Here are the highlights from the current issue of Development:

Human embryos: a mixed bag

Human embryonic stem cells (hESCs) are conventionally derived from embryos that are at the blastocyst stage of development. Now, to gain insight into lineage allocation in early human development, Susan Fisher and colleagues report the derivation of various hESC lines using single blastomeres from 8- and 12-cell human embryos (p. 4010). The characterisation of these lines reveals that, at the transcriptome level, they differ from blastocyst-derived lines. Notably, blastomere-derived hESCs are enriched for genes expressed by trophoblasts and the ectoplacental cone. The researchers further show that blastomere-derived lines are hypomethylated in genomic regions that control trophoblast differentiation and early developmental processes, indicative of trophoblast competence. Variations in gene expression profiles are also observed among the various blastomere-derived lines (all of which are derived from embryos from a single couple), highlighting the existence of blastomere heterogeneity. In line with this, the authors show that levels of EOMES, T, GDF15 and active b-catenin differ among the blastomeres of 8- to 10-cell human embryos. Finally, the researchers report the derivation of the first human trophoblast stem cell line, which could be used in the future to model placental development and disorders.

Cilia: at the heart of chamber maturation

Increasing evidence suggests that blood flow and biomechanical forces generated by the developing heart feedback to regulate cardiac chamber formation and maturation. How such forces are sensed and translated, however, remains unclear. Here, Jiandong Liu and co-workers show that, in zebrafish, cardiac contraction activates Notch signalling in the endocardium to control chamber maturation (p.4080). By analysing embryos deficient for troponin T type 2a (tnnt2a), which encodes an essential component of the cardiac contraction apparatus, the researchers first show that cardiac contraction is required for the formation of cardiac trabeculae – the luminal protrusions that are indicative of cardiac chamber maturation. They further show that cardiac contraction controls Notch signalling in the endocardium; notch1b expression is reduced in tnnt2a^–/– embryos. Notch activation, they report, induces the expression of ephrin b2a and neuregulin 1 to promote trabeculation. Finally, the authors demonstrate that shear stress controls notch1b expression in a primary cilia-dependent manner, suggesting that primary cilia in this context are responsible for detecting fluid flow. In summary, these findings highlight a molecular mechanism that links flow sensing to the transcriptional changes that regulate cardiac development.

Vascular development in full flow

Vascular development and angiogenesis are known to be regulated by various signals, but the roles played by blood flow and biomechanical signals are unclear. This is mostly because the ability to image and measure changes in blood flow has been limited. Now, Elizabeth Jones and colleagues develop a method to simultaneously image blood flow dynamics and vascular remodelling (p. 4158), and use this technique to show that flow dynamics control sprout location and elongation in quail embryos (p.4151). Their imaging approach uses micro-particle image velocimetry: embryos are injected with a fluorescent dye that labels endothelial cells and with fluorescent microspheres that act as tracers of fluid motion. Subsequent imaging via a high-speed camera allows changes in blood flow dynamics and vessel geometry to be quantified. Using this method, the researchers demonstrate that sprout location can be predicted based on flow dynamics; sprouts form from vessels that are at a lower pressure towards vessels at a higher pressure and localise to points where shear stress, a force created by flow, is at a minimum. In addition, the rate of sprout elongation is proportional to the pressure difference between the two vessels. These studies provide insights into the hemodynamic forces at play during vascular development and open the door to further studies of the biomechanical control of vascular remodelling.

Con-Nek-ting cilia biogenesis and resorption

The left-right organiser (LRO) is a transient ciliated structure that plays a key role in establishing left-right (LR) asymmetry in the vertebrate embryo. However, the mechanisms that control the formation and resorption of cilia on this structure are unclear. In this issue (p. 4068), Martina Brueckner and colleagues reveal that Nek2 regulates cilia biogenesis and resorption at the Xenopus LRO. They show that both the knockdown and the overexpression of nek2, which encodes a NIMA-like kinase, result in reduced cilia numbers and motility at the LRO and hence abnormal LR development. Nek2 is known to play a role in centriole separation and, in line with this, the authors reveal that the knockdown of nek2results in centriole defects in the LRO. They further show that Nek2 acts upstream of the tubulin deacetylase Hdac6, and that it interacts with the nucleoporin Nup98, to control cilia resorption. Together, these findings demonstrate that Nek2 is involved in multiple stages of the cilia life cycle. Given that NEK2 has previously been implicated in abnormal laterality in humans, these findings also provide further evidence that links Nek family kinases to human ciliopathies.

An appendage to Hox gene function

Hox genes are best known for their role in axial patterning during embryogenesis, but they have also been implicated in the development and patterning of cutaneous accessory organs, such as hair follicles and mammary glands. How do they function in this context? Here, by showing that Hoxc8 can initiate an ectopic mammary gland programme in mice, Lara Carroll and Mario Capecchi propose that Hox genes regulate the distribution of cutaneous appendages (p. 4056). They first show that Hoxc8 is transiently expressed in the early surface ectoderm prior to mammary line formation. The researchers then show that the conditional overexpression of Hoxc8 – to express it in regions where it is not normally expressed – results in the formation of ectopic mammary placodes. These ectopic rudiments express known mammary placode markers, such as Tbx3 and Wnt10b. The authors further report that the ablation of ectodermal Tbx3 prevents the formation of both normal and ectopic mammary rudiments, suggesting that Tbx3 is directly regulated by Hoxc8 during mammary development. Together, these and other findings highlight a role for Hoxc8 in the initial stages of mammary development and suggest that Hoxc8 and other Hox genes play roles during the regional specification of cutaneous appendages.

PLUS…

How to make an oligodendrocyte

The loss of oligodendrocytes – the cells that produce the myelin sheath of axons – can result in a broad array of diseases including cerebral palsy and multiple sclerosis. Accordingly, replacing lost oligodendrocytes holds great promise as a therapeutic strategy. Here, Goldman and Kuypers describe the molecular events regulating oligodendrocyte development in vivo and discuss how our understanding of this process has led to the establishment of methods for producing oligodendrocytes in vitro. See the Primer on p. 3983

*Also see the other articles in the “How to make…” series here

Morphogen rules: design principles of gradient-mediated embryo patterning

The Drosophila blastoderm and the vertebrate neural tube are archetypal examples of morphogen-patterned tissues that create precise spatial patterns of different cell types. Here, James Briscoe and Stephen Small compare these systems in the context of gene regulatory networks and dynamical systems theory. This comparison reveals several shared features that suggest that a set of common design principles underpins the patterning of both tissues. See the Review on p. 3996

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It’s getting to that time of year when we think about the year to come and the things we hope it will bring: love, health, money… and maybe a little bit of help with our research.

There are many things that could make the year 2016 better for a developmental scientist: new equipment, better protocols, an easier way to maintain a model organism, or quite simply some more time to do experiments! So, in the spirit of the festive season, this month we are asking:

What is your research wish list for the New Year?

Share your thoughts by leaving a comment below! You can comment anonymously if you prefer. We are also collating answers on social media via this Storify. And if you have any ideas for future questions please drop us an email!

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Here is some developmental biology related content from other journals published by The Company of Biologists.

Genome-wide lacZ profiling in the mouse

Tuck, Estebel and colleagues have produced an open access adult mouse expression resource with the expression profile of 424 assessed in up to 47 different organs, tissues and substructures using a lacZ reporter gene. Read the paper here [OPEN ACCESS].

A method for labelling leukocytes infiltrating the mouse retina

Sim and colleagues describe a method to image myeloid cells infiltrating the mouse retina in vivo using a depot injection of indocyanine green dye (ICG). Read the paper here [OPEN ACCESS].

Urine-derived stem cells predict patient response to cholesterol-lowering drug

Si-Tayeb, Idriss and colleagues have shown that cells derived from patient urine samples can be reprogrammed to generate patient-specific models of hypercholesterolemia, providing a tool to predict patient response to cholesterol-lowering drugs. Read the paper here [OPEN ACCESS], and read the press release here.

Mass and drought resistance linked in seedlings

Seedlings of large-seeded plants are considered to withstand abiotic stress more efficiently. Meng and colleagues show that the integration of drought stress response into the regulation of mass is mediated by the transcription factor ARF2. Read the paper here.

New role for eIF6 in myofibroblast differentiation

Myofibroblast differentiation is regulated by TGF-β1. In this paper, Yang and colleagues show that eukaryotic initiation factor 6 can modulate myofibroblast differentiation by altering the occupancy of the TGF-β1 promoter by H2A.Z and Sp1, affecting TGF-β1 transcription. Read the paper here.

High-salt exposure increases cardiovascular defects in early chick embryos

In chick, embryonic mortality at early stages is usually due to vascular malformations. Wang and colleagues show that high-salt exposure results in angiogenesis and heart defects, possibly due to excess generation of reactive oxygen species (ROS). Read the paper here.

The effects of rearing amphibious fish out of water

Wells, Turko and Wright rear an amphibious fish in and out of water, and find that embryos reared in aqueous environments consume more energy than their faster developing terrestrial counterparts. Read the paper here.

Hatching success does not decrease with higher nest temperatures in flatback turtles

Elevated nest temperatures caused by climate change could harm the viability of ectotherm eggs. Howard and colleagues show that high nest temperatures do not decrease hatching success in flatback turtles, and observe a high pivotal sex-determining temperature in these turtles. Read the paper here.

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The electroporation technique is widely used in developmental biology to deliver foreign DNA into cells and study gene function. The chick embryos exhibit a remarkable easy access to perform electroporation and follow in ovo development.

Electroporation of limb somites allows the misexpression of genes in limb somite derivatives, like myogenic and endothelial cells, while electroporation of the lateral plate targets other limb cells like cartilage, bone and tendons.

We previously performed limb somite electroporation using two plasmids, one containing the gene-of-interest and one containing the reporter gene. However, this approach does not allow analysis at the cellular level, which constitutes a limitation of the technique.

To overcome this limitation and perform analysis at the cellular level, we aimed to design bicistronic vectors to misexpress, in the same cell, the gene-of-interest and the reporter gene. To do this, we took advantage of the 2A peptide that allows expression of a bicistronic mRNA. In these vectors, a single peptide is produced by the bicistronic mRNA, and auto-cleavage of the 2A peptide subsequently produces equal amounts of the two proteins. To test our vectors, we used control plasmids expressing two fluorescent proteins, Tomato and GFP, separated by the 2A peptide. We further used the Tol2 transposon system to allow genomic integration of the construct and enable analysis at late developmental stages.

One focus of our research is skeletal muscle and tendon formation. Therefore, we designed a set of stable bicistronic vectors containing different promoters to target muscle cells at different stages of differentiation. After performing limb somite electroporation using these vectors, we observed a simultaneous cellular expression of Tomato (membrane) and GFP (nuclei) at the different stages of muscle differentiation. In electroporated limbs, the ubiquitously expressed CMV/βactin promoter targeted both muscle progenitors (Pax7+ cells) and differentiated cells (myosin+ cells). The p57MRE/βactin promoter, which drives expression in differentiated myoblasts, targeted mononucleated (myosin-) cells and muscle fibres (myosin+). Finally, the MLC (myosin light chain) promoter targeted differentiated cells (myosin+). Lateral plate electroporation with the vector containing the CMV/βactin promoter allowed Tomato and GFP co-expression in cartilage, tendons and connective tissue of the limbs, but never in myogenic (Pax7+ or myosin+) cells.

We believe that this set of tools can be used to efficiently misexpress genes at different time points of myogenic cell differentiation and analyse the consequences for muscle development. Moreover, because these vectors can be integrated into the genome, the analysis at late developmental time points can be performed. Finally, the combination of limb somite and lateral plate electroporation can provide us with a tool to study the molecular and cellular interactions between the different components of the musculoskeletal system.

Full article at : http://www.biomedcentral.com/1471-213X/15/39

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15—17 March 2016
St. Catharine’s College, Cambridge, UK

The aim of this Biochemical Society focused meeting is to explore the mechanisms of BMP signal transduction and regulation of signalling, and discuss how genetic and epigenetic alterations result in aberrant signalling and how this leads to cancer. The BMP signalling pathway is a key therapeutic target and we will consider how to manipulate the pathway with small molecules or biologics. This conference will bring together researchers from academe and industry, and will be a great opportunity for those working on BMP signalling, cancer, stem cells, structural biology and drug discovery to share data and interact in a vibrant, but informal environment.

Organizers

• Caroline Hill (The Francis Crick Institute, United Kingdom)
• Alex Bullock (University of Oxford, United Kingdom)
• Gareth Inman (University of Dundee, United Kingdom)

Topics

• Mechanisms of BMP signalling
• Deregulation of BMP signalling in cancer and therapeutic approaches
• Genetic and epigenetic alterations in BMP pathway components
• BMP signalling in disease

Details of the event and a list of speakers can be found here.

There will be opportunities for abstracts to be elevated to short oral talks, particularly from early-career scientists.

Abstract deadline: 12 January 2016

Earlybird registration deadline: 12 January 2016

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We are seeking for highly motivated and enthusiastic candidates interested in understanding how tissue compartmentalization and cell fate decisions take place in the Central Nervous System during embryonic development. The project will focus in understanding the genetic mechanisms involved in the control of the cellular fate using zebrafish embryos as a model. Experience on molecular biology, imaging, and developmental biology will be an advantage.

We are located in the Department of Experimental and Health Sciences of the Universitat Pompeu Fabra in Barcelona. Interested candidates are encouraged to contact Cristina Pujades for more information. To apply, send a letter of interest, CV, a short description of your research background, and contact details of 2 referees to cristina.pujades[at]upf.edu          http://pujadeslab.upf.edu/

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Two positions are available as part of two Research Council-funded doctoral training programmes at The University of Manchester, the first one supported by the BBSRC and the second one by the MRC. Both projects involve work on the fruit fly Drosophila as a highly efficient and relevant model organism to study fundamental mechanisms of neuronal ageing and degeneration with unique detail and depth and delivering understanding of high biomedical applicability. Further information about fruit flies as a model organism is available here. Note that Andreas Prokop, who supervises on both projects, drives active programmes of science outreach and public engagement (see here), and students will have unique opportunities to develop transferable skills in science communication, which is of increasing relevance in modern science and an important category on a researcher’s CV. Full details on how to apply for these positions can be found on the UoM BBSRC DTP website.

Position 1: Advanced imaging and mathematical modelling of ageing and neurodegeneration in the nervous system

Position 2: An interdisciplinary approach to unravel mechanistic understanding of Frontotemporal lobar degeneration

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In 2014, the British Society of Developmental Biology (BSDB) has initiated the Gurdon Summer Studentship program with the intention to provide highly motivated students with exceptional qualities and a strong interest in Developmental Biology an opportunity to engage in practical research. Each year, 10 successful applicants spend 8 weeks in the research laboratories of their choices, and the feedback we receive is outstanding. Please, read the student reports, kindly sent to us by Oliver Davis from Brighton and Sussex Medical School who was hosted in summer 2015 by Jean-Paul Vincent at the Crick Institute.

Dying for a pattern

This summer, I had the glorious opportunity of undertaking a BSDB funded research project in the laboratory of Jean-Paul Vincent at the Francis Crick Institute in Mill Hill, London. My project took place in the lab of Dr Jean-Paul Vincent (www.jpvincentlab.com) under the patient and inspiring tutelage of one of his PhD students, Sam Crossman. Our project investigated the mechanism of apoptosis in the model organism Drosophila melanogaster.Apoptosis is a form of programmed cell death and is an important process in the development of all multi-cellular organisms. Understanding why cells die in certain situations and not in others is of relevance to many areas of health, including embryological disorders and cancer, and my overall research aim was to investigate the role of apoptosis in the developing fly embryo. To do this, I worked with strains carrying mutations in genes required for patterning the anterior-posterior axis. Mutation of these so-called patterning genes can trigger extensive apoptosis in the embryonic epidermis (figure. 1) and therefore provides a useful model to investigate the apoptotic machinery in Drosophila.

The cause of the ectopic apoptosis observed in patterning mutant embryos is not fully understood. One previously suggested explanation is that the cells of the epidermis can sense their ability to adopt the correct fate and undergo apoptosis if they lack the required patterning inputs to do so (Werz et al, 2005). However, if this were the case, there would have to exist an unknown machinery that would allow individuals cells to detect patterning errors and initiate apoptosis as a result.

In order to determine if cells are truly capable of detecting patterning errors, I planned to use a light inducible form of Cre recombinase to clonally remove a lox flanked allele of the ftz gene in a small subset of cells within each segment. If these small clones survive in an otherwise wild type embryo, it would argue against a cell-autonomous system where individual cells monitor their ability to differentiate correctly and would suggest that an alternative mechanism could be in play.

 

 

One difficulty with this plan is that ftz is activated very early on in embryogenesis. As a result, I set out to generate an early acting form of Cre, which could be used to remove ftz before it has carried out its function. To achieve this I spent the first part of my project cloning the Cre enzyme into a plasmid containing the actin promoter. As actin is an important protein in every cell, it is expressed from very early stages of embryogenesis. As a result, we hoped that by using the actin promoter to drive expression of Cre, we could produce the enzyme early enough to remove our lox flanked ftz allele in a timely manner and create mutant cells.

Unfortunately, cloning proved frustrating. Fortunately, it also proved educational. I learnt a lot about the way that experiments work, and how progress in science is more staccato than smooth. One issue I faced was the purification of my final plasmid using an Invitrogen maxiprep column. Each time I purified the plasmid I ended up with a lower yield than required, as I needed enough DNA to send to a company that would use it to generate a transgenic fly. I adjusted a parameter each time, but in the end it may have just been a plasmid with a low copy number as during my last attempt I purified it from a much larger bacterial culture, which finally gave me a sufficient amount of DNA.

The second part of my project was spent optimising a fluorescent in-situ hybridisation (FISH) protocol. I planned to use FISH to label cells expressing the pro-apoptotic gene hid in a series of patterning mutant embryos to characterise the regions where cell death occurs. As a control, I first conducted the protocol with a probe against the segment polarity gene wingless, which is expressed in a row of cells in every segment. This probe was made by someone else in the lab and is known to work, so I used it to learn the steps of the in-situ protocol.

My FISH experiments with the wingless probe worked like a treat (figure 3), but difficulties soon followed when I attempted to make a new probe to label cells expressing hid. The stainings I conducted with the hid probe I had made repeatedly failed to work, and every attempt to generate a new probe proved unsuccessful. Disappointingly, I reached the end of my project before I managed to develop a protocol that worked, but I at least learnt plenty of science along the way!

My time in J.P.’s lab has been great for a number of reasons. I’ve come to love the problem solving nature of science and the freedom you get to explore what really interests you. It really is an adventure! However, I’ve also realised how difficult a career in science can be. Whilst this project has inspired me to become a scientist, I wonder if there’s a route into academia that would better suit my background as a medical student and my wider interests in medicine.

Sources

(1) Werz C, Lee TV, Lee PL, Lackey M, Bolduc C, Stein DS, Bergman A. Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila. Development, 2005. 132(24): p. 5343-5352.

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Applications are invited from highly motivated and creative individuals who are interested in studying fundamental mechanisms of neuronal migration and axon guidance. The main focus of our research is to understand the molecular and cellular mechanisms underlying the development of neural circuits using the embryonic spinal cord as a model system (http://www.ucmm.umu.se/english/research/sara-wilson/). The fellowship is currently funded for two years and is available immediately. The laboratory is located at the Umeå Centre for Molecular Medicine (UCMM), Umeå University, Sweden. UCMM is an interdisciplinary department, which focuses on questions in basic medical sciences and developmental biology and provides an interactive modern environment with good core facilities.

Requirements: Individuals with a background in developmental biology, neuroscience, molecular and cell biology or related discipline and with a keen interest in developmental neuroscience are encouraged to apply. The successful candidate will have or about to receive a Ph.D. in a relevant discipline and be proficient in written and spoken English.

Technical experience with imaging, vertebrate embryonic model systems – especially chick or mouse electroporation, mouse handling and genetics is a big advantage although training will be given. Experience with molecular, cellular and/or evolutionary biology will be positively considered. The most successful candidate will have a high level of motivation, be creative, organized and rigorous and have the ability to work both independently and within a team.

Please submit your application (reference 2015SW100) by 16^th December 2015 to sara.wilson@umu.se by sending the following documents as pdf files:

1) A short cover letter (not more than 1 page) to include a description of your research experience and suitability for the position.

2) Curriculum Vitae including: publication list, technical expertise, names and contact information for three referees.

Informal enquiries may be directed to Dr. S.I. Wilson (sara.wilson@umu.se).

We look forward to your application!

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