48 anatomical structures of the presented mouse embryo atlas are shown in 3D.

Here is the backdrop for our recent paper in Development, “A novel 3D mouse embryo atlas based on micro-CT”.   With the human genome project complete, the sequence and the location of each gene in the genome is understood.  However, the understanding of gene function and the corresponding expressed phenotype for all the genes in the human genome is still in its infancy.  Most of the research aimed to tackle this question will be carried out in the mouse due to the 99% genetic homology between mice and humans and the available techniques to manipulate mice genetically.  Over the last decade, the efforts of a world wide consortium, the International Knockout Mouse Consortium (IKMC, www.knockoutmouse.org), has embarked on a mission to knock out each of the ~23,000 genes in the mouse genome, one at a time, and generate the resultant mice.  With this effort now close to completion another world-wide effort, the International Mouse Phenotyping Consortium (IMPC, www.mousephenotype.org), has been established and the plan on how to phenotype the resultant mice from the IKMC project is being formulated.  What is well understood is that ~30% of the gene knockout mice strains will be embryonic lethal, further accentuating the need for an assay to phenotype mouse embryos throughout development.

If you have two groups of mouse embryos, one wild-type and one mutant, with a single gene knockout, how do you find out what’s different about them?  How do you get clues to the function of the knocked out gene and its role in mouse embryo development?  The most intuitive answer would be to look at the two groups of mouse embryos with a microscope and see if you can find any gross differences in morphology in the mutant group.  You could hypothesize that the organ or structure that shows an aberration in comparison with the wild-type group is an area where that particular gene function is important and carry on with more focused phenotyping assays from there.

This is the exact premise of our recent paper in Development.   Our aim was to eliminate the human bias and time needed to parse through thousands of high-resolution images by developing automated computer methods that could export volume measures of each of the major organ structures within the mature mouse embryo.   We used advanced high-resolution 3D imaging called Micro-CT to image 35 E15.5 C57/Bl6 mouse embryos and developed sophisticated computer software to automatically calculate the mean volumes and standard deviation of 48 structures inside the mouse embryo.  To achieve this, each of the 48 structures within a representative average image of all 35 mouse embryo images were manually painted by one individual, totalling ~400 hours of work.  Through this we acquired baseline volumetric measurements of wild-type mice to determine how tight the variation is among controls.  The resulting labeled data set (the above figure) exists as an E15.5 mouse embryo atlas for which all future mutant strains can be compared with and automatic volume measurements can be executed.  The results presented in this paper, in our opinion, is an important step in demonstrating the feasibility of using 3D imaging as a primary screen in the IMPC pipeline and provides a robust tool that can handle and analyze the large volume of images that will be acquired.

Wong MD, Dorr AE, Walls JR, Lerch JP, & Henkelman RM (2012). A novel 3D mouse embryo atlas based on micro-CT. Development (Cambridge, England), 139 (17), 3248-56 PMID: 22872090

(11 votes)

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I just came across a press release that looked like it might be interesting to some of you: 1DegreeBio has launched a Stem Cell Portal on its site, that allows you to easily find user reviews and protocols for antibodies used in stem cell research.

1DegreeBio is a relatively new company and the database of reviews is still incomplete, but they do have long lists of antibodies for various targets, and an easy interface for search queries. For example, of the more than two hundred listed antibodies against POU5F1, only two have currently been reviewed by users, and another three were linked to relevant publications.

The search screen

The resource is free to use, though, and if you submit your own reviews of the antibodies you use, you can earn points that you can then spend in their shop, on Amazon gift cards or geeky toys, or donate to charity.

As the number of reviews on the site grow, you’ll be able to make more accurate assessments on the functionality of various antibodies. It seems especially useful for antibodies from certain companies that don’t do any quality testing themselves, but that are sometimes unavoidable when no other supplier sells the antibody you need. I do wonder how 1DegreeBio will be able to ensure that all reviews are done by actual independent researchers, and not left by employees of the companies selling the products, but that is something that might also be less of an issue as the number of reviews grows.

If any of you are using the 1DegreeBio site and their stem cell portal, feel free to leave a comment about your experience. I didn’t sign up myself, because I’m no longer using antibodies, but I’ve gone through many, many vials during my PhD, and have emailed several companies to check species specificity (“Does your human antibody also work with mice?”). If this kind of “Yelp for antibodies” had existed back then I’m sure I would have left many reviews about extreme variation in batch strength, unidentified cross-reactivity, and the few unexpectedly perfect antibodies that saved my thesis.

(6 votes)

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POSTDOCTORAL  POSITIONS  in  Cell  and  Developmental  Biology  is available to study the cellular and molecular mechanisms controlling the development of the lymphatic vasculature using available mouse models  and  its  functional  roles  in  health  and  disease.  Highly motivated individuals who recently obtained a PhD. or MD degree and  have  a  strong  background  in  molecular  and  developmental biology 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, Ph.D (guillermo.oliver@stjude.org) Member

Department of Genetics

St. Jude Children’s Research Hospital

332 N. Lauderdale

Memphis, TN 38105

USA

www.stjude.org/departments/oliver.htm

(2 votes)

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Here are the highlights from the current issue of Development:

eIF4E-3 puts a cap on spermatogenesis

Gene expression is translationally regulated during many developmental processes. Translation is mainly controlled at the initiation step, which involves recognition of the mRNA 5′ cap structure by the eukaryotic initiation factor 4E (eIF4E). Eukaryotic genomes often encode several eIF4E paralogues but their biological relevance is largely unknown. Here (p. 3211), Paul Lasko and co-workers report that Drosophila eIF4E-3, one of eight fly eIF4E cognates, is essential for spermatogenesis. The researchers show that eIF4E-3 is a testis-specific protein and that male flies lacking eIF4E-3 are sterile. eIF4E-3 is required for meiotic chromosome segregation and cytokinesis, they report, and for nuclear shaping and sperm individualisation. The researchers also show that eIF4E-3 physically interacts with other components of the cap-binding complex. Furthermore, many proteins are expressed at different levels in wild-type and eIF4E-3 mutant testes, suggesting that eIF4E-3 has widespread effects on translation. These results add to the evidence that alternative forms of eIF4E add complexity to the control of gene expression during eukaryotic development.

SnoN regulates mammary alveologenesis and lactation

Mammary epithelial cells undergo structural and functional differentiation at late pregnancy and parturition to initiate milk secretion. TGF-β and prolactin signalling act antagonistically to regulate this process but what coordinates these pathways? On p. 3147, Kunxin Luo and colleagues report that SnoN, a member of the Ski family of pro-oncogenic and anti-oncogenic proteins, regulates both TGF-β and prolactin signalling to control alveologenesis and lactation in mice. The researchers show that the expression of SnoN, a negative regulator of TGF-β signalling, is induced at late pregnancy through the coordinated actions of TGF-β and prolactin. Heightened SnoN expression, they report, represses TGF-β signalling, which relieves TGF-β inhibition of the prolactin pathway. SnoN also directly promotes prolactin signalling by stabilising Stat5, a mediator of prolactin signalling. Consistent with these results, alveologenesis and lactogenesis are severely disrupted in SnoN^–/– mice and mammary epithelial cells from these mice fail to undergo proper morphogenesis in vitro unless rescued by active Stat5 expression. Together, these results identify a new role for SnoN in the regulation of lactation.

Co-operative neuronal migration

During the development of the central nervous system, neurons and/or neuronal precursors travel along diverse routes from the ventricular zones of the developing brain and integrate into specific brain circuits. Neuronal migration has been extensively studied in the forebrain but little is known about this key developmental event in the embryonic midbrain (mesencephalon). On p. 3136, Kwang-Soo Kim, Anju Vasudevan and co-workers remedy this situation by studying the migration of dopaminergic (DA) and GABAergic (GABA) neurons in the mouse mesencephalon. They show that DA and GABA neurons follow similar paths to the ventral mesencephalon (VM) in a temporally sequential manner. Interestingly, they report that in Pitx3-deficient (aphakia) mice, which have a defective DA neuron architecture, DA neuron migration is abnormal, stalled DA progenitors fail to reach the VM and GABA neurons also fail to migrate to the VM. These results suggest that pre-existing DA neurons modulate the migration of GABA neurons, thereby providing new insights into neuronal migration and the establishment of brain connectivity during mesencephalon development.

Out on a limb: HoxD chromatin topology

Anterior-posterior patterning of both the primary embryonic axis and the secondary body axis (limbs and digits) in mammals requires regulated Hox expression. Polycomb-mediated changes in chromatin structure control Hox expression during the first patterning event but are they also involved in the second? Here (p. 3157), Robert Hill, Wendy Bickmore and colleagues analyse the chromatin topology of the HoxD gene cluster in immortalised cell lines derived from posterior and anterior regions of distal E10.5 mouse limbs and in dissected E10.5 limb buds. They report that there is a loss of polycomb-catalysed histone methylation and a chromatin decompaction over HoxD in the distal posterior, compared with the anterior, limb. Moreover, the global control region spatially localises with the 5′ HoxD genomic region specifically in the distal posterior limb, a result that is consistent with chromatin looping between this long-range enhancer and its target genes. Thus, the researchers conclude, the development of the mammalian secondary body axis involves anterior-posterior differences in chromatin compaction and looping.

Developmental roles for ribosomal biogenesis genes

Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene, which functions during maturation of the large 60S ribosomal subunit, cause a disorder characterised by exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. Steven Leach and colleagues have now refined a zebrafish model of this ‘ribosomopathy’ (see p. 3232). Knockdown of the zebrafish sbds orthologue, they report, fully recapitulates the developmental abnormalities of the human syndrome but, interestingly, unlike in other ribosomopathies, loss of p53 does not rescue these developmental defects. The researchers show that impaired proliferation of pancreatic progenitor cells is the primary defect underlying the pancreatic phenotype and report that loss of sbds results in widespread changes in the expression of genes related to ribosome biogenesis, rRNA processing and translational initiation, including ribosomal protein L3 and pescadillo. Notably, inactivation of either of these genes also impairs expansion of pancreatic progenitor cells in a p53-independent manner. Together, these results suggest new p53-independent developmental roles for ribosomal biogenesis genes.

Mapping the mouse embryo

The sequence and location of every gene in the human genome is now known but our understanding of the relationships between human genotypes and phenotypes is in its infancy. To better understand the role of every gene in the development of an individual, the International Mouse Phenotyping Consortium aims to phenotype targeted gene knockout mice throughout the genome (∼23,000 genes). Because many of these mice will be embryonic lethal, methods for phenotyping mouse embryos are needed. Michael Wong and colleagues are developing such an approach and, on p. 3248, they present a new three-dimensional atlas of the mouse embryo. To produce their atlas, the researchers combined micro-computed tomography images of 35 E15.5 mouse embryos into an average image using automated image registration software, and then manually segmented 48 anatomical structures. This atlas establishes baseline anatomical phenotypic measurements against which mutant mouse phenotypes can be assessed; in the future, a mutant embryo image can be registered to the atlas and its organ volumes calculated automatically.

Plus…

Making waves: the rise and fall and rise of quantitative developmental biology

The tenth annual RIKEN Center for Developmental Biology symposium ‘Quantitative Developmental Biology’ held in March 2012 covered a range of topics. As reviewed by Davidson and Baum, the studies presented at the meeting shared a common theme in which a combination of physical theory, quantitative analysis and experiment was used to understand a specific cellular process in development. See the Meeting Review on p. 3065

A computational image analysis glossary for biologists

Meyerowitz and colleagues present a glossary of image analysis terms to aid biologists and  discuss the importance of robust image analysis in developmental studies.

See the Primer article on p. 3071

Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies

Members of the MADS-box transcription factor family play essential roles in almost every developmental process in plants. Kaufmann and colleagues review recent findings on MADS-box gene functions in Arabidopsis and discuss the evolutionary history and functional diversification of this gene family in plants.

See the Review article on p. 3081

(1 votes)

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With an overwhelming majority, this crisp fly embryo staining won the third round of cover voting, in which readers of the Node chose from images taken in the 2011 Woods Hole Embryology course to select a cover for an upcoming issue of Development.

It shows a 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).

The winning image was captured by Julieta María Acevedo of the Fundacion Instituto Leloir in Argentina, and Lucas Leclere of the Sars International Centre for Marine Molecular Biology in Norway. Congratulations!

The runner-up was an immunostaining of a butterfly wing disk, taken by Alessandro Mongera, Maria Almuedo Castillo, and Jakub Sedzinski. The image of a C. elegans head, taken by Eric Brooks and John Young, got third place, and the final image, of a 3rd instar Drosophila wing disk, was the work of Lynn Kee.

Meanwhile, you’ll see the winner of the previous round on the cover of Development tomorrow, so keep an eye on the journal website.

(1 votes)

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British researchers working with human embryos for IVF have been wondering about the effects of components of the culture medium they use.

In the UK, culture media used for IVF and fertility research are regulated by the Medicines and Healthcare Regulatory Agency (MHRA) and at European level. Regulation ensures that all researchers and clinicians in the field are using industry standard medium, but manufacturers can change the composition of the media at any time without input from its end users.

When IVF researchers became curious about the role of the various components in the culture medium, and whether the manufacturers could justify changes to the medium, they contacted the HFEA (Human Fertilization & Embryology Authority) – an independent regulatory body in the UK that oversees IVF and related research.

Among many of its other tasks, the HFEA has been trying to make sure that culture medium used for in vitro maturation and preimplantation cultures is safe and optimized for use with human embryos. For example, in 2001, they surveyed IVF clinics about the culture medium they use, mainly to investigate any potential infection risks.

In December 2011, the authority’s Scientific and Clinical Advances Advisory Committee (SCAAC) published a report outlining the current knowledge about the effect of culture media components on embryo viability and development. It’s available on their website.

This most recent report notes that “Although generally considered to be safe based on past and current experience, there are still uncertainties about the long term effects of the culture media used for in vitro fertilisation, and questions remain about the effect of varying components in media. Varying concentrations of components such as growth factors, amino acids, energy substrates, and antibiotics may potentially impact on early embryo development and may have longer term health implications.”

The main conclusion of the report is that, at the moment, the research is inconclusive. They recommend more research into effects of growth medium on early and late development, as well as further investigation into long-term health effects.

The report summarizes some of the studies that have been done to date, which suggest that it’s crucial to keep an eye on what manufacturers add to the media. In one example, an article by Harper et al in Human Reproduction points out that culture medium used in human embryo development was designed based on studies in animal development. It’s not optimized for human development, because for many conditions we simply don’t know what the optimal human condition is. What we do know is that the conditions used in embryo cultures may have long-term effects. They cite a study by Dumoulin and colleagues that showed that different preimplantation media result in differences in birth weight after successful IVF pregnancies. Since birth weight may be associated with disease risk later in life, this research in particular highlights the importance of studying the effect of the various components of culture media.

Studies such as these suggest that any changes in culture media for human embryos need to be assessed very closely. However, the manufacturers of culture media are not obliged to disclose the exact composition of the media they sell. That makes it very difficult to understand the effect of various factors on development.

In an ongoing effort, the HFEA is collecting reports and studies such as these, and is gathering feedback from IVF clinics and researchers. They hope to find out what can be done to ensure that the effects of the culture media used in this field are well-regulated and understood, and plan to communicate their findings to the MHRA.

These studies may take years to complete, especially those assessing long-term effects, but a stronger scientific understanding of the role of in vitro culture medium in human development will eventually translate to a higher success rate and lower risk of IVF procedures.

Harper J, Magli MC, Lundin K, Barratt CL, & Brison D (2012). When and how should new technology be introduced into the IVF laboratory? Human reproduction (Oxford, England), 27 (2), 303-13 PMID: 22166806

Dumoulin JC, Land JA, Van Montfoort AP, Nelissen EC, Coonen E, Derhaag JG, Schreurs IL, Dunselman GA, Kester AD, Geraedts JP, & Evers JL (2010). Effect of in vitro culture of human embryos on birthweight of newborns. Human reproduction (Oxford, England), 25 (3), 605-12 PMID: 20085915

(3 votes)

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We are seeking to appoint post-doctoral researchers to study microenvironmental regulation of stem cells in mammalian skin.

The aim of our group is to gain a better understanding of the mechanisms underlying the ways in which tissue microenvironments are regionally specialized, and how the specialized microenvironments instruct cellar behaviour and communication, and organ formation. We are particularly interested in the role of the extracellular matrix (ECM) in the formation of the stem cell microenvironment or niche.
http://www.cdb.riken.jp/en/02_research/0202_creative30.html

A recent study by our team has shown that the molecular composition of the basement membrane in mouse hair follicle stem cell niche, the bulge, is highly specialized. One stem cell-derived component, nephronectin, is important for the development and positioning of the bulge-residing arrector pili muscles, which, among other functions, are responsible for goosebumps (Fujiwara et al. 2011. Cell 144, 577-589). This was the first report to show that stem cells regulate the fate and positioning of surrounding niche cells through the specialization of the basement membrane.

To gain further insight into fundamental aspects of the microenvironmental regulation of stem cells, we use mouse skin as a model and seek to better understand 1) the molecular landscape of basement membrane specialization in the stem cell niche, 2) mechanisms by which the basement membrane in the stem cell niche is regionally specialized, and 3) how the specialized basement membrane controls stem cell niche formation, stem cell behaviour and the conversation between stem cells and their neighbouring cells.

Successful candidates will receive an excellent salary commensurate with qualifications and experience. Our Centre, the Center for Developmental Biology (CDB), is a world-leading research institute in the fields of developmental and regenerative biology and has state-of-the-art research facilities. The Centre provides a truly international, collegial, and supportive environment for its nearly thirty laboratories, and the freedom and resources to pursue their research toward deeper understanding of developmental biology. All necessary communications can be conducted in English, and support services are available for non-Japanese-speakers.

Please contact Hironobu Fujiwara, PhD (hironobu.fujiwara@cancer.org.uk) for further information.

To apply, please send 1) a cover letter, 2) a CV with publication list, 3) names and contact details of two referees, 4) a brief summary of research achievements and future research interests (two page maximum) to Hironobu Fujiwara (hironobu.fujiwara@cancer.org.uk) via email. Please send the application as a single PDF file. This call will be closed when the positions are filled.

(No Ratings Yet)

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What was new on the Node this past month?

“Developments in development” essay competition
Congratulations to Joanna Asprer and Máté Varga, whose essays were selected by the judges in our recent essay competition. Read both essays on the Node, and then vote for your favourite. The final winning essay will be published in Development.
–An Excitingly Predictable ‘Omic Future – Joanna Asprer
–There’ll Be dragons? – The coming era of artificially altered development – Máté Varga

Research
Karuna Sampath’s lab has a paper out in the most recent issue of Development, and shares the backstory on the Node. They discovered that knockdown of squint affected dorsal axis formation in zebrafish, but their initial findings didn’t seem to match the prevailing knowledge about the role of squint, or even the stages at which it was expressed. “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 zebrafish embryo!” writes Karuna, while graduate student Shimin Lim gives the student perspective in a second post: “… 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.”
In their Node posts and the paper, Karuna and Shimin describe how they solved the puzzle to match both their new observations and the existing literature in the field. It’s a story of science in action!

Conferences and courses
This month we heard from Gi Fay Mok, who attended the 12th International Conference on Limb Development and Regeneration in Mont-Tremblant, Canada. It sounds like it was a great conference!

A few weeks later, developmental biologists gathered in Canada again, for the annual SDB meeting, which was held in Montreal this year. Patricia Gongal summarized the meeting’s highlights and we collected tweets from the conference on Storify.

We also received more updates from the Woods Hole Embryology Course. Priti wrote about making connections and meeting people while Manuela reflected on the course after it finished.

Meanwhile, there is currently an open voting round to choose a Development cover from these images from last year’s embryology course. Which image is your favourite?

Also on the Node
– Clare Cox reviewed the film “Stem Cell Revolutions”. This is an educational documentary about advances in stem cell research, produced by Amy Hardie and Clare Blackburn. Read Claire’s review, and then watch the documentary!

(No Ratings Yet)

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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:

(more…)

(1 votes)

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