with the electricians, it would be clear that the people were separated by shirt color, not profession. Analogously, eye-fated cells forced to express ephb4a localize out of the eye field but continue to express mab 21/2, an eye field marker (see reprinted Figure 6B). Similarly, cells fated as telencephalic cells but forced to express efnb2a localize to the eye field though they never express mab 21/2 (see reprinted Figure 6C). These data suggest that eph/ephrin signaling does not impact the ultimate fate of the cells, but is essential for eye and telencephalic cells to sort into the necessary morphogenetic clusters that are required for eye and brain development.

Figure 6 reprinted from Cavodeassi et al., 2013. Eye field cells incorrectly expressing ephs localize to the telencephalic area but continue to express eye field marker mab21/2 (B/B’), while telencephalic cells incorrectly expressing ephrins localize to the eye field but fail to express eye field markers (C/C’). A/A’ are control experiments showing distribution of transplants in both the eye field and the telencephalic area. The dashed white line delineates the eye field.

Conclusions: Cavodeassi and colleagues nicely demonstrate that cell segregation behavior is dependent on Eph/Ephrin signaling, but independent of cell fate. The signaling pathways that determine eye field versus other forebrain fates are still mostly unknown, although a recent paper by Bielen and Houart (2012) suggests that BMP may play a role in this switch. With the cellular and molecular data provided by these two papers, the scientific community is that much closer to shining a flashlight into the darkness of the unknown.

References:

Arvanitis, D. & Davy, A. (2008) Eph/Ephrin signalling: networks. Genes and Dev 22, 4112-429.

Bielen, H. & Houart, C. (2012) BMP signaling protects telencephalic fate by repressing eye identity and its Cxcr4-dependent morphogenesis. Developmental Cell 23:4, 812-822.

Cavodeassi, F., Ivanovitch, K. & Wilson, S. (2013) Eph/Ephrin signaling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis. Development 140, 4193-4202.

(3 votes)

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Organisation: College of Life Sciences, University of Dundee

Supervisors: Dr Kim Dale and Dr Philip Murray

Studentship starting: 1st October 2014

The BBSRC East of Scotland Bioscience Doctoral Training Partnership provides training for postgraduates with diverse background (including biosciences, physical sciences, mathematics) to address biological questions using a range of technologies. This 4-year programme consists of a PhD project supplemented by Biosciences and Generic Skills Training, including a 3-month professional internship outside of academia

Application Deadline: 10th April 2014

Project

Oscillators are ubiquitous throughout biology (e.g. cardiac rhythms, circadian rhythms, the cell cycle). By definition they are dynamic and nonlinear in nature, making their behaviour nontrivial to quantify and understand.

During vertebrate embryonic development, the formation of segmented blocks of mesodermal tissue (known as somites) occurs according to a strict temporal and spatial sequence and is regulated by a molecular oscillator known as the somitogenesis clock. The somites are transient structures that proceed to form essential segmented trunk tissues, such as the ribs and vertebrae of the skeleton, skeletal muscle, tendons and dermis. The process of somitogenesis is currently a field of high impact multidisciplinary research for a variety of reasons: for example, aberrations that arise during the segmentation process can give rise to medical conditions, such as scoliosis, in which the curvature of the spine is abnormal and while the etiology of many of these syndromes is largely unknown, linkage analyses have attributed some of these pathologies to mutations in key, highly conserved, segmentation clock genes; the system allows one to probe the fundamental questions of how heterogeneous spatial structure can emerge in an embryo and how the emergence of structure is coupled to embryo growth; the strict, regular, and cell co-ordinated spatio-temporal ordering with which somite formation occurs provides a unique means to quantitatively probe fundamental biological processes, such as gene transcription and mRNA processing, in in vivo contexts; modern observations demonstrate that a rich dynamical system, that is both amenable to and requiring of mathematical analysis, underlies the formation of morphological structure during somitogenesis.

It is now widely accepted that the spatio-temporal periodicity by which somites form is governed by oscillatory patterns of gene expression, regulated by a molecular oscillator known as the somitogenesis clock.  Recent advances in the field have demonstrated that small groups of cells, taken from the most immature region of the pre-somitic tissue of a mouse embryo, undergo emergent patterning when cultured ex vivo in a plastic culture dish, a process that can be visualised in real time using genetically-modified reporter mice. These observations require the analysis of large datasets (i.e. real-time movies) and the use of mathematical models to interpret the spatio-temporal dynamics.

The goal of this study is to improve upon the current protocol by using lithographic techniques to fabricate a microfluidic channel that will mimic the 3D geometry in which the explanted tissue resides in vivo. This system will be used to house the tissue explant and will allow us to probe the underlying emergent behaviour that regulates somitogenesis in previously impossible ways. For example, recent work in the Dale lab has demonstrated that particular drugs modify the pace of the somitogenesis clock oscillations. Whilst these studies have been limited to snap shot views of the process, the developed technology will enable careful control of drug delivery and real-time monitoring of effect. There are numerous other means by which the developed toolkit will be used to probe fundamental questions regarding the emergence, propagation, degree of cell autonomy, and maintenance of somitogenesis clock oscillations.

The prospective student will benefit from respective expertise available at CLS (KD), Mathematics (PM) and Physics (DMG). KD will provide training in embryological techniques, PM will provide training in the use of computational software and mathematical modelling and DMG will provide training in the development and use of the microfluidic devices that will be used to house the explant. The student will benefit from being part of vibrant research groups in each of the individual disciplines and having access to a wide range of resources in the individual divisions

The project will generate high-quality, quantitative datasets that, together with mathematical  modeling techniques, will enable us to measure and probe the somitogenesis clock oscillator. The developed techniques will allow us to integrate understanding of the molecular networks that generate oscillatory phenomena at the subcellular scale with observed emergent tissue-scale patterns. Using a predict-measure-refine workflow, the project will enable us to test and refine existing models of somitogenesis clock oscillations.

This project is ideal for a candidate with strong interests in cell/developmental Biology and the use of mathematical modelling to study biological questions in vivo. An enthusiasm for science and an enquiring mind is essential. No prior knowledge of chick or mouse development is required. This will involve a significant amount of imaging using confocal microscopy, alongside standard cell biological techniques such as whole mount immunostaining and in situ hybridisation. It will also involve image analysis and quantitation and modelling of the data sets.

Entry Requirements

Candidates must have a first or upper second class honours degree .

To apply

Interested candidates should in the first instance contact Kim Dale (j.k.dale@dundee.ac.uk).

For formal applications, visit:http://www.lifesci.dundee.ac.uk/phdprog/apply

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The BSDB Gurdon Studentship scheme funds highly motivated undergraduate students to perform developmental biology summer projects in the labs of BSDB members.

Closing date for applications is the 31st March 2014.

Please pass the news on to any keen undergrads who are thinking of doing a PhD in Developmental Biology!

More information: http://bsdb.org/awards/gurdon-studentships-for-summer-vacation-work/

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Science communication using social media is becoming a very popular way of making science more accessible to the public, as well as a way to get your research noticed.  This is true in developmental biology as it is in other fields. Can we use social media for “knowledge translation”, to make the latest in developmental biology news to assessable to everyone?   Can we get more people interested in Science and developmental biology research?

Anat456 is a 4^th year developmental biology paper taught in the Anatomy Department at the University of Otago, taken by post-graduate students (in the 4^th year of a BSc Honours, MSc or Postgraduate Diploma in Science degree). We introduced a social media-based science communication assignment into the course last year, as a means of introducing blogging as a form of science communication, as well as using it as a way to assess their understanding of developmental biology. Anecdotally, some undergraduate and postgraduate students have little insight into social media as a science communication platform, as distinct from the broader uses social media is put to.

The assignment set within the course is to blog about a high-impact paper published in the field of developmental biology in the last year.  The students are told to pitch their writing at the level of an audience with  with first year university or high school level scientific understanding.

The particular skills we are looking to assess in the students through this exercise include:

–       Their ability to put research into a broader context

–      Their understanding of the research problem and aim of the paper and how the researchers tried to answer it. This requires an ability to interpret research methods and data correctly. Communicating Developmental Biology research requires a good understanding of it in the first place!

–       Their written and visual communication skills (can they make a blog that attracts your attention).

–       The ability to get and give constructive feedback to each other (through comments)

Within the class room there is limited opportunity for students to present their written work outside of the course or even to their own classmates in larger classes.  Doing a blog assignment allows our students to present their work to a much broader audience. With that, we would like to welcome and encourage NODE readers to visit the Anat456 blog site https://blogs.otago.ac.nz/anat456/

Feel free to ask questions via comments to the Anat456 students. By doing so, you will help polish the next generation of developmental biologists!

Dr Megan Wilson

Anatomy Lecturer

(5 votes)

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A study by Sofia J. Araújo, a Ramón y Cajal researcher with the Morphogenesis in Drosophila lab at the Institute for Research in Biomedicine (IRB), elucidates the genetic regulation of cell migration. Published today in the scientific journal Plos One, the research is part of the thesis work performed by Elisenda Butí, first author of the article.

Cell migration is highly coordinated and occurs in processes such as embryonic development, wound healing, the formation of new blood vessels, and tumour cell invasion. For the successful control of cell movement, this process has to be determined and maintained with great precision. In this study, the scientists used tracheal cells of the fruit fly Drosophila melanogaster to unravel the signalling mechanism involved in the regulation of cell movements.

The research describes a new molecular component that controls the expression of a molecule named Fibroblast Growth Factor (FGF) in Drosophila embryos. The importance of FGF in cell migration was already known but little information was available on its genetic regulation. In the study, Araújo and her team have discovered that a protein called Hedgehog, known to be involved in morphogenesis, regulates FGF expression.

“This is the first time that a direct connection has been demonstrated between the Hedgehog pathway and an increase in FGF during cell migration,” says Araújo.

“The results are really interesting for biomedicine,” explains the researcher, “as the Hedgehog pathway is overexpressed in some of the most invasive tumours, such as the most common kind of skin cancer.”

The team explains that this is a step forward for research into cell migration mechanisms and that future applications will emerge as further investigation and studies are conducted.

Reference article:
Hedgehog is a positive regulator of FGF signalling during embryonic cell migration
Elisenda Butí, Duarte Mesquita and Sofia J. Araújo
Plos One (2014) 10.1371/journal.pone.0092682

This article was first published on the 21st of March 2014 in the news section of the IRB Barcelona website

(4 votes)

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A new method to study the beauty and relevance of cellular lineage

The origin of living beings has always interested, intrigued and fascinated curious researchers during the history of mankind. In the XIX^th century, along with the flourishing Cell Theory and its “all living cells arise from pre-existing cells by division” the discipline of developmental biology was born. This new research area tried to understand the mechanisms by which a founder cell divides a limited number of times to give rise to a new organism. Gaining knowledge of cell lineage from progenitors in different tissues of the body became the “Holy Grail” of developmental biology. Lineage during embryonic development encloses all relevant biological information about formation and differentiation of the tissues and organisms under normal conditions, and it can explain a multitude of pathological conditions.

Unfortunately the cells of adult individuals don’t reveal much about their embryological past and therefore cell lineage is difficult to analyse without experimental intrusion. Cells, not just the ones that live in UK, don’t possess ID cards that contain their family names and their birthdates.

We considered these issues so important that we invested the last few years to establish a new labelling method named CLoNe for the study of cell lineages that can be used virtually in all types of tissues and amniote species (Garcia Moreno et al., 2014). In order to understand how it works I will make use of an analogy among the study of cell lineages and genealogy, the study of familiar lineages.

As mentioned above, adult cells don’t easily show information about their birthdate, birthplace, lineage relationships, such as surnames do, but some methodologies do actually allow distinguishing some cells from others. We can insert exogenous DNA into embryonic cells by means of viral particle vectors or through transfection by electroporation (a well-extended method in developmental neurobiology in which a low electric current transiently opens cell pores and guide and introduce through them DNA vectors previously injected in the tissue). This DNA usually carries a reporter gene, typically the easily detectable fluorescent protein GFP, which we could consider as the surname in the ID card of the cell. When a transfected cell expressing GFP divides, the daughter cells acquire, or should we say inherit, the expression of GFP. Therefore, we could define all GFP-expressing cells as the heir of a common cell lineage, couldn’t we?

Actually not, since this simple system entails two major limitations. First that a single reporter gene doesn’t generate variability of surnames that would be enough to reliably discriminate among the billions of cells comprising a whole organism, e.g. someone is called Jones in Wales, or Molnár in Hungary. If we were all called Smith in UK, or García in Spain, it would be impossible for a genealogist to reveal the familiar origins and relationships of any of us. In our method CLoNe, inspired by the multi-coloured fluorescent Brainbow mice (Livet et al., 2007), we tackled this weakness by transfecting numerous distinct reporter genes, a total of twelve different fluorescent proteins. These 12 proteins are capable of generating thousands of different combinations on the basis of combinatorial hue, subcellular expression pattern of the fluorophores and intensity of the fluorescence (Fig. 1, observe the variability of fluorescent hues of the transfected cells in the chick brain). This extension in the number of reporter genes and their countless permutations is able to generate a vast amount of distinguishable surnames. Now, in addition to the Smiths we can distinguish the Jones and the Clayworths, or even the multiple-barrelled names (e.g. Smith-Jones-Clayworths–Howshams) that are truly unique. This is an idea that has been utilised in Spain for centuries (e.g. García-Moreno, or Manuel-Duarte-de Bendito) and now driving Pubmed Citations into a much more secure grounds.

The second important drawback of the transfection by electroporation method implies the dilution of the fluorescent proteins till they are finally lost by successive cellular divisions (e.g. in Spain the family name is changing as you go down on generations, rather than sticking to the same multi-barrelled names). The transfected DNA constructs remain episomal, in the cellular cytoplasm, and distribute to the daughter cells during mitosis. Due to the finite number of DNA copies transfected, daughter cells inherit fewer and fewer DNA copies after each cell cycle and after a certain and unknown number of divisions, they don’t inherit the mark anymore. As a consequence, cells generated after the loss of the reporter gene are indistinguishable from non-transfected cells. So an unspecified portion of the cell lineage would be erroneously considered not belonging to the lineage. Imagine that after several generations the Smiths or the Clayworths forget their surnames. Our imaginary genealogist wouldn’t be able to relate individuals from different generations. In our method, influenced by Star Track method for labelling astrocytic clones (García-Marqués et al., 2013), we recombined the reporter genes into the nuclear genome of the transfected cells by means of the piggyBac transposon (Ding et al, 2005). Transposons are genetic elements that can jump to different locations within the genome. In our case, a random number and combination of the various reporter genes jump from the episomal constructs into the host genome of the transfected progenitor cell. During cell cycle, DNA synthesis duplicates the combination of reporter genes present in the genome and daughter cells inherit identical copies of the cellular surname, the combinatorial fluorescent expression (Fig. 2, progenitor cells at the germinal zone of the chick brain; chains of cells derived from the same stem cell share a common colour palette of fluorescent proteins). Back to genealogy (or should I say gene analogy?), our transposition system perpetuates the Clayworth surname in the family to all descendants; every new generation will always be called Clayworth. However, do we know that all the Clayworths are related and descend from the same original Clayworth?

Although random integration by piggyBac is very capable to create a substantial number of surnames and though these are perpetuated in the genome the problem is not completely solved, since our ambitious genealogist doesn’t study a small town, but the biggest of the largest capitals, several magnitude orders over London, Mumbai or Mexico DF. There are millions of cells in the nervous system of a mouse, not to speak of the billions of cells in the human body. The countless combinations of surnames are simply not enough when considering billions of cells at once because the same random surname could occur independently in several progenitors during transposition. To avoid it, we apply two complementary strategies in CLoNe. First we reduce the number of transfected cells through the dilution of the Cre recombinase-expressing construct (which is responsible for the activation of the fluorescent labelling). Therefore in our particular cellular London, a small proportion of citizens do have a surname while a vast majority of the population doesn’t have a surname, so we can claim two individuals named Clayworth as relatives. Additionally, just as every genealogist does when refining the study to medieval kings, with CLoNe we decide a priori (before transfection) what cellular population we study the lineage in. During embryonic development stem cells express a variety of genes in order to perform their proliferative and regional identification functions. These genes are eventually very specific and identify a given population of progenitor cells. By associating the expression of Cre, and therefore the appearance of cellular surnames, to the expression of these identitary genes, CLoNe allows us to provide surnames to only our population of interest (Fig. 3, in the example we chose to selectively label in red cells expressing the transcription factor Dbx1). Remarkably this could be the main distinctive feature of CLoNe, the previous choice of the progenitor to be studied, instead of the a posteriori deduction of the labelled lineage. Ending the analogy, thanks to the employment of regulatory sequences of identitary genes, with CLoNe we can target and refine our study to the genealogy of the Clayworths from Notting Hill, or the Smiths from Kensington. And since we generate a great number of surnames we can always study the lineage of multiple cellular clones in the same piece of tissue (of multiple families in the same neighbourhood). The Clayworths, the Smiths, the Howshams AND the Skywalkers from Kensington can be studied at the same time, even though they are next-door neighbours. This cannot be achieved by other commonly used ways, such as viral methods, that only employ a single reporter gene.

CLoNe has been designed for a multitude of tissues and species, and has been tested in the nervous system, muscular and epithelial tissues of chick and mouse embryos. Its use can be extended to a large variety of experimental paradigms therefore it could be of interest to a variety of developmental biologists. By means of this new method we can generalise the study of cell lineage as always interesting, intriguing and fascinating. Not just biology or genealogy, in many aspects and disciplines the knowledge of lineages and kinships plays a crucial role, for otherwise Star Wars would have been only another space war movie.

References

Ding, S., Wu, X., Li, G., Han, M., Zhuang, Y. and Xu, T. (2005). Efficient Transposition of the piggyBac (PB) Transposon in Mammalian Cells and Mice. Cell 122, 473–483.

García-Marqués, J. and López-Mascaraque, L. (2013). Clonal identity determines astrocyte cortical heterogeneity. Cereb. Cortex 23, 1463-1472.

García-Moreno, F., Vasistha, N., Begbie, J. And Molnár, Z. (2014). CLoNe is a new method to target single progenitors and study their progeny in mouse and chick. Development 2014 141:1589-1598; doi:10.1242/dev.105254.

Livet, J., Weissman, T. A., Kang, H., Draft, R. W., Lu, J., Bennis, R. A., Sanes, J. R. and Lichtman, J. W. (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56-62.

(10 votes)

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Here are the highlights from the current issue of Development:

A vascular scaffold for islet nerve growth

In many organs, the processes of vascularisation and innervation are frequently interdependent. In the pancreas, islet endocrine cells secrete vascular endocrine growth factor (VEGF) to direct vascularisation, but little is known about how islet innervation is regulated. On p. 1480, Marcela Brissova, Alvin Powers and colleagues analyse the role of VEGF, and the vasculature, in controlling nerve fibre growth in the developing mouse pancreas. They find that, although neural crest-derived nerves surround the islets during embryonic development, they do not penetrate them until postnatal stages. This late step of islet innervation is dependent on VEGF, but indirectly – via its effects on the vasculature. Axon guidance molecules and extracellular matrix factors expressed by endothelial cells, and growth factors produced by endocrine cells are likely involved in promoting innervation; the authors propose that the islet vessels may act as a scaffold for nerve fibre growth. Such insights into how islet formation, vascularisation and innervation are coordinated are key for developing strategies to produce functional islets for therapeutic purposes.

Sorting out the hindbrain with Hox

The Hox genes are best known as regulators of anterior-posterior identity along the embryonic axis, including in the central nervous system. Here (p. 1492), Alex Gould and co-workers identify a function for Hox proteins in regulating cell segregation and boundary formation between rhombomeres of the mammalian and chick hindbrain. Hox4 family members show an anterior expression border at the r6/7 boundary, and Hox4 depletion inhibits formation of this boundary. Conversely, cells ectopically expressing Hox4 cluster together and display boundary-like features at their borders. These data suggest that the presence of an interface between Hox4-expressing and non-expressing cells is required for cell segregation at the r6/r7 boundary. Mechanistically, the authors show that Eph/ephrin genes known to be involved in boundary formation are regulated by Hox4, and Hox4 appears to control cell shape in a non-autonomous fashion – promoting apical enlargement on either side of the rhombomere interface. Other Hox proteins appear to have similar activities, suggesting a conserved function for this protein family in regulating cell segregation.

Integrin internalisation in angiogenesis

Clathrin-mediated endocytosis regulates the signalling activity and turnover of multiple plasma membrane proteins. Interfering with endocytosis can therefore have complex effects on developmental processes. William Sessa and colleagues (p. 1465) investigate the role of the Dynamin 2 (Dnm2) GTPase – a key component of the endocytic machinery – during angiogenesis in mice. Using endothelial cells in culture, they find that downregulation of Dnm2 impairs angiogenesis, even though vascular endothelial growth factor (VEGF) signalling is enhanced – due to increased surface levels of the receptor. This is in contrast to previous work suggesting that endocytosis might promote VEGF signalling. The authors ascribe the Dnm2 knockdown-induced defect in vessel formation at least partially to disrupted integrin turnover: focal adhesion size is increased and inactive integrin receptors appear to accumulate on the cell surface. Importantly, these observations hold true in vivo: conditional deletion of Dnm2 in mouse embryos causes severe angiogenic phenotypes that are consistent with impaired integrin function.

Measuring signal RAtio for limb patterning

The vertebrate limb is an important model for understanding developmental patterning processes. Along the proximo-distal axis, the limb is segmented into stylopod (upper limb), zeugopod (lower limb) and autopod (hand/foot) regions, marked by the expression of particular homeobox genes – Meis1/2 most proximally and Hoxa13 most distally. Fibroblast growth factor (FGF) is a key inducer of distal fate, while the role of retinoic acid (RA) in promoting proximal fate has been controversial. Miguel Torres and co-workers (p. 1534) now show that RA can inhibit distal identity in both chick and mouse, and that downregulation of RA signalling by Meis1/2 is required to permit Hoxa13 expression and specify the autopod. Thus, opposing gradients of FGF and RA pattern the proximo-distal axis. However, distal cells only become competent to express Hoxa13 at later time points, and the authors provide evidence for a timing mechanism that involves regulation of the chromatin state. The authors therefore propose a dual mechanism for regulating proximo-distal identity: antagonistic signalling gradients and an underlying temporal constraint.

A new cell type in the frog skin

The Xenopus embryonic epidermis is a mucociliary epithelium – analogous to that found in mammalian airways. This tissue is characterised by the presence of multiciliated cells (MCCs) and goblet cells. A third cell type, the ion-secreting cell, was recently discovered in the Xenopus epidermis. Two papers now identify a final cell type in the frog embryonic skin, the small secretory cell (SSC). Eamon Dubaissi and colleagues (p. 1514) show that SSCs are specified by the transcription factor Foxa1, are characterised by the presence of large secretory vesicles containing mucin-like (glycosylated) proteins and are important for immune defence: tadpoles lacking SSCs die from bacterial infection. Axel Schweickert and co-workers (p. 1526) find that these cells also secrete serotonin and provide evidence for serotonin-mediated regulation of ciliary beat frequency in MCCs, as in other systems. Given the value of the Xenopus epidermis as a model for mucociliary epithelia and disease, the identification of SSCs provides important insights into the nature and function of this epithelium.

No master regulator for EMT

Epithelial-mesenchymal transition (EMT) is a cell state change used repeatedly during development across evolution, while aberrant EMT is associated with cancer metastasis. The transcriptional control of EMT has been extensively studied, and several transcription factors (TFs) shown to play crucial roles; notably, TFs such as Twist and Snail have been proposed to be ‘master regulators’ of EMT. On p. 1503, Lindsay Saunders and David McClay challenge this concept by analysing the specific functions of TFs implicated in the EMT gene regulatory network (GRN) of early sea urchin embryos. The authors analyse five features of EMT – basement membrane remodelling, de-adhesion, apical constriction, loss of apico-basal polarity and directed motility – and find that different TFs show varying effects on each of these processes. They then use these data to build sub-circuits within the GRN for each feature. Strikingly, none of the TFs are involved in all five sub-circuits, implying that – in sea urchin at least – the idea of a master regulator for EMT does not hold.

PLUS…

Haploid animal cells

Haploid genetics holds great promise for understanding genome evolution and function. Much of the work on haploid genetics has previously been limited to microbes, but possibilities now extend to mammals. Here, Anton Wutz examines the potential use of haploid cells and puts them into a historical and biological context. See the Development at a Glance poster article on p. 1423

Switching on cilia: transcriptional networks regulating ciliogenesis

Cilia play many essential roles in fluid transport and cellular locomotion, and as sensory hubs for a variety of signal transduction pathways. Here, Sudipto Roy and colleagues review our understanding of the transcriptional control of ciliary biogenesis, highlighting the activities of FOXJ1 and the RFX family of transcriptional regulators. See the Review article on p. 1427

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