Here are the highlights from the current issue of Development:

Dynamics of human thymus development

The thymus is the primary organ responsible for generating T cells. Although thymus development has been studied in mice, little is known about how the human thymus develops. Here (p. 2015), Clare Blackburn and colleagues provide a comprehensive analysis of human thymus organogenesis. Using gene expression analyses, the researchers show that the spatial and temporal expression patterns of factors involved in thymus development in mice are conserved in humans. They also demonstrate that the human thymus arises from the third pharyngeal pouch, as in mice and in contrast to previous suggestions. Furthermore, they report, thymic epithelial cell differentiation as well as the immigration of mesenchymal cells and vascular progenitors into the human thymus occur after the onset of FOXN1 expression, consistent with the timing of these events in mice. Finally, the authors define precisely when the human thymus becomes colonised with haematopoietic cells, which are CD45⁺/CD7⁺/CD34^int cells. Collectively, this study provides key insights into the conservation of thymus development between mice and humans, which has major clinical implications for enhancing or replacing thymus function.

Coordinating head-to-tail with front-to-back

During embryogenesis, the anterior-posterior (AP) and dorsal-ventral (DV) axes are specified by the activity of key signalling pathways. FGF, Wnt and retinoic acid together pattern the AP axis: high activity defines more posterior tissues, which are specified later in development than anterior tissues. The BMP pathway specifies ventral fate; low BMP activity defines dorsal. Whether and how these pathways intersect to coordinate patterning of the two axes is poorly understood. On p. 1970, Megumi Hashiguchi and Mary Mullins provide evidence for synchronisation of DV and AP patterning in the zebrafish embryo. Upon temporally restricted inhibition of BMP in embryos anteriorised by inhibition of FGF or Wnt, the dorsalised tissue takes on the AP fate appropriate to the anteriorised embryo, rather than that corresponding to the time of BMP inhibition. The authors identify one mechanism mediating pathway cross-talk: MAPK, activated downstream of FGF, phosphorylates and inhibits the BMP effector Smad5. Thus, this work establishes the close temporal coordination of AP and DV patterning, and provides insights into how this is achieved.

miR-203 drives progenitor cell differentiation

MicroRNAs are important for the regulation of gene expression in a vast array of processes. In the skin, miR-203 has been shown to be crucial for the proper differentiation of the interfollicular progenitor cells, although the specific mechanism of this has remained elusive. In this issue (p. 1882), Rui Yi and colleagues investigate the precise timing of miR-203 activation during epidermal differentiation. They show that miR-203 is transcriptionally activated in the differentiating progeny of interfollicular progenitor cells upon asymmetric cell division. Using keratinocytes derived from miR-203-inducible mice, the authors found that miR-203 functions to promote an immediate exit from the cell cycle, leading to a complete loss of self-renewal after just 72 hours. They further identify a multitude of novel miR-203 targets in vivo, and demonstrate that co-repression by miR-203 of many of these, including Skp2, Msi2 and p63, is necessary for the function of miR-203 in inhibiting self-renewal. These data provide novel insights into the widespread role of miR-203 in differentiating interfollicular progenitor cells in the skin.

Leaf patterning: AS1 far AS2 we know

The correct establishment of adaxial-abaxial patterning is crucial for leaf expansion and growth. The AUXIN RESPONSE FACTOR (ARF) family of proteins are key determinants of organ symmetry and abaxial patterning in Arabidopsis thaliana and are subject to complex regulatory control at both the transcriptional and translational level. Here (p. 1958), Chiyoko Machida and colleagues uncover an additional, dual mechanism for the regulation of ETT (also known as AFR3) by an ASYMMETRIC LEAVES1 (AS1)-AS2 complex. First, the authors show that the AS1-AS2 complex directly represses ETT expression via binding of AS1 to the promoter region. Second, they provide evidence for an indirect mechanism of regulation via miR390- and RDR6-dependent post-transcriptional gene silencing of both ETT and ARF4. The authors also suggest a possible epigenetic mechanism, as AS1-AS2 maintains the methylation status of ETT within the coding region. These discoveries shed light on the molecular framework of early leaf patterning events and help to uncover the events that lead to the specification of distinct adaxial and abaxial fates during leaf development.

PAR-alell pathways for polarity

In the C. elegans embryo, anterior-posterior polarity is defined at the one-cell stage, via asymmetric and reciprocal localisation of cortex-associated PAR protein complexes: PAR-3, PAR-6 and aPKC localise to the anterior, whereas PAR-1, PAR-2 and LGL-1 are enriched at the posterior. Polarity maintenance involves mutual antagonism between the anterior and posterior complexes and may also involve CDC-42-dependent regulation of myosin activity. Kenneth Kemphues and co-workers (p. 2005) now provide evidence for multiple and partially redundant pathways acting at the posterior to maintain polarity once it has been established. Both PAR-2 and CDC-42, acting in separate pathways, have dual functions in independently regulating both anterior PAR complex localisation and myosin activity, whereas LGL-1 appears to have a buffering role in controlling PAR-6 protein levels. Although the molecular details of these pathways remain incomplete, the complex and overlapping mechanisms operating to maintain polarity in the early embryo underscore the importance of robust and efficient polarisation for subsequent development.

Stem cells go out of bounds

Many animal tissues maintain populations of slowly proliferating stem cells that contribute to tissue homeostasis and repair. In Drosophila, for example, stem cells reside throughout the midgut and within the hindgut and renal tubules. But how and when do these cells arise? Volker Hartenstein and colleagues now show that Drosophila gut progenitors migrate across tissue boundaries and adopt the fate of the organ in which they come to reside (p. 1903). Using lineage tracing, the researchers demonstrate that a subset of adult midgut progenitors, which are initially located in the larval midgut, migrate posteriorly during development and contribute to the adult ureter and, subsequently, the renal stem cell population. In addition, they report, a population of hindgut progenitors migrates anteriorly into the midgut territory to differentiate and give rise to midgut enterocytes. These findings suggest that a stable boundary between the midgut (an endodermal tissue) and the hindgut/renal tubules (ectodermal tissues) does not exist and instead multipotent progenitors are able to cross the boundaries between these domains.

Plus:

Molecular pathways regulating mitotic spindle orientation in animal cells

Orientation of the cell division axis is essential for both symmetric cell divisions and for the asymmetric distribution of fate determinants during, for example, stem cell divisions. Lu and Johnston review both the well-established spindle orientation pathways and recently identified regulators to provide a updated view of how positioning of the mitotic spindle occurs. See the Review article on p. 1483

Getting out and about: the emergence and morphogenesis of the vertebrate lymphatic vasculature

New insights into lymphatic vascular development have recently been achieved thanks to the use of alternative model systems, new molecular tools, novel imaging technologies and a growing interest in the role of lymphatic vessels in human disorders. Here. Hogan and colleagues review the most recent advances in lymphatic vascular development, with a major focus on mouse and zebrafish model systems. See the Review article on p. 1857

[Ed. comment: This is the first of a number of posts by a few people who attended this year’s BSDB/BSCB meeting. Look out for more on the meeting over the coming days]

As every year, the joint BSDB/BSCB spring meeting was taking place on the 17th – 20th of March 2013 on the vast campus of Warwick University. Over three days, an exciting line-up of speakers presented their latest research findings, accompanied by more than 200 scientific posters on display!

Among the first events of the conference was a workshop focused on alternative careers for scientists, mainly aimed at PhD and early post-doctoral researchers. Five speakers gave an insight into the paths they have taken since finishing their PhDs and talked about their current positions.

Jana Voigt, currently a research strategy analyst at the University of Cambridge, spoke about her contact with pharmaceutical consulting, which she jokingly called “the dark side”. She then mainly focused on her experience as a research programme manager at the MRC, where she was responsible for dealing with funding applications and reviews.

Ann Wiblin is a business development associate at Abcam at the moment. She described her responsibility in selecting, testing and managing Abcam product lines and also gave valuable advice to those who aspire to this career. Most notably, she recommended learning as many scientific techniques as possible and stressed the importance of networking.

Roli Roberts, currently associate editor at PLOS Biology, called himself a “recovering academic”. After going all the way from his PhD to a senior lecturer position at King’s College London, he decided that he “wasn’t having much fun anymore”. In his current role, he mostly handles manuscripts submitted to the journal, assesses their quality and arranges peer review. For people who want to enter this industry, he pointed out that publishers prefer to hire candidates with several years of post-doctoral experience.

Daniela Peukert is currently a science policy officer for the Society of Biology, where she provides evidence-based opinions for the government and funding bodies, for example in policy decisions. This involves interacting with experts of different fields of biology and arranging meetings with those who seek information. Her main advice was to remain flexible, since she was confronted with many new situations and unfamiliar tasks in her position.

Sam Gallagher introduced her talk with a slideshow of her career to date and the rock song “Summer of ’69” by Bryan Adams. From hereon, her talk boiled down to “Dear god, what did I think I was doing?” as she walked the audience through her career in academic research, the pharmaceutical industry and consulting. As one of the key ingredients to a successful career, she emphasised the importance of taking responsibility and stepping up.

I was particularly impressed with the breadth of careers and personalities that the organisers recruited for this workshop. Each individual entered their line of work in a different manner, but all stressed that it is critical to think outside of one’s own specialised field of research. Every speaker highlighted their broad interest in science, which is rarely compatible with research in academia. Moreover, many took on responsibilities which had little to do with their PhD or post-doctoral work, such as organising meetings, becoming involved in societies and volunteering. The most important message that I took away from this workshop is that there is a life outside of academia and that job opportunities do exist for those science-lovers who do not wish to spend their careers writing grants or managing a lab.

We had another busy month on the Node, with posts and comments relating to research articles, upcoming meetings, resources for developmental biologists…and, of course, the stem cell image contest.

Here’s a round-up of some of the highlights:

Stem cell image contest

Voting for the stem cell image contest opened earlier this month, and we’ve had close to 2000 votes in so far. It looks like a close race between a couple of the images – which is your favourite? Get your vote in if you haven’t done so already! Voting will close at noon GMT on Wednesday 10th April.

The winning image will appear on a cover of Development as well as on the new stem cell pages we’re developing for the journal’s website.

Research

– Over the last couple of years, Kim Cooper (a post-doc in the Tabin lab) has been providing us with updates (see her intro post here) from her multiple trips to China, during which time she has been collecting jerboas. This month, Kim shared with us the news of the first research article using jerboas to answer a fundamental question in developmental biology.

– Cat Vicente highlighted an cracking (!) paper on the development of crocodile head scales.

– We heard more about a recent paper that identified hair cell progenitors in the postnatal cochlea.

Resources

As always, the Node  proved to be a great place to find and share useful resources. This month we were introduced to:

– an updated toolkit of stem cell resources.

– CiteAb: a search engine for antibodies.

– PostPostDoc: a website for PhDs and postdocs.

Meetings

Mariana Delfino-Machin reported back from the RIKEN CDB Symposium: “The Making of a Vertebrate”

A number of upcoming meetings were also announced:

– the EMBO Workshop on Morphogen Gradients

– a Wellcome Trust meeting on Regenerative Medicine

– a University of Maine Developmental Biology Teaching Workshop

Finally, it was also a busy month in terms of job postings – a good sign really, in light of the current economic climate. To see the most recent job ads, click here. Don’t forget that it’s easy to post a job advert…or anything else that is relevant for the community…simply register and get posting!

POSTDOCTORAL POSITIONS are available to study the cellular and molecular processes controlling the early development and patterning of the brain and visual system using in vivo mouse models and organ culture systems.

Highly motivated individuals who recently obtained a PhD or MD degree and have a strong background in developmental neurobiology are encouraged to apply. Interested individuals should send their curriculum vitae, a brief description of their research interests, and the names of three references to:

Guillermo Oliver, PhD

Member

Department of Genetics

St. Jude Children’s Research Hospital

262 Danny Thomas Place,

Memphis, TN 38105-3678

E-mail: guillermo.oliver@stjude.org

http://www.stjude.org/oliver

Asymmetric cell division of neural stem cells

Funding for a PhD and a postdoc position is immediately available in the lab of Jens Januschke at the College of Life Sciences (University of Dundee, UK) to study asymmetric cell division in Drosophila. The projects address how stem cells produce two daughter cells with different fates through a single division. The basic aims are to mechanistically dissect how polarity of stem cells and cell fate choice are linked using genetics and state-of-the-art live imaging as well as OMX super resolution microscopy.

Asymmetric cell division is of critical importance for development and normal tissue maintenance. We use Drosophila neural stem cells, called neuroblasts, as a model to identify the mechanisms that underpin this fundamental process. Defective cell fate choice is implicated in many diseases particularly cancer. Neuroblasts offer the exciting possibility to study the mechanisms that govern cell fate choices in living stem cells and document and measure their activity over time.

If you are interested in the project, want to deepen your background in cell and developmental biology and have a high interest in microscopy please contact us.  Applicants should have a strong background in cell or developmental biology. Experience in quantitative image analysis and previous experience with Drosophila are advantageous. Please provide  a short letter of motivation, CV and the email addresses of three references. Funding for specialised training courses in microscopy is also available.

References

Januschke, J. et al. Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts. Nature cell biology 15, 241-248,(2013).

Januschke, J., Llamazares, S., Reina, J. & Gonzalez, C. Drosophila neuroblasts retain the daughter centrosome. Nature communications 2, 243,(2011).

Januschke, J. & Gonzalez, C. Drosophila asymmetric division, polarity and cancer. Oncogene 27, 6994-7002,(2008).

http://www.lifesci.dundee.ac.uk/people/jens-januschke-1

http://www.lifesci.dundee.ac.uk/research/cdb/

http://www.dundee.ac.uk/main/colleges-services/

http://microscopy.lifesci.dundee.ac.uk/index.html

See below a posting from our website (http://amapress.gen.cam.ac.uk/) on the discussion that took place at the BSDB on whether to change or not the name of the society to include Stem Cell Biology. Some of the people who have read it have encouraged me to post it here to see what people think and I believe is a good idea (to open it up to discussion). In many ways it follows the spirit of Olivier Pourquie’s editorial at the beginning of the year in Development (http://dev.biologists.org/content/139/1/1.full)

With my wishes for a good Easter

Stem Cells in Developmental Biology: a debate at the BSDB

Last week the BSDB (British Society for Developmental Biology) celebrated its annual gathering at Warwick. Always a good place to go for quality developmental biology which is enhanced by the arrangement of holding the meeting together with the BSCB (British Society for Cell Biology): these days there is much cell biology in developmental biology. One of the BSDB sessions focused on Stem Cells and highlighted the clear connection between this area of research and developmental biology, or so it seems to some of us but ……perhaps not all.

The AGM of the BSDB had, for the second year running, a ballot to change the name of the society to include “stem cells’ in its name. Thus, the proposal was to change the name from “British Society of Developmental Biology” to “British Society of Developmental and Stem Cell Biology”. The proposal had been flagged last year and, after a vigorous discussion, was rejected, but by a narrow margin which allowed the subject to be brought up again to the AGM this year, where it was resoundingly rejected. But read on……….

The discussion preceding the vote was heated and highlighted several misconceptions about research in stem cells which, perhaps, represent some reality.

The ones in favour argued, correctly I think (I was and am in favour of the change) that Stem Cell research is part of developmental biology and that while there is much that has to do with medicine, the links between Developmental and Stem Cell Biology are strong and essential for both fields, that Developmental Biology can bring rigour and direction to Stem Cell Biology and that Stem Cell Biology can bring challenges, new ways and possibilities for Developmental Biology. They (we) argued that, after all, Stem Cell biology has always been part of Developmental Biology, albeit somewhat cryptically. Including Stem Cells in the name of the society is an acknowledgement of the times and can have its benefits because there is no denying that Stem Cell Biology is a central and key element of research these days; including it explicitly in the Societie’s agenda would allow the BSDB to have a strong voice in policy, funding and education in these increasingly influential area of research.

The ones against the change argued that Stem Cell Biology is different from Developmental Biology, that it has a clinical slant which would attract a different crowd to the meeting, force different content in sessions and, overall, distract us from the main business: the workings and evolution of embryos and systems. It was suggested that such a change would alienate several established constituencies within the society that would abandon the group. Mpre significantly, that, eventually, with another vote a few years down the line, Developmental Biology would be booted out of the title for the society to become the British Society for Stem Cell Biology.

As far as a I am concerned this was a missed opportunity. While I appreciate many of the points made by the ‘noes’ , I feel that their arguments are based on fear for a future that will always take over no matter what we do and that, in the long term, there will be consequences from not seeing this. We live in an increasingly corporate world in which lobbies are important and, in the context of our business, provide the basis for funding and policy. For the Society of Developmental Biology Society to have an explicit voice in the Stem Cell community is not just an extension of its natural remit and interests, but it is a way to bring some real science into a field that is increasingly interested in applications without having covered the bases. The foundations of Stem Cell Biology lie in Developmental Biology and it is important that developmental biologists have a say on decision making in that important field. Stem Cell Biology is not exactly, as some people claim, a new area of research (developmental biologists have been working with stem cells and their lineages for years) but it is certainly an area that recently has come of age to carve its own intellectual niche like, in many ways, Developmental Biology did in the 1970s (let us not forget that Developmental Biology is an offshoot of Embryology). It was argued that Stem Cells are born with Till and McCulloch (1964 Proc Nat Acad Sci. 51, 29–36). True but what they were looking at is the question of the origin of the blood, a problem in Developmental Biology whether one likes it or not. At certain places, and stem cell research is one of them, boundaries blur. Is genomics and bioinformatics genetics? Yes it is. In my book, Stem Cell Biology is part, and a very important part of Developmental Biology.

But let us move away from the heart of the question (of course the scientific content) and look, briefly, to the context of the discussion. The boundary, as I have said is blurred, and a situation can develop (and in certain places is happening) that some people, funding bodies, society, come to see Developmental Biology through the eyes of an unbridled Stem Cell Biology. After all, is it not organs out of cells that is the goal of stem cell biology? And is it not understanding these processes the goal of Developmental Biology? Then, what is the difference? The answer is simple, Stem Cell Biology wants to do, Developmental Biology wants to understand. It would be a pity not to bring them together. One can see here history trying to repeat itself: throughout the XVIII and XIX century engineers and inventors were making steam engines with little knowledge of physics, and they worked, but it is when the knowledge of thermodynamics is brought into the frameowork of the engineers that the engines become efficient. The same can be said of computing where, again, it is physics that makes the hardware that we have today. The fundamental science will always help the more applied side and needs it. So, much to be gained from Developmental Biology having a say in Stem Cell Biology. But there is a second more difficult question: what will be the consequences of the agenda of stem cells running that of developmental biology?

I can see a marginalization of model organisms and a biasing of the agenda towards applied science, applied in a trial and error way, rather than in the tradition of Science. I might be wrong in the extreme formulation of these concerns but I am certain that some of this will happen.

In the end, my impression was that the ‘noes’ were afraid, afraid of the future without realizing that he future will happen and that by not seeing the trends and joining them, we shall always be left to mercy of those trends, without a voice to influence them. I worry that model systems that have taught us so much about basic biology will slowly be squeezed into corners because we do not have a voice to explain that flies have stem cells, that stem cells are part of the make up of an organism which cannot be understood outside its context, that stem cells are a problem of evolutionary biology, that stem cells are a linguistic twist of linage analysis and lineage analysis has always been a problem of developmental biology, from Roux and Driesch to Garcia Bellido with Till and McCulloch in the middle. Incorporating Stem Cell Biology in the name of the society would have been a way of having a strong voice in a trend that is rapidly gaining momentum.

We shall see what the future harbours. The BSDB is a strong society which represents a vibrant and engaging community so there is no reason why things will change rapidly. However, one thing is clear: there is a need for the voice of developmental biologists to be heard in the Stem Cell community. A mechanism needs to be found for this. It is necessary as much to have a representation to remind that community where their real roots lie and the benefits of listening to the fundamentals of their field. There is a drift which was, unfortunately, at the heart of many of the speeches for the no, that Stem Cell Biology is more clinically that basic research orientated. One can see how this can be construed but, decisions like the one we have taken will increase this gap and foster this misunderstanding. It would be good (it is always good) to take lessons from history. As the interactions between physics and engineering prove, there is much to be gained from the interactions between a field that tries to find practical solutions and one that explain the causes of the problems. Let us hope that the BSDB can find a way to influence some of the directions of Stem Cell biology. For the moment it is as if two twins have been separated; each with their own mind but with shared genetics.

I’ve just sent out EuroStemCell’s monthly newsletter and thought I’d share a couple of the big items that might interest all of you.

It’s been another busy month, with lots of events ranging from schools activities to discussions with policy makers about changes to the EU Clinical Trials Directive. Plus, we’ve given our toolkit of educational and outreach resources a facelift to make it even easier to find the public engagement tool you need for your event.

In Italy stem cells have hit the headlines as the Italian Ministry of Health has overruled regulators to allow use of an unproven stem cell therapy in public hospitals. Stem cell researchers in Italy and beyond have expressed alarm about the decision. We explain the issues and share quotes from leading scientists in our news article.

You can read the full March newsletter on our website. Below a bit more on this month’s big items.

Our stem cell toolkit has had a facelift!

Our toolkit of downloadable stem cell resources and activities has been growing so fast it’s getting hard to keep up with all the new additions. We launched with just five tools, but that number has since grown to 13 tools for teaching and talking about stem cells, in the classroom, science centre, open day, festival and other educational settings. We’ve had great feedback from teachers and others from around Europe: “[The] toolkit web page … is very easy to use. Everything appeals to me: not only the “surface”, icons and links, but also the contents are very useful and interesting.” Teacher, Italy.  Read more

Sproing! Sproing! Sproing! If there is one animal that deserves its own cartoon sound, it is the jerboa – a bipedal desert rodent with extraordinarily elongated hindlegs, fused foot bones, and loss of the first and fifth toes. I blogged here from China last spring during the most recent field collection of jerboa embryos, and now I’m excited to share news of the first research article using jerboas to answer a fundamental question in cell biology and skeletal growth and evolution: how do growth plate chondrocytes enlarge, and how do growth plates adjust cell size to contribute to differences in rates of skeletal growth?  I have been derelict about entries, not sure what people would find interesting to read, so there is a lot of backstory that I will try to summarize. If anyone wants to see some of this story fleshed out, I would be happy to take blog post requests :)

I decided to postdoc with Cliff Tabin because of my interest in the limb as a structure to study morphological diversity, but “I am interested in limb diversity” isn’t a focused way to start a project. We spent a few months tossing around ideas for a specific animal to study wherein I laughingly proposed dinosaur, dolphin, and horse. That’s one great thing about Cliff – he took each of my crackhead ideas seriously and never said “no”. That meant my joking suggestion of horses turned to “Here’s your horse” when a student at Harvard introduced us to the jerboas. The jerboa and horse have converged on similar adaptations including loss of toes and fusion and elongation of remaining elements. I am actually starting to address convergence in horses as well since it turns out horse embryology isn’t impossible. But that’s a story for another day.

The early years were focused on acquiring specimens involving multiple trips to China to collect pregnant females and harvest the embryos. The benefit of going to China is that the species there hibernate through the winter and breed almost synchronously in the spring. This means that if we hit the timing right, we can get tons of embryos from a short collection. Unfortunately it takes some trial and error to get that timing right. When you work on a seasonal animal that means it can take several frustrating years to achieve success.

Meanwhile I embarked on an adventure to get the first research colony of jerboas established, and everything I’d read online said they would not take care of their offspring in captivity. It was the tiger mauling at the San Francisco Zoo in 2007 that brought to my attention the Association of Zoos and Aquariums and, more importantly, the International Species Information System. I sent an email to ISIS to ask if they had a husbandry manual or holdings records for any species of jerboa, and they responded with no husbandry manual but with the contact information for 11 zoos and conservation centers that had jerboas in their records at some point since the 1960s. I emailed all of them and got one amazing response from the Breeding Centre for Endangered Arabian Wildlife in Sharjah, UAE. This species of jerboa isn’t endangered, but that whole story and how I learned to raise jerboas is probably also best saved for another entry or this will get ridiculously long.

I started investigating mechanisms of digit loss and mechanisms of rapid skeletal elongation in parallel, but the embryos to study digit loss were in short supply until this last (amazing) collection. Meanwhile, the colony started to breed, and I realized that the significant time window for increased hindlimb elongation was in early postnatal stages – easy to get from my burgeoning colony without decimating the breeding population. I started with an analysis of BrdU labeling index…nothing exciting there. One day as I was looking at sections that were stained with H&E it struck me: those hypertrophic chondrocytes in the jerboa foot are HUGE.

So I dug through the literature and found a paper by Norm Wilsman and colleagues in 1996 that quantified the percent contribution of a number of factors to the daily rate of skeletal elongation (growth in microns per day). They determined that the process of volume enlargement during hypertrophy of the terminally differentiated chondrocytes contributes most significantly to skeletal elongation. Additionally, the size of those hypertrophic chondrocytes varied most between skeletal elements that elongate at different rates within an animal (ie the fast proximal tibia growth plate versus the slow proximal radius).

Hypertrophic chondrocyte size contributes significantly to skeletal growth and to differences in rates of growth, so how is cell size regulated? But that question turns out to be the second question in the pipeline. The first question was “How do these cells get big?” There was some discussion in the literature, primarily from Joseph Buckwalter and Peter Bush, suggesting that chondrocytes may enlarge by cell swelling – a disproportionate increase in cytoplasmic fluid volume. However when I talked to a couple of cell biologists at Harvard, I was met with some resistance. Plant cells swell, but they also have cell walls to contain the pressure. Animal cells don’t swell. If the neurons in your brain swell by as little as 10%, they rupture which causes some serious problems. Regulated volume increase and regulated volume decrease are so important that our bodies have developed mechanisms to stabilize cell volume in cases of shifts in blood plasma osmolarity. But hypertrophic chondrocytes looked really empty in transmission electron micrographs…

I decided to address this question of whether chondrocytes swell and put together a K99 proposal (which missed the payline by one point in the only submission I could make before hitting the 5 year mark). In the lead up to grant submission, Cliff put me in touch with Tim Mitchison in Systems Biology who introduced me to Seungeun Oh in Marc Kirschner’s lab. Seungeun had just finished her PhD in Spectroscopy at MIT and had joined Marc’s lab to apply microscopy methods to questions of cell volume control in the cell cycle. She had the perfect methodology to answer my question – using the retardation of a wavelength of light to quantify cellular dry mass contents. The very first experiment we did was on her old set up at MIT the day before my grant deadline to provide proof of principle. I had dissociated postnatal day 5 mouse tibia chondrocytes, and we carried the dish on the M2 shuttle across town to the basement of MIT. (Isn’t all of MIT a long basement corridor?) One of the first images we took was a perfect cluster of 3 cells of varying sizes showing a small cell, an intermediate cell that was more red on the phase shift heat map, and a third cell that was enormous and less red on the phase shift heat map indicating it hadn’t increased in total mass as would be expected for a larger cell maintaining high density. Even though we still had to collect data from many more samples and show that the cells remained spherical and didn’t just flatten out (which could also decrease the phase delay), we were so excited and gave each other a good high five. That image from the very first day became panel “a” of Figure 1, because we never again saw such a perfect cluster of cells together demonstrating the effect of cell swelling on phase shift in a single image. It’s like they were waving at us with a taunting “You’re right!”

That first day led to 2 ½ years together in a small dark room imaging cells, countless hours of clicking on images in Matlab, writing, re-writing, reworking figures, and discussions of osmolarity and properties of light in the hybrid language of an organismal biologist and a physicist. It was a perfect merging of two disciplines. From that initial observation in the mouse proximal tibia, we went on to discover that there are three phases of chondrocyte enlargement: an initial phase where chondrocytes increase in volume while maintaining a “normal” dry mass density of about 18%, a second phase where cells swell and dilute their dry mass to about 7%, and a final phase where cells continue to get larger but maintain low dry mass density. Larger cells get bigger by extending the third phase, and smaller cells truncate the process that’s shared by all chondrocytes observed in this study. To get at a genetic mechanism controlling this process, we investigated the cell phenotype in animals that were conditional mutants for insulin-like growth factor 1. We found that not only do the growth plate specific differences in cell size disappear, but that igf1 controls the third phase that is most variable in growth plates elongating at different rates. This puts the igf1 pathway in a perfect place to investigate the genetics underlying evolutionary differences in growth rate. Stay tuned.

But for all of the answers this paper provided, it opened a whole new can of question mark shaped worms. How do animals establish different skeletal proportions? (The original question I set out to answer.) How does the cell carry out metabolic functions in the face of a 3-fold decrease in dry mass density? How does the cell regulate its membrane surface area? And a big one – how in the heck do these cells drive all that water in to dilute the cytoplasm? As I’m preparing to set off and establish my own lab, I’m looking forward to where these questions and more will lead.

Here are the highlights from the new issue of Development:

Molecular map of posterior hypothalamus

The hypothalamus is a key integrative centre in the vertebrate brain that regulates many essential functions, including homeostasis and stress responses. Several transcription factors that are essential for hypothalamic development have been identified but the production of diverse neuron types in this complex brain region is poorly understood. Here (p. 1762), Andrea Wolf and Soojin Ryu identify the transcription factors required for the specification of two distinct neuron types in the zebrafish posterior hypothalamus. They show that the transcription factor Fezf2 is important for the early development of the posterior hypothalamus. Furthermore, the differential expression of Fezf2, Otp, Foxb1.2 and Sim1a defines distinct subdomains in the posterior hypothalamus during neuronal specification. The neuron types that produce the hypothalamic hormones Vasoactive intestinal peptide (Vip) and Urotensin 1 (Uts1) develop in these different subdomains, they report, and Vip neuron specification requires Otp and Sim1a whereas Uts1 neuron specification requires Fezf2, Sim1a and Foxb1.2. Together, these findings provide mechanistic insights into the generation of neuronal diversity in the hypothalamus.

Afadin: making and shaping tubules

The formation and elongation of polarised epithelial tubules is essential for the structure and function of several metazoan organ systems but the molecular mechanisms that regulate tubulogenesis are largely unknown. Here (p. 1774), Denise Marciano and colleagues provide new insights into tubulogenesis by studying the developing mouse nephron. The researchers show that kidney mesenchymal cells contain Par3-expressing microdomains on adjacent cells. These microdomains coalesce to form a continuous lumen, which elongates by extension and by additional de novo lumen formation. Both lumen formation and elongation require afadin, a nectin adaptor protein that is implicated in adherens and tight junction formation. Using mice that lack afadin in nephron precursors, the researchers demonstrate that afadin is required for the coalescence of Par3-expressing microdomains, which is needed to establish apical-basal polarity and generate a continuous lumen. These results reveal a novel mechanism for lumen formation and morphogenesis in vivo in which afadin plays a central role through its recruitment of polarity and junctional proteins to sites of lumen formation.

Ubiquitylation promotes planar polarity

Planar cell polarity depends on the asymmetric localisation of core planar polarity proteins at apicolateral junctions. This asymmetric distribution probably develops through amplification of an initial asymmetry and seems to require the regulation of core protein levels. Now, Helen Strutt, Elizabeth Searle and co-workers (p. 1693) show that two distinct ubiquitylation pathways regulate the junctional levels and asymmetry of core planar polarity proteins in Drosophila. The researchers report that a Cullin-3-Diablo/Kelch ubiquitin ligase complex and the deubiquitylating enzyme Fat facets regulate the levels of the core planar polarity proteins Dishevelled and Flamingo, respectively, at apicolateral junctions but have no effect on the total cellular levels of Dishevelled and Flamingo. Notably, both increases and decreases in the junctional levels of core proteins caused by disruption of the ubiquitylation machinery reduce core protein asymmetry and disrupt planar cell polarity. Thus, the researchers suggest, ubiquitylation maximises the asymmetric localisation of core planar polarity proteins by fine-tuning their levels at junctions.

Tcf7l1 sets the stage for lineage specification

During mammalian embryogenesis, substantial cell proliferation occurs before the establishment of the body plan during gastrulation. Thus, before gastrulation, individual embryonic cells must be pluripotent. In vitro experiments with embryonic stem cells (ESCs) have indicated that the transcription factors Oct4, Sox2 and Nanog are components of a gene regulatory network (GRN) that stimulates self-renewal of pluripotent cells and have identified Tcf7l1 (Tcf3) as an inhibitor of GRN activity. But what is Tcf7l1’s function during embryonic development? To find out, Bradley Merrill and colleagues have been examining embryogenesis in Tcf7l1^-/- mouse embryos (see p. 1665). They report that mesoderm specification is delayed in these embryos, thereby uncoupling it from primitive streak induction. Moreover, in vitro, Tcf7l1 activity is necessary to switch the response of pluripotent ESCs to Wnt/β-catenin signalling from self-renewal to mesoderm specification. Thus, the researchers suggest, Tcf7l1 prepares pluripotent epiblast cells in gastrulating mouse embryos for lineage specification and ensures that lineage specification is coordinated with the dynamic cellular events that occur during gastrulation.

β-catenin helps ES cells stick to pluripotency

Canonical Wnt signalling and E-cadherin-mediated cell adhesion are both involved in mouse embryonic stem (mES) cell maintenance. β-catenin (Ctnnb1) is central to both these processes – it mediates the transactivation of Wnt target genes and also connects E-cadherin to the actin cytoskeleton via α-catenin. But which β-catenin function is absolutely required for mES cell self-renewal and pluripotency? On p. 1684, Ignacio del Valle and colleagues investigate this controversial question. The researchers use Ctnnb1^-/-Eα mES cells in which the constitutive expression of an E-cadherin-α-catenin fusion protein maintains cell adhesion. Preservation of cell adhesion, the researchers report, is sufficient to promote the leukaemia inhibitory factor (Lif) signalling pathway, which is required for mES cell maintenance, and the transcriptional factor network that controls the mES cell state. They also implicate E-cadherin in the activation of Lif signalling by showing that it stabilises the Lifr-Gp130 co-receptor complex. Together, these findings suggest that only the adhesive function of β-catenin is absolutely required for the propagation of mES cells in culture.

Hopx marks hair follicle stem cells

Adult tissue-specific stem cells both self-renew and generate functional progeny. Mammalian hair follicles, which are characterised by cyclical phases of growth (anagen), regression (catagen) and rest (telogen), are an ideal system in which to investigate the homeostasis of an adult stem cell population. On p. 1655, Jonathan Epstein and co-workers show that Hopx, which encodes an atypical homeodomain protein, is specifically expressed in long-lived stem cells in the basal bulge of the mouse telogen hair follicle. Hopx⁺ cells, they report, contribute to all the lineages of the mature hair follicle. In addition, the researchers identify a previously unknown progenitor population in the lower hair bulb of anagen-phase follicles and show that these Hopx-expressing cells contribute to the cytokeratin 6-positive inner bulge niche cells in telogen that regulate the quiescence of adjacent hair follicle stem cells. Because Hopx expression also marks other adult stem cell populations, the researchers suggest that tissue-specific stem cell populations might share homeostatic mechanisms.

PLUS:

Brassinosteroid signalling

Zhi-Yong Wang and colleagues provide an overview of the highly integrated BR signalling network and explain how this steroid hormone functions as a master regulator of plant growth, development and metabolism.

See the Development at a Glance poster article on p. 1615

Morphogen transport

Alex Schier et al analyze various morphogen transport models using the morphogens Nodal, fibroblast growth factor and Decapentaplegic as case studies. They propose that most of the available data support the idea that morphogen gradients form by diffusion that is hindered by tortuosity and binding to extracellular molecules.

See the Hypothesis article on p. 1621

The University of Maine Darling Marine Center will once again host a Developmental Biology Teaching Workshop, June 25-29, 2013.  Openings are still available!

This workshop is geared toward university faculty, postdocs, and graduate students interested in teaching developmental biology.  It provides basic hands-on experience with organisms commonly studied in teaching laboratories. These include sea urchins and sand dollars, planaria, Drosophila, chick embryos, Spirostomum, Hydra, Lumbriculus, and flowering plants. Techniques will range from classical microsurgical techniques to fluorescence microscopy and applications of reporter gene technology.

Course Instructors:
Dr. Leland Johnson, Augustana College, SD
Dr. Eric Cole, St. Olaf College, MN
Guest Instructor, Dr. Mark Spiro, Bucknell University, PA

This workshop is the recipient of a Non-SDB Educational Activities Grant from the Society for Developmental Biology.  For more information visit the course website here: http://www.dmc.maine.edu/workshops.html#devbiocourse