Here I share the background story on my graduate work that was recently published in Development, “Dorsal activity of maternal squint is mediated by a non-coding function of the RNA”: I first joined Karuna Sampath’s group at Temasek Life Sciences Laboratory in 2005 during my undergraduate days. It was an exciting year in the lab because Aniket Gore, a former student, had published an interesting story on the identification of asymmetrically localized maternal sqt RNA as the earliest dorsal marker in zebrafish. I found the role of sqt in dorsal specification immensely intriguing, therefore I started working on it as a graduate student.

However, very soon, my project was shrouded with controversy.  Colleagues in the field had challenged Aniket’s findings, and I was following up on his work.  The first hurdle I encountered was to convince Karuna (yes, you are reading this correctly) that sqt RNA has a function independent of Sqt protein.  The sqt insertion mutants were thought to be nulls. I was stunned when I saw that sqt^cz35 RNA elicited transient dorsal expansion in wild-type embryos. I remember showing Karuna the sqt^cz35 RNA-injected embryos stained for goosecoid and chordin, excited that I had identified a non-coding function. But the first thing she asked was, “Are you sure your sqt^cz35 prep is not contaminated with traces of wild-type sqt?”  I re-transformed pCS2+sqt^cz35, made countless mini-preps, sequenced them, synthesized capped RNA, injected and assayed each for dorsal expansion in the embryos to show there was no contamination.  In any case, wild-type sqt RNA did not show transient dorsal expansion, so I knew this was something unusual.

The series of experiments that followed was rather straight-forward. I tested different forms of non-coding sqt RNA (sqt^mut RNAs) and found that even heterologous sequences fused with sqt UTR could induce dorsal, and this does not require Oep-dependent Nodal signaling. Since the dorsal activity was in the sqt UTR, Steve Cohen and Mark Featherstone, who serve on my thesis committee, asked if microRNAs had any part to play. MZdicer embryos were still able to respond to the sqt^mut RNAs, ruling out miRNAs in this process.  This was a new function I had found in the sqt 3’UTR.

No words can describe my excitement at being able to share my findings with the readers of Development. Although the journey was quite arduous, I am pleased that my work has addressed some of the issues surrounding the debate regarding maternal sqt and DV axis formation, and that I have found an interesting area of biology that can be further mined upon.  My focus is now to understand the precise mechanism by which maternal non-coding sqt functions in dorsal axis formation.

Shimin Lim

Shimin Lim, Pooja Kumari, Patrick Gilligan, Helen Ngoc Bao Quach, Sinnakaruppan Mathavan, & Karuna Sampath (2012). Dorsal activity of maternal squint is mediated by a non-coding function of the RNA Development, 139 (16) : 10.1242/dev.077081

(13 votes)

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You’ve probably already read Patricia Gongal’s summary of the recent SDB meeting (and if not, go do that now!) but many others have also reported from Montreal through Twitter. We’ve collected some of the tweets from the conference below:

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Here is the backdrop for our recent paper in Development, “Dorsal activity of maternal squint is mediated by a non-coding function of the RNA”:  This work follows up a previous publication from my laboratory where we showed that knock-down of maternal squint (sqt) or ablation of sqt-containing cells led to loss of dorsal structures in zebrafish (Gore et al., 2005).  It was a surprising finding, and did not fit with the prevailing view of sqt function or the sqt and one-eyed pinhead (oep) mutant phenotypes.  Furthermore, authors of two papers (Pei et al., 2007 and Bennett et al., 2007) reported that they could not detect sqt RNA in early embryos.  It seemed as though we were ascribing a function to an RNA no one else could see – it was The Emperor’s New Clothes of the zebafish embryo!  Our view was that the sqt insertion mutants used by the other groups were not RNA nulls.  We also couldn’t understand why they did not detect maternal sqt RNA in the mutants when it was readily visible to us (Gore et al., 2007).  There were many unresolved questions, and I was encouraged (by my institute’s scientific advisory board and other colleagues) to get to the bottom of the debated issues.  So we set out to understand the basis for the differences in the findings from the various groups, and the mechanism of maternal sqt in dorsal axis formation.

As it turns out, the differences in the datasets between the groups were very informative, and uncovered really interesting biology, which we report in Shimin Lim’s paper.  We think the initial lack of processing of maternal sqt RNA (which explains some of the differences in the RNA expression data), is likely important for other aspects of sqt function such as localization and translation.  We were very surprised to find that over-expression of mutant sqt RNA (that is incapable of making a functional signaling protein) or the sqt 3’UTR fused to any reporter could expand the expression domain of dorsal genes.  Shimin had uncovered a new non-coding activity of the RNA.

However, the dorsal expansion disappeared later in gastrulation, which was really perplexing, difficult to track, and it took us a while to figure out what was happening.  My colleague, Steve Cohen, then pointed out to us the example of bicoid transgenes that transiently expand the head anlagen in Drosophila embryos.  Shimin’s finding that the requirement for oep is not absolute for dorsal expansion by non-coding sqt RNA was another piece of the puzzle that fit well, given the differences in the phenotypes of the signaling mutants and our morphants.  That the sqt morpholinos block both RNA localization as well as protein synthesis, and lead to loss of nuclear beta-catenin helped us make sense of our previous results showing loss of dorsal structures in sqt morphants; the maternal sqt puzzle seemed to be coming together.

But there are still several pieces missing in the puzzle.  The findings we report in Shimin’s paper explain many, but not all the differences in the RNA expression data between the groups.  Alleles that are RNA nulls are required to unequivocally demonstrate the function(s) of maternal sqt.  And the biggest piece of the puzzle remains to be found – how exactly does sqt RNA function in dorsal axis formation?  So there is more interesting biology to be uncovered.

Despite the shadow of skepticism surrounding our sqt work, and the frustration of trying to make sense of data that simply doesn’t fit with prevailing models, my enthusiastic young colleagues who did this work remain excited about uncovering the unexpected, discovering novel findings, and I hope they will see the satisfaction that comes from solving a challenging puzzle.

Shimin Lim, Pooja Kumari, Patrick Gilligan, Helen Ngoc Bao Quach, Sinnakaruppan Mathavan, & Karuna Sampath (2012). Dorsal activity of maternal squint is mediated by a non-coding function of the RNA Development, 139 (16) : 10.1242/dev.077081

Gore, A., Maegawa, S., Cheong,A., Gilligan,P., Weinberg,E.  and Sampath, K. The zebrafish dorsal axis is apparent by the four-cell stage. Nature 438: 1030-1035 (2005).

Bennett, J. T., Stickney, H. L., Choi, W. Y., Ciruna, B., Talbot, W. S. and Schier, A. F. Maternal nodal and zebrafish embryogenesis.  Nature 450(7167): E1-2 (2007).

Pei, W., Williams, P. H., Clark, M. D., Stemple, D. L. and Feldman, B. Environmental and genetic modifiers of squint penetrance during zebrafish embryogenesis.  Dev Biol 308(2): 368-78 (2007).

Gore A.V., Cheong A., Gilligan P.C., Sampath K.   Gore et al. reply.  Nature 450(7167):E2-E4 (2007).

Karuna Sampath

(14 votes)

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Do you need to learn a new technique?  Are you planning a collaborative visit?  If so please have a look at our Travelling Fellowships – http://www.biologists.com/fellowships.html.  The next deadline is the 31st August.

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Open University – Stazione Zoologica Anton Dohrn International Ph.D. Program: 7 Positions on offer, 2012 – 2013

7 Ph.D. fellowships are available to carry out interdisciplinary training in Biological Sciences at the Stazione Zoologica ‘Anton Dohrn’ Naples Italy.

Applications are invited from suitably qualified postgraduate candidates (see details)

The closing date for applications is August 31st 2012

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Here are the research highlights from the current issue of Development:

Less air, more muscle repair

Oxygen levels in stem cell niches are often lower than those in surrounding tissues, and hypoxia is known to regulate the self-renewal and proliferation of multiple stem cell types. Now, Shihuan Kuang and co-workers report that hypoxia regulates the ‘stemness’ of satellite cells (muscle stem cells) (see p. 2857). Upon muscle injury, quiescent satellite cells, which lie below the basal lamina of muscle fibres, are activated to proliferate, differentiate and fuse into myofibres, which regenerate the damaged muscle. The researchers show that hypoxic conditions favour satellite cell quiescence by promoting self-renewal divisions without affecting the overall proliferation of primary myoblasts. Hypoxia, they report, activates Notch signalling, which suppresses the microRNAs miR-1 and miR-206, thereby upregulating Pax7, a key regulator of satellite cell self-renewal. Moreover, hypoxic conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells in vivo. These results identify oxygen level as a physiological regulator of satellite cell activity and suggest that manipulation of oxygen exposure could improve muscle regeneration after injury.

Embryonic stem cells achieve XEN state

Early mammalian embryogenesis is characterised by a gradual restriction in the developmental potential of embryonic cells. By the blastocyst stage, embryonic and extra-embryonic cells have diverged in their fate and function. However, on p. 2866, Kathy Niakan and co-workers describe a robust method for converting mouse embryonic stem cells (mESCs) derived from the inner cell mass of the blastocyst into extra-embryonic endoderm (XEN) stem cells. Previous studies have derived XEN from mESCs by overexpression of Gata4 or Gata6, but here the researchers achieve the same outcome by adding growth factors to a standard culture medium. They confirm that downregulation of the pluripotency transcription factor Nanog and expression of primitive endoderm-associated genes are necessary for XEN cell derivation, and they show that FGF signalling is required to exit mESC self-renewal but not for XEN cell maintenance. Intriguingly, the researchers also show that not all pluripotent stem cells respond equivalently to differentiation-inducing signals. Together, these results suggest that mESCs have a broader differentiation capacity than previously appreciated.

Gut feelings about mesothelial development

The vertebrate body cavity (coelom) and its internal organs are lined with a simple squamous epithelium called the mesothelium. Mesothelia, which generate the vasculature of coelomic organs, are relatively quiescent in healthy adults but are implicated in pathogenic conditions such as peritoneal sclerosis. The mesothelium of the heart develops from the proepicardium, a localized exogenous population of cells that migrates to and envelops the myocardium; but what are the developmental origins of other mesothelia? Here (p. 2926), David Bader and colleagues identify a novel mechanism for the generation of mesothelia by investigating intestinal development in avian embryos. Using long-term lineage tracing, they show that mesothelial progenitors of the intestine are intrinsic to the gut tube anlage. Moreover, using a new chick-quail chimera model of gut morphogenesis, they show that mesothelial progenitors are distributed throughout the gut primordium and are not derived from a localized, exogenous proepicardium-like source of cells. Thus, cardiac and intestinal mesothelia have strikingly different developmental histories, despite their similarities in ultrastructure and developmental potential.

Neural lineage pools: REST assured

During nervous system development, coordinated activation and suppression of transcriptional activators and repressors regulates a network of lineage-specific genes that maintains the neural stem/progenitor (NS/P) pool and ensures the orderly acquisition of neural cell fates. Here (p. 2878), Nurit Ballas and colleagues characterise the role played by REST (RE1 silencing transcription factor, a known master repressor of neuronal genes) during neural development. By analyzing the function of REST at different stages of mouse neural development, the researchers show that, although it represses neuronal genes in embryonic stem (ES) cells, it is not required for the maintenance of ES pluripotency or for the conversion of ES cells to neurogenic or gliogenic NS/P cells. REST is required, however, for NS/P cell self-renewal. Moreover, it maintains the proper ratio of neurons and glia during differentiation and regulates genes involved in both neuronal and oligodendrocyte specification and maturation. Thus, REST plays a central role during neural development by regulating the pool size of different neural lineages.

The long and short of developmental cell migration

Long-distance cell migration is an important feature of embryonic development, but the mechanisms that underlie these migratory phenomena are unclear. Now, by combining computational modelling and experimental analysis of neural crest (NC) cell migration in chick embryos, Paul Kulesa and co-workers provide a new mechanistic explanation for long-distance cell migration (see p. 2935). The researchers begin by hypothesizing that chemotaxis drives cells to move in a directional manner towards a distant target. This simple model is insufficient, however, to explain their new experimental data. Instead, model simulations that include tissue growth predict that directed NC cell migration requires leading cells to respond to long-range guidance signals and trailing cells to respond to short-range cues. The researchers subsequently identify two NC cell subpopulations with distinct gene expression profiles and cellular orientations within migratory streams, and experimentally confirm model predictions of the effects of exchanging leading and trailing cell positions. Thus, they conclude, a two-component mechanism that includes chemotaxis and cell-cell contact drives long-distance cell migration.

miR-7 fine-tunes pancreatic development

MicroRNAs (short non-coding RNAs that post-transcriptionally silence target mRNAs) are essential for early pancreas development. But how specific microRNAs are intertwined into the transcriptional network that controls endocrine differentiation is poorly understood. On p. 3021, Eran Hornstein and colleagues investigate the involvement of miR-7, which is highly expressed in the endocrine pancreas of several vertebrates, during mouse endocrine cell differentiation. They show that miR-7 is expressed in mouse endocrine precursors and mature endocrine cells, and that the transcription factor Pax6, which is required for endocrine cell differentiation, is an important miR-7 target. The overexpression of miR-7 in developing pancreas explants or in transgenic mice downregulates Pax6 and inhibits α- and β-cell differentiation, they report, whereas miR-7 knockdown has opposite effects. Notably, Pax6 downregulation reverses the effects of miR-7 knockdown on insulin promoter activity. These findings, which suggest that miR-7 and Pax6 are wired into a transcriptional network that ensures the precise control of endocrine cell differentiation, may help in the development of cell-based therapies for diabetes.

Plus…

Vascular instruction of pancreas development

Recent studies suggest that blood vessels provide organs with non-nutritional signals that control their development and homeostasis.  In this issue, Ondine Cleaver and Yuval Dor review the contribution of the vasculature to developing tissues, with a focus on the pancreas.

See the Review article on p. 2833

Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage

In this issue, Frederic Relaix and Peter Zammit review recent recombination-based studies that have furthered our understanding of satellite cells – muscle stem cells. The clear consensus is that skeletal muscle does not regenerate without satellite cells, confirming their pivotal and non-redundant role.

See the Review article on p. 2845

(1 votes)

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Montreal is open and friendly, and this spirit was captured by the 71st annual SDB meeting that just wrapped up in this great city. The conference atmosphere was relaxed and open, with a lot of unpublished work presented, and thoughtful and productive question periods. Many different fields were represented by developmental biologists hailing from all over the world, but it was a particularly great meeting for those interested in gene regulation, enhancers, and transcription factor function. The full program and abstracts are publicly available on the meeting website here.

There were some really excellent talks coming from surprising model systems. The meeting started out Thursday evening with Nicole King giving a convincing argument for the importance of an ancestral choanoflagellate-like cell and its interactions with bacteria to the development of multicellularity and numerous modern cell types. Elaine Ostrander made a compelling case for the genetic utility of purebred dogs, and how they can help identify genes that control different traits. Not to be left behind, the mouse model stood its ground in Andy McMahon‘s presentation about his lab’s latest work on the hedgehog pathway.

Friday, Saturday, and Sunday had jam-packed schedules with concurrent sessions and poster sessions every day. The venue was really well-suited to jumping between concurrent sessions, which was great. I was really amazed by the quality of all of the talks. It’s sometimes easy to get lost in the nitty-gritty details of which methylated histone marks what, or which transcription factor binds where and is a co-factor for what else, but speakers like Eileen Furlong, Scott Barolo, Benoit Bruneau, and many, many others gave outstanding talks about epigenetic regulation in different developmental contexts. It’s clear that the field of enhancer function and gene regulation is going in the direction of chromatin-based mechanisms. Many teams are looking at chromatin modifications in specific cell types and stages, which is a major advance in understanding enhancer function in the embryo.

The imaging session was also particularly impressive. My favorite was Maria Barna‘s talk on visualizing Sonic hedgehog particles in vivo in the mouse embryo. Her lab’s beautiful live images may completely change our model of Shh signaling.

The late-night session on trends in publishing was also really interesting, with 6 representatives of different journals giving their opinions on where developmental biology is going, and how journals are changing. The journals Development, Developmental Biology, Current Biology, Nature Reviews Genetics, Science, and the new journal eLife were there. Overall, integrating fields that have traditionally been separate (for example, development and stem cells, molecular biology and evolution) are becoming more and more important. eLife was also a hot topic of discussion, with its unique peer-review process and business model up for debate. Beverly Purnell mentioned that Science is experimenting with changes to peer-review, such as asking reviewers to weigh in on the others reviewers’ comments.

The conference wrapped up Sunday evening, first with the awards ceremony. Steve Farber gave a heart-warming presentation about BioEYES, an education program that won the Viktor Hamburger Outstanding Educator Prize this year. Cliff Tabin, winner of the Edwin G. Conklin Medal, presented the talented students and postdocs that have brought his lab to life, and Antonio Garcia Bellido, winner of the Developmental Biology-SDB Lifetime Achievement Award spoke about his life’s work. The meeting came to a close with a classy banquet and lively dance floor- the idea for a Santana tribute band was clearly a success!

(7 votes)

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I take on the story where my colleagues left it, one week now after leaving MBL and the embryology course behind. Settled into my normal life, I look back and I am sure I am yet to process what we have just experienced.  It would be redundant, for my colleagues have already done this, to write in detail about the long hours; the accessibility to resources and equipment one could not even dream of; the support; the teaching assistants and faculty members available round the clock; the drive and enthusiasm with which we seemed to get through the weeks; the mix of cultures, languages and personalities that, somehow, just worked… I would instead like to go through the last stretch of the course, provide a very personal evaluation of its transcendence, and ask that the subtle references to our time at the MBL be excused.

We start the last two weeks and take a big breath. We have ethics discussions and learn about each other’s views and countries, our uncertainties and our realities, about just how similar our goals are. We swim in the sea in the day and at night, watching the bioluminescence around us. We show each other our national dishes, and eat, drink and sing by the water.  We march, laugh and play on the fourth of July and, back in the lab, we go through a week of magic. Limbs being lost and regrown within hours, grafted hydra which welcome their new tentacles, neoblasts in planaria: so efficient, so fast.  Fun discoveries and funny moments: worms with two heads and an instant thought “let’s give them a posterior end!”, but “oh, no! it’s all anterior” and yet more heads…  A revelation of how tough and resilient, these seemingly simple and fragile creatures can be.

Week six and suddenly, the embryology course being held at the MBL makes more sense than ever, as we get to play with ctenophores, squids, cuttlefish,  tunicates, annelids, marine gastropods, and everything the sea wants to show us. We have boat trips, nets, light hunting, Matt and his plankton, under the sea… Some of the most beautiful forms of life presenting themselves to us, shining, glowing, swimming, and as we have come to see them… dancing. We harvest their embryos and their beautiful larvae and we stain, grow, inject and film them. We do cell lineage tracing, injecting, ablating, we unravel their function. But mostly we look, admiring the complexity with which the “simplest” forms of life put themselves together. So many similarities with the “higher” forms at the early stages of development make it even more difficult to understand how so much diversity is possible.

From a personal perspective, I do not know where my research is going. I have learnt the importance of the detail and the in depth knowledge of each component within the big picture. How to go back to the way I was doing things, knowing there is so much more! So many possibilities! So much to do, to learn, to discover, to play with! As we were assured when we started the course, this is certainly a life changing opportunity. I am yet to discover, or decide, how it will change. So for now, good bye Woods Hole, and many thanks to all the student of the embryology course, from whom I learnt so much, and to the directors, for being scientists we look up to, as well as great colleagues and friends.

(10 votes)

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Now that you’ve had a chance to read both shortlisted essays in the “Developments in development” essay competition, it’s time to vote for the one that you think should be published in Development.

Here are the links to the full essays:

An Excitingly Predictable ‘Omic Future – by Joanna Asprer

There‘ll be dragons? – The coming era of artificially altered development – by Máté Varga

The poll closes August 15, noon GMT.

And while you’re here, why not also pick a cover for Development?

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This week you don’t only get to decide which essay, from our competition, will appear in Development (see nominations, and the poll later today), but it’s also time to choose another cover from images from the 2011 Woods Hole embryology course. Vote in the poll below the images for the one you would like to see on the cover of Development. (Click any of the images to see a bigger version.) Poll closes on August 6, noon GMT.

1. 5th instar imaginal hindwing disk from the Painted Lady butterfly, Vanessa cardui. Immunostained for Engrailed in red. All nuclei are revealed by DAPI staining (blue), and trachae are shown in green. This image was taken by Alessandro Mongera, Maria Almuedo Castillo, and Jakub Sedzinski.

2. 3rd instar wing disk from Drosophila melanogaster. Triple flip-out clone system (courtesy of Melanie Worley and Iswar Hariharan) was used to reveal various cell lineage clones shown in yellow, blue, and purple. All nuclei shown in gray (DAPI). This image was taken by Lynn Kee.

3. Head of an adult C. elegans. DiI staining (red) reveals environmentally exposed neurons, while the JR797 GFP line allows visualization of all neurons (blue). This image was taken by Eric Brooks and John Young.

4. Ventral view of stage 16 Drosophila melanogaster embryo immunostained for Tropomyosin (green; muscle), Pax 3/7 (blue; segmentally repeated nuclei in CNS and ectoderm), and anti-HRP (red; cell bodies and axons of the nervous system). All nuclei shown in gray (DAPI). This image was taken by Julieta María Acevedo and Lucas Leclere.

While you’re here, why not also vote for the winner of our essay competition?

(5 votes)

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