Cecilia Lara-Mondragón and Mario Arteaga-Vázquez

Hello, my name is Cecilia Lara. I am an undergrad student working under the supervision of Dr. Mario Arteaga-Vazquez at the Laboratory of Epigenetics and Developmental Biology. Our lab is part of the Institute for Biotechnology and Applied Ecology at the University of Veracruz (INBIOTECA-UV). The INBIOTECA-UV is located in Xalapa, a rainy, damp, green and beautiful city in the heart of the state of Veracruz, Mexico. Its foggy nights remind you of those in horror movies where a huge guy with an axe can jump out of the bushes and chase you at any time! Right there in the south of the city is our little institute and our second home- the epilab (as we call it). At the epilab we study an emerging model, a liverwort called Marchantia polymorpha (Marchantia), which is a non-vascular plant that belongs to the most basal lineages of land plants.

Marchantia polymorpha is descendant of the first plants that colonized terrestrial environments 

Plants are fundamental for life in our planet. A major event in earth’s history was the colonization of the terrestrial environment by plants. Land plants (embryophytes) colonized terrestrial environments about 480 millions years ago. Bryophytes include the oldest extant land plant lineages (liverworts, mosses, and hornworts).There is an intense debate over the phylogenetic relationships between different bryophytes, mainly regarding which group is sister to either land plants or vascular plants, but evidence from the fossil record, molecular, systematic and phylogenomic data strongly suggests that liverworts were some of the first plants that colonized the landscape. This makes Marchantia a very interesting model to address many questions from an evolutionary perspective. For example: How did plants evolve from an aquatic ancestor? What kind of molecular and developmental innovations played an essential role during the evolution of embryophytes, resulting in the extraordinary radiation of land plants? What has been the contribution of epigenetic regulation during the evolution of land plants? This and many more questions can make more than one biologist lucubrate night after night!

Evolutionary developmental biology of paramutation and RNA-based gene silencing pathways

The main goal of our lab is trying to understand the molecular basis, evolution and developmental implications of paramutation. Paramutation is the most amazing and extreme example of transgenerational epigenetic inheritance. Paramutation is an interaction between alleles (or homologous sequences) that leads to mitotically and meiotically heritable changes in gene expression. During the last decade we started to gain insight into the genetic and molecular mechanisms of paramutation. We now know that in plants, paramutation is mediated by components of an RNA-directed DNA methylation pathway that is involved in the epigenetic regulation of transposable elements. In our lab, we are studying the function and evolution of RNA-directed gene regulation in both Marchantia and maize. Our adventure in Marchantia started in 2009 when Dr. Arteaga visited Dr. John Bowman’s laboratory in Australia. Three years later he attended a Marchantia Workshop in Kyoto, where he officially established a collaboration with Dr. Takayuki Kohchi and Dr. Bowman, both pioneers of Marchantia research and leading the Marchantia genome sequencing initiative. This collaboration has extended to many other labs in Japan, including Dr. Kimitsune Ishizaki’s group in Kobe University and Dr. Katsuyuki T. Yamato’s group in Kinki University.

General anatomy of Marchantia polymorpha

Marchantia plants are morphologically simple. They grow as a thallus (flat-sheet tissue reminiscent of leaves) (Figure 1) with rhizoids (single cell root-like filaments) growing on the lower surface and repetitive units adapted for photosyntesis on the upper surface. Marchantia reproduces both sexually and asexually. Asexual propagules (gemmae) are formed inside specialized structures (gemmae cups) (Figure 1) which are dispered by abiotic mechanical factors (e.g raindrops).

Figure 1. Marchantia thallus. A mature thallus of M. polymorpha showing gemmae cups and gemmae.

Sex in Marchantia is determined by the presence of cytologically distinct sex chromosomes, males having one very small Y chromosome while females have one X chromosome. The male and female thalli look alike, but they can be easily distinguished based on the morphology of the sexual structures they produce (Figure 2). Male antheridiophores (Figure 2A and Figure 3) or female archegoniophores (Figure 2B and Figure 4) arise from the upper surface of the thallus. Antheridiophores produce sperm-forming antheridia and archegoniophores produce egg-forming archegonia. In contrast to what is observed in angiosperms, sperm in Marchantia consist of a single motile cell capable of traveling in an aqueous environment towards the egg cell. The embryo resulting from a cross is enclosed in the gametophyte. It lacks an indeterminate meristem but grows a number of different cell types, including a sub-epidermal population of cells on the apical pole that correspond to the precursors of the germ cells that will undergo meiosis to form the spores.

Figure 2. Marchantia male and female strains. Populations of male (A) and female (B) plants with reproductive structures already induced and ready to be crossed.

Figure 3. Marchantia anteridiophores. A. Anteridiophores primordia. B. Young developing anteridiophores. C. Mature anteridiophore with sperms ready to be collected (note the cloudiness in the droplet of water).

Figure 4. Marchantia archegoniophores. A. Archegoniophores primordia. B. Young developing archegoniophores. C. Mature archegoniophore 

Figure 5. Marchantia growth room. Shelves with Marchantia growing on rockwool under white and far-red lights (top). Both lights are required to induce reproductive structures.

Figure 6. Marchantia sporangium. A-C. Developmental series of early sporogenesis. D. Mature sporangium. E. Close up of spores from panel D. F. Archegoniophore with closed sporangia. G. Open sporangium showing recently released spores. H. Archegoniophore with open sporangia.

 Confocal 3D reconstruction of DAPI stained Marchantia sperms, archegonium and egg-cell.

Genetic crosses in Marchantia polymorpha

Genetic crosses are very easy to perform. A drop of water is incubated on top of an antheridiophore and after a couple of minutes the water will become cloudy. This is the sign to collect the drop of water (loaded with sperm) which can now be added on top of a developing archegoniophore. That’s it! You just made a genetic cross that will produce thousands of spores per sporangium.

How to perform genetic crosses in Marchantia?

A typical day at the epilab

A typical day in the lab starts with a 30 minutes walk from my house to the lab. Then I check on the plants, making sure they have enough water and that the AC unit is working properly. Xalapa is hot and humid most of the year so it is extremely important to keep the plants in an optimal temperature range from 20 to 22 °C. Marchantia grows nicely on a number of substrates including, vermiculite, turface (baked clay), ground brick, rockwool and it can also be grown in vitro in both solid and liquid media. Marchantia protocols including in vitro plant growth, induction of reproductive structures, cryopreservation and genetic transformation of spores have been developed by Dr. Kohchi’s group over decades of work. Recently, new protocols for genetic transformation of gemmae and thalli were developed by Dr. Yutaka Kodama’s group at the Utsunomiya University. This might sound like a cliché, but it is true, we are standing on the shoulders of giants.

During the last couple of months I have spent nights and days doing molecular biology benchwork, cloning genes and making all sorts of genetic constructs for my B.S. thesis. My thesis is part of a large scale comparative genomics project that involves the molecular characterization of small RNA-based gene silencing mechanisms in Marchantia. I am particularly focused on the characterization of the Argonaute gene family and the miRNA repertoire of Marchantia. One of the greatest features of Marchantia is that it is very easy to grow and to handle. You can grow large populations of Marchantia in very little space (Figure 2 and 5). My own special assignment in the lab is to keep our spore stocks safe and sound. For this, I select the most promising young antheridiophores and archegoniophores for crossing, and three or four weeks later I collect sporangia with thousands of spores that I dry and keep at either 4° C (if we plan to use them within a couple of months) or -80° C (for long term storage) (Figure 6).

I came to the epilab two years ago as a scholar of the Research Summer Program of the Mexican Academy of Science. I was and still am extremely excited to work with Marchantia. I have had the opportunity to learn a lot of things about plant biology, genomics, evolution and developmental biology. In spite of the ever changing weather and the ferocious population of arthropods (mostly mosquitoes) that populate Xalapa during the summer, this experience has been one of the most frutiful times in my life both academically and personally.

Marchantia polymorpha is rapidly developing into a remarkable experimental model with a powerful toolkit for comparative studies, molecular genetics and functional genomics thanks to the hard work of a number of pioneer scientists.

If you are interested in our work just visit the epilab webpage: epilab.weebly.com

Acknowledgements

We would like to thank all friends and colleagues who helped us start the epilab in Mexico. We are particularly in debt with Ana Dorantes-Acosta, Liliana Arteaga-Dorantes, Elena Arteaga-Dorantes, Vicki Chandler, Xuemei Chen, Daniel Grimanelli, Hervé Vaucheret, John Bowman, Takayuki Kohchi, Kimitsune Ishizaki, Katsuyuki Yamato, Eduardo Flores, Rebecca Mosher, Blake Meyers, Efraín de Luna, Luis Herrera-Estrella, Félix Recillas-Targa, Alfredo Cruz-Ramírez, Juan Caballero-Pérez, Alfredo Herrera-Estrella, Patricia León-Mejía, Mario Zurita-Ortega, Alejandra Covarrubias-Robles, Federico Sánchez-Rodríguez, José Reyes-Taboada, Noé Duran-Figueroa, Andrés Cruz-Hernández, Shih-Shun Lin and Francisco Díaz-Fleischer. We thank past and current members of the epilab and the INBIOTECA-UV. We also thank our friend Luis Alberto Cruz Silva, Research Specialist of the Microscopy Unit in Biomimic^TM at the Institute of Ecology A.C. (INECOL). We are grateful to the University of Veracruz (Cuerpo Académico CA-UVER-234) and the following funding agencies: UC MEXUS Collaborative program (Grant 2011-UCMEXUS-19941-44-OAC7), Consejo Nacional de Ciencia y Tecnología (CONACYT) (Grants: CB-158550 and CB-158561), JEAI- Institut de Recherche pour le Développement (IRD) (Grant: COSEAMX1- EPIMAIZE).

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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This interview first appeared in Development.

Juergen Knoblich is a senior scientist and deputy scientific director of the Institute of Molecular Biotechnology of the Austrian Academy of Sciences in Vienna. We met Juergen at the 56th Annual Drosophila Research Conference, where we asked him about his work in this model system and, more recently, on human cerebral organoids, and about his thoughts on recent technological developments and the funding situation.

How did you first become interested in biology? Was there someone who inspired you?

I was always very interested in chemistry, but at some point my chemistry teacher told me that there was not much more to be discovered in chemistry, and that the new trend was to study biochemistry – which is what I did at university. It was really during my master’s thesis research that I became interested in genetics, and I got my training as a scientist during my PhD in the lab of Christian Lehner. He essentially taught me everything that I know about flies.

The Drosophila neuroblast has been the focus of your research for many years. Why did you choose this system?

I started my Drosophila career looking at cell cycle progression. As a PhD student I worked on cyclins and the transcriptional regulation of cell cycle exit. The logical next step for me was to look at the cell biology of mitosis itself. It so happened that my postdoc lab, the lab of Yuh Nung Jan at the University of California, San Francisco (UCSF), had just discovered the phenomenon of asymmetric cell division, which I then studied, mostly in the peripheral nervous system. The honest reason why I shifted to neuroblasts was that it allowed me to connect my cell biological research with something that is of very great medical relevance, i.e. stem cell biology. It is a fantastic cell biological system and you learn things from it that you can use and translate into higher organisms.

How do you feel that Drosophila research has changed over the course of your career?

Drosophila research over the past 10, 15 years has changed dramatically. If I had, as a PhD student, the technological tools available in flies now, I would have been the happiest person on Earth! Things are so much faster now. It started with the sequencing of the fly genome, which changed things completely. And now we have CRISPR-Cas, which is another revolution.

What is also very good about Drosophila as a model system is that there are a lot of people in the community who are fascinated by technology. They generate these absolutely fantastic collections, the latest being the MiMIC collection (http://flypush.imgen.bcm.tmc.edu/pscreen/index.php), that are available to the entire fly community and speed up our research so much.

Do you think that Drosophila as a genetic model system is being threatened by other model systems catching up with genomic tools?

I don’t think so. My lab uses not only Drosophila, but also mouse and human systems. So I honestly think that it is not just the technology that is better in Drosophila. There is a fundamental design difference between Drosophila and vertebrates. The enormous optimization of Drosophila development has eliminated many redundancies in the genome, and that comes in very handy for a geneticist. So when you make a certain mutation in Drosophila you typically get a very clear answer, and that is not usually the case in the mouse.

What is a threat to Drosophila research is that the interest of funding organizations and young scientists is starting to shift. Funding organizations are much more interested in direct medical translation. This is reflected in the interest of students. Drosophila as a system needs to switch to more disease-oriented research, and a lot
of groups are actually doing this. I think this enormous trend towards application is the real threat to Drosophila research. But it will survive.

Drosophila is famous as a genetic model, but you have been quite involved in RNAi screening efforts in this organism. What do you think is the relationship between knockout versus knockdown approaches, especially in the context of the recent developments in genome technology?

My lab makes extensive use of the genome-wide RNAi collection at the Vienna Drosophila Research Centre (VDRC). The collection was originally made by Barry Dickson and it is an absolutely invaluable resource for my lab. However, in some of the recent VDRC board meetings we discussed whether we have to prepare ourselves to stop maintaining this resource. Personally, I do not think RNAi will be replaced by CRISPR, and the reason for this is that RNAi is very versatile. You can make tissue-specific knockdowns, you can control them over time, you can make RNAi lines that have different knockdown levels, and they are a very successful tool to perform genome-wide screens.

CRISPR is a very interesting phenomenon. The technology is less than two years old and there has never been, to my knowledge, a technology that has so quickly transformed the entire field of genetics. It has effectively replaced other techniques to generate gene knockouts in flies, which were always difficult to use. In Drosophila, CRISPR is a great technology to generate stable loss-of-function point mutations, or for making insertions and tagging genes. But it cannot generate genome-wide loss-of-function resources that have the same versatility as the RNAi collection. Genome-wide loss-of-function screens using CRISPR-Cas will be possible at some point but will have their own problems, namely the fact that you do not have complete control over all the mutations that you make. So I think CRISPR and RNAi are complementary
techniques.

More recently, your lab has been making important contributions to the field of organogenesis, generating cerebral organoids in vitro. This is a shift away from the core focus of what your lab has been doing for many years…

It is quite an adventure for a Drosophila lab to all of a sudden work on human genetics, but I have a lot of fun trying out new things. We actually started shifting to the mouse a few years ago, and my lab now has almost five to six years of research experience in mouse brain development. The work of my lab is very much driven by the interest of the postdocs, and I typically develop projects with them, rather than telling them what to do. The organoid system was the project of Madeline Lancaster, who joined my lab as a postdoc initially to work on two-dimensional culture in mouse. We both then decided that it might actually be a really good idea to shift to humans, and to a three-dimensional culture. But she should take all the credit for the actual experimental protocol.

There was also a specific scientific question behind this project. As a postdoc I characterized a gene in Drosophila called inscuteable. Inscuteable is a molecular switch for spindle orientation. In mouse, changing the orientation of the mitotic spindle can change the number of neurons that are generated in the cortex, and a number of recent findings make inscuteable a really good candidate for the cortical enlargement you see as you go from the mouse to human. The one critical experiment that is missing is to
make a human inscuteable mutant. That was for me the real reason for developing this organoid system, and I hope that we will have this mutant very soon.

The cerebral organoids have a range of potential applications, from trying to understand how the brain develops at a more basic level to model human disease. Where would you like your lab to go next?

The core interest of my lab is lineage specification in stem cells. Recent findings have shown that there is a very strong difference in the cortical lineage between rodents and primates. Understanding this lineage change in evolution is one of the key questions that fascinate me, and I would like to address this question in my lab. However, modelling disease in organoids is not something that I want to neglect, and we have started to set up a translational research unit. But this is not going to be the core interest of my lab.

The cerebral organoids, often called minibrains, attracted a lot of media attention.What was your experience interacting with the press?

I should first say that the term ‘minibrains’ was not invented by us, but we were not surprised that they gained this name!

Nothing I did before ever generated such a high degree of media attention. I was quite well-prepared for it, and the press conference that was organized by Nature helped a lot in thinking about what the message was that I wanted to get across and how to best do that. I think that preparation for this kind of discussions is very important. Talking to the high-level news channels (such as the BBC or CNN) is very easy. It becomes more difficult when the tabloid press gets interested, but by taking the journalists seriously, and by preparing how to get the core of our message across, it can also work. Overall, it was a very good experience, and the press coverage was generally very positive. Everyone realized the enormous potential of what we did, and we addressed well the understandable concerns regarding what could be done with this system.

So you would encourage other scientists to interact with journalists?

I should say that for the organoids I had no choice! But in general, yes. Our research is funded by the public, and the public has a right to know what we are doing and why we are doing it. It is absolutely essential if we want to continue to be funded. This of course means going to the media. It also means accepting the rules of the media, and accepting that they have a different understanding of what is true or is not true. Conversely, we should also have a certain level of tolerance towards our colleagues when they explain things to the media or the lay public and deliberately use less accurate language.

How do you feel about the funding situation in Europe at the moment, particularly with the recent threat to the European Research Council (ERC) budget? Do we have reasons to worry?

Yes, I do think we have reasons to worry. For a long time there was a dogma that, even in times of reduced financial prosperity, the one thing that would not be touched were the long-term future investments, such as science and education. All of a sudden that changed, and I do not really understand why. The recent threat to the ERC project is, in my view, nothing less than a scandal. The ERC is one of the success stories of Europe; for once something in which the European Union has become a role model for other funding organizations. It has united European sciences. To cut the budget of this organization in favour of short-term investments is the wrong decision. Although I think the ERC will survive, any minor cut threatens the whole system, given the very low acceptance rate for ERC grants. There is no final decision on this yet, and I still hope the European Union will reconsider its decision.

In this context of funding difficulties, what is your advice for young scientists?

I think what is happening at the moment is a transient phase, and as such we must distinguish between those whowant to go into science and those who are currently young scientists. Science funding, particularly for the biological sciences, grew strongly for a long time, but we have now reached a point where funding rates are constant, or even going down. There are a number of reasons for this. One of the reasons is that you cannot grow forever. The other is that biological sciences have made all sorts of promises, such as cures for a variety of diseases. At some point the public got impatient and said “now really deliver those cures”. This is why there is a strong shift towards translational research at the moment. This is, I think, a bit short-sighted, as we all know that the great discoveries in science do not come from a direct targeted discovery, but are to some degree serendipitous.

When you ask me what my advice is to young scientists, whether they should go into science, I think yes, by all means. They should pick the area by their interests, and not by the funding situation. My feeling is that we are undergoing a shrinking process, but this is a transient period. Indeed,when I started my PhD the situation was very similar. There was even a whole department dedicated to biologists at the Stuttgart unemployment office, because, among the natural scientists, biologists were the ones with the highest unemployment rate. This changed dramatically afterwards. On the other hand, if you ask me about the advice to give to people who are about to start their own lab, then it is tough at the moment and one really has to be very dedicated to being a scientist. But most of the people I know who really wanted to become a scientist succeeded in the end.

(4 votes)

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Here are the highlights from the current issue of Development:

Capping off sesamoid bone development

Sesamoid bones are small, flat bones that are embedded within tendons. To date, it has been thought that these bones develop within tendons in response to mechanical signals, but now (on p. 1831) Elazar Zelzer and colleagues challenge this assumption, focussing on the patella (the kneecap), which is the largest sesamoid bone in the human body. They show that that, in mice, the patella initially develops as a bony process that is part of the adjacent femur bone. Subsequently, they report, the patella is separated from the femur during the process of joint formation. This process is regulated by mechanical load; patella separation is perturbed in mutant embryos that are devoid of contracting muscle. The authors further demonstrate that, similar to bone eminences – superstructures that mediate bone-tendon attachment – the patella arises from progenitors that express the chondrocyte marker Sox9 and the tendon marker scleraxis (Scx) and that are regulated by TGFβ/BMP signalling. Together, these findings provide a new model for patella development and highlight that a high degree of plasticity exists during skeletal patterning and development.

FGF receptors sculpt synaptogenesis

A balanced network of excitatory and inhibitory synapses is required for correct brain function, and any perturbations to this balance can give rise to neurological and psychiatric disorders. It has been shown previously that FGF22 and FGF7 promote excitatory or inhibitory synapse formation, respectively, in the hippocampus, but how do these ligands mediate their synaptogenic effects? Here, Hisashi Umemori and co-workers use various FGF receptor knockout mice to address this question (p. 1818). They first show that excitatory presynaptic differentiation is impaired inFgfr2b and Fgfr1b mutant mice. Following on from this, they reveal that both FGFR2b and FGFR1b act downstream of FGF22 and are required for FGF22-dependent excitatory presynaptic differentiation. The authors further show that the kinase activity of FGFR2b as well as its ability to bind to FRS2 and PI3K is required for it to respond to FGF22. By contrast, they report, inhibitory presynaptic differentiation is defective only in Fgfr2b, and not Fgfr1b, mutants. In line with this, they demonstrate that FGF7 requires FGFR2b and not FGFR1b to mediate its effect on inhibitory presynaptic differentiation. Together, these findings indicate that distinct but overlapping sets of FGF receptors sculpt excitatory and inhibitory synapse formation in the mammalian brain.

ATRX: keeping quiet in the embryo

Human embryos are particularly susceptible to chromosome instability (CIN) and errors in chromosome segregation, but the molecular mechanisms that regulate and sense CIN in mammalian embryos are unclear. Here, on p. 1806, Maria Viveiros and colleagues investigate the role of the chromatin remodelling protein ATRX in early mouse embryos. They first show that ATRX, which is transmitted to the early zygote through the maternal germ line, localises to pericentric heterochromatin (PCH) within the maternal pronucleus, where it is required for the transcriptional repression of major satellite transcripts. The loss of ATRX hence leads to the abnormal expression of maternal satellite transcripts. The authors also demonstrate that the maternal inheritance of ATRX helps to set up an epigenetic asymmetry between the maternal and paternal chromosomes, which might be implicated in facilitating chromosome segregation. In line with this, ATRX loss, they report, causes abnormal centromeric mitotic recombination and an increase in double-strand DNA breaks. Overall, these data highlight an important role for ATRX in the early mouse embryo and provide new insights into how CIN is controlled in early mammalian development.

Divide but stay together: cytokinesis in tubes

During development, epithelial tubes often need to grow while still maintaining their barrier properties. How can cells divide without disrupting the integrity of the tubular epithelium? On p. 1794, Markus Affolter and colleagues address this question in the Drosophila larval tracheal system. In a particular subset of tracheal tubes, there is extensive remodelling during the early third instar, such that unicellular tubes, in which a single cell encircles the lumen and creates junctions with itself, transform into multicellular tubes, a process accompanied by proliferation. The authors demonstrate that this transition involves cell intercalation to replace the autocellular junctions with intercellular ones. Depending on cell length, mitosis may occur either before or after this junctional remodelling is complete, thus generating two major classes of cytokinesis events. In both cases, cytokinesis is asymmetric, with the new membrane extending from the side of the cell where the nucleus is located. In rare cases, this can lead to the formation of a binucleate and an anucleate daughter. The authors further find that Dpp signalling is required for appropriate junctional remodelling and cell division. Together, these data provide insights into how barrier integrity can be maintained through cell division in these tubular structures.

A double take on the segmentation clock

The segmentation clock, which controls the periodic formation of somites along the vertebrate body axis, involves the oscillating expression of clock genes in presomitic mesoderm (PSM) cells. Oscillations are synchronised between cells, giving rise to a sweeping wave of gene expression throughout the PSM. This cyclic wave of gene expression is known to slow as it moves anteriorly, but the causes and implications of this slowing have remained unclear. Here, Sharon Amacher and co-workers investigate segmentation clock dynamics in zebrafish embryos (p. 1785). Using a her1:her1-venus reporter to visualise clock gene oscillations in real-time, the authors show that the periodicity of oscillations slows as PSM cells become displaced anteriorly. This slowing gives rise to a situation in which cells that are one somite apart are actually in opposite phases of the clock. Thus, they report, a one-segment periodicity is observed in the posterior of the embryo, whereas a two-segment spatial periodicity is seen in the anterior. The researchers further demonstrate that neighbouring cells oscillate synchronously in both the posterior and anterior PSM until they are incorporated into somites. Based on these findings, the authors propose an updated model of the segmentation clock.

PLUS:

An interview with Juergen Knoblich

Juergen Knoblich is a senior scientist and deputy scientific director of the Institute of Molecular Biotechnology of the Austrian Academy of Sciences in Vienna. We met Juergen at the 56^th Annual Drosophila Research Conference, where we asked him about his work in this model system and, more recently, on human cerebral organoids, and about his thoughts on recent technological developments and the funding situation.

Hematopoietic development at high altitude: blood stem cells put to the test

In February 2015, scientists gathered for the Keystone Hematopoiesis meeting, which was held at the scenic Keystone Resort in Colorado, USA.  During the exciting program, field leaders and new investigators presented discoveries that spanned developmental and adult hematopoiesis within both physiologic and pathologic contexts.  See the Meeting Review on p. 1728

Building the backbone: the development and evolution of vertebral patterning

The segmented vertebral column comprises a repeat series of vertebrae, each consisting of the vertebral body and the vertebral arches. Despite being a defining feature of vertebrates, much remains to be understood about vertebral development and evolution. Particular controversy surrounds whether vertebral structures are homologous across vertebrates, how somite and vertebral patterning are connected, and the developmental origin of vertebral bone-mineralizing cells. Here, Roger Keynes and colleagues consider these issues. See the Review on p. 1733

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This week I have the pleasure of attending the “Getting Into and Out of Mitosis” Workshop organised by The Company of Biologists. On Sunday, renowned experts and early-career scientists alike travelled to Wiston House in West Sussex, UK, to listen to the latest research, ask questions and discuss results in the field of cell division.

It is certainly unlike any meeting I have attended in the past. Due to the small size of the Workshop, all participants get to know each other from the very first day. Here, early-career scientists have a unique opportunity to interact closely with leaders in the field. There are many opportunities for such interactions during meals and coffee breaks, whilst admiring the beautiful period house where the meeting is held and during the organised walk through the stunning English countryside.

Another unique and much appreciated feature of the Workshop is the generous 15-minute question period after each talk. Combined with inclusive atmosphere, these have stimulated some great discussions involving not only the senior scientists, but also the students, postdocs and junior group leaders. There are also general discussions built into the programme about the open questions in the field. Even though I’ve left the bench, I can feel a contagious research energy that is sure to spark many collaborations.

A meeting report, with more details of topics covered, will soon be published in Journal of Cell Science. For now, I look forward to another day and a half of exciting talks about mitosis!

By Anna Bobrowska, Editorial Intern, Journal of Cell Science

The next Company of Biologists workshop will be on Transgenerational Epigenetic Inheritance. For more details click here.

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Deadline for application: 15 May 2015.

BUILDING, REPAIRING AND EVOLVING BIOLOGICAL TISSUES

Roscoff (Brittany), France, September 13-17, 2015

This conference will bring together Developmental Biology, Evolutionary Biology and Maths/Physics, disciplines that are often combined pair-wise, but rarely all together. This will elicit creative discussions, not only on classical questions in developmental biology and morphogenesis but also about regeneration, repair and the behavior of stem cells in their natural environment.

There will be 5 thematic sessions:

(1) Specifying and evolving patterns, size and shape; (2) Collective behavior; (3) Self-organisation; (4) Regeneration, repatterning and repair; and (5) Tissue homeostasis.

The conference will be held in picturesque Roscoff, on the north coast of Brittany. The atmosphere will be convivial, allowing junior researchers and students to interact with world class scientists. The number of attendees will be limited to 100.

Deadline for application: 15 May 2015.

Details at http://www.cnrs.fr/insb/cjm/2015/Vincent_e.html and http://jpvincentlab.com/news/

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The first Boston Young Embryologist Network talks event will be held in the Warren Alpert Building at Harvard Medical School Thursday May 14th, 6-8pm. Food and drink will be provided for discussions and mingling afterwards.

Please register with eventbrite to give an estimate of numbers for food.

Two postdocs, Vaibhav Pai from Tufts, and Stan Artap from Beth Israel, will give short talks about their work investigating bioelectric patterning in Xenopus laevis brain development, and the role of transcription factors in facilitating endocardial-myocardial interactions in the developing mouse heart.

We look forward to seeing you there, and stay tuned for the next meeting on June 18th!

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Last year we interviewed Niteace Whittington, who won the Society for Developmental Biology (SDB) poster prize at the 2014 meeting in Seattle. Niteace’s prize was attendance at the joint meeting of the British Societies for Cell Biology and Developmental Biology (BSCB and BSDB). Continuing the interview chain, Niteace interviewed Wendy Gu, who won the BSDB poster prize there. As a prize, Wendy will be attending the 2015 SDB meeting this July, in Snowbird, Utah. Unfortunately they did not get a chance to meet in person, so Niteace interviewed Wendy over Skype a few days later.

NW: Congratulations on your achievement. You weren’t at the conference dinner when your prize was announced, so how did you feel when you found out?

WG: I found out at breakfast the following morning. The first person who told me was a PI who used to be based in my department. She sat down and said ‘Congratulations!’ I thought ‘For what?’ I thought she meant ‘Congratulations, you are about to submit your dissertation next week’. So I said ‘Yes, I am very relieved’. She had a strange look on her face, thinking ‘Why are you relieved that you won a poster prize?’ It was only afterwards that she explained to me what had happened the night previously, and it was then that it hit and the news made any sense to me. I’m sure it was the same for you when you won your poster. The standards are so high, and it could have gone to any number of other equally capable scientists, so I know how lucky I am!

NW: Yes, when I won I was in such a state of shock. I was thinking ‘Are you sure that you said the right name?’!

In which lab did you do your PhD and what does your lab work on?

WG: I am based in the lab of Matthias Landgraf in the Department of Zoology at the University of Cambridge. As a group we are interested in how neural circuits are specified, how they function, and the behavior of the animal once the nervous system is built. The model organism we work on is the fruit fly, Drosophila melanogaster, an insect that undergoes complete metamorphosis. But most of our work focuses on the embryonic, and larval stages of development.

NW: What was the title of your poster?

WG: The role of Wnt5 ligand and the Ryk family Wnt receptors in positioning neurites along the anteroposterior axis of the developing Drosophila ventral nerve cord. It is a very technical title!

NW: Could you give a brief summary of what you presented?

WG: The biological question I addressed during my PhD is how axons make a choice between growing anteriorly or posteriorly in the developing nerve cord. The ventral nerve cord of Drosophila is analogous to the vertebrate spinal cord, and within it neurons have to decide where to terminate within a 3-Dimensional space. We know from work done previously in both vertebrate and invertebrate systems that these decisions are axon guidance mechanisms, which involve guidance molecules and the receptors expressed in the neurons. In the medio-lateral axis,the positional cue system Slit/Robo determine the extent to which axons grow medially or laterally. Another positional cue system dictates how the dorso-ventral axis is specified: the Sema/Plexins.

When I started four and a half years ago we didn’t know what positional cue system, i.e. which signal and receptors, was acting in the anterior-posterior axis. What I managed to do in the last few years was to show that Wnt5 is the ligand, or the positional cue, that provides information to the sensory neurons when they grow into the central nervous system. One class of partner receptors of Wnt5 are Ryks, and these include Derailed (Drl), Derailed-2 (Drl-2) and Doughnut on 2 (Dnt). I have shown that sensory neurons express Dnt receptors but not Drl or Drl-2 receptors. Dnt receptors are required for the afferent terminals that project posteriorly. So, DNT needs to be expressed in those neurons in order for them to grow in the proper direction. However, the molecular mechanism that underpins selective growth of axon terminals either anteriorly or posteriorly is unclear. The second biggest finding is that although the other two receptors are not normally expressed, if you exogenously provide either Derailed or Derailed 2 you can also force them to grow and shift their terminals more anteriorly. So one of the receptors is necessary, while the other two are sufficient, in a developmental context.

NW: Had you presented your data previously?

WG: I presented a less complete story at the 2013 Neurobiology of Drosophila meeting at Cold Spring Harbor Laboratory. My current poster has a complete narrative, or as much as I can do within the time frame of a UK PhD. It contains some of the data that will go into a publication.

NW: Do you plan to submit a publication soon?

WG: I would like to but I have to write it first! I think I am going to treat myself to a week or two of break after the intensive writing of a PhD (I submitted on Tuesday!). Once I have celebrated properly I will write it in a journal format. The difficulty is deciding where to submit! Luckily, my topic can be published either in a neuroscience journal or a development biology journal, so in a way I am lucky that I have more choices in that respect.

NW: So you are ready to finish up?

WG: I am. I’m interviewing for postdoc positions. My first interview is happening in less than two weeks and then I have two other ongoing applications. In fact, I met one of the PIs during the BSDB meeting in Warwick, so it was a very productive meeting on many levels!

NW: Are you thinking of staying in the same area of research or are you looking to branch out a little?

WG: I’m ready to venture out. I think after working on this system for four years I want to do something different. The three labs I have applied to are very different in their research scopes. The first one works on neurogenesis and neurodifferentiation in the zebrafish. The second project involves engineering the epigenome. It would involve the use of genome editing tools such as CRISPRs, TALENs and Zinc-finger nucleases to alter the epigenomic code and see what effects it has on various model systems and cell lines. The third option is a complete change and that is to work on plant development. As you can see, I am torn between three very different subjects, all of which I am very excited about. We will see! How about you? Did you stay within your discipline or have you branched out slightly?

NW: I’m in a new model organism, using mouse instead of frog, and I am also looking at a different area of neurogenesis. My graduate work focused more on the brain and the central nervous system and in my new lab (Susan Wray, NIH) I am looking at olfactory development. So I branched out a little bit, but in baby steps!

Are you excited to attend the SDB meeting?

WG: I am very excited. I have never been to that part of America before, and Utah and Snowbird in the summer sounds quite enticing.

NW: Would you be interested in a postdoc in the States?

WG: Not at this point in my life. Two of the positions that I had mentioned are based in Europe and the other in Australia. I am actually from North America, and I have done research in the USA previously, so I am looking to get more exposure to the world before returning. I am sure at some point in my career I will be back in North America, and in the USA in particular. So much of the exciting research is coming from where you are based! Maybe not in the near future, but certainly if I am in a position to look for a second postdoc or perhaps a tenure-track position…

NW: I am sure you will meet some really interesting people while you are at the SDB meeting here. You may find some collaborators or networks that could help with potential jobs in the future. These meetings are really cool because you get to really see what is going on in the other side of the world.

WG: Speaking of which, what did you think of our British Society for Developmental Biology meeting?

NW: I think the biggest difference was the time zone! I was a little bit jetlagged. But I had a really good time, and it was a really good experience, interacting with different people in different areas of development.

WG: I am glad to know that we hosted you well here in the UK!

NW: That’s all of my questions! Thank you for your time and congratulations again. I wish you nothing but the best for your future work!

WG: I’m really sad that we couldn’t meet in person, but we are lucky that we live in an age in which technology can come to the rescue!

(2 votes)

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by Peg McCarthy and Bridget Nugent

The biological phenomenon of hormonally induced sexual differentiation of the brain has been an empirical topic of study for over 50 years¹ but much remains to be discovered in terms of both mechanism and functional impact. In the McCarthy lab we exploit the many advantages of the laboratory rat as our model because of its well-characterized neuroanatomy and behavioral repertoire which, as in humans, differs markedly in males and females. In particular we have drilled deeply into the mechanistic bedrock controlling male copulatory behavior with a focus on one brain region, the preoptic area (POA), which is often associated with the hypothalamus but is actually telencephalic in origin². If this brain region is lesioned, males loose all interest in sex³ and if it is stimulated they loose all interest in anything but sex⁴. The importance of the POA to male reproduction cuts across species from newts to humans, but the mechanisms underlying its importance are only beginning to be understood in the rodent.

In the beginning, most studies justifiably focused on neurotransmitter production, release and binding, assuming that a change in behavior must be the result of changes in neural activity. But finding precisely what about neurons was different in males versus females proved frustrating and largely unproductive until attention turned to other factors. In particular we determined that prostaglandins, a normally inflammatory mediator, was both necessary and sufficient to masculinize the brain if elevated during a critical perinatal “sensitive period”⁵. Even more surprising, non-neuronal cells that constitute the brain’s immune system called microglia, are a principle source of the prostaglandins that masculinize the POA and male copulatory behavior⁶. Cell-to-cell communication that involves neurons, astrocytes and microglia helps to shape the synaptic profile of this brain region by increasing the density of excitatory spine synapses on dendrites of POA neurons.

In the course of our studies we noted that the sex difference in synaptic density, males having twice the number of spine synapses per unit of dendrite length as females, was stable across the lifespan despite the juvenile hiatus during which there are little to no gonadal steroids in circulation in either males or females. This led us to ponder the question of how this cellular memory was maintained, and to answer that we turned our attention to epigenetics.

Steroid hormones are obvious candidates as genome modifiers because their transcription factor receptors are associated with co-factors that possess histone deacetylase activity. But to our surprise we found that rather than directly interacting with DNA or chromatin, steroids were instead (or more likely in addition to) decreasing the activity of a class of enzymes called DNA methyltransferases (DNMTs), lowering their ability to methylate DNA⁷. Reduction in the activity of these actively methylating enzymes results in demethylation of DNA through mechanisms not well understood. Regardless, the impact is an emancipation of genes that then direct masculinization of the POA resulting in male-typical synaptic patterning and copulatory behavior in adulthood. Moreover, if we allowed demethylation to occur outside of the sensitive period for sexual differentiation of the brain, females were still capable of being masculinized. The end of the sensitive period is operationally defined as the point at which giving masculinizing hormones to a female no longer has any effect on sexual differentiation of her brain. We confirmed that the sensitive period ends before day 10 after birth, and we also showed that hormones no longer reduce DNMT activity at this time. Thus the end of the sensitive period appears to be due to the loss of effect of steroids on DNMT activity. To our knowledge this is the first ever report of a mechanism for the end of the sensitive period.

The next big question of course is – what genes are demethylated in the bipotential brain leading to masculinization? Here we have a tale of woe as this is far harder to answer than it might at first seem. Our initial approach was to categorize gene expression profiles in males and females with and without DNMT inhibition using RNA-Seq. To our surprise there were a relatively small number of genes that showed overall expression differences in males and females, less than 100, and they were evenly distributed between males and females. We then looked to see what genes were turned on in females following demethylation. The expression of many genes was induced in the female POA follow DNMT inhibition, and importantly the majority that had been higher in males were now upregulated in females. These genes we considered our masculinization genes, which after eliminating unknowns was reduced to less than 10 candidate genes. The obvious next step was to characterize methylation of CpG islands found in the promoters of each gene. We chose to use the highly quantitative approach of 454 sequencing. However, there were several challenges inherent to this technique: its only good for amplicons of ~300-600bp, and so for large CpG islands several amplicons are required, meaning multiple pairs of primers per promoter. The book keeping alone is a nightmare and determining CpG methylation requires bisulfite conversion from a large number of biological replicates (in our case three groups with n’s of 8-10 per group). Thus the potential sources of error were insomnia inducing. But despite all this, with all quality controls in place, we found absolutely no meaningful sex differences in CpG methylation in any of our candidate genes. Nothing. At first this was very hard to believe, much less accept, but in retrospect, it makes sense.

The first clue that we should not have expected to detect differences in these candidate genes was hiding in our own data. The observation that DNMT activity is higher in the neonatal female POA was matched by global methylation levels of DNA extracted from the POA, such that females had twice the amount of methylation as males. Twice the amount of methylation on the entire genome should not reduce down to differences in the promoters of a handful of genes. Either our measure of DNA methylation was flawed, or most of the action was outside of the promoters. Turns out both are at least partially true.

Extraordinary claims require extraordinary evidence – a quote attributed to Carl Sagan and used by the dreaded Reviewer #3 to insist that if we were going to claim that females had double the DNA methylation in the POA as males we needed better proof than an antibody based colormetric assay kit. There are only a few roads to complete methylome analysis and most of them are full of potholes. The only superhighway is Genome Wide Bisulfite Sequencing (GWBS) but the tolls are very high and the data generated could circle the globe many times over. So what if we took the on ramp but crashed and burned, finding no sex differences in DNA methylation? These are the kinds of decisions one should not contemplate for long or you will talk yourself out of it.   So we did it; collecting and bisulfite converting the DNA from the POA of newborn males, females and females masculinized by hormone treatment two days earlier, with 3 biological replicates per group, the industry standard. We shipped our samples off for sequencing and waited. It was agony until the data arrived by UPS, as the files were too large to send electronically, and then it was shear terror. Fortunately, one of us (BN) had gained sufficient bioinformatics experience to conduct an initial analysis and found that indeed, females do have twice the level of DNA methylation as males, but only at sites that are very highly methylated, not across the genome. In fact females also had more sites that were entirely unmethylated compared to males and masculinized females, suggesting tight epigenomic regulation in females. Analyses of where in the genome differences in methylation status between males and females can be found showed that most differences are in the intragenic regions, but of course this is where most methylation is found and so the significance of this remains unclear at the moment. Relatively few sex differences were found within CpG islands or shores, consistent with our failure to find any differences in the promoters of our candidate genes in our earlier attempts at 454 bisulfite sequencing.

The journey to publishing this paper had begun over two years earlier and we were anxious to put it to rest, submitting the final draft with the bare minimum of analyses of the GWBS data. But there is a treasure trove of information; we are anticipating more surprises reside therein. These will surely provide insight into the proximate mechanisms establishing and maintaining sex differences in this brain region, but will not answer the big picture question of why the brains of males and females evolved this way. At this point we can only speculate, and our speculations fall along two lines of thinking. The first is the process of canalization, which Waddington proposed as the function of epigenetic modifications and a process since considered widely in the context of evolution and the robustness of species in the face of challenge⁸. The robustness of sex differences in neuroanatomical endpoints (NOT behavior) is reminiscent of canalization and the marked differences in epigenetic marks we have observed may be a mediating factor. Second is a more tautological explanation based on the notion that the reproductive strategy of females requires close guarding of precious and limited gametes while males have a continuous and plentiful supply that they are eager to share. If females were to begin to play like males the consequences would be costly if not disastrous, therefore they actively suppress the gene profile that if activated, leads to masculinization of brain and therefore behavior.

More information on the McCarthy lab here.

1. PHOENIX CH, GOY RW, GERALL AA, & YOUNG WC (1959). Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology, 65, 369-82 PMID: 14432658

2. Puelles, L., Harrison, M., Paxinos, G., & Watson, C. (2013). A developmental ontology for the mammalian brain based on the prosomeric model Trends in Neurosciences, 36 (10), 570-578 DOI: 10.1016/j.tins.2013.06.004

3. Heimer, L., & Larsson, K. (1967). Impairment of mating behavior in male rats following lesions in the preoptic-anterior hypothalamic continuum Brain Research, 3 (3), 248-263 DOI: 10.1016/0006-8993(67)90076-5

4. Malsbury, C. (1971). Facilitation of male rat copulatory behavior by electrical stimulation of the medial preoptic area Physiology & Behavior, 7 (6), 797-805 DOI: 10.1016/0031-9384(71)90042-4

5. Amateau, S., & McCarthy, M. (2004). Induction of PGE2 by estradiol mediates developmental masculinization of sex behavior Nature Neuroscience, 7 (6), 643-650 DOI: 10.1038/nn1254

6. Lenz, K., Nugent, B., Haliyur, R., & McCarthy, M. (2013). Microglia Are Essential to Masculinization of Brain and Behavior Journal of Neuroscience, 33 (7), 2761-2772 DOI: 10.1523/JNEUROSCI.1268-12.2013

7. Nugent, B., Wright, C., Shetty, A., Hodes, G., Lenz, K., Mahurkar, A., Russo, S., Devine, S., & McCarthy, M. (2015). Brain feminization requires active repression of masculinization via DNA methylation Nature Neuroscience, 18 (5), 690-697 DOI: 10.1038/nn.3988

8. WADDINGTON, C. (1942). Canalization of Development and the Inheritance of Acquired Characters Nature, 150 (3811), 563-565 DOI: 10.1038/150563a0

(3 votes)

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Research technician position is available to work in the laboratory of Dr. Guillermo Oliver in projects related to mammalian organogenesis using mouse models and cultured ES cells.

Mouse experience and molecular biology skills are required

To apply write to

Guillermo Oliver, Ph.D (guillermo.oliver@stjude.org)

Feinberg Cardiovascular Institute, Northwestern Medical School

Chicago, USA

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