Hi! I’m Tanvi, a third year PhD student in the Ettensohn Lab at Carnegie Mellon University in Pittsburgh, Pennsylvania, USA. The big question our lab is concerned with is how the genome encodes morphogenesis. We use transcriptional gene regulatory networks as a tool to study how transcription factors and signaling pathways regulate the expression of downstream genes involved in developmental anatomy. We use the sea urchin embryo as a model system as it is remarkably well-suited for regulatory network analysis and is a wonderful system for studying cell behaviors during development. The sea urchin embryo has a relatively simple morphology and is composed of only 10-12 different cell types. Large numbers of synchronously developing embryos can be obtained easily, and several specific cell types can be isolated relatively easily in large quantities. Just as importantly, these embryos are transparent and detailed cell behaviors can be observed in vivo, either during normal development or after different kinds of molecular manipulations, such as gene knockdowns. Detailed gene regulatory networks have already been established for various cell types of the embryo. The Strongylocentrotus purpuratus, or purple sea urchin, genome has been sequenced and a high quality transcriptome profile is available for different developmental stages. The genomes of several other echinoderms are at different stages of assembly and provide a terrific resource for comparative studies.

Our lab works with two different sea urchin species – the purple sea urchin, which is maintained at 15˚C, and the green sea urchin, which requires a warmer 23˚C. Both species are kept in temperature-controlled seawater tanks that are well-aerated and clean. The graduate students of the lab do most of the routine maintenance such as handling incoming urchin shipments, cleaning tanks, checking salinity and ammonia levels, feeding the urchins, and fishing out dead animals from time to time. Our extremely handy advisor deals with occasional tank breakdowns.

Purple sea urchins (S. pupuratus). Photo courtesy: ALAMY       

Green sea urchins (L. variegatus). Photo courtesy: R. Wallocombe

We receive our adult sea urchins from California or Florida, depending on the species. When we need urchins, we simply send an email to a commercial supplier and a few days later we receive FedEx boxes containing fertile sea urchins cooled on ice! The urchins tolerate overnight shipping fairly well, but some release their gametes (or “spawn”) along the way. This could be a mild annoyance or a nightmare depending on how many animals spawn. Once the urchins arrive, we place them in separate cups (usually old yogurt containers) with seawater and float them on the tanks. We change the seawater every few hours. This acclimates the urchins to the tank temperature and water and minimizes stress. Any urchins that may have spawned require additional water changes and are kept isolated until they stop spawning.

Purple sea urchins recently transferred into their tank

After 6-8 hours, when all the urchins are stable, they are transferred into the tank.  It is absolutely essential that urchins placed in the tank are not actively spawning, since seawater containing gametes (even a small number) will induce mass spawning in all the urchins! The next morning, I check on the tanks with fingers crossed, hoping that the urchins have not all spawned and died in a cloudy smelly mess at the bottom of the tank (this happens only rarely but is even more unpleasant than it sounds). The task of transferring newly arrived urchins to the tank is shared within the lab, which greatly reduces the stress and anxiety associated with it!

A day or two later, the urchins are delightfully low-maintenance. They cling to the walls of the tank and stay more-or-less stationary until we pluck them out to collect eggs or sperm. They can survive without feeding for many weeks, but we feed them sea kelp to keep them happy and full of gametes.

Spawning urchins shedding eggs (left) and sperm (right)

I work with embryos of the purple sea urchin. I usually work on embryos at the early gastrula stage, and set up embryo cultures about 24 hours before my experiment. Collecting gametes is a simple matter of shaking the urchins vigorously. This stresses them and causes them to release a fraction of their gametes, which I collect “dry” (sperm) or “wet” (eggs). I then plop the urchins into floating cups and later transfer them back into the tank. If I need more gametes than they provide willingly, I inject them with a 0.5M KCl solution, which induces them to shed all their gametes. Unfortunately, they usually do not survive this treatment. It is impossible to tell male purple sea urchins from females from external morphology alone, so picking the right urchin out of the tank is a matter of chance. I usually don’t need to go through more than 3-4 urchins before getting both eggs and sperm. The yield of gametes can be as high as a whopping 10-15 ml of eggs and 1 ml of sperm, if the animals are ripe. Sperm stays fresh for one week at 4˚C, which is convenient. Eggs are usually used the same day but can be stored overnight at 4˚C.

To set up an embryo culture, I simply mix eggs and sperm in a glass bowl filled with seawater! A small drop of sperm is more than enough to fertilize ~1 ml of eggs. This would, theoretically, give me about 1 million synchronously developing embryos! I check the culture under the microscope to ensure that almost all the eggs are successfully fertilized and then cover the bowl and keep it at 15˚C for 24 hours. The culture needs no maintenance and the embryos do not need to be fed unless a developmental stage later than the 72-hour larva is required.

The next day, I start my experiment by checking on the embryos. A good culture will have embryos that have developed synchronously with a morphology characteristic of their developmental stage. The culture may not develop well if the embryos are too concentrated or the quality of the eggs was poor. When the embryos reach the desired stage, I collect them by spinning in a clinical centrifuge.

My current experiments involve isolating a specific cell type of the embryo, the primary mesenchyme cells (PMCs), which build the embryonic endoskeleton. For this, I collect large amounts of 24-hour embryos and subject them to various washes using embryo-dissociation solutions. The embryos can withstand the mechanical forces of repeated centrifugation, which makes my life very easy! I separate the primary mesenchyme cells from other embryo cell types using a simple sucrose gradient. I am usually able to quickly isolate large amounts of PMCs. It is exceptionally rare to be able to isolate a specific population of early embryonic cells in such large quantities! The PMCs remain viable in culture for days, and carry out their developmental program autonomously. Needless to say, isolated PMCs have been an extremely useful model for me to study the development of a specific cell lineage. I am interested in understanding how genes involved in the morphogenesis of PMCs are regulated at the transcriptional level. I have been able to identify hundreds of genes differentially expressed in PMCs at the early gastrula stage using isolated PMCs. I am now working towards locating cis-regulatory elements mediating the expression of these PMC-enriched genes. My ultimate goal is to construct a detailed and comprehensive gene regulatory network that will explain how PMC morphogenesis is encoded in the genome.

Other lab members routinely set up and use embryo cultures for many interesting experiments involving injection of morpholinos or DNA constructs, transplantation or removal of various cell types, drug treatments to block certain developmental pathways, in situ hybridizations to analyze spatial gene expression patterns, and other kinds of experiments. The ease of working on sea urchins enables us to spend less time worrying about obtaining embryos and more time focusing on uncovering the mysteries of development!

This post is part of a series on a day in the life of developmental biology labs working on different model organisms. You can read the introduction to the series here and read other posts in this series here.

(6 votes)

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Here is our monthly round-up of some of the interesting content that we spotted around the internet:

News & Research:

– A new paper in Nature claims that cellular reprogramming can be achieved by exposing cells to stress.

– The winners of the 2014 SDB awards were announced. Richard Harland, Christopher Wylie and Janet Heasman are among those honoured.

– ‘A Fred Sanger would not survive today’s world of science’- Sydney Brenner discusses Sanger’s career.

– The first European bank for induced pluripotent stem cells was announced.

– An article in the Guardian suggests that the axolotl, a great model for regeneration studies, may be extinct in the wild.

– An excellent article by Ed Yong discusses a recent paper by Kondo and Yamanaka on zebrafish stripe formation.

– And the 12th of February was Darwin’s birthday- happy Darwin day everyone!

Weird & Wonderful:

– Do you like knitting and science? Join the Glasgow city of science project to beat a world record by knitting your own microbe!

– Are you a fan of the game ‘Cards against humanity’? Then the new edition ‘Cards against scientists‘ is for you!

– Valentine’s day is upon us! If that special person in your life is a scientist, you may want to follow #AcademicValentines for a few ideas.

– And we bet you never thought that sushi could teach you about developmental biology…

Beautiful & Interesting images

– We found a website with images of 8 DNA sculptures from around the world.

– Phil Gates (@SeymourDaily) tweeted a series of beautiful plant cross-sections.

– If you ever wanted to know what scientists think of each other, this helpful and funny diagram will answer all your questions.

– Ultrasound scans, small cameras and computer graphics were used to generate these beautiful images of animals in the womb.

– And this figure explaining what scientists really say in the lab was one of our most popular tweets this month:

 Keep up with this and other content, including all Node posts and deadlines of coming meetings, by following the Node on Twitter.

Axolotl image by Orizatriz, wikimedia commons

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Dear The Node readers,

We are Alicia (1st year PhD student) and Marion (3rd year PhD student) and we work in an ascidian lab at CNRS, Montpellier in France. Alicia’s aim is to compare gene regulatory networks of Ciona intestinalis and Phallusia mammillata during early embryogenesis. Ciona is much better characterised, therefore, Alicia has so far only worked with Phallusia. Marion works on enhancers and aims to better understand the sequence determinants of their activity, using Ciona intestinalis as a model organism.

Phallusia mammillata in a jazzy setting: they are a brilliant model organism to study developmental biology. Unfortunately, we no longer have the blue backdrop for our tanks but we do have a starfish!

Working with ascidians

In the world, more than 70 labs work with ascidians[1]. Ascidians belong to the tunicate group; these are marine invertebrates that belong to the phylum Chordata. Tunicates are the sister group of vertebrates with whom they share some features of chordate embryonic developmental programme such as the formation of tadpole larvae. Due to their small and rapidly evolving genome, they are powerful organisms to study chordate evolution. Ascidians are simple genetically because they have not undergone whole genome duplication events and exhibit limited gene redundancy and compact regulatory regions.

The sea squirt Ciona intestinalis is a good model to study developmental biology; they have rapid development and the embryos have invariant lineages. Specialised databases such as Aniseed[2], which was created by the Lemaire lab, have catalogued an atlas of expression profiles, cell lineages and single cell morphological properties and also gene, transcript and protein data. One can retrieve information about each cell in the embryo through different identification markers (name, position), which facilitates the analysis and interpretation of results.

Phallusia mammillata and Ciona intestinalis are thought to have diverged about 300 million years ago but they still share a common invariant embryonic cell lineage. Phallusia arrived in our lab ~3 years ago because of its experimental advantages over Ciona: they have more eggs, transparent embryos and show much less seasonality in embryo production. After a few optimisations, we can now perform all of Ciona’s routine experiments on Phallusia. Comparative genomics approaches can be undertaken to study ascidian developmental programs as many ascidian genomes have now been sequenced.

Ascidians are simple organisms making them easy to analyse. They have a much lower cell number during embryogenesis when compared to vertebrates: 110 to 112 at the onset of gastrulation and about 2600 cells at the tadpole stage. Due to their invariant cell lineage, a phenotype can be characterised with single cell resolution. Electroporation provides a strong tool to easily and quickly make hundreds of transgenic embryos making ascidians a powerful model to study gene regulation.

No Ciona were harmed in the making of this video – our colleague Mathieu playing around with their siphons.

The routine in the lab

One of our favourite practicals is collecting the eggs and sperm from our dear Phallusia; this is done first thing in the morning in our 17°C embryo room. It is always exciting to see the volume of eggs that we manage to collect; it is cause for celebration when the Phallusia has 1.5ml or more. Sperm is not quite as exciting as we only require 10µl; nevertheless, it is still much appreciated.

Dechorionation is performed to remove the chorion and follicular cells which allows a better visualization of the embryonic development and facilitates any further experiments. Phallusia dechorionation lasts a couple of hours. In Ciona, this process is very fast and it is performed after fertilisation. Eggs are now very fragile and should be gently manipulated. Fertilisation is rather fast: sperm is activated (under the stereoscope, we can see the sperm become agitated), and then mixed with eggs. Most of the eggs are fertilized at the same time and thousands of embryos will develop in a synchronous way. From this point on, 20 minutes remain before the first cleavage. During this short time frame, electroporations can be performed.

Collecting our embryos at each stage is a day-long process; it takes the embryos up to 5 hours to reach early gastrula. We try to disperse the embryos as much as possible in the plates as they develop to avoid that they touch and fuse together to create what we call little monsters. Once they have reached the stage of interest, we fix the embryos and colour them if needed. From here on, our embryos can be kept for many months.

Microinjection is a very common technique in ascidian laboratories. It is used for 2 main goals: exogenous expression (microinjection of mRNA) and gene expression knock down (microinjection of morpholinos). Ascidian oocytes are particularly sensitive to microinjection when compared to other species and easily explode or get stuck to the material during microinjection until you get the technique right (we are lucky to have our post-doc Ulla). It is a rather slow technique demanding a high degree of patience and precision but its potential is fantastic. Unlike electroporation, microinjection allows for ubiquitous expression without mosaicism. You can also use interesting control situations with a single embryo. Microinjection of mRNA in a single cell at the 2 cell-stage allows to have half of an embryo expressing an exogenous mRNA whilst the other half remains wild type. Ascidians have bilateral symmetry until late embryonic stages. Although in terms of analysis microinjection techniques are very appealing they provide a much lower number of transgenics to work with than electroporation techniques.

Our ascidian tanks – a main attraction within our building where many come to admire our animals.

Shipment and animal care

We get a weekly shipment of Phallusia and Ciona. Before they are transferred to the tanks, the tanks are thoroughly cleaned during which time the animals are kept in the water in which they were transported. This waiting period means that their water temperature can gradually warm up to match the temperature of the tank water. We do not want to disturb the animals by transferring them before they are ready. We have to order each shipment a week in advance because a special boat and diving expeditions are organised to collect the animals by Roscoff Marine station. We thus need to plan ahead how many animals we need and order only what is required.

Our tanks are in the lab where we work so we regularly check how our animals are doing. To assess if the animals are healthy, we give the tanks a little tap to see if the animals respond by closing their siphons. The colour (and the smell) of the animals can also be an indicator of their health as they turn darker when they die. We have to remove dead animals as soon as possible because they raise nitrite levels in the tanks, which is bad for the other animals. Our sea squirts are kept in natural sea water which is delivered by an oyster farmer.

Working with wild animals

We have not yet started to culture ascidians in our lab; therefore, we work with wild animals delivered from Brittany. Ciona’s fertility depends greatly on sea water temperatures; the period during which they produce eggs spans from April to November in France. So we have to take this into account when planning our experimental work. Funnily enough, our experiments greatly depend on the climatic conditions 1000km from our lab! At each delivery, we could be in for a surprise: did our animals survive the trip? Do they have eggs? Will they develop properly? One pleasant surprise we occasionally get is clandestine animals attached to the Phallusia such as starfish, brittle stars, urchins, worms, annelids or porcelains and the list goes on. At the moment, we have a beautiful purple starfish as a lab pet and we feed her mussels.

Acknowledgments – We would like to thank our postdoc Ulla-Maj Fiuza for her time helping us write this article (and for her microinjection knowledge).

[1] www.tunicate-portal.org

[2] www.aniseed.cnrs.fr

This post is part of a series on a day in the life of developmental biology labs working on different model organisms. You can read the introduction to the series here and read other posts in this series here.

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Dear Node colleagues,

This is a call for the registration to the EMBL Master Course Bioimage Data Analysis to be held from Monday, 12 May – Friday, 16 May 2014.

This course will focus on computational methods for analyzing images of proteins, cells and tissues, to boost the learning process of participants who have an immediate need to deploy image analysis in their own research. The course extends from the basic foundations of image processing and programming to the actual implementation of workflows using scripting in ImageJ macro and Matlab languages. The students will be guided to design such scripts to address some practical bioimage analysis projects. Among those course-projects, topics that are interesting for developmental biologists could be:

• Quantitative Evaluation of Multi-cellular Movements in Drosophila Embryo
• Tumor Blood Vessels: 3-D Tubular Network Analysis

For details on other topics, please visit the course website. We aim to gather expert knowledge to organize a world-leading course for image analysis in the fields of biophysics, cell biology and developmental biology.

The course will take place in Heidelberg, Germany at the EMBL Advanced Training Centre. Registration and motivation letter deadline is February 25, 2014. Please visit our course website for more details:

http://www.embl.de/bias2014/

You are welcome to circulate this announcement to interested members and groups within your institution.

We look forward to welcoming you to Heidelberg, Germany.

Scientific Organisers

• Kota Miura (EMBL Heidelberg, Germany)
• Sébastien Tosi (Institute for Research in Biomedicine – IRB Barcelona, Spain)
• Perrine Paul-Gilloteaux (Institut Curie, France)

If you have any questions, please do not hesitate to contact:

Diah Yulianti
Conference Officer
European Molecular Biology Laboratory
Email: diah.yulianti@embl.de

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The early bird deadline for the BSCB/BSDB spring meeting registration is today (Friday 7th of Feb).

http://www.bscb-bsdb-meetings.co.uk/reg.htm

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Why iodine deficiency during pregnancy may have disastrous consequences

Higher mammals, such as humans, have markedly larger brains than other mammals. Scientists from the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden recently discovered a new mechanism governing brain stem cell proliferation. It serves to boost the production of neurons during development, thus causing the enlargement of the cerebral cortex – the part of the brain that enables us humans to speak, think and dream. The surprising discovery made by the Dresden-based researchers: two components in the stem cell environment – the extracellular matrix and thyroid hormones – work together with a protein molecule found on the stem cell surface, a so-called integrin. This likely explains why iodine deficiency in pregnant women has disastrous consequences for the unborn child, affecting its brain development adversely – without iodine, no thyroid hormones are produced. “Our study highlights this relationship and provides a potential explanation for the condition neurologists refer to as cretinism”, says Wieland Huttner, Director at the Max Planck Institute in Dresden. This neurological disorder severely impairs the mental abilities of a person.

In the course of evolution, certain mammals, notably humans, have developed larger brains than others, and therefore more advanced cognitive abilities. Mice, for example, have brains that are around a thousand times smaller than the human one. In their study, which was conducted in cooperation with the Fritz Lipmann Institute in Jena, the researchers in Dresden wanted to identify factors that determine brain development, and understand how larger brains have evolved.

A cosy bed for brain stem cells
Brain neurons are generated from stem cells called basal progenitors that are able to proliferate in humans, but not in mice. In humans, basal progenitors are surrounded by a special environment, a so-called extracellular matrix (ECM), which is produced by the progenitors themselves. Like a cosy bed, it accommodates the proliferating cells. Mice lack such ECM, which means that they generate fewer neurons and have a smaller brain.

The scientists therefore conducted tests to see whether in mice, basal progenitors start to proliferate if a comparable cell environment is simulated. The result: “We simulated an extracellular matrix for the brain stem cells using a stimulating antibody. This antibody activates an integrin on the cell surface of basal progenitors and thus stimulates their proliferation”, explains Denise Stenzel, who headed the experiments.

Because a requirement of thyroid hormones for proper brain development was previously known, the researchers blocked the production of these hormones in pregnant rats to see if their absence would inhibit basal progenitor proliferation in the embryos. Indeed, fewer progenitors and, consequently, neurons were produced, likely explaining the abnormal brain development in the absence of thyroid hormones. When the action of these hormones on the integrin was blocked, the ECM-simulating antibody alone was no longer able to induce basal progenitor proliferation.

A combination of ECM and thyroid hormones thus appears necessary for basal progenitors to proliferate and produce enough neurons for brain development. Human brain stem cells produce the suitable environment naturally. “That is probably how, in the course of evolution, we humans developed larger brains”, says Wieland Huttner, summing up the study. The research produced another important finding: “We were able to explain the role of iodine in embryonic brain development at the cellular level”, says Denise Stenzel. Iodine is essential for the production of thyroid hormones, and an iodine deficiency in pregnant women is known to have adverse effects on the brain development of the unborn child.

Original publication:
Stenzel, Denise; Wilsch-Bräuninger, Michaela; Wong, Fong Kuan; Heuer, Heike; Huttner, Wieland B.:
Integrin αvβ3 and thyroid hormones promote expansion of progenitors in embryonic neocortex.
Development (2014)
doi: 10.1242/dev.101907

Stem cells in the cortex of a mouse embryo (cell nuclei: blue).

 
This article was first published on the 4th of February 2014 in the news section of the Max Planck Institute of Molecular Cell Biology and Genetics website

(3 votes)

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–  Researchers at IRB and IBMB-CSIC, in Barcelona, and at the University of Wageningen, in the Netherlands, reveal how auxin hormone-regulated proteins activate developmental genes in plants.

– Auxins are key components of plant growth and have many applications in agriculture. The biomedical application of these hormones are also being addressed.

– The study is published today in the scientific journal Cell.

A joint study published in Cell by the teams headed by Miquel Coll at the Institute for Research in Biomedicine (IRB Barcelona) and the Institute of Molecular Biology of CSIC, both in Barcelona, and Dolf Weijers at the University of Wageningen, in the Netherlands, unravels the mystery behind how the plant hormones called auxins activate multiple vital plant functions through various gene transcription factors.

Auxins are plant hormones that control growth and development, that is to say, they determine the size and structure of the plant. Among their many activities, auxins favor cell growth, root initiation, flowering, fruit setting and delay ripening. Auxins have practical applications and are used in agriculture to produce seedless fruit, to prevent fruit drop, and to promote rooting, in addition to being used as herbicides. The biomedical applications of these hormones as anti-tumor agents and to facilitate somatic cell reprogramming (the cells that form tissues) to stem cells are also being investigated.

The effects of auxins in plants was first observed by Darwin in 1881, and since then this hormone has been the focus of many studies. However, although it was known how and where auxin is synthesized in the plant, how it is transported, and the receptors on which it acts, it was unclear how a hormone could trigger such diverse processes.

At the molecular level, the hormone serves to unblock a transcription factor, a DNA-binding protein, which in turn activates or represses a specific group of genes. Some plants have more than 20 distinct auxin-regulated transcription factors. They are called ARFs (Auxin Response Factors) and control the expression of numerous plant genes in function of the task to be undertaken, that is to say, cell growth, flowering, root initiation, leaf growth etc.

Using the Synchrotron Alba, in Cerdanyola del Vallès (Barcelona), and the European Synchrotron, in Grenoble, structural biologist Dr. Miquel Coll and his team analyzed the DNA binding mode used by various ARFs.

For this purpose, the scientists prepared crystals of complexes of DNA and ARF proteins obtained by Dolf Weijers team in Wageningen, and then shot the crystals with high intensity X-rays in the synchrotron to resolve their atomic structure. The resolution of five 3D structures has revealed why a given transcription factor is capable of activating a single set of genes, while other ARFs that are very similar with only slight differences trigger a distinct set.

“Each ARF recognizes and adapts to a particular DNA sequence through two binding arms or motifs that are barrel-shaped, and this adaptation differs for each ARF,” explains Roeland Boer, postdoctoral researcher in Miquel Coll’s group at IRB Barcelona, and one of the first authors of the study.

The ARF binding mode to DNA has never been described in bacteria or animals. “It appears to be exclusive to plants, but we cannot rule out that it is present in other kingdoms. Our finding is highly relevant because we have revealed the ultimate effect of a hormone that controls plant development on DNA, that is to say, on genes.” says Miquel Coll.

Reference article:

Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors
D. Roeland Boer, Alejandra Freire-Rios, Willy van den Berg, Terrens Saaki, Iain W. Manfield, Stefan Kepinski, Irene López-Vidrieo, Jose Manuel Franco, Sacco C. de Vries, Roberto Solano, Dolf Weijers, and Miquel Coll
Cell (2014) 156, 577-589 http://dx.doi.org/10.1016/j.cell.2013.12.027

This article was first published on the 30th of January 2014 in the news section of the IRB Barcelona website

(3 votes)

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Research Associate

(2 positions)

University College London -MRC Laboratory for Molecular Cell Biology

Full Time : The appointment will be on UCL Grade 7. The salary range will be £32,699 – £39,523 inclusive per annum, inclusive of London Allowance.

The Mao lab studies the mechanics of tissue growth and regeneration, combining genetics, in vivo live imaging, automated image analysis, experimental biophysics and computational modelling. Current projects in the lab include interdisciplinary approaches to study how mechanical forces affect tissue growth, tissue architectural changes, cell shape changes during mitosis and tissue regeneration in the Drosophila wing disc.

We are looking for an experienced developmental or cell biologist to study the mechanics of in vivo 3D tissue growth, using initially the Drosophila wing as a model system.

Available from March 2014, one post is offered for a period of three years in the first instance and the other for one year in the first instance.

Candidates should possess / or shortly be awarded a PhD degree in biology, biophysics, or a related biological sciences discipline. A strong background in advanced live imaging, cell / tissue mechanics, quantitative image analysis, molecular and cell biology are required. Experience with Drosophila genetics, tissue mechanical measurements and tissue manipulation techniques are advantageous.

To access further details about the position (ref 1399630) and how to apply please visit: http://www.jobs.ac.uk/job/AID186/research-associate/

If you have any queries regarding the application process, please contact Ione Karney i.karney@ucl.ac.uk.

Highly committed candidates are encouraged to contact Yanlan Mao by email at y.mao@ucl.ac.uk.

Closing Date: 20/2/2014

Latest time for the submission of applications: 5.00pm.

UCL Taking Action for Equality

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I, Narender Kumar, am a graduate student in Prof John C. Larkin’s lab. Our lab is located in the Life Sciences Building on the main campus of Louisiana State University (LSU) Baton Rouge. LSU is located on the banks of the historical Mississippi River and the river levee is the one of the best places to walk or bike on a sunny winter’s day.

Arabidopsis thaliana (Fig.1A) is a model used to study plant genetics, molecular biology and biochemistry. This plant was first discovered by Johannes Thal in the sixteenth century and initially named “Pilosilla siliquosa”, but since then it has gone through a number of name changes and is now widely known as Arabidopsis thaliana, or by the common name thale cress or mouse-ear cress. The genus Arabidopsis has many species, but A. thaliana is the most commonly used in labs. This plant belongs to an agronomical important plant family- Brassicaceae (Cruciferae), though it does not have any agronomical importance in itself.

Arabidopsis trichome development is the main focus of our lab. The Arabidopsis trichome, or shoot epidermal hair, is a giant, unicellular polyploid cell on the leaf epidermis (Fig.1B). Trichomes are polyploid as a result of a process called endoreplication, a form of mitotic cell cycle in which DNA replicates without cytokinesis causing the cell to become polyploid. Our lab is trying to understand the mechanism of endoreplication using trichome development as a model.

Over the last 30 years, Arabidopsis use as a model has increased dramatically because of the following suitable characteristics: –

1)      It has a short life cycle- Arabidopsis completes its life cycle in 8-12 weeks from germination to harvesting.

2)      It easily grows in a restricted space and is very easy to maintain in an indoor growth chamber.

3)      It produces many seeds. Each silique (seed capsule) contains 30-60 seeds, and each plant has around 50-60 siliques, so a plant can produce thousands of seeds.

4)      It has a sequenced and comparatively small genome in the plant kingdom (135 megabases, and approximately 25000* genes). It has 5 chromosomes and has genes similar to those of agronomically important crops, so it is a good model for crop plants.

5)      Transgenic lines are easily produced by infection with Agrobacterium tumefaciens (and the floral dip method used is easy and fast).

6)      Numerous mutants are available in the stock centers: Arabidopsis Biological Resource Center (ABRC), Nottingham Arabidopsis Stock Center (NASC), and RIKEN Bioresource Center (BRC)/SENDAI Arabidopsis Seed Stock Center (SASSC) etc.

7)      Arabidopsis is a self-pollinated plant and cross-pollination is easy to do in the lab.

 Fig-1. A) Arabidopsis plant in Growth chamber. B) Scanning Electron Microscopic Image (SEM) of Arabidopsis trichome

I am writing under the title “a day in the life of an Arabidopsis lab”, but it is very difficult to explain everything by just telling about one day. Therefore, to make it more understandable, I will explain step by step how I maintain and work with Arabidopsis throughout its life cycle.

Growth Chamber: – We have a very nice habitat (a climate-controlled chamber, Fig-2) for our favorite Arabidopsis plants in the basement of the Life Sciences Building. This chamber always maintains a 21-22 °C temperature and is divided into different shelves to house the different groups of plants. Besides the growth chamber, we have a preparation room in which we do all Arabidopsis related chores including sowing the seeds, harvesting and washing etc.

Fig-2. Climate-controlled growth chamber

Sowing the seeds: – In our lab, we grow Arabidopsis plants in big rectangular flats. Each flat can accommodate three black trays, and as each tray can hold 12 pots and each pot can have up to three plants, one flat can accommodate 36-108 plants (Fig-3A). However, when looking at seedling phenotypes, we plant approximately 30 seeds in each pot. The Arabidopsis life cycle starts with sowing the seeds on the soil. We fill the pots with ready-made dirt, water, and vermiculite to keep the soil well aerated.

Fig-3. A) A flat with 36 pots. B) MS media plates showing five days old Arabidopsis plants

Soil works best when growing transgenic plants that need to be sprayed (see later), but if I have any other marker (such as kanamycin) or need a clean root without soil or dirt to study root development then MS medium plates (Fig.3A) are ideal. Arabidopsis in MS plate plants are transferred to soil later. Arabidopsis seeds are very tiny and can easily be scattered around and contaminate other seeds.

Arabidopsis seeds need high humidity to germinate efficiently so the flats are covered with humidity domes until seeds germinate and start turning green, which takes five to six days (Fig-4A). I check the plants and growth chamber twice a day, in the morning and in the evening before I leave the lab. Plants must be watered properly as both too much water or too little water both can adversely affect plant growth. Too much water can lead to the appearance of white mold on the soil, which can infect the plants. Too little water can lead to drought stress. Therefore, proper watering and optimum temperature is necessary for a happily growing Arabidopsis plant. It is also important to protect the plants from insects, so we hang pest-traps in the growth chamber to catch fungus gnats.

Fig-4. A) Five-six old day plants. B) 15-day old plants. C) 1 month old plants

Flowering: – At approximately four-five weeks, plants start flowering and are ready for transformation to produce transgenic lines (Fig-4C). This means it is time to switch them from top to mid shelves. Only healthy looking flowering plants are used for transformation.

Transformation and separation of lines: – Tranformation is achieved by infecting seeds with Agrobacterium, which transfers the DNA of interest to the the developing ovule and produces transgenic seeds. Arabidopsis seeds are contained within siliques (seed capsules), so before every transformation I make sure to cut all the already-formed siliques to increase the efficiency of getting transgenic seeds. After transformation, I lay the plants down in a flat (Fig-5A)  for two days, after which they are placed upright and watered with nutrient solution. One week after transformation the plants need to be stacked and tied (Fig-5B), both to support them and to avoid any contact or entanglement with other plants. Contamination is the main issue while maintaining a transgenic line, so I use Aracons (Fig-5C), which provide proper air to the plants and help avoiding the mixing of seeds to other lines.

Fig-5. A) Plant after transformation. B) Tied plants after transformation. C) Plants in Aracons

Harvesting and store seeds: – After 3 or 4 weeks of transformation, plants become brown and dried, indicating that they are ready for harvesting (Fig-5B).  Using scissors I carefully cut the plant from the pot, rub them with my fingers and strain the seeds to remove pods and plant debris (Fig-6). One plant can give thousands of seeds.

Fig-6. Harvesting tools

I always collect seeds in tubes containing 1 or 2 desiccant pellets. For long-term use, we store seeds in a desiccator at -20c. If seeds are not properly dry, over time their germination efficiency decreases.

Transgenic plants: – After collecting the transgenic seeds I and sow them in a big black tray (not in pots). Transgenic plants are resistant to the herbicide glufosinate-ammonium, so I spray the plants with this herbicide to select for successful transformants.

Fig-7. A) Seeds are planted for selection. B) Transgenic plants after selection

Advantages and disadvantages: – There is no need to be present around the clock, but the growth chamber has to be monitored frequently. There is no fear of any infection, blood or need for surgery, and it is a very clean and friendly model organism. With proper care, seeds will remain viable for years, so it is possible to return to old projects easily. The only major disadvantage is the chance of cross-contamination between seed lines. One line can be contaminated easily when other groups or two researchers are working on different project in the same area. Also, the generation time of six to eight weeks is a bit slower than other systems. But in the meantime, there are always molecular experiments to do.

*The number of genes in the Arabidopsis genome was edited from 2500 to 25000 (17/02/14)

This post is part of a series on a day in the life of developmental biology labs working on different model organisms. You can read the introduction to the series here and read other posts in this series here.

(14 votes)

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This 7 day workshop is intended for wet lab researchers who want, at least, to extend their understanding of computational analysis tools and methods, but will probably also want to acquire grounded computational skills to enable them to work independently. They may be PIs, post docs or PhD students, but are less likely to be research technicians.

Registration for the 3rd Xenopus Bioinformatics workshop is now open.

The course will be highly practical and will involve working with or analyzing real data to illustrate all of the skills, techniques and approaches covered. All of this data will be drawn from real experimental work in Xenopus.

Although geared towards Xenopus researchers, individuals working on other models are welcome to attend. Last year we had several people working on other systems (including axolotl and the three-toed jerboa) who participated.

‘No biologist left behind.’ Skills will be taught soundly and progressively, and with sufficient practice, so that no reasonably competent and attentive person has trouble keeping up with the basic skills being taught.

The best kit for delivering the course for maximum impact and take-out value will be the users own laptop.

Attendees will be encouraged to bring/develop their own mini-projects during the course, but there will always be relevant data available for people who do not bring their own.

We will try and in advance to determine which high level methods are likely to be of most interest, but should not necessarily be limited to only teaching things that people know in advance that they want to learn.

There will be a relatively low emphasis on touring ‘useful web sites’, external data sources will be explained and referenced as they are needed as source of data or targets of analyzed data for visualization.

More information can be found at the National Xenopus Resource web page under the workshops tab.

Instructors
Leonid Peshkin, Harvard University
Taejoon Kwon, Univerity of Texas at Austin
Virginia Savova, Harvard University
Ian Quigley, Salk Institute

(1 votes)

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