The eggs of domestic birds have been used in the study of developmental biology, leading to the extensive accumulation of knowledge on embryonic development. However, the early events involved in bird development, particularly the mechanism underlying fertilization, have not been elucidated in as much detail as those of other species of animals. The ooplasm in avian eggs is opaque, which makes it difficult to manipulate in vitro for artificial fertilization systems such as intracytoplasmic sperm injection (ICSI). Furthermore, a hen must be sacrificed to obtain a single egg due to the difficulties associated with the constant induction of multiple ovulations.

In our previous study published in Biology of Reproduction (69:1651-1657, 2003), ‘‘Fertilization and development of quail oocytes after intracytoplasmic sperm’’, we successfully established the first avian ICSI system; however, live chicks had never been produced until recently. Although fertilization in birds is polyspermic, namely the entry of many sperm (approximately 60 in chickens) is permitted but only one sperm nucleus participates in zygotic formation with the female pronucleus, the single ejaculated sperm of a quail or chicken was capable of activating the egg for the fertilization process and subsequent blastoderm development 24 hr after ICSI. However, the rate at which the blastoderm developed (16%) was limited, and did not go beyond blastoderm stage X even when the period of cultivation of quail embryos was extended to 72 hr after ICSI. We could not determine why fertility was low even though a single chicken or quail sperm was able to effectively activate the mouse egg. I subsequently joined Shizuoka University in 2011, and the research performed there revealed that the rate of pronuclear formation in quail ICSI-eggs was very low, and also that first cleavage of the ICSI-egg took 1.5 hours longer than that with in vivo fertilization. However, the rate of pronuclear formation increased when the egg was injected with a single sperm in combination with calcium chloride or strontium chloride. These findings prompted us to speculate that a single quail sperm may not be sufficient to induce increases in Ca²⁺ concentrations in the egg cytoplasm for the pronuclear formation of quail eggs. Polyspermy may play a pivotal role as a Ca²⁺source in egg activation.

I was fortunate to meet with Drs. Inaba and Shiba in the University of Tsukuba, who are experts on Ca²⁺ imaging analysis, and collaborated with them to image increases in intracellular Ca²⁺ concentrations in the quail ooplasm. At that time, I successfully produced the first live quail chick by ICSI with sperm extracts (SE). This baby was named ‘‘Megumi’’, which means “The grace of God” in Japanese. The delayed cleavage of ICSI-eggs was prevented by microinjecting 2 ng SE (equivalent to 200 sperm) and the embryo underwent normal development beyond stage X at a high rate. The microinjection of 2 ng SE into quail egg evoked two phases of Ca²⁺ changes; multiple, long-lasting spiral-like Ca²⁺ waves that followed an initial transient increase in Ca²⁺ concentrations (See Fig), which have never been demonstrated in other species. By the end of 2012, I had finished a pilot study involving biochemical analyses to identify the sperm-derived egg-activating factors (sperm factors) that triggered these spiral-like Ca²⁺ oscillations. Phospholipase Czeta (PLCZ) was easily determined to be an inducer of transient increases in Ca²⁺ concentrations. However, difficulties were associated with identifying the sperm factors that induced spiral-like Ca²⁺ oscillations because a single microinjection of each candidate factor failed to evoke these oscillations. We unintentionally discovered that a simultaneous injection of aconitate hydratase (AH) and citrate synthase (CS) induced spiral-like Ca²⁺ oscillations, similar to those caused by the injection of SE.

Megumi and her offspring

Regarding the functions of the two characteristic increases in Ca²⁺ concentrations in quail fertilization, PLCZ-induced transient increases in Ca²⁺ concentrations were found to be necessary for the resumption of meiosis, similar to that in mammals, whereas spiral-like Ca²⁺ oscillations alone did not complete the fertilization process. However, the microinjection of neither PLCZ cRNA alone nor AH and CS cRNAs induced the normal development of ICSI-derived zygotes, whereas the simultaneous injections of these 3 factors enabled them to hatch. Therefore, AH and CS-generated Ca²⁺ signaling suggests a novel role for fertilization cellular event independent of PLCZ-induced Ca²⁺ signaling in birds. Spiral-like Ca²⁺ oscillations may function as the major driving force for cell cycle progression in early embryos. However, it has not yet been determined how they enhance the development of the early embryo.

It is our hope that the successful production of healthy chicks after ICSI will lead to significant advances in the introduction of genes into birds as well as cloning technology. ICSI–mediated gene transfer would not only streamline the procedure, but also overcome the low efficiency of avian transgenesis by conventional techniques such as the production of germline chimera. In addition, the development of somatic cell nuclear transfer techniques, in combination with 3 sperm factors, should contribute to the protection of endangered species as well as restoration of extinct species.

Mizushima, S., Hiyama, G., Shiba, K., Inaba, K., Dohra, H., Ono, T., Shimada, K., & Sasanami, T. (2014). The birth of quail chicks after intracytoplasmic sperm injection Development, 141 (19), 3799-3806 DOI: 10.1242/dev.111765

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“In scientific thinking are always present elements of poetry. Science and music requires a thought homogeneous.” ~Albert Einstein.

This year the biannual Zebrafish meeting started out in the best possible fashion, combining music with science. The exceptionally talented organist Dr. Graham Lieschke performed three pieces composed by Bach, and Overture Hall never sounded better! Having a musical background myself, I was enthralled. Not to mention such a beautiful set of pipes deserves to be showcased. It was blissful.

I do hope this is repeated in the future! But getting back to the meeting.

Every two years the Zebrafish community descends upon Madison, WI to discuss the recent advances in the field, new technologies pertinent to our model system, and just catch up with old friends. This year was no different. Being my third Madison meeting, plus one international zebrafish meeting, I have a lot of friends to catch up with. It is especially great seeing grad student friends who are now postdocs, and postdoc friends who have moved on to start their own labs. Some friends I only see at this meeting, as they have moved to other countries.

Here are some of the current and former Vanderbilt folks, catching up between sessions!

One thing that strikes me every time I attend this meeting is the vast array of biological systems that researchers are using zebrafish to study. Originally established as a model to study development, zebrafish are also well adapted for many other areas of study including cancer and growth control, neural degeneration, genetics and genomics, signaling and gene regulation, chemical biology, and behavior. There is never a problem finding something of interest. In fact, I often wanted to be in two places at the same time! Being in the field of neurodevelopment, I had lots of options, from sessions on neural degeneration, regeneration and disease; neural circuits, neurophysiology and behavior; and neural development, just to name a few. While the sessions tended to be organized by subject, the workshops were more technique based that could be utilized by many research areas. These techniques ranged from genome editing and lineage tracing to neural and vascular imaging to maternally controlled development and post embryonic development.

This year we had two Keynote Speakers, Dr. Sarah Tishkoff and Dr. John Postlethwait. Dr. Tishkoff spoke about her work studying human genetic variation in Africa using genome-scale genetic variation analyses conducted across diverse African populations. Also using genome analysis, Dr. Postlethwait uses Teleost fish as a model for understanding vertebrate development and disease by determining the origin of genes and gene families across Teleost species. In addition to these renowned researchers, there were also five plenary sessions running the gamut from imaging, genetics and genomics, to physiology and disease, to morphogenesis and organogenesis.

Each year I most look forward to learning about the latest and coolest technologies emerging in the field, and especially for me, the advances in imaging technologies. Zebrafish are so well suited for imaging studies and I can always count on a session/workshop or two at these meetings discussing the recent advances, complemented by beautiful timelapse movies. Of particular interest to me this year was the Workshop “Emerging Technologies in Neural Circuit Analysis” organized by Filippo del Bene and Marnie Halpren. Here several prominent researchers were invited to speak on new advances using transgenic tools to correlate neural activity to behavior and the some of the challenges that remain. Some of these new tools include optogenetic probes, genetically encoded voltage and calcium indicators, and new and interesting behavioral models. The use of zebrafish for behavioral studies is increasing and Erik Duboué discussed means to quantify the response of zebrafish larva to aversive stimuli. Additionally, Kurt Marsden uses a head-restrained method to map neural circuits activated during the startle response. Two esteemed researchers from Janelia Farm spoke on imaging neural activity; Douglas Kim discussed the optimization of fluorescent probes while Misha Ahrens described his research using calcium indicators to create functional maps of the entire fish brain. Other researchers also use adult zebrafish, including Hitoshi Okamoto who utilizes optogenetics, and has generated a plethora of valuable transgenic lines, and Koichi Kawakami who uses the Gal4-UAS system to genetically dissect the adult brain. Other researchers are studying neural connectivity, including Claire Wyart who studies the spinal cord using Channel Rhodospin and Alex Schier who discussed an immunohistochemical approach to study neural activity. Finally, other exciting techniques involve manipulating the activity of the neural circuit, as discussed by Harold Burgess who uses transgenic tools, and David Prober who uses TRP channels to ablate or activate specific neurons. One element I especially appreciated from this workshop was the extended question and answer time following the presentations.

Another really neat element of this year’s meeting was seeing the use of new technologies leading to great research! Two years ago the relatively new technology of genome editing via TALENs was the “it” topic of the fish meeting. Everyone was either already trying it, or going to. Every poster presenter who used the traditional morpholino approach was asked if they had considered TALENs, and entire workshops and sessions were devoted to discussing the new technology, alternative approaches, and troubleshooting some of the difficulties. This year it was great to see new data based on TALEN induced mutants! Just a wonderful example of the zebrafish community at large embracing and sharing a new technique which leads to the advancement of research across biological disciplines.

As it’s become the tradition, the last night of the meeting involves dinner on the waterfront Memorial Union Terrace with live music from Terrace After Dark, the past two meetings this has been Natty Nation. The waterfront area is perfectly setup to not only mingle with other conference attendees, but also get a local flavor with plenty of outdoor seating and an awesome view of the sunset over the water.

These festivities were followed by DJ Mike Carlson for a conference party, where everyone from PIs to graduate students can get their groove on. Overall, it’s just a great time to hang out with fellow researchers, enjoy the view, and just have some fun, as it has been for many many years. Although the next US zebrafish meeting is slated to occur in Orlando with a joint GSA model system meeting and I do not know the locational fate after that, I do know that Madison will always have a warm spot in the zebrafish community’s heart.

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The Wythe Lab at Baylor College of Medicine favors a cross disciplinary approach to solving key problems in cardiovascular development. Rather than wed ourselves to a singular model system or technique, we attack biological questions from multiple fronts, using gold standard genetic models-such as the mouse, cutting edge imaging techniques, genome-wide analyses, and classical biochemistry.

We have funding and are actively recruiting for people to pursue projects in multiple areas (again, from multiple angles), but are currently seeking expertise in developmental biology (zebrafish or mouse), molecular profiling methods (particularly genomics and proteomics), bioinformatics, and imaging. Our efforts center around the goal of elucidating the key transcriptional and molecular nodes that establish and maintain a functional cardiovascular system.

Baylor College of Medicine (an equal opportunity employer) is located in the heart of the Texas Medical Center in Houston, Texas. The TMC is a world-class research environment, consisting of more than 40 non-profit institutions, with one of the highest densities of clinical, translational, and basic science research facilities in the world.

Please visit http://www.wythelab.com/ for more information:

If you are passionate about biology, motivated to kick butt in the lab, and interested in joining our group, please send a CV, a cover letter explaining your interest in the work of the lab and how you would fit into the group, as well as a list of three to five references and their contact information all as a single, combined PDF if possible) to joshua.wytheATbcm.edu.

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Here is October’s round-up of some of the interesting content that we spotted around the internet:

News & Research:

– Bananas, Jesus on toast and polar bear disguises- some of the 2014 IgNobel Prizes! And since then the real Nobel Prizes have also been announced, with two prizes for Biology. The prize in Physiology or Medicine was awarded to the discovery of the positioning system of the brain, and the Prize in Chemistry to the developers of super-resolution microscopy.

– ‘Sometimes, the brightest stars in science decide to leave’- on life outside the lab, in Nature News & Comment.

– A patient is for the first time implanted with iPS cells, in Japan- here and here.

– Nature launched a new website devoted to science software, apps and online tools.

– ‘The man who grew eyes’- a feature article on Yoshiki Sasai’s research in Mosaic.

– A movie based on the Woo Suk Hwang cloning scandal has been a success in the Korean box office.

– Can you explain your research using an infographic? The Society of Biology is running a competition! The deadline is the 5th of November.

– And you can vote for who you think should be the Stem Cell Person of the Year 2014 until the 22nd of October!

Weird & Wonderful:

– Crocket is an unusual way to determine authorship order in a paper!

– Ever wanted to 3D print a frog dissection model? One of the several models available at the NIH 3D print Exchange website!

– Explaining how science works… using cookies!

– Cute DNA plush toys… with magnets for hydrogen bonds!

– And follow the ups and downs of life in the lab with the Lego Academics twitter account!

Peer 1: Brilliant! Accept with no changes; Peer 2: Groundbreaking! Accept with no changes; Peer 3: Reject. pic.twitter.com/KbLPuT24vu

— Lego Academics (@LegoAcademics) August 12, 2014

Beautiful & Interesting images:

– Chromosome sweets

– The winners of the FASEB BioArt image competition have been announced

– Beautiful chicken embryo

Chicken embryo RT @Powerfulpixs via @embryoproject pic.twitter.com/2rokc3L0DT

— the Node (@the_Node) September 3, 2014

Videos worth watching:

– A great animation video on the New York Times illustrates the discoveries of van Leeuwenhoek and the beauty of the microbial world.

– See the winners of the Stem Cell Video contest launched by the Knoepfler lab blog

– And we spotted this great video on chick development:

 Keep up with this and other content, including all Node posts and deadlines of coming meetings and jobs, by following the Node on Twitter

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Last Friday we came across the blog of the Sauka-Spengler lab in Oxford, and noticed that their PCR machines are called Kit and Kat. This made us think- who else gives pet names to their lab equipment? We asked the twittersphere for suggestions, and collated them in the storify below. How about your lab? Share the fun names of your lab equipment by leaving a comment!

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Researchers generate for the first time Drosophila melanogaster with intestinal cancer and reveal key genetic factors behind human colon cancer.

The scientists identify a human gene that favours the proliferation of tumour cells in early stages of colon cancer.

Furthermore, the flies are useful for faster and more economic drug screening.

Researchers at the Institute for Research in Biomedicine (IRB Barcelona) have managed to generate a fruit fly (Drosophila melanogaster) model that reproduces human colon cancer. With two publications appearing in PLoS One and EMBO Reports, the IRB team also unveil the function of a key gene in the development of the disease.

“The breakthrough is that we have generated cancer in an adult organism and from stem cells, thus reproducing what happens in most types of human cancer. This model has allowed us to identify subtle interactions in the development of cancer that are practically impossible to detect in mice with the current technology available,” explains the biologist Andreu Casali, Associate Researcher at IRB Barcelona and leader of the Drosophila project.

Although the flies do not have a colon, they have an intestine that includes a colon and rectum and that works in the same way as the human colon. The scientists generated mutant flies for two genes that are altered in most human colon tumours, namely APC and Ras. Thanks to the ease with which genetic studies can be performed in Drosophila, the researchers were able to examine the effect of 250 genes that are altered in these types of tumour and found that, of these, 30% affected growth while the others had no significant effects.

“The advantage of the model is that it allows us to explore genetic alterations more quickly, to distinguish between those that are important and those that are not, and to see what role they play,” explains Òscar Martorell, first author of the paper that appears in EMBO Reports published today. “Performing these genetic experiments in mice is time-consuming and costly and the Drosophila model allows us to rapidly analyse new pathways that could be relevant for colon cancer,” adds the co-author of the study, Francisco Barriga, a postdoctoral fellow working on colon cancer in vertebrate models. Undertaken over five years, the study is the result of collaboration between the Development and Morphogenesis in Drosophila Lab and the Colorectal Cancer Lab, both at IRB Barcelona.

Of all the genes that have a relevant function, the group focused on one called Mirror in Drosophila and lrx in humans. The experiments with flies led to the finding that this gene favours tumour growth in early stages of human cancer. “The problem with human cancer is that we know very little about what happens in the early stages. Our models allows us to better study its development.” Also, Casali goes on to speculate that the human gene lrx may become a good drug target “for example, to prevent benign adenomas from developing further.” However, first the validity of the gene as a therapeutic target has to be tested in mice.

A good in vivo guinea pig for drugs

The researchers also expound that flies can be used to study candidate drug molecules to combat cancer. Drosophila would serve as an intermediate step between the in vitro phase and testing in vertebrates. On the one hand, this model has the in vitro advantages because many molecules can be tested at a minimum dose, and on the other, it shares the advantage of animals models because, as it is a living organism, toxic molecules or those with poor absorption could be omitted very quickly.

“If there are 2000 promising molecules among a million tested in vitro, instead of testing them in mice, Drosophila could offer a good alternative to identify the two or three that are most appropriate. Both time and costs would be reduced,” explains Casali.

With this aim, Casali has start collaborating with the group headed by Ernest Giralt (IRB Barcelona)—an authority on pharmacological chemistry and peptide design—to use flies to test new families of molecules against cancer.

Reference articles:

Iro/Irx transcription factors negatively regulate Dpp/TGF-b pathway activity during intestinal tumorigenesis

Òscar Martorell, Francisco M. Barriga, Anna Merlos-Suárez, Camille Stephan-Otto Attolini, Jordi Casanova, Eduard Batlle, Elena Sancho and Andreu Casali

EMBO Reports (2014 Oct 8). 10.15252/embr.201438622

Conserved mechanisms of tumorigenesis in the Drosophila adult midgut

Òscar Martorell, Anna Merlos-Suárez, Kyra Campbell, Francisco M. Barriga, Christo P. Christov,

Irene Miguel-Aliaga, Eduard Batlle, Jordi Casanova, Andreu Casali

PLoS One (2014 Feb) doi: 10.1371/journal.pone.0088413

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There is something exciting about biologists joining forces with physicists and/or mathematicians, and finding a common language to solve biological problems that are just too complex to understand without stepping outside the realm of ‘traditional’ biology. At the recent EMBO conference on plant development, interdisciplinary studies were the main focus. And as the organiser of the meeting, Ottoline Leyser, stated ‘developmental biology is at the vanguard of this revolution because of its inherently multiscale focus’.

The location of the conference was the Sainsbury Laboratory, a new institute at the University of Cambridge in the UK (so not so far from the Node office!). This was a perfect location for the meeting. The Sainsbury Laboratory focuses on interdisciplinary plant developmental biology (the conference organisers is also the director the institute!) and is set in the grounds of the Cambridge University Botanical Gardens, where the beauty of the plant world is in display. Indeed, tours of the gardens where available at the end of the meeting, although unfortunately I didn’t have the opportunity to join. Here, I will highlight a few of what I found the most exciting presentations at the meeting.

The Sainsbury Laboratory in Cambridge, UK

Although the keynote lecture that kicked off the meeting was interdisciplinary, it was not about plant development. Or indeed about plants at all. The first speaker was Kristian Franze (University of Cambridge), who works on neural development. Ottoline Leyser explained the rationale: many years ago she persuaded the organisers of the British Society for Developmental Biology meeting to include a pollen tube researcher in the neurobiology session. It is was time, therefore, the do the opposite. Kristian started his talk by drawing parallels. Ramification of branches in tree must involve forces. Neurons also undergo ramification, but the neuro field has not been as good as the plant field at appreciating the importance of forces in development. His talk focused on his lab’s work understanding the forces and mechanics underlying neural development. Another speaker in the session was Olivier Hamant (École Normale Supérieure Lyon); according to Ottoline: ‘you can’t possibly have a session on mechanical forces in plants without Olivier’. Using atomic force microscopy his lab was able to examine the stress pattern of pavement cells, and how the organisation of stress in these cells impact on the orientation of MTs (recently published in eLife).

In the ‘self-organising tissue systems’ session, Jennifer Nemhauser (University of Washington) took interdisciplinarity to the ultimate consequences. Interested in understanding how auxin signalling works, but limited by the redundancy and complexity of the pathway, Jennifer collaborated with an engineer at her university to create a synthetic system in yeast that recapitulates auxin signalling (published in PNAS). Her talk demonstrated the power of synthetic systems to provide insight into developmental processes.

Poster session in the corridors of the institute- you can see the labs on the right handside!

In the ‘self-organising cell systems’ session, Claire Grierson (University of Bristol) talked about the self organisation of root hair morphogenesis. Her talk ended on an emerging theme- the importance of studying developmental biology to understand (and modify) ecology and the environment. As she explained, 30% of arable land is lost due to soil erosion. Her lab is starting to focus on how root architecture can play a role in preventing this phenomenon. José Feijó (Gulbenkian Institute/ University of Maryland) works on pollen tubes, one of the fastest growing cells in nature. He showed some beautiful microscopy of these cells in action, and showed how calcium signalling is involved in pollen tube growth and morphogenesis (published in Science). Also in this session, Ray Goldstein (University of Cambridge) talked about cytoplasmic streaming, the persistent circulation of fluid in eukaryotic cells that is driven by molecular motors. Fluid mechanics plays an important role in understanding this process, and Ray made a good case for the contributions of maths and physics in understanding self-organising systems in nature.

In the ‘comparative approaches’ session, Ken Shirasu (RIKEN) focused on Striga, a parasitic plant with incredibly destructive effects on agriculture. Part of its success relies on the ability of the thousands of seeds that each plant produces to only germinate when within 1mm of a host plant (such as wheat). These parasites are then able to connect their vasculature with that of the host via the formation of a structure called the haustorium. His lab has established P. japonicum has a model to study this type of infection. Meanwhile, Angela Hay (Max Planck Institute, Cologne) tried to understand the biology and mechanics behind the explosive seed dispersal method used by C. hirsuta, which can launch seeds up to 2 metres from the mother plant. Finally, Steve Smith (University of Western Australia) talked about karrikins, compounds that are present in the soil following forest fires, and which are able to trigger germination and influence seed photomorphogenesis.

Interdisciplinary science can apply to many biological questions. This conference highlighted just that, with collaborations with other fields helping solve questions in a wide range of biological problems, from basic developmental questions to environmental problems and crop issues. Interdisciplinarity will probably have an increasingly strong presence in Biology, and this meeting shows that plant researchers are at the forefront of this new trend.

One of the conference’s dinners, at Downing college

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Here are the highlights from the current issue of Development:

Small (molecule) steps to making bone

The repair of cartilage and bone following damage remains a clinical challenge. Current cell-based therapies rely mostly on adult mesenchymal stromal cells, but the expansion of these into correctly differentiated and functionally competent chondrocytes, which give rise to cartilage and then bone, remains problematic. Here, Naoki Nakayama and colleagues develop a small molecule-based approach that mimics the embryonic somitic chondrogenesis programme and can be used to differentiate mouse embryonic stem cells (ESCs) into chondrocytes in vitro (p. 3848). The authors first show that activation of Wnt signalling using a small molecule inhibitor of Gsk3 (CHR99021), together with inhibition of BMP signalling using a BMP type I receptor inhibitor (LDN193189), is sufficient to induce ESCs to form paraxial mesoderm-like progeny. This population, they report, expresses trunk paraxial mesoderm and somite markers but fails to express markers of sclerotome, which gives rise to cartilage. However, knowing that sonic hedgehog (Shh) and the BMP antagonist noggin are required for sclerotome induction in vivo, the researchers then demonstrate that short-term treatment of the mesodermal progeny with an Shh receptor agonist (SAG1) and the BMP inhibitor LDN193189 results in a sclerotome-like intermediate, leading to functional chondrocyte formation. When ectopically transplanted into immunocompromised mice, these chondrocytes were able to mineralise and form pieces of bone that contain marrow. This readily scalable and chemically defined method for directing chondrogenesis thus offers a promising approach for cartilage-mediated bone regeneration.

OTX2 gets a head start

The gene orthodenticle homologue 2 (Otx2) encodes a paired-type homeodomain transcription factor that is known to play a role in head morphogenesis. In the mouse, Otx2 is expressed in the anterior neurectoderm, where it is required for the differentiation of anterior neural tissues. Otx2 is also expressed in the anterior mesendoderm (AME) but its role here is unknown. On p. 3859, Patrick Tam and co-workers investigate the role of Otx2 in the AME. Using Otx2 AME conditional knockout embryos, the researchers show that Otx2 activity in the AME is essential for head formation. They further demonstrate that the expression of Dkk1 andLhx1, which are known regulators of head formation, is impaired in the AME of the Otx2conditional knockout embryos. Dkk1 is a direct target of Otx2, and the researchers further identify regulatory regions in the Lhx1 locus to which Otx2 can bind, suggesting that Lhx1 is also likely to be a direct target of Otx2. Finally, the analysis of AME-specific Otx2;Lhx1 andOtx2;Dkk1 compound mutant embryos reveals that Otx2 acts synergistically with Lhx1 andDkk1 in the AME during head formation. In summary, these findings uncover a crucial role for Otx2 during head and forebrain development.

A new TALE of PU.1 function

Numerous transcription factors (TFs), including PU.1 and Scl, are known to play important roles during haematopoiesis, but how these act within wider TF networks is unclear. Now, Berthold Göttgens and colleagues use transcription activator-like effectors (TALEs) to manipulate the expression of PU.1 and Scl and determine how these TFs function during developmental haematopoiesis (p. 4018). They first show that the modulation of PU.1 expression affects cell fate decisions during embryoid body haematopoiesis; PU.1 upregulation, for example, drives haematopoietic commitment but causes a loss of proliferative ability, whereas PU.1 repression inhibits the maturation and differentiation of early haematopoietic cells. They further report, using single-cell gene expression analyses, that TALE-induced PU.1 expression is associated with changes in the expression of several other haematopoietic genes, suggesting that early activation of PU.1 expression drives a haematopoietic programme at the expense of endothelial gene expression. Following on from this, the researchers show that the PU.1-14kb enhancer is active in the mid-gestation dorsal aorta in vivo, and that PU.1 is detectable in the early haemogenic endothelium. Together, these studies uncover a novel role for PU.1 during haematopoietic specification and highlight the use of TALEs in understanding developmental TF networks.

Modelling morphogen-controlled gene expression

Pattern formation during development often depends on the differential regulation of gene expression in response to a morphogen gradient, but how such gradients govern gene expression is unclear. A simplified view suggests that the morphogen activates a transcriptional activator, and that differential gene expression is dependent on the affinity or number of binding sites for this activator within target genes. However, this model does not account for bifunctional transcriptional effectors – those that function as activators and repressors – and has also been questioned by recent experimental results. Here, James Briscoe and colleagues describe a unifying mathematical model of morphogen-dependent gene expression that can explain recent counterintuitive findings (p. 3868). Using sonic hedgehog (Shh)-dependent patterning of the mouse neural tube as an example, the researchers develop mathematical models, based on statistical thermodynamic principles, that account for competitive binding of the active and repressive isoforms of Gli, the transcriptional effector of Shh, and that also represent other inputs that are known to regulate Shh target gene expression. Their modelling predicts that, for each Gli target gene, there is a neutral point in the Shh gradient, either side of which altering Gli binding affinity has the opposite effect on gene expression. They further report that inputs other than the morphogen determine the transcriptional response. Together, these analyses help reconcile conflicting results in the field and provide a theoretical framework that can be used to examine differential gene expression in other contexts.

PLUS…

 

The T-box gene family: emerging roles in development, stem cells and cancer

The T-box family of transcription factors exhibits widespread involvement throughout development in all metazoans. Here, Virginia Papaioannou provides an overview of the key features of T-box transcription factors and highlights their roles and mechanisms of action during various stages of development and in stem/progenitor cell populations. See the Primer on p. 3819

 

New insights into the maternal to zygotic transition

The initial phases of embryonic development occur in the absence of de novo transcription and are instead controlled by maternally inherited mRNAs and proteins.  Following this period of transcriptional silence, zygotic transcription begins, the maternal influence on development starts to decrease, and dramatic changes to the cell cycle take place. Here, Steven Harvey and colleagues discuss recent work that is shedding light on the maternal to zygotic transition. See the Review on p. 3834

 

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To form complex organs, somatic stem cells proliferate and then differentiate during development. In this process, intrinsic factors, i.e. the sequential expression of transcriptional genes, and extrinsic factors, i.e. extracellular microenvironment, are intimately involved. Recent in vitro studies have revealed that the physical properties of the extracellular niche, possibly tissue stiffness, may play an important role in cellular behavior, growth and differentiation. This is referred to as “mechanotransduction”. However, the procedure by which physical cues are sensed and translated into gene expression, and their physiological significance in vivo,are essentially unknown.

To understand the role of mechanotransduction in cellular behavior and fate specification during development, one of the critical points is to determine whether there are any spatiotemporal shifts in stiffness in a given developing tissue. Atomic force microscopy (AFM) is a strong tool for this purpose. Using this system, the stiffness in certain postnatal tissues including the brain has been previously examined. In our recent paper in Development, we combined this system with a structural support to measure stiffness in the embryonic mouse brain, one of the softest tissues in our body. We further combined this technique with immunostaining, to increase the spatiotemporal resolution of the measured tissue based on anatomical information (Figure 1A).

As a result, we obtained hitherto unknown results about the shift in stiffness in the developing brain tissue (Iwashita et al, 2014; Figure 1). First, stiffness in the proliferative zones including ventricular zone (VZ) and subventricular zone (SVZ) gradually increases during embryogenesis (Figure 1B). During brain development, gliogenesis starts around birth, after the neurogenic period takes place. Interestingly, previous studies showed in vitro that the lineage shift from neural to glial cells was influenced by a shift in stiffness. Our results provide an attractive scenario in which the extracellular environment, i.e. the stiffened tissue in the later stages of the embryonic brain, may be arranged for better production of glial cells in vivo.

Secondly, we found that there is a sharp peak in stiffness in the intermediate zone (IZ) and a gentle peak in the cortical plate (CP), at the middle stage of neurogenesis (Figure 1B, C). In addition, the stiffness in the IZ tends to be higher than other regions at the mid-stage of brain development. In the IZ, massive cell migration is observed during brain development. Although the physiological significance of the higher stiffness in the IZ is still unclear, it may contribute to directed migration of neural cells toward the CP.

To summarize, we described for the first time the spatiotemporal shift in stiffness that is observed in the developing brain. In this study, we measured the developing brain tissue and cellular stiffness as an experimental model. Our strategy, however, can be applied to profile various types of tissues and cells, and could help understanding the role of tissue stiffness as a physical cue for cell fate determination of somatic stem cells.

 Figure 1. Summary of spatiotemporal measurement using AFM (click image to see a bigger version)

(A) Immunohistological images of developing brains. Cortical layers consist of VZ, SVZ, IZ and CP. Preplate (PP) is a structure transiently appears in early phase of brain development. Red, Pax6; blue, Tbr2; green, Tuj1. This data is modified from Iwashita et al., 2014, Figure 2A.

(B) Schematics of the temporal shifts in stiffness in each layer. The vertical axis shows tissue stiffness. The horizontal axis shows the developmental time course.

(C) Schematics of the spatial shifts in stiffness of cortical layers in each developmental stage. The horizontal axis shows stiffness.

Misato Iwashita and Yoichi Kosodo

Iwashita, M., Kataoka, N., Toida, K., & Kosodo, Y. (2014). Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain Development, 141 (19), 3793-3798 DOI: 10.1242/dev.109637

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