The community site for and by
developmental and stem cell biologists

A day in the life of a larval-microbe lab

Posted by , on 21 March 2017

Hello, my name is Tyler Carrier and I am a second year PhD student in the laboratory of Adam Reitzel at the University of North Carolina at Charlotte. Broadly speaking, this laboratory focuses on the ecology, evolution, and development of marine invertebrates and their life history stages. The basis of this work uses echinoderms and cnidarians, with most efforts pertaining to the intertidal starlet sea anemone Nematostella vectensis. I, however, have gone against this common thread and work with larval stages of echinoids (sea urchins); namely, the contribution of associated-microbiota in their development, evolution, and response to environmental variation.

 

Echinoids and their larvae

Sea urchins are part of the Phylum Echinodermata (Class: Echinoidea) and are close relatives to sand dollars (Echinoidea), sea stars (Asteroidea), brittle stars (Ophiuroidea), sea cucumbers (Holothuroidea), and feather stars (Crinoidea). Sea urchins are a group of bottom-dwelling (benthic) invertebrates found throughout the world’s ocean and are the primary grazers of sub-tidal kelp beds. In many cases, urchins spawn annually by releasing gametes into the water column where fertilization and larval development take place.

 

Left: Purple sea urchin, Strongylocentrotus purpuratus; Center: Red sea urchin, Mesocentrotus franciscanus; Right: Green sea urchin, S. droebachiensis. Larval forms for each are below.

 

Urchin larvae, termed the echinopluteus, develop in the water column for weeks to months, during which they are at the mercy of ocean currents and, as a result, may be transported great distances from their parental origin. While developing the echinopluteus faces a multitude of environmental variants, including salinity, temperature, and food quantity. Over the course of evolutionary time, the echinopluteus has coped with these variants by altering their physiology and developmental trajectory.

 

My dissertation aims to shed light on if and how associated microflora provide larvae the genomic compliment to cope with abiotic and biotic environmental variables. Microbes associated with the animal host often differ considerably in the laboratory from that in nature. Therefore, to most accurately assess my research questions, I journey from the comforts of my bench to the field, where I collect and spawn adult urchins and rear their larvae.

 

A typical day in the field

Days in the field do not, in any way, reflect those at the bench. Time in Charlotte is consumed by processing samples: DNA extractions, PCRs (and re-doing PCRs because of contamination – there are non-larval bacteria everywhere!), gel electrophoresis, using QIIME and R to analyze 16S rRNA datasets, and writing grants and manuscripts. When compared to the field, those days are boring! And, as with many field-oriented biologists, the few weeks spent in the field dictate productivity for the year. Below I will outline a day from recent fieldwork at the Friday Harbor Laboratories (University of Washington), a field station located on an island wedged between the Olympic Peninsula and Vancouver Island.

 

Days in the field are often never-ending, but the natural beauty between the land and sea are indescribable.

 

06:00: My alarm goes off, I stumble out of bed, and, as it was the day before, find that the sun is just rising. To start the day refreshed, I shower and head to breakfast.

 

07:00: Prior to caring to my larvae, I make a list of what must be done today and another for what I’d like to do if I find some spare time. The first list typically includes a tally of which cultures of larvae need to be sampled, imaged on the resident compound microscope, and need a water change and to be fed. Depending on the day or week, I tend to note that adult urchin cultures need feeding (incase I need more larvae) and to do so need to get in a row boat and collect drift kelp around the dock.

 

07:30: I begin by using a hemocytometer to approximate the density of my phytoplankton cultures (for feeding larvae).

 

08:00: One by one, I reverse filter larval cultures (three liter glass jars) down to ~100 mL. Each takes approximately ten minutes, and, depending on the day, I filter 12 or 24 cultures.

 

After each jar is reverse filtered, I transfer the remaining seawater to a finger bowl and, using a dissecting microscope, count ~100 larvae and transfer these to a 1.5 mL pre-labeled Eppendorf tube. This is one of the most important steps and focal points because here I am able to survey larval health, and am able to make anecdotal observations that may be interwoven with empirical results.

 

Following reverse filtration and sampling, larvae caged in Eppendorf tubes are spun into a pellet and seawater is replaced with a fixative (RNAlater) to preserve most DNA and RNA. These fixed samples are then moved into a -20 °C freezer.

 

12:30: With sampling complete, it is time to break for lunch and to catch up with resident and visiting scientists, their students, and those taking summer courses.

 

13:30: Having been fed, it is time to make sure the larvae are, as well. Based on phytoplankton counts this morning, I subsample phytoplankton cultures and allocate food quantities to their respective jars.

 

14:00: Now that husbandry is done, it is time to image larvae from each treatment. A small aliquot of larval cultures are transferred to finger bowls and are brought to the resident compound microscope.

 

16:00: To clear the mind from a taxing daytime session and to start de novo for the nighttime shift, I trade my laboratory attire for running gear and explore the island in a 45-minute to an hour run before dinner. Although this may take away from research time, it allows the rest of the day to be more focused and productive. Plus, the views aren’t half bad!

 

Jackson Beach, Friday Harbor, WA

 

18:00: To be able to distinguish larval-specific microbes from environmental microbes, it is essential to sample the seawater, as well. While waiting, this semi-free time allows me to read a paper or two.

 

19:30: To monitor the effect of my experimental treatments on larval growth, the evening begins by analyzing the images taken before dinner. For this I use ImageJ and a cup of coffee. Hey, at least the larvae are adorable!

 

22:00: One activity that hasn’t been featured yet is e-mail and writing, and that’s because larvae take priority. To conclude the day, I catch (back) up on e-mail and try to write a couple paragraphs for upcoming grants and manuscripts in preparation. This takes me until about mid-night, by which time I should make my way towards bed… but tend to get distracted with a game or two of billiards or ping-pong.

 

00:30: Until tomorrow…

 

Thumbs up (5 votes)
Loading...

Tags: , , , ,
Categories: Lab Life

Navigate the archive

Use our Advanced Search tool to search and filter posts by date, category, tags and authors.

The people behind the papers: Holly Voges, Enzo Porrello & James Hudson

Posted by , on 15 March 2017

The use of organoids – which can be defined as artificially grown masses of cells or tissue that resemble organs – in basic and clinical research has snowballed in recent years, providing insight into fundamental developmental processes and disease etiology. Today’s paper comes from the new Special Issue of Development devoted entirely to organoids, and reports the use of human cardiac organoids to address the regenerative capacity of the immature heart. We caught up with first author Holly Voges, and her co-supervisors Enzo Porrello and James Hudson, who jointly run the Cardiac Regeneration Lab in the University of Queensland.

 

James, Enzo and Hannah in the lab

 

So James and Enzo, how did you come to co-run the Cardiac Regeneration lab in the University of Queensland?

JH I graduated with a Bachelor of Engineering (Chemical & Biological) from the University of Queensland in 2006. Rather than getting a job in the mining/oil and gas industry I decided to do a PhD in tissue engineering as I found this a more rewarding career path for my inquisitive personality. I graduated with a PhD in 2011 under the guidance of Prof Justin Cooper-White who was one of the prominent Australian researchers at the forefront of bridging the engineering and biology disciplines. From here I really wanted to apply my skills and propel my research in cardiac tissue engineering to the next level. I therefore approached Prof Wolfram-Hubertus Zimmermann one of the world leaders in cardiac tissue engineering to do a postdoc in his lab. My work in Prof Zimmermann’s lab from 2011 to 2012 was in part supported by a postdoctoral research fellowship from the German Cardiology Society, which enabled me to develop new cardiac differentiation protocols and tissue engineering strategies as part of the team Prof Zimmermann had put together. I was then awarded a National Health and Medical Research Council of Australia Early Career Fellowship for 2013-2016 and therefore decided to move back to Australia to continue my research in cardiac tissue engineering. Upon returning to Australia my goal was now to build on my previous work and use human cardiac organoids (hCOs) to find novel therapeutics for patients with cardiac disease. This is when I teamed up with Dr Enzo Porrello who also had the same goal with his work in mice on cardiac regeneration. Ever since then we have been working together to decipher how cardiomyocytes regeneration is regulated in the hope of finding novel regenerative therapeutics for cardiac disease.

 

“We have been working together to decipher how cardiomyocytes regeneration is regulated in the hope of finding novel regenerative therapeutics for cardiac disease”

 

EP I completed my PhD at The University of Melbourne and Baker Heart and Diabetes Institute. My PhD thesis focused on the developmental origins of cardiac hypertrophy, which is when I first became interested in understanding the mechanisms controlling cardiomyocyte cell cycle arrest in the neonatal period. I was subsequently offered a postdoctoral position in Eric Olson’s lab at UT Southwestern Medical Center and I moved to Dallas in 2009. During my time in the Olson lab, working together with Hesham Sadek, we uncovered a previously unappreciated regenerative capacity of the neonatal mouse heart. My research since then has focused on understanding the mechanisms that control cardiac regenerative capacity during the neonatal period in mammals. When I returned to Australia in 2012 to set up my independent laboratory at The University of Queensland, one of the questions I was very interested in was whether the human heart retained a similar regenerative potential during foetal/neonatal life. At the time, pluripotent stem cell-derived cardiomyocytes were emerging as a new technology platform to study human cardiac biology. When I first met James Hudson in 2013 we discussed numerous aspects of cardiac developmental and regenerative biology for several hours and realised that we had a unique opportunity to combine our skill sets to address some important questions in the field. We merged our labs in 2014 and our joint research program takes advantage of our respective skill sets in human cardiac tissue engineering and in vivo mouse models to develop much needed regenerative therapies for heart disease.

 

What is Brisbane, and Australia in general, like for stem cell and regeneration research?

EP & JH There are world class researchers in Brisbane in this area, not only in cardiac but in different fields of research. Australia wide the community is also strong as the Australian government has supported a big stem cell and regeneration initiative “Stem Cells Australia” which started in 2011 ($21 million). The funding will run out soon so hopefully another program is funded to continue to move our research forward, as there has been some exciting progress. We also have an annual meeting run by “Australasian Stem Society for Stem Cell Research” which is attended each year by Australian and also prominent international researchers each year. The International Society for Stem Cell Research (ISSCR) annual meeting is the largest in our field, and next year it will be in Melbourne Australia so hopefully there will be a strong Australian contingent at this meeting next year.

 

PDMS molds and poles used in the study, from Figure 1, Voges et al, 2017

 

And Holly, how did you come to join the lab?

HV I was lucky enough to join the lab as an undergraduate student when Enzo was setting up at UQ. I came across the lab profile online and thought his work was really interesting so I contacted him to see if I could do a 6 month project and here are we now, five years later!

 

Before you started your work, what was known about the regenerative capacity of human hearts of different ages?

EP, JH & HV Landmark papers from Enzo’s earlier work for the first time demonstrated that the regenerative capacity is lost during development in mammals. In contrast to the adult, newborn mice are capable of fully regenerating their hearts after injury (resection and myocardial infarction), a finding which is now widely supported by data from multiple labs. It has been known by clinicians for some time that in early life patients undergoing cardiac insults have better outcomes and heart recovery than adults. There has also been a recent case study demonstrating full recovery from myocardial infarction in a newborn. However, it was not fully understood whether this recovery/regeneration was due to cardiac regeneration or clinical management of these patients.

 

And what does organoid technology bring to the table for research into heart development and repair?

EP, JH & HV It provides us with an in vitro human model of cardiac regeneration we can use to complement our in vivo studies in mice. Mice and humans have many differences in their biological and physiological properties and models such as this give us an additional tool in our quest for regenerative therapeutics.

 

Morphological features of human cardiac organoids, from Figure 1, Voges et al. 2017.

 

Can you give us the key results of the paper in a paragraph?

EP, JH & HV Our hCO display properties and maturity consistent with fetal-like human heart tissue. We developed an injury model in this paper to determine how the hCO would respond to injury. In this study we chose to model disease using cryoinjury because it enables us to injure part of the tissue whilst keeping the remainder of the tissue healthy and viable. If we used ischemia-reperfusion or a chemical insult (eg. cardiotoxin) it would have affected the entire tissue. We then showed that the cryoinjured tissues have a high cell cycle activity and they can regenerate and recover their functionality following injury.

 

Do you have any idea why regenerative capacity is lost with heart maturation? Would more ‘mature’ organoids be poor regenerators?

EP, JH & HV In our on-going work in the hCO and in vivo in the mouse we are starting to understand more about the maturation process and how this regulates cardiomyocyte cell cycle and regeneration. Part of our work is finding the key drivers of maturation in hCO and whether mature hCO have the capacity to regeneration. We believe that more mature hCO will lose their regenerative potential.

 

Localized loss of α-actinin and nuclear staining in cryoinjured cardiac organoids, from Figure 2, Voges et al, 2017

 

Will your model have relevance for clinical avenues to cardiac repair in adults?

EP, JH & HV Our hope is to use this model to find key regulators of cardiac regeneration (together with our in vivo mouse models). Through these studies, hopefully we can find factors that can be used to facilitate cardiac regeneration in adult hearts and patients with cardiac disease.

 

And Holly, when doing the research, did you have any particular result or eureka moment that has stuck with you?

HV There were a number of really exciting moments along the way and it is great to be surrounded by people who share this excitement. One memory that stands out was when I finished collecting the organ bath data. This proved to be a time-consuming and painful process, so when I finally saw the exciting results I immediately ran down the hallway to show James and Enzo.

 

Expression of Ki-67+ cells in cryoinjured cardiac organoids, from Figure 3, Voges et al. 2017

 

And what about the flipside: any moments of frustration or despair?

HV I definitely had many moments of frustration throughout this project. Most of the time it felt like the moments of despair outweighed the eureka moments, but that only made the eureka moments more exciting.

 

What about your plans for the future following this paper?

HV I’m currently working on my next paper, which will enhance our hCO system to be a step closer to resembling native heart tissue by incorporating other endogenous cell types.

 

And what next for the lab?

EP & JH We are working together and also with academic and industry collaborators to try and study the cardiac regeneration process and the molecular mechanisms governing cardiomyocyte cell cycle at different stages of development. The holy grail is to find novel regenerative therapeutics for cardiac disease.


 

Holly K. Voges, Richard J. Mills, David A. Elliott, Robert G. Parton, Enzo R. Porrello, James E. Hudson. 2017. Development of a human cardiac organoid injury model reveals innate regenerative potential. Development, 144(6): 1118-1127

Browse the People behind the Papers archive here.

 

Thumbs up (4 votes)
Loading...

Tags: , ,
Categories: Research

On Growth and Form conference – Call for Papers

Posted by , on 14 March 2017

On Growth and Form 100 logo

ON GROWTH AND FORM CENTENARY CONFERENCE

13-14 October 2017

University of Dundee and University of St Andrews

2017 marks 100 years since the publication of D’Arcy Thompson’s landmark book On Growth and Form.

To mark the occasion, a two-day interdisciplinary conference is being organised at the Universities of Dundee and St Andrews, where D’Arcy spent most of his career and where his surviving collections are held. It will feature a range of presentations covering every aspect of D’Arcy’s own work and the various fields that it has influenced. The conference will also include visits to the D’Arcy Thompson Zoology Museum and the Bell Pettigrew Museum of Natural History and there will be a special preview of a new exhibition exploring On Growth and Form and its legacy.

We would like to invite you to submit proposals for any of the following:

  • 30-40 min presentations giving an overview of D’Arcy’s influence in a particular field (eg cybernetics or anthropology)
  • 20 min papers focusing on a more specific area of influence – eg a particular research area (past or present) that has been illuminated by his ideas, or a particular aspect of D’Arcy’s own career
  • 10 min spotlight talks describing your own current research in an area that connects to D’Arcy’s work
  • Themed sessions combining three 20 min or six 10 min presentations, as outlined above. Session chairs would be expected to get commitment from individual speakers in advance of their submission, and provide separate abstracts for each.

Proposals should take the form of an abstract of not more than 300 words, which should be sent to Matthew Jarron, University of Dundee Museum Services on museum@dundee.ac.uk by 2 May 2017.

ADDITIONAL EVENT

The conference will be followed by a Newton Institute workshop hosted by the University of Dundee 16-20 October 2017 on Growth, Form and Self-Organisation in Living Systems – see https://www.newton.ac.uk/event/gfsw02

Thumbs up (1 votes)
Loading...

Categories: News

Choosing Sides: Two steps in establishing the polarisation vector in algal zygotes

Posted by , on 14 March 2017

Comment on “Two-step cell polarization in algal zygotes”, Nature Plants, 3, 16221, (2017).

Department of Biology, Ghent University, Krijgslaan 281 S8, 9000 Ghent, Belgium

VIB-UGent Center for Plant Systems Biology, Technologiepark 927, B-9052 Ghent, Belgium

Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium

 

Complex multicellular life has evolved from unicellular organisms along at least five independent paths, giving rise animals, plants, fungi, red algae and brown algae, respectively. Asymmetric divisions are key in this process as a means to create diverse cell types. At the molecular level, the uneven distribution of molecular components such as mRNA, proteins or organelles determines a polarisation vector. A subsequent cell division along this polarization vector results in two daughters cells which are no longer equivalent and receive different cell fates.

In recent years, insights in the molecular mechanisms determining cell polarisation and asymmetric cell division have mainly emerged from investigations on animals. With exception of yeasts, the mechanisms of polarity establishment are much less well established in other groups of eukaryotes. While many model cell polarity systems from land plants lend them to the research of polarity signalling, polarity is often either predetermined (e.g. pollen) or the cells are enclosed in the surrounding tissue (e.g. zygotes), which renders them less suitable for studies examining polarity establishment. Conversely, eggs of brown algae are released in the surrounding seawater as radial symmetric spheres and polarity is established after fertilization. Since the middle of the 19th century, there has been interest in the gametes, zygotes and embryos of fucoid brown algae (Fucus and Silvetia) and a considerable effort has been invested in the study of their gamete recognition and fertilisation, cell wall biosynthesis and polarity acquisition. One asset of these organisms is their broadcast spawning nature. Gamete releases can be experimentally induced resulting in large populations of synchronously developing embryos. In addition, the zygotes are also large (60–100 µm) and therefore easy to (micro-)manipulate.

In fucoids the mechanism of cell polarisation has been well established. Upon fertilization the apolar egg cell develops a cell wall and starts the polarization process. The sperm entry site and subsequently the light direction provide cues for establishing the polarization vector de novo and both determine the direction and sense of the polarisation axis simultaneously. Unfortunately, it is very hard to culture fucoid brown algae. Hence, they are less amenable to genetic and molecular studies. Similarly the development of the cultivable, but isogamous, brown alga Ectocarpus siliculosus as a successful model for genomic research has been accompanied by a shift towards the developmental research questions related to life cycle and fertilisation biology, but away from polarity establishment.

 

[Scheme - Fucus cell polarisation]
Figure 1. The polarization process in fucoid zygotes. A. Fertilized zygote, showing a sperm induced F-actin patch. B. Zygote polarizing according to the light direction. C. Polarized zygote showing a photopolarized F-actin patch, polarized endomembrane cytoskeleton and polarized MT cytoskeleton. D. Asymmetrically divided embryo with a thallus and rhizoid cell. Adapted from Bogaert et al. 2013.
 
 

 

Since many years, the Phycology Group of Prof. Dr. Olivier De Clerck is studying diverse biological aspects of the brown alga Dictyota for which laboratory culture are more easy to maintain. Fascinated by plant developmental biology I approached Prof. Dr. Olivier De Clerck to inquire for a master dissertation. Coincidentally while studying the mechanism of gamete recognition in Dictyota in the summer of 2007, Prof. Dr. Olivier De Clerck observed that the spherical egg cells deform into elongated rugby ball shaped spheroids after fertilization. Propelled by this finding a master dissertation subject was discussed over some Belgian beers and we teamed up with Prof. Dr. Tom Beeckman of the Root Development group VIB – PSB in Ghent. Early development of the Dictyota embryo quickly turned out to be different from Fucus and the collaboration ultimately matured in a PhD fellowship funded by the FWO (Research Foundation – Flanders).

 

[habitus Dictyota]
Figure 2. Female gametophyte of Dictyota dichotoma in the field (credits: H. Verbruggen).
 

I vividly remember the excitement, observing that the egg cells of Dictyota elongate minutes after addition of the male gametes. Within three minutes a population of spherical egg cells was transformed into a homogenous population of rugby ball shaped cells, with the elongation observable under the stereomicroscope. From that moment, it was clear that the direction of the elongation must have been predetermined; no process could be so fast to determine an elongation direction after fertilisation in those mere seconds. This was later corroborated by TEM sections showing a heterogenous organisation of the organelles suggesting the presence of a preformed axis in the unfertilized eggs. During a research stay in the lab of Dr. Susana Coelho and Dr. Mark Cock at the SBR Roscoff I managed to visualize the autofluorescence of the chloroplasts of eggs being fertilized and provided in vivo evidence that the preformed axis indeed represents the elongation axis.

It is peculiar that this notion has been left unnoticed for so many years given the many efforts of John Lloyd Williams in the late 19th century describing the periodicity of gamete release and the parthenogenetic development of Dictyota dichotoma. Prof. Dr. Dieter G. Müller, best known for his ground breaking work on Ectocarpus siliculosus, his early work also concentrated on Dictyota dichotoma describing the release periodicity of the female gametophytes in laboratory conditions. Only later we heard that Prof. DG Müller observed in the 60s the very same elongation of the eggs, while working for the developmental biologist Prof. Dr. Lionel F. Jaffe. The observation was received with much excitement and Jaffe suspected fundamental differences with the fertilisation response of fucoids, but a technical difficulty in the culturing withheld them from following up this observation. Most probably priorities must have laid in detangling the fertilisation and polarisation of the zygote of Fucus at that time.

 

[chlorooplast distribution - elongation]
Figure 3. Chloroplast autofluorescence showing the preformed axis predicts the elongation axis. Adapted from Bogaert et al. 2017.

The notion to walk in the footsteps of two of the most iconic early developmental biologists working on brown algae was most exciting to us. Quickly we realized that the elongation had implications for the way the cell polarize. While the egg cell has a predetermined elongation axis the direction of the polarisation vector, (aka ‘the sense of the polarization vector’) is not predetermined. In other words, it is not decided which side is going to develop into the leaf-like upper part or the lower root-like part of the alga. This was best illustrated by the fact that while the elongation direction was unresponsive to the direction of the light, which of the two poles will develop into the rhizoid is determined by the direction of the unilateral light. Moreover, toluidine blue O staining – a marker for permanent fixation of the rhizoid pole in fucoids – was only observed hours after fertilization. Therefore determination of the polarisation vector in Dictyota is inherently a two-phased process where initially the direction of the polarisation vector is determined and only later also the sense of the polarization vector.

 

[scheme Dictyota, Fucus, Arabidopsis]
Figure 4. A scheme comparing zygote polarization in Dictyota, Fucus and Arabidopsis. Timing of the two phases of polarisation are denoted in blue. Red dashed line represents direction of the polarisation vector (without sense). Red line with arrow denotes a polarisation vector with both direction and sense. Adapted from Bogaert et al. 2017.
 

These two phases are not only completed with a different timing, they also rely on at least two different mechanisms because they depend on two different cues. While the direction is maternally determined, the sense of the polarisation axis is determined by the light direction. Due to the different timing, the two phases even occur in different life stages: the direction in the polarisation vector in the oogonium and the sense is determined hours after fertilization in the diploid sporophyte. In animal systems it is however well known that developmental processes during the first cell divisions are under control of the transcriptome transcribed before egg arrest because de novo transcription (zygotic genome activation) is postponed. This is termed the maternal-to-zygotic transition. The broadcast spawning nature makes it relatively easy to yield large populations of cells, because we did not have to rely on microdissection techniques and FACS techniques. Therefore with some moderate effort we could obtain three biological replicate libraries of mRNA of gametes, zygotes and asymmetrical divided embryos. This allowed us to show that there is a large degree of zygotic genome activation taking place already during the first cell cycle, showing that the next generation is indeed acquiring developmental control over the cell polarisation.

I am thrilled with this publication, because it once more shows that research on oogamous brown algae is not from the past. The availability of an oogamous broadcast spawning brown alga that can be easily cultured and releases its gametes into the external medium opened up the opportunity for developing it into a research model complementary fucoids. This publication however shows that different and complementary insight can be gained from Dictyota early development.

References

Bogaert KA, Arun A, Coelho SM, De Clerck O (2013) Brown algae as model for plant organogenesis. In Methods in Molecular Biology: Plant Organogenesis (De Smet I ed). Humana Press. Springer Protocols, Heidelberg, 959: 97-125.

Bogaert KA, Beeckman T, De Clerck O (2017) Two-step cell polarisation in algal zygotes. Nature Plants 3: doi:10.1038/nplants.2016.221.

Thumbs up (3 votes)
Loading...

Tags:
Categories: Research

Register for Cellular Dynamics: Membrane-Cytoskeleton Interface meeting by 21st of March!

Posted by , on 14 March 2017

The deadline for applications for this exciting meeting is approaching fast.

For more information, or to apply, click here.

Organisers: Michael Way, Elizabeth Chen, Margaret Gardel and Jennifer Lippincott-Schwartz
Date: 21 – 24 May 2017
Location: Southbridge Hotel & Conference Center, Massachusetts, USA

Speakers:
Anna Akhmanova (Utrecht University, The Netherlands)
Daniel Billadeau (Mayo Clinic, USA)
Anthony Bretscher (Cornell University, USA)
Gaudenz Danuser (University of Texas Southwestern Medical Center, USA)
Cara J. Gottardi (Northwestern University Medical School, USA)
Kathleen J. Green (Northwestern University Medical School, USA)
Erika Holzbaur (University of Pennsylvania, USA)
Johanna Ivaska (University of Turku, Finland)
Tomas Kirchhausen (Harvard Medical School, USA)
Sophie Martin (University of Lausanne, Switzerland)
Mark Peifer (University of North Carolina, USA)
Jenny Russinova (Ghent University, Belgium)
Erik Sahai (The Francis Crick Institute, UK)
Giorgio Scita (IFOM Foundation & University of Milan, Italy)
David Stephens (University of Bristol, UK)
William Trimble (Sick Kids Research Institute, Canada)
Kristen Verhey (University of Michigan, USA)
Gia Voeltz (University of Colorado, USA)
Kenneth M. Yamada (NIH/NIDCR, USA)

Thumbs up (No Ratings Yet)
Loading...

Categories: Uncategorized

In Development this week (Vol. 144, Issue 6) – SPECIAL ISSUE ON ORGANOIDS

Posted by , on 14 March 2017

The current issue of Development – our Special Issue on Organoids – features a collection of review- and research-based articles focusing on organoids. Here are some of the highlights. Happy reading (and thanks to everyone who contributed)!

 

Organoids: a Special Issue

In her Editorial, Melissa Little provides an overview of the entire contents of the Special Issue, highlighting some of the important findings and major themes therein.

 

The hope and the hype of organoid research

In their Spotlight article, Meritxell Huch and Juergen Knoblich, together with Matthias Lutolf and Alfonso Martinez-Arias, discuss the exciting promise of organoid technology, as well as the current limitations and what it will take to overcome them.

 

Ethical issues in human organoid and gastruloid research

Megan Munsie, Insoo Hyun and Jeremy Sugarman summarize some of the important ethical issues associated with research involving human organoids and other complex self-organized structures. Read their Spotlight article.

 

The physics of organoids: a biophysical approach to understanding organogenesis

Svend Dahl-Jensen and Anne Grapin-Botton highlight some interesting applications of physics in organoid research, from computational modeling of organoid biology to understanding mechanical aspects of organ development. Read their Spotlight article.

 

Using brain organoids to understand Zika virus-induced microcephaly

In their Spotlight article, Hongjun Song, Guo-li Ming and colleagues summarise the latest advances in using cerebral organoids to model Zika virus infection and the resulting pathology. 

 

Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish

This Development at a Glance poster article by Heather McCauley and James Wells summarises how knowledge gained from developmental biology can be used to guide human in vitro organogenesis, and discusses the potential applications of this technology.

 

Translational applications of adult stem cell-derived organoids

Jarno Drost and Hans Clevers discuss how adult stem cell-derived organoids can be used to model human diseases, create personalized cancer therapies and further efforts in regenerative medicine. Read the Primer article.

 

 

 

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

In their Review article, Mijo Simunovic and Ali Brivanlou discuss the basic physical and biological principles that underlie the self-organization of embryonic stem cells into organoids, and how this informs human development.

 

 

Lung organoids: current uses and future promise

Brigid Hogan and colleagues explore the latest advances in both adult and embryonic stem cell-derived lung organoid culture, and discuss how these systems can be used to understand homeostasis and regeneration. Read their Review.

 

Dissecting the stem cell niche with organoid models: an engineering-based approach

In their ReviewZev Gartner and co-workers highlight how organoids have been used to model and characterize stem cell-niche interactions and how new engineering approaches enable systematic study of the stem cell niche.

 

PLUS:

 

This Special Issue also contains a number of Research Reports, Research Articles and Techniques & Resources Articles – click here for a full listing!

 

 

 

 

Thumbs up (1 votes)
Loading...

Categories: Research

Postdoctoral scientist in genomics of human stem cell differentiation at Cincinnati Children’s

Posted by , on 9 March 2017

Closing Date: 15 March 2021

We seek a highly motivated postdoctoral fellow to spear head research investigating the genomic basis of transcriptional specificity of cell signaling during the directed differentiation of human pluripotent stem cells into the digestive and respiratory organoids. The goal is to understand how different transcriptional programs are activated by the same cell signaling pathways, at different times in development and disease. You will join a multidisciplinary team in the Zorn Lab, Division of Developmental Biology at Cincinnati Children’s Hospital Research Foundation.

 

Qualified applicants will have a PhD with peer review research publications, a demonstrated expertise in genomic analysis, and a keen interest to develop an independent research program in the area of genomics, development and stem cell biology.

 

Please submit your application to aaron.zorn@cchmc.org with the following information: a cover letter, statement of interest, and CV with contact details for 3 referees.

 

Closing Date: May 31st, 2017

The Cincinnati Childrens Hospital Medical Center, and the University of Cincinnati are Affirmative Action/Equal Opportunity Employers. Qualified women and minority candidates are especially encouraged to apply.

Thumbs up (No Ratings Yet)
Loading...

Categories: Jobs

BBSRC iCase 4 Year PhD Studentship (Lee – Fellows)

Posted by , on 8 March 2017

Closing Date: 15 March 2021

Department/Location: Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Reference: PS11650

Closing date: 31 March 2017

Collaborative PhD Studentship with Astra Zeneca

Elucidating cellular behaviours and potential oncogenicity utilising in vitro organoid culture system Project Description

Department name: Cambridge Stem Cell Institute, University of Cambridge Supervisors: Primary: Dr Joo-Hyeon Lee (University of Cambridge) and Dr Mick Fellows (AstraZeneca)

Assessment of cell transformation (i.e. the acquisition of malignant characteristics in morphology, growth control or function) has proven problematic to model in vitro. The soft agar cell transformation assay has been validated, but only in a limited number of immortalised or mixed population embryonic cells and the specificity of the assay has been questioned. Furthermore, the genetic basis of cell transformation in this model has not been fully delineated (Harvey et al 2015, Creton et al 2012). The use of genetically and phenotypically stable organoids derived from single human stem cells (Schwank et al 2013) should provide a better model for cell proliferation and transformation.

Organoids have the advantage of 3D morphology, being able to be derived from several major organs and can be engineered to demonstrate proliferative effects by introducing a cell type specific ki67 fluorescent protein to tag. Organoid stability is also dependent on several growth factors and inhibitors e.g. EGF, Wnt, BMP inhibitor, TGFb inhibitor. Growth following withdrawal of these factors, which are known pathways implicated in cancer genesis, will be indicative of cell phenotypic and/or transformative changes. It is also proposed that this model could be used to investigate mechanisms of pro-oncogenicity and also the potential off-target effect of genome editing by CRISPR/Cas9, for which there is currently an unmet need for in vitro assays to assess concerns around inappropriate editing in oncogenes.

The project will develop organoids from a variety of tissue sources, including organoids with the ki67 tag, and assessment of proliferative and morphological changes following withdrawal of growth factors after treatment with reference carcinogens and following transfection with specific and promiscuous guide RNA and associated Cas9 protein. Comparative data will be generated from alternative in vitro methodologies to analyse cell transformation e.g. the soft agar assay. Reference carcinogens will be analysed in this assay along with the potential of CRISPR/Cas9 editing to induce cell transformation (which has not been previously assessed). The student will also use next generation sequencing technologies to identify specific genes involved in morphological changes in both cell and organoid cultures. Success will provide new and more relevant model for understanding and assessment of morphological changes and carcinogenesis.

Eligibility and Funding

This studentship covers 4 years’ UK/EU tuition fees (see below for EU eligibility requirements) and a maintenance stipend.

BBSRC funding is available for UK nationals and EU students who meet the residency requirements. Further information about eligibility for funding can be found on the BBSRC website: http://www.bbsrc.ac.uk/documents/studentship-eligibility-pdf/

How to Apply

Please visit our website to find out more, and about the application process itself: http://study.stemcells.cam.ac.uk/study/otheropportunities/#BBSRC-iCASE-ASTRAZENECA.

Deadline for receipt of applications: midnight on Friday 31st March 2017.

References

Harvey, Howe et al. Mutagenesis. 2005 Jan;20(1):51-6. http://mutage.oxfordjournals.org/content/20/1/51.long

Creton, Aardema et al. Mutagenesis. 2012 Jan;27(1):93-101. http://mutage.oxfordjournals.org/content/27/1/93.long

Schwank, Koo et al. Cell Stem Cell . 2013 Dec 5;13(6):653¿58. http://www.sciencedirect.com/science/article/pii/S1934590913004931

Thumbs up (No Ratings Yet)
Loading...

Categories: Jobs

Funded PhD projects in Mathematical Modelling of stem cell dynamics at University of Southampton

Posted by , on 8 March 2017

Closing Date: 15 March 2021

Fully-funded PhD studentships (3 years) for projects in Mathematical/Computational Biology are available at the University of Southampton (Mathematical Sciences). The PhD projects will be supervised by Drs Philip Greulich and Ben MacArthur and will involve mathematical and computational modelling of stem cell dynamics in biological tissues, in particular related to cancer and development. The candidate should have, or should expect, an upper second-class degree or higher, and a genuine interest in biological problems.

An outline of two potential PhD project topics can be found here:

https://www.findaphd.com/search/ProjectDetails.aspx?PJID=80983&LID=1703

https://www.findaphd.com/search/ProjectDetails.aspx?PJID=82402&LID=1703

For indicating interest and further information, please contact P.S.Greulich@soton.ac.uk and see www.southampton.ac.uk/maths/about/staff/psg1u16.page for information about the supervisor

Thumbs up (No Ratings Yet)
Loading...

Categories: Careers, Education, Funding, Jobs

Organelle Assembly in Vivo: The Love-Hate Relationship of Thermodynamic and Active Processes

Posted by , on 6 March 2017

Comment on ”Independent active and thermodynamic processes govern nucleolus assembly in vivo”, Proceedings of the National Academy of Sciences, 114 (6), 1335-1340, (2017).

Hanieh Falahati, Lewis–Sigler Institute for Integrative Genomics, Princeton University.

Eric Wieschaus, Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University.

 

The whole universe is moving toward disorder; this is the second law of thermodynamics in simple terms. Yet, living organisms have found a way to keep themselves organized by spending energy. Cells need this organization in order to provide specialized microenvironments for different cellular functions. Two types of intracellular organizations can be distinguished in biological systems: The first type is membrane-bound organelles such as ER, or lysosome. The composition of these organelles is maintain by spending energy and active transport of molecules across a membrane. The second type of intracellular organization comes from membrane-less organelles such as nucleoli, histone locus bodies, and stress granules that are high-concentration assemblies of different proteins and RNAs. A puzzling question is that without a membrane, how are cells able to form and maintain these organelles.

 

A historical perspective

The question of how membrane-less organelles form immediately captured my attention the first time I heard about it in an introductory meeting with my doctoral advisor, Prof. Eric Wieschaus. Of course this question has fascinated many scientists since the initial observation of the nucleolus, the quintessential membrane-less organelle, in the 18th century. In 1898, Montgomery performed a very comprehensive study of the nucleoli of different cell types, hand-drawn in 346 figures (a sample is shown in Fig. 1), and concluded that the nucleolus forms when a substance from the cytoplasm enters the nucleus and is deposited there “in the form of masses of varying dimensions, which may be either globular or irregular in shape, according as they are fluid or viscid in consistency”. He further describes that nucleoli form via “coalescence of numerous small portions of nucleolar substance”, consistent with its fluidity (Montgomery 1898). However, this model faded away with the advancement in cell biology and genetics which showed that the nucleolus forms around ribosomal DNA repeats and is the site of active transcription and processing of ribosomal RNA, and ribosomal biogenesis. By the end of the 20th century, nucleolus was commonly thought to form actively, in the process of making ribosomes, until a number of influential works from Hyman and Rosen groups redirected the focus to the liquid nature of the membrane-less organelles (Brangwynne et al. 2009; Li et al. 2012). Based on this liquid property, it was proposed that formation of membrane-less organelles is a spontaneous liquid-liquid phase separation (LLPS), similar to the separation of oil from water.

 

Figure 1. Hand drawing of nuclei with nucleoli (n) of different cell types drawn by Montgomery in 1898. N. Gr.: nuclear granule. Chr.: Chromatin.

 

By the time I joined the Wieschaus lab at Princeton, two main hypotheses existed for the formation of membrane-less organelles: 1. The LLPS model, in which the components of these organelles assemble spontaneously in a thermodynamically-driven process due to their favorable intermolecular interactions, and form a liquid phase; 2. The active assembly model which suggests that these organelles form as a result of an active process, which happens inside the cells or inside the organelles. Such active processes are reactions carried out by enzymes that couple these reactions to an energy source such as ATP. The evidence for the LLPS model comes from experiments showing that many of the proteins which localize to the membrane-less organelles have the physical properties to phase separate in vitro. However, in most cases the phase separation is only observed at concentrations much higher than cellular levels. Therefore, a pressing question is whether the physical properties of these components would also drive their spontaneous assembly in vivo, or active processes are responsible for their congregation. Accordingly, we decided to develop an in vivo test that would allow us to distinguish between the LLPS and the active mechanism, and apply it to study nucleolus assembly as a model for the formation of membrane-less organelles.

 

Working out a path – How to test the phase separation model in vivo?

Developing such an in vivo test was not trivial, since compared to in vitro systems, living cells are far more limited in the ways they can be manipulated. But considering the expertise of my advisor, Eric Wieschaus, in manipulating Drosophila embryos, his lab was the best place to do this type of research. It was not the only reason for us to study this question in fly embryos: Development in Drosophila starts without a nucleolus, and the nucleolus forms at a particular developmental stage (Video 1). This allows us to manipulate the system and study its effect on the nucleolus assembly. In addition, nucleolus forms during the 13 synchronous divisions at the beginning of development, therefore at each time-point there are multiple nuclei at the exact same developmental stage, which helps with statistics. Finally, during these 13 division the nuclei share the cytoplasm, which means that all the nuclei have access to the same concentration of most molecules. These features make fly embryos an ideal system for studying nucleolus assembly.

 

Video 1. Nuclei of D. melanogaster embryos start development without a nucleolus, and form the nucleolus (bright foci) for the first time at nuclear cycle 13.

 

How can you test whether the formation of an organelle in vivo is a thermodynamically driven LLPS, or an active assembly process? In general, LLPS processes are affected by two factors: concentration and temperature (Fig. 2). Increasing the concentration results in an increase in the size of assemblies: The more oil you add to the water, the bigger the oil droplets. However, active processes are also affected similarly with concentration: The higher the concentration of the reactants, the more products (assemblies) are formed. Therefore, changes in the concentration cannot distinguish between an LLPS and an active assembly. On the contrary, temperature often has a differential effect on these two processes: In general, lowering the temperature causes more condensation and enhances phase separation, but slows down active enzymatic reactions. This means that by quantifying the effect of various temperatures on the assembly formation, one can distinguish between an LLPS and an active assembly model.

 

Figure 2. (Left) Phase separation processes are affected by concentration and temperature. Increasing the concentration and decreasing the temperature results in the formation of larger assemblies. (Right) Active processes are carried out by enzymes which couple them to an energy source. Therefore, the rate of these processes is determined by the rate of enzyme activity, which is reduced at lower temperatures.

 

At the time, our strategy to examine the effect of temperature on the formation of assemblies was received with skepticism among the community. The main concern was whether in the temperature range tolerable by biological systems, an effect would be detectable. A reassurance, however, was an exquisite work by Sarah Veatch, who showed that plasma membrane vesicles isolated from living cells exhibit phase transitions between 15 and 25°C (Veatch et al. 2008). Subsequently, we employed a microfluidic device that allowed us to control the temperature of a fly embryo between 6 and 31°C (Fig. 3). We used this microfluidic device to examine the effect of temperature on the first time formation of assemblies, and also to test the reversibility of assembly formation, for six different nucleolar proteins.

 

Figure 3. A microfluidic device was used to control the temperature of an embryo in a range of 6 to 31°C by flowing in warm or cold water.

 

The unexpected result of two independent mechanisms

Much to our surprise, the results of our test showed that two independent mechanisms govern the assembly of different subsets of nucleolar proteins: While four of the studied proteins depict properties of LLPS, the two others are recruited actively. At lower temperatures, the phase separating proteins assemble at an earlier developmental time, and dissolve upon heating (Video 2). The latter is consistent with the reversibility of thermodynamic processes. On the contrary, the formation of active proteins is inhibited at low temperatures, and is irreversible, an exclusive property for active processes.

 

Video 2. The nuclei of intact fly embryos are subjected to temperature changes in the surrounding fluid. As the temperature is shifted from low to high, the phase separated proteins dissolve, as can be seen in the disappearance of the bright spots.

 

rDNA: Come together, right now, over me

Our unexpected result that two independent mechanisms govern nucleolus assembly in vivo raised a new question: How are these two different mechanisms coordinated to form a single organelle? In a previous paper, we had shown that transcription of rDNA dictates the spatiotemporal precision in the formation of assemblies by the phase separating proteins (Falahati et al. 2016). Interestingly, we were also able to show that rDNA is necessary for the recruitment of the two actively assembling proteins. This suggests that rDNA can function as a coordinator between the two independent mechanism. In addition, it highlights the fact that in vivo, even the assembly of phase separating proteins is regulated by active processes such as transcription.

 

We are very enthusiastic about the publication of this paper as it is a significant advancement in the field both from a methodological and mechanistic standpoint. We took an interdisciplinary approach and developed an invaluable tool for closing the gap between the current knowledge of the in vitro self-assembly and the formation of membrane-less organelles in vivo at its full complexity. From a mechanistic perspective, the large number of proteins studied here allowed us to unravel the unappreciated complexity in the formation of intracellular assemblies, as our results show the presence of at least two independent mechanisms for the recruitment of nucleolar proteins. We are confident that the method and the results introduced in this paper will set a framework to better understand how normal or pathological intracellular assemblies form.

 

 

References:

  1. Brangwynne, CP et al. 2009. “Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation.Science 324(5935): 1729–32.
  2. Falahati, H., Pelham-Webb, B., Blythe, S., & Wieschaus, E. 2016. “Nucleation by rRNA Dictates the Precision of Nucleolus Assembly.Current Biology 26(3): 277–85.
  3. Falahati, H., & Wieschaus, E. 2017. ”Independent active and thermodynamic processes govern nucleolus assembly in vivo.” Proceedings of the National Academy of Sciences, 114 (6), 1335-1340.
  4. Li, P., Banjade, S., Cheng, H.C., Kim, S., Chen, B., Guo, L., Llaguno, M., Hollingsworth, J.V., King, D.S., Banani, S.F. and Russo, P.S. 2012. “Phase Transitions in the Assembly of Multivalent Signalling Proteins.Nature 483 VN-(7389): 336–40.
  5. Montgomery, Tho S. H. 1898. “Comparative Cytological Studies, with Especial Regard to the Morphology of the Nucleolus.” Journal of Morphology 15(2): 265–582.
  6. Veatch, S. L., Cicuta, P., Sengupta, P., Honerkamp-Smith, A., Holowka, D., & Baird, B. 2008. “Critical Fluctuations in Plasma Membrane Vesicles.” ACS Chemical Biology 3(5): 287–93.
Thumbs up (2 votes)
Loading...

Tags: , ,
Categories: Lab Life, Research