We have an offering an exciting post-doctoral opportunity to image and quantitatively analyse tiny transparent ascidian embryos with a brand new multi view light sheet microscope. Apply now!

Preimplantation development establishes the founding cell population of the adult mammal in the epiblast. This naïve pluripotent state employs a unique hand of transcription factors to ensure epigenetic resetting and unbiased embryonic potential. In rodents, naïve pluripotency can be captured in the form of embryonic stem (ES) cells^1-4, however other mammals have proven more refractory. This raises important questions such as why can naïve pluripotency in rodents be readily stabilised in culture and what is the difference to other mammals, in particular primates, like ourselves? Furthermore, it remains unclear which factors control the rise of naïve pluripotency in the embryo and how primates initiate lineage segregation.

Figure 1: (A) Lineage-specific profiling of the emergence and progression of naïve pluripotency in the mouse embryo. (B) Diffusion map of lineage-specific RNA-seq samples from embryo, diapause and ESC. 

We started to tackle some of these questions by scrutinising the emergence and dissolution of naïve pluripotency in vivo⁵. Taking advantage of Pdgfr::GFP knock-in mice⁶, we performed lineage-specific RNA-seq from the 8-cell stage to the postimplantation embryo (Figure 1A). This allowed us to define dynamically expressed genes and to derive the transcriptional signature of each developmental stage. Naïve pluripotency markers (Klf2, Klf4, Esrrb, Tfcp2l1) are tightly restricted to preimplantation development, with the majority being expressed in both, embryonic and extraembryonic lineages. Epigenetic modifiers associated with a permissive chromatin state (Tet1, Tet2, Prdm14) were predominant during preimplantation development. These factors were exchanged for regulators mediating de novo DNA methylation (Dnmt1, Dnmt3a, Dnmt3b) and transition to more closed chromatin configuration (Hdac5, Hdac11) after implantation.

Naïve pluripotency is a transient state during normal development. We wanted to relate this to stable pluripotent states in vivo (diapaused epiblasts) and in vitro (ES-cells). Therefore, we profiled diapaused epiblasts and ES-cells using the same protocol. This was essential to allow direct comparison between the minute RNA quantities from the embryo and conventional bulk culture. Analysis of ES-cells and diapause in relation to normal development reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast (Figure 1B). Interestingly, diapaused epiblasts showed strong JAK-STAT and WNT signalling pathway component expression. It is tempting to speculate that the pathways evolved to mediate developmental arrest in vivo may facilitate capture of naïve pluripotency in vitro in mouse.

However, what are the differences between naïve pluripotency in the mouse embryo compared to other species, in particular primates? To address this, we turned to the common marmoset (Figure 2), a small New World monkey and emerging primate model for biomedical research^7-9. ICMs of early, mid- and late marmoset blastocysts (Figure 3) were isolated by immunosurgery and profiled by RNA-seq. The majority of pluripotency-associated genes was expressed in the ICM and naive markers NANOG, KLF4 and TFCP2L1 colocalised in the putative epiblast. However, the absence of KLF2, NR0B1, FBXO15 and BMP4 suggests primate-specific adaptations in the wider pluripotency network.

Figure 2: Challithrix jacchus (common marmoset )

The specific pathways driving lineage commitment and segregation in the primate embryo are poorly understood, therefore we assessed signalling pathway component expression during normal mouse development, diapause and in the primate ICM. RNA-seq datasets were intersected with annotated signalling pathways for side by side comparison between mouse and marmoset. Ready to use summaries are available to the community through our online resource:

Mouse and Marmoset Pathway Expression Atlas for Early Development

http://pathway-atlas.stemcells.cam.ac.uk/

Figure 3: Immunofluorescence of marmoset late blastocyst for NANOG (green), GATA6 (white), CDX2 (red) and DAPI (blue).

At the early ICM cell stage, we observed substantial differences in NODAL, FGF and WNT signalling. To test the functional relevance, mouse and marmoset morulae were cultured to the late blastocyst stage in the presence of specific inhibitors of these pathways. The experiments demonstrate that lineage specification in primate preimplantation development is regulated by both WNT and FGF pathways, in contrast to mouse, where FGF/ERK is the primary and sufficient driver. Furthermore, the data shows that NANOG expression in the ICM does not require FGF, WNT or NODAL signals. In particular, we noted an increase in NANOG positive cells when ERK activation is inhibited by PD03. Robust expression of Nanog in the absence of FGF/ERK signalling is reported in a variety of species, including mouse, rat, bovine¹⁰ and human blastocysts¹¹ and may present a general feature of naïve pluripotency in mammals.

Finally, we would like to emphasise that all lineage-specific RNA-seq data is available as formatted tables in the supplementary to make the data readily accessible for biologists without bioinformatical support.

Literature

1              Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156 (1981).

2              Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78, 7634-7638 (1981).

3              Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519-523, doi:nature06968 [pii] 10.1038/nature06968 (2008).

4              Boroviak, T., Loos, R., Bertone, P., Smith, A. & Nichols, J. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification. Nat Cell Biol 16, 516-528, doi:10.1038/ncb2965 (2014).

5              Boroviak, T. et al. Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis. Dev Cell 35, 366-382, doi:10.1016/j.devcel.2015.10.011 (2015).

6              Plusa, B., Piliszek, A., Frankenberg, S., Artus, J. & Hadjantonakis, A. K. Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst. Development 135, 3081-3091, doi:135/18/3081 [pii]

10.1242/dev.021519 (2008).

7              Sasaki, E. et al. Generation of transgenic non-human primates with germline transmission. Nature 459, 523-527, doi:nature08090 [pii]

10.1038/nature08090 (2009).

8              Hanazawa, K. et al. Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus). Theriogenology 78, 811-816, doi:10.1016/j.theriogenology.2012.03.029 (2012).

9              Mansfield, K. Marmoset models commonly used in biomedical research. Comp Med 53, 383-392 (2003).

10           Kuijk, E. W. et al. The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos. Development 139, 871-882, doi:10.1242/dev.071688 (2012).

11           Roode, M. et al. Human hypoblast formation is not dependent on FGF signalling. Dev Biol 361, 358-363, doi:10.1016/j.ydbio.2011.10.030 (2012).

And the winner of the latest round of images from the Woods Hole embryology course is… the short-tailed fruit bat embryo!

Here are the full results:

– Pig embryo: 102 votes

– Longfish inshore squid embryo: 67 votes

– Short-tailed  fruit bat embryo: 296 votes

– Mouse embryo: 129 votes

Many congratulations to Idoia Quintana-Urzainqui (University of Santiago de Compostela, Chile), Paola Bertucci (EMBL, Heidelberg, Germany), Peter Warth (Universität Jena, Germany) and Chi-Kuo Hu (Stanford University, USA) who took this image at the 2014 course. It shows a stage 19 short-tailed fruit bat (Carollia perspicillata).  The  left side of the image shows the fixed embryo before staining, imaged using a Zeiss AxioZoom with ApoTome.   The right side of the image shows the embryo after staining for cartilage (Alcian Blue), imaged using a Leica M80 Stereomicroscope. This image will feature in the cover of a coming issue of Development.

The other great images in this round were taken by Agne Kozlovskaja-Gumbriene and Anne-Marie Ladouceur (pig embryo), Michael Piacentino (longfish inshore squid embryo) and Raymond Yip (mouse embryo).

Sorry for all the messing about, but there are now a number of ways of accessing  my latest polemic which is called The last 50 years: mismeasurement and mismanagement are impeding scientific research.

Here is one

http://making-of-a-fly.me/files/pdf/Lawrence-2016.pdf

PhD Program on Quantitative Biology – Open call:

LabEx inform is a interdisciplinary and international research consortium on Quantitative Biology of Signaling, in Marseille, France (http://labex-inform.com/).

LabEx INFORM has its own PhD program, providing interdisciplinary training and excellent education in renowned local institutions with competitive stipend.

We encourage the application of graduate students (master degree or equivalent) with backgrounds in biology but also engineering, physics and mathematics.

This PhD program is integrated in the international training program called BIOTRAIL, providing excellent support to welcome and train international students.

The LabEx INFORM (interdisciplinary program) opens up to 5 positions this year. Recruitment campaign is now open until March 20th 2016!

Information and application procedures : http://sciences.univ-amu.fr/biotrail/apply

Flyer_BioTrail_LabexInform

This editorial by Monica J. Justice and Paraminder Dhillon was first published in Disease Models & Mechanisms.

ABSTRACT

Experiments that use the mouse as a model for disease have recently come under scrutiny because of the repeated failure of data, particularly derived from preclinical studies, to be replicated or translated to humans. The usefulness of mouse models has been questioned because of irreproducibility and poor recapitulation of human conditions. Newer studies, however, point to bias in reporting results and improper data analysis as key factors that limit reproducibility and validity of preclinical mouse research. Inaccurate and incomplete descriptions of experimental conditions also contribute. Here, we provide guidance on best practice in mouse experimentation, focusing on appropriate selection and validation of the model, sources of variation and their influence on phenotypic outcomes, minimum requirements for control sets, and the importance of rigorous statistics. Our goal is to raise the standards in mouse disease modeling to enhance reproducibility, reliability and clinical translation of findings.

It seems an obvious point, but the model used should be appropriate for the question being addressed. An ideal disease model accurately mimics the human condition, genetically, experimentally and/or physiologically. At DMM, we require that the similarities to human disease be rigorously validated, preferably by proof-of-principle experiments demonstrating response to treatment. The controversial study described above compared microarray gene expression data from humans and mice. In one example, data from human blunt-trauma patients were analyzed together with data from a mouse inbred strain that had been exsanguinated. Losing a large amount of blood does not equate to blunt trauma, and so this could be perceived as comparing apples to oranges. Furthermore, inbred mouse strains represent limited genetic diversity and might not reflect the responses generated in a genetically polymorphic human population. The conclusions drawn in this manuscript did not take into account these potential sources of experimental differences between the mouse and human, and raise the possibility of bias in data analysis.

In a different study, a mouse model was reported to display the key motor symptoms seen in humans with amyotrophic lateral sclerosis (ALS) (Wegorzewska et al., 2009). On the basis of this, the model was used in preclinical studies and promising drug candidates were tested in clinical trials; however, these drugs ultimately failed in humans (Perrin, 2014). It was then shown that this mouse is a poor genetic and phenotypic model of the human condition (Hatzipetros et al., 2014). This example illustrates how relevance to the human disease being studied, supported by strong data to validate the use of the model, is crucial for clinical translation. Humanized models – mice expressing human transgenes or engrafted with functional human cells or tissues – can provide important tools to bridge the gap between animals and humans in preclinical research.

Postdoctoral position to study organogenesis and regeneration using the mouse hair follicle as a model system. The Morgan lab exploits genetically engineered mice to study the role of the mesenchyme in the development and regeneration of the hair follicle with a focus on how feedback between the epithelial and mesenchymal compartments is modified to regulate regeneration and to achieve distinct morphogenetic outcomes. Preference will be given to recent Ph.D. graduates with a strong background in developmental or stem cell biology and molecular biology skills. To apply, send a c.v. and statement of research interest to Bruce Morgan <bmorgan@mgh.harvard.edu>.

Applications are invited for a highly motivated post-doctoral research associate to join the laboratory of Dr Raman Das in the Faculty of Life Sciences at the University of Manchester, UK. The successful candidate will exploit cutting-edge live tissue imaging techniques to dissect the cell biological mechanisms regulating establishment of neuron polarity and axon extension during vertebrate neurogenesis.

This project builds on our recent discovery of a new form of cell sub-division, apical abscission, which regulates detachment of new-born neurons from the ventricle of the neural tube and facilitates progression of neuronal differentiation. (Das, R.M. & Storey, K.G. (2014) Apical Abscission Alters Cell Polarity and Dismantles the Primary Cilium During Neurogenesis. Science 343, 200-204.).

This MRC funded position is available for 3 years (in the first instance). Applicants should have recently obtained (or shortly expect to gain) a PhD in cell and developmental biology or a related field. Experience with vertebrate embryos and live imaging techniques would be an advantage, as would an interest in the cellular mechanisms directing neuronal differentiation. The successful candidate will benefit from hands-on training and close interaction with the principal investigator and will benefit from the world-class facilities available in the lab and at the University of Manchester.

Informal enquiries are encouraged and should be directed to Dr Raman Das at raman.das@manchester.ac.uk.

For more details please visit: https://www.jobs.manchester.ac.uk/displayjob.aspx?jobid=10997

This is an terrific Shelf Life video made by Erin Chapman at the American Museum of Natural History. It features our work on a symbiosis between the classic model salamander Ambystoma maculatum and its algal symbiont, Oophila amblystomatis.

Does anyone have 21st century ideas on how to run a diverse (not just developmental biology) departmental journal club?