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Postdoctoral Research Associate (Hendrich Lab)

Posted by , on 3 May 2016

Closing Date: 15 March 2021

Department/Location: Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, University of Cambridge

Salary: £28,982-£29,847

Reference: PS08402

Closing date: 22 May 2016

Fixed-term: The funds for this post are available for 2 years in the first instance.

The Wellcome Trust – Medical Research Council Stem Cell Institute at the University of Cambridge provides outstanding scientists with the opportunity and resources to undertake ground-breaking research into the fundamental properties of mammalian stem cells (http://www.stemcells.cam.ac.uk/).

Transcriptional control of lineage decisions in embryonic stem cells.

Applications are invited for a postdoctoral position to investigate the molecular control of embryonic stem cell lineage commitment and differentiation. The successful applicant will be part of an interdisciplinary collaboration between The Cambridge Stem Cell Institute and Microsoft Research to understand how information is processed by individual stem cells to bring about cell fate decisions.

For this position demonstrated experience in the analysis of transcriptional mechanisms will be required. The candidate is expected to have considerable expertise in molecular biological and biochemical techniques, basic mammalian cell culture, and to be familiar with basic programming and computational methods. Previous experience in higher-level programming, mammalian stem cell biology, and/or chromatin biochemistry is highly desired. The position will be based in the Hendrich laboratory and is available immediately.

You should have been awarded a PhD degree or equivalent and have several years laboratory experience.

To apply online for this vacancy and to view further information about the role, please visit: http://www.jobs.cam.ac.uk/job/9561. This will take you to the role on the University’s Job Opportunities pages. There you will need to click on the ‘Apply online’ button and register an account with the University’s Web Recruitment System (if you have not already) and log in before completing the online application form.

The closing date for all applications is the Sunday 22 May 2016.

Please upload your Curriculum Vitae (CV) and a covering letter in the Upload section of the online application to supplement your application. If you upload any additional documents which have not been requested, we will not be able to consider these as part of your application.

Informal enquiries are also welcome via email to: Dr Brian Hendrich Brian.Hendrich@cscr.cam.ac.uk, Dr Sara-Jane Dunn Sara-Jane.Dunn@microsoft.com or to jobs@stemcells.cam.ac.uk.

Interviews are most likely to take place at the end of May 2016.

Please quote reference PS08402 on your application and in any correspondence about this vacancy.

The University values diversity and is committed to equality of opportunity.

The University has a responsibility to ensure that all employees are eligible to live and work in the UK.

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In Development this week (Vol. 143, Issue 9)

Posted by , on 3 May 2016

Here are the highlights from the current issue of Development:

 

Making inroads into spermatogonial differentiation

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Differentiation of spermatogonial cells is a crucial part of spermatogenesis. Many of the key signalling pathways and molecules that are involved in spermatogonial differentiation have been identified, but their precise function at the cellular level as well as their downstream targets are not well understood. In this issue (p. 1502), Ming-Han Tong and colleagues address this with an in-depth look at the role of retinoic acid (RA) in spermatogonial differentiation. The authors specifically block retinoid signalling by introducing a dominant-negative mutant of RA receptor alpha (RARα) targeted to the spermatogonia of the transgenic mice. With this model, they show how a lack of RA signalling completely blocks spermatogonial differentiation in homozygous mice, which is due to the arrest of the undifferentiated cells in the G1/S phase. The authors then use RNA-Seq to probe for possible downstream targets of RA signalling in this context, and identify a role for replication-dependent core histone genes in promoting spermatogonia differentiation. These data make a significant contribution to our understanding of the mechanisms underlying spermatogonial differentiation, and the creation of a novel mouse mutant will be a valuable tool for the field.

 

Surprising role for CP110 in cilia biogenesis

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Primary cilia are antenna-like cellular organelles that act as sensory receptors and also play an important role in signal transduction. Formation of these structures occurs as cells exit the cell cycle, whereupon centrioles migrate to the apical domain and become the basal bodies that anchor the new cilia as it forms. Centrosomal protein CP110 is a crucial regulator of centriolar division during the cell cycle and is thought to act as a key suppressor of ciliogenesis, based on in vitro studies. In this issue (p.1491), Anand Swaroop and colleagues add a new twist to this theory and show that, in vivo, the absence of CP110 results in a failure to make cilia in a Cp110−/− mouse model. The authors show that ablation ofCp110 causes lethality shortly after birth due to organogenesis defects that are similar to those observed in ciliopathies. Using serum-starved embryonic fibroblasts derived from Cp110−/− mice, they further demonstrate a failure of basal body docking to membranes during cilia formation. These data challenge the prevailing view and demonstrate a more complex role of CP110 in the ciliogenic pathway, and highlight the importance of in vivo studies for our understanding of ciliogenesis in a physiologically relevant setting.

 

New model for organ growth termination

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Robust growth termination is essential to ensure that organs reach their correct size and grow no further. The precise mechanism of growth termination and the relative contributions of reduced cell proliferation and increased cell differentiation are elusive, and it is not known to what extent these mechanisms may be conserved in different evolutionary contexts. In this issue (p. 1482), Dagmar Iber, Fernando Casares and colleagues combine quantitative three-dimensional measurements with mathematical modelling to investigate growth dynamics in the Drosophila eye disc. The authors show that, much as in other organs and species, the growth rate declines continuously in the eye disc. Moreover, they computationally evaluate how well different candidate growth laws fit with the observed kinetics of organ growth and differentiation, and find that both an exponential and an area-dependent decline in the growth rate fit the data, although the latter offers the most parsimonious explanation. By testing this model prediction in a Drosophila strain with smaller eyes, they confirm experimentally that the area growth rate declines inversely proportional to the total eye disc area, even when the growth rates and relative areas are very different. The area-dependent growth mechanism proposed by the authors is an alternative model to explain the still unresolved issue of how organs know when to stop, and to stop consistently.

 

Improved protocol for purification of differentiated hepatocytes

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Directed differentiation of pluripotent stem cells (PSCs) into hepatocyte-like cells (HLCs) shows great promise for disease modelling as well as regenerative medicine. Unfortunately, current differentiation protocols result in heterogeneity in differentiation efficiency as well as the production of immature and undesirable cell types. In this issue (p. 1475), Chad Cowan and colleagues report an in-depth transcriptional and functional analysis of mature HLCs purified using the membrane marker asialoglycoprotein receptor 1 (ASGR1) from amidst the heterogeneous population of differentiating cells. The authors perform microarray profiling as well as functional assays for albumin and urea secretion and cytochrome activity, and find that the ASGR1+ cells exhibit a gene profile and functional characteristics similar to primary human hepatocytes, as compared with the HLCs negative for ASGR1. Although the cells isolated by this method are not perfect mimics of primary adult hepatocytes, the observed increase in homogeneity represents a substantial improvement in the differentiation of HLCs. This approach might therefore serve as a means to overcome the variation in the efficiency of HLC differentiation when starting from different PSC lines.

 

PLUS…

 

Heartbreak hotel: a convergence in cardiac regeneration

DEV1435In February 2016, The Company of Biologists hosted an intimate gathering of leading international researchers at the forefront of experimental cardiovascular regeneration, with its emphasis on ‘Transdifferentiation and Tissue Plasticity in Cardiovascular Rejuvenation’. As discussed by Michael Schneider, participants at the workshop revealed how understanding cardiac growth and lineage decisions at their most fundamental level has transformed the strategies in hand that presently energize the prospects for human heart repair. See the Meeting Review on p. 1435

 

Plant regeneration: cellular origins and molecular mechanisms

DEV1442Compared with animals, plants generally possess a high degree of developmental plasticity and display various types of tissue or organ regeneration. Here, Keiko Sugimoto and colleagues summarise how plants control these various types of regeneration and how developmental and environmental constraints can influence plant regenerative regulatory mechanisms. See the Review on p. 1442

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This month on the Node- April 2016

Posted by , on 3 May 2016

Here are some of the posts we featured on the Node in the last month!

 

Research

– Chen-Hui wrote about skinbow,  a new system to study cell dynamics during epithelial regeneration in zebrafish.

– Our latest evo devo post was by Andrew, who discusses his recent Development paper highlighting the similarities between Shh in the gill arches and the tetrapod limb.

– Icha shared a recent video protocol on how to use light sheet microscopy to image zebrafish eye development.

– Mark described  how high pressure freezing can be used to study the Drosophila trachea.

– And we highlighted two classic papers recommended by James Briscoe that examine how the mouse embryo regulates its size.

 

20160413-Chen-fin

 

Meetings

Valeria and Mathew– Last month we were at the Spring Meeting of the British Society for Developmental Biology, where we interviewed the winner of the Beddington Medal for best PhD thesis Elena Scarpa, whose thesis work focused on contact inhibition in the neural crest. We also featured a new instalment of our ongoing poster interview chain, with SDB poster winner Valeria Yartseva interviewing BSDB poster winner Mathew Tata. If you weren’t at the meeting you can check out the full list of award winners here, and several of the talks, including the Waddington Medal Lecture by Enrico Coen and the Cheryll Tickle Medal lecture by Abigail Tucker are now available on YouTube!

-Also last month, Isabella reported from the Canadian Developmental Biology conference, which took place in Banff, Alberta.

– How do you mend a broken heart? A recent Company of Biologists workshop brought together experts in the field of heart development, regeneration and tissue engineering to discuss this topic, and Juliane wrote for the Node about it!

 

Interviews

– Last month we featured two connected interviews. The first one was with limb developmental biologist Cheryll Tickle, who told us about how the field changed during her long career as a developmental biologist. Our second interview was with  Abigail Tucker, the winner of the first Cheryll Tickle medal. Abigail told us about her work on craniofacial research, the challenges and rewards of working with funky critters and the importance of science outreach.

– We also featured an interview with Drosophila genetics pioneer Gerry Rubin, originally published in Disease Models & Mechanisms.

Cheryll and Abigail

 

Also on the Node

Bento DNA– Plagiarism, data manipulation, author disputes… what are the biggest ethical issues in life science publishing at the moment? And how can we prevent them? Share your thoughts!

– And Bento lab is a low-cost, portable DNA laboratory that aims to bring genetic analysis to everyone. Read more about this interesting project!

 

Happy reading!

 

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Categories: Highlights

From our sister journals- April 2016

Posted by , on 29 April 2016

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

 

CoB_DisModMech_AW_RGB

 

 

 

 

New neural crest EMT reporter

DMM EMTStewart and colleagues describe a novel neural crest EMT reporter for rapid in vivo drug screening in zebrafish. They use to identify a small-molecule EMT inhibitor that blocks this process by activating retinoic acid signaling. Read the paper here [OPEN ACCESS].

 

 

Untangling developmentally programmed obesity

5-hydroxytryptamine (5-HT) is a trophic factor whose synthesis is nutritionally regulated. Martin-Gronert and colleagues show that maternal protein restriction increases fetal brain 5-HT and might contribute to changes in production and function of hypothalamic 5-HT2C and 5-HT2A receptors in the offspring later in life. Read the paper here [OPEN ACCESS] and find out more about this work on this Node post.

 

DMM heart fieldA role for LRP2 in cardiac development

DeRuiter and colleagues examine the role of the second heart field and neural crest cells in outflow tract formation in the mouse embryo. They show that depletion of the LPR2 results in a disturbed contribution pattern and subsequent common arterial trunk. Read the paper here [OPEN ACCESS].

 

 
Journal typography

 

 

 

 

CPEB1 and DAZL cooperate in oocytes

JCS179218S1Conti and co-workers devise a new strategy to quantify the ongoing translation of specific mRNAs by measuring the extent of their co-immunoprecipitation with tagged ribosomes. Using this method, they show that the RNA-binding proteins DAZL and CPEB1 cooperate to regulate mRNA translation and protein synthesis during the meiotic cell cycle in mouse oocytes. Read the paper here.

 

JCS midgutSeptate junction formation in Drosophila midgut

Izumi, Furuse and colleagues show that a tetraspanin family protein, Tsp2A, is an essential component of septate junctions in the Drosophila endodermal epithelia and is involved in intestinal barrier function. Read the paper here.

 

 

Journal typography

 

 

 

 

Surviving hypoxia

JEBRundle and co-workers examine whether the timing of cardio-respiratory development in the marine gastropod Littorina obtusata is important for determining whether embryos survive hypoxia. They show that individuals that develop their adult cardiovascular system early survive low oxygen conditions. Read the paper here.

 

Famished bee larvae cope better with starvation in later life

Two  papers examined whether honeybees can capitalise on the experience of food shortages as larvae to prepare for times of scarcity when adults. They show that bees that experienced deprivation during development were better prepared to survive starvation in later life. Read the papers here and here.

 

 

CoB_BioOpen_AW

 

 

 

LawrencePlanar Cell Polarity

Lawrence and colleagues show that Drosophila utilises the Dachsous/Fat system differently as it develops. They also show that the localised expression of four-jointed in the tendon cells may help polarise all rows of denticles in late larval stages. Read the paper here [OPEN ACCESS].

 

 

 

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Developmental Biology, Neuroscience and Reproduction PhD Projects in New Zealand.

Posted by , on 28 April 2016

Closing Date: 15 March 2021

Applications are invited for PhD projects in the Department of Anatomy at the University of Otago, New Zealand.

Competitive PhD scholarship funding is available.

See the following link for project description and the application process. Applications due by the 17th of MAY 2016

Dunedin, Otago Peninsula & Harbour, & Pacific Ocean - aerial
Dunedin, Otago Peninsula & Harbour, & Pacific Ocean – aerial

 

http://anatomy.otago.ac.nz/phd-opportunities

St Clair, Dunedin, NZ
St Clair, Dunedin, NZ

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Question of the month- Ethical issues in life sciences publishing

Posted by , on 28 April 2016

In the last few years, the life sciences have been plagued by cases of scientific misconduct which led to corrections,  retractions and, to some extent, in a lack of trust on the scientific record. This has encompassed a variety of issues, from  manipulation to fabrication of data, from inappropriate use of statistics (unintentional or otherwise) to the inability to reproduce results, from authorship disputes to plagiarism. Some of these practices are clearly misconduct, while others may have become almost common practice under the current publishing and funding pressures. Which of these do you think is most widespread? Which do the most damage? And what can we do to prevent them? This month we are asking: 

 

What do you think are the biggest ethical issues in life science publishing at the moment?

 

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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Untangling developmentally programmed obesity: role of the serotonin system

Posted by , on 27 April 2016

This post highlights the approach and findings of a new research article published in Disease Models and Mechanisms (DMM): ‘5-HT2A and 5-HT2C receptors as hypothalamic targets of developmental programming in male rats’. This feature was written by Richard Seeber as part of a graduate level seminar at The University of Alabama (taught by DMM Editorial Board member, Prof. Guy Caldwell) on current topics related to use of animal and cellular model systems in studies of human disease. The course is designed to expose students to recent research in a variety of diseases, and for this assignment, students were asked to read and provide a scholarly summary of an assigned research article ‘in press’ at DMM. Richard’s summary was selected by the editorial team for publication at the Node. The text has been edited and shortened by DMM in conjunction with the author.

 

Over the past three decades, the global incidence of obesity, a condition characterized by excess body fat, has more than doubled. According to the WHO, obesity now affects over two billion people and is fast becoming a global epidemic. Although about 13% of the world’s population now lives with some degree of obesity, elucidating the molecular underpinnings of the disorder has proven challenging.

In 1994, the discovery of a gene encoding leptin, the so-called ‘satiety hormone’, provided a framework for explaining the mechanisms underlying obesity – in part. Leptin acts on different neuronal cell populations, including the pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus, to regulate appetite and modulate energy homeostasis. Previous research suggests that chronic overproduction of leptin results in leptin resistance, leading to decreased feelings of satiety and thereby increased risk of developing obesity (Myers et al., 2008).  Although defects in leptin signaling are important contributors to the development of obesity, these alone do not provide a complete picture of the underlying molecular mechanism, and there is evidence for leptin-independent programing of obesity. For example, low-birth-weight mice that undergo subsequent rapid growth (‘recuperation’) demonstrate adult obesity, increased food intake, and increased fat pad size, irrespective of leptin levels (Cottrell et al., 2011).

Research has uncovered such a leptin-independent mechanism of obesity: defects in 5-hydroxytryptamine (‘5-HT’ or ‘serotonin’) signaling result in lasting increases in food consumption and weight in mice (Tecott et al., 1995). Such lasting changes in food consumption and weight could be developmentally programmed by a congenital deficiency of serotonin receptors, as fetal serotonin receptor expression decreases in response to high levels of serotonin itself (Pino et al, 2004). Poor prenatal nutrition has been demonstrated to affect fetal developmental programming and has also been tied to obesity later in life, although molecular mechanisms underpinning such obesity have remained poorly characterized (Godfrey et al., 2000). Connections among prenatal nutrition, serotoninergic signaling, and feeding behavior were elucidated in a recent study published in Disease Models and Mechanisms, in which a team probed the effects of prenatal nutritional challenge followed by rapid postnatal growth on the serotoninergic system in rats (Martin-Gronert et al., 2016).

To establish rat models for further analysis, the team fed pregnant rats with a protein-restricted diet. As expected, the offspring of these nutritionally limited mothers weighed significantly less than the pups born from normally-fed control counterparts. Both experimental and control pups were allowed to nurse on normally-fed control mothers, leading to rapid ‘catch-up’ growth of the low-birth-weight pups, which were termed recuperated rats. Of note, this model is clinically correlated to observations made in human neonates, as low birth weight human babies also often experience rapid postnatal ‘catch-up’ growth (Ong et al., 2002).

To examine the effect of nutritionally-induced high 5-HT levels on expression of the corresponding receptor, 5-HT2CR (encoded by Htr2c in rats), the authors analyzed levels of Htr2c mRNA. As predicted by previous findings (Pino et al. 2004), the highly-plastic transcriptome profile of 5-HT2CR changed in response to altered levels of 5-HT in nutritionally challenged fetal and neonatal brain tissues, which displayed a significant decrease in Htr2c mRNA when compared to controls. This difference in mRNA levels abated after the nutritionally-challenged rat offspring underwent rapid growth during nursing and subsequent weaning; however, even after nursing and weaning from control mothers, nutritionally challenged pups showed significantly decreased hypothalamic 5-HT2CR protein levels. This suggests a possible explanation for why low birth weight coupled with rapid growth could have long-lasting defects in the regulation of hunger and food consumption.

To validate the biological significance of altered expression of 5-HT2CR in recuperated rats, the authors directly administered D-fenfluramine to the brains of control and recuperated rats. Previously used in the treatment of human obesity, D-fenfluramine is metabolized by the liver to D-norfenfluramine, a potent 5-HTR agonist capable of exerting anorectic effects (Gibson et al., 1993). Control rats experienced a decrease in food consumption in response to treatment; however, recuperated rats showed impaired sensitivity to D-fenfluramine-driven redunction in food intake. This suggests that alterations in serotonin receptor-mediated signaling result in resistance to pharmacological modulation of feeding behavior, possibly through lowered 5-HT2CR levels.

Next, the authors studied whether early nutritional challenge followed by rapid growth resulted in alterations to the expression of other genes that are involved in regulating appetite. Using laser-capture microdissection, the authors isolated cells of the arcuate nucleus, the hypothalamus’s ‘hunger center’, from 3-month-old control and recuperated rats and performed global transcriptome analysis on isolated samples. The team’s microarray analysis revealed several significantly upregulated and downregulated genes in recuperated mice. Importantly, the most upregulated gene identified was Htr2a, which encodes another serotonin receptor family protein, 5-HT2AR. This significant upregulation, however, does not occur until birth and subsequent nursing of nutritionally-challenged rat pups: a change in Htr2a expression wasn’t observed in neonatal and fetal brains. Thus, the increase in nutrients and sudden growth brought on by nursing could serve as a stimulus for alternative serotonin receptor production when 5-HT2cR levels are altered by poor prenatal nutrition.

The authors then sought to determine whether upregulated 5-HT2AR localized to satiety-signalling POMC neurons in the arcuate nucleus. Using in situ hybridization histochemistry, the authors demonstrated the presence of 5-HT2A receptors on POMC-expressing neurons of the hypothalamus. Given the significant increase in 5-HT2AR expression in recuperated mice and the localization of those receptors to regulatory POMC-expressing neurons in the hypothalamus, the authors suggest that the upregulation of this alternative serotonin receptor could serve to balance diminished 5-HT2C receptor levels in recuperated mice by offering an alternative mechanism through which to regulate feeding behavior. In support of this hypothesis, they show that treatment with a 5-HT2AR agonist leads to suppressed food intake in 3-month-old recuperated rats, demonstrating that these rats are sensitive to pharmacological modulation of this pathway.

Using a clinically relevant rat model of postnatal recuperation following prenatal diet restriction, Martin-Gronert et al. have offered a molecular mechanism through which feeding behavior could be altered for life through perturbations in serotonin signaling. It has previously been reported that low-birth-weight human neonates who undergo rapid postnatal growth are at increased risk for obesity; thus, this new study could provide insight to a molecular mechanism of developmentally-programmed obesity in humans. As the relative contributions of serotonin receptor subfamily-mediated and leptin receptor-mediated signaling to obesity remain unknown, future work could make use of serotonin receptor and leptin receptor double mutants, with particular attention paid to mutagenesis of various serotonin receptor subtypes. These mutants could be fed varying diets to further probe the relative contributions of each receptor type to the genotype-by-environment interactions at play in the etiology of obesity.

Additionally, the authors offered strong evidence for the upregulation of the 5-HT2A receptor, which could be a valuable druggable target in the treatment of obesity caused by disrupted 5-HT2C signaling secondary to prenatal nutritional challenge and accelerated postnatal growth. As of yet, most selective 5-HT2A receptor agonists often cause psychiatric side effects. Future efforts should be made to identify additional drug-like 5-HT2A agonists from compound libraries, which could be filtered, optimized, and then screened in vivo for improved efficacy in the treatment of obesity with less severe side effects – work to which this rat model would be amenable.

 

References

Cottrell, E.C., Martin-Gronert, M.S., Fernandez-Twinn, D.S., Luan, J., Berends, L.M. and Ozanne, S.E. (2011). Leptin independent programming of adult body weight and adiposity in mice. Endocrinology 152, 47

Gibson, E.L., Kennedy, A.J., and Curzon, G. (1993). d-Fenfluramine- and d-norfenfluramine-induced hypophagia: differential mechanisms and involvement of postsynaptic 5-HT receptors. Eur. J. Pharmacol. 242(1), 83-90.

Godfrey, K.M. and Barker, D.J.P. (2000). Fetal nutrition and adult disease. Am. J. Clin.   Nutr. 71(5), 1344-52.  

Martin-Gronert, M.S., Stocker, C.J., Wargent, E.T., Cripps, R.L., Garfield, A.S., Jovanovic, Z.J., D’Agostino, G., Yeo, G.S.H., Cawthorne, M.A., Arch, J.R.S., Heisler, L.K., and Ozanne, S.E. (2016). 5-HT2A and 5-HT2C receptors as hypothalamic targets of developmental programming in male rats. Dis. Model. Mech. 9(4), 401-12.

Myers, M.G., Cowley, M.A., and Münzberg, H. (2008). Mechanisms of Leptin Action and Leptin Resistance. Annu. Rev. Physiol. 70, 537-56.

Ong, K.K. and Dunger, D.B. (2002). Perinatal growth failure: the road to obesity, insulin resistance and cardiovascular disease in adults. Best Pract. Res. Clin. Endocrinol. Metab. 16, 191-207.

Pino, G.D., Moessner, R., Lesch, K.P., Lauder, J.M. and Persico, A.M. (2004). Roles for serotonin in neurodevelopment: more than just neural transmission. Curr. Neuropharmacol. 2, 403–417.

Tecott, L.H., Sun, L.M., Akana, S.F., StraRoles for serotonin in neurodevelopment: more than just neural transmissiock, A.M., Lowenstein, D.H., Dallman, M.F. and Julius, D. (1995). Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors. Nature 374, 542- 6.

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Scratching the surface of a rainbow

Posted by , on 26 April 2016

Chen-SEC-scale top-2

 

Why some vertebrates like salamanders and zebrafish are able to regenerate complex tissues while humans cannot is a question that has fascinated biologists for centuries. Understanding how and why regeneration occurs in these animals can inspire novel treatment strategies for regenerative medicine. At the cellular level, the regeneration process is driven by dynamic activities of cell migration, cell proliferation, and cell assimilation between old and new tissues. All of these events must be orchestrated in a precise order and at appropriate locations along the proximal-distal axis in order to restore a flawless, complex tissue (e.g. limbs or fins) from an amputation stump. With current imaging tools and platforms, it remains challenging to capture these dynamic, intricate cell behaviors in regenerating tissues from live adult vertebrates.

In 2012, my colleague in Dr. Ken Poss’s laboratory at Duke University, Vikas Gupta, had just successfully applied the “Brainbow” technique to the zebrafish heart to study cell behaviors during heart development and regeneration (Gupta and Poss, 2012). Since its debut in 2007, this elegant, multicolor cell labeling technique was mostly used to untangle the neuronal circuits in the brain (Livet et al., 2007). Vikas’s study demonstrated that this technique can also be applied to cell types other than nerve cells. At the time, biology aside, I was amazed by the beauty of the images he captured and started to wonder how I can apply this technique to fins, the tissue I study. The idea was that by tagging cells with diverse colors using the Brainbow cassette, I would be able to retrospectively determine contributions of distinctly labeled cells and their progeny in regenerating tissues, a key mechanistic question in understanding appendage regeneration. In addition, because fins are external, flat, and optically translucent, I might be able to uncover novel cell dynamics during regeneration by following these bar-coded cells in live animals. To label most cell types in fin tissue, I naively employed the ubiquitin promoter to drive the expression of Cre recombinase, while using the beta-actin2 promoter to drive the Brainbow cassette. To impose precise temporal control of Cre activity, I constructed a dual-inducible system that combines both the Tet-on system and an inducible Cre (CreERT2) in the transgene. The activity of Cre recombinase would require exogenous addition of both Doxycycline and Tamoxifen, limiting the possibility of leaky recombination. Such transgene design appeared to work nicely in injected, mosaic embryos.

Several months later when I began to screen through transgenic founders, I was at first disappointed to find that leaky recombination still occurred in many lines, and the expression domain of the Brainbow cassette was quite variable. However, I also noticed that progeny from one particular founder consistently displayed an unexpected, dazzling pattern (Figure 1) that was restricted to the outermost layer of the skin. Amazed by diverse hues displayed in this stable transgenic line, I assessed color stability of these labeled, post-mitotic cells by time-lapse imaging. Much to my surprise, multicolor tagging on this population of epithelial cells was rather stable, making tracing these cells over long time periods possible. Ken and I began to see that this “skin-bow” line may serve as a tool to study cell dynamics during skin turnover and regeneration. With hopes of tracing hundreds of cells in a large field of view, we were very fortunate to team up with two terrific quantitative biologists: Stefano Di Talia, who at the time just had established his laboratory at Duke, and Alberto Puliafito, a postdoctoral scientist in Luca Primo’s group in Italy to tackle this challenge. Alberto developed customized algorithms to segment our images, quantify and transform diverse cell behaviors that we just had a glimpse into compelling numbers.

 

Figure 1. Fin epithelium of adult skinbow zebrafish   

 

With the skinbow system, we showed that regeneration of skin can be dissected into the most basic building block (i.e. cells), and each cell can be accurately monitored at the population level as regeneration takes place (Chen et al., 2016). Our findings identified diverse cell behaviors in response to different injuries that we would not have anticipated or discovered in fixed samples (click on video link below). The skinbow system provides a quantitative readout for studying these cell behaviors and their underlying mechanisms, many of which may be perturbed in aging, infected, or malignant skin tissues. As a proof of concept, we demonstrated that skinbow can be coupled with other transgenic lines to study cell-cell interactions during epithelial regeneration, or be employed as a screening platform to uncover molecular influences on certain cell behaviors. Among many future directions, I and others in the field are positioned to apply similar approaches (i.e. combination of cell barcoding, live imaging, and quantitative analysis) to illuminate activities of basal epithelial cells, bone cells, and mesenchymal cells in regenerating zebrafish tissues. The skinbow system might well be the first step to establish a complete, three-dimensional map of cell dynamics during vertebrate appendage regeneration. We merely scratched the surface of the subject at this point (literally!). New transgenic strains and analysis tools need parallel development to quantify cell behaviors in their respective z-positions, including in deep tissues. Nevertheless, I expect that new Brainbow cassettes that were recently developed in Jean Livet’s group (Loulier et al., 2014) would allow more flexibility in tagging and tracing different cell types in vivo, as now one can choose to paint either entire cells, or just nuclei and/or cell membranes in multicolor.

One thing I have learned to appreciate from this project is to be always on the lookout for unexpected findings, which can turn out to be more colorful than your best-laid plans.

 

See videos at: https://www.youtube.com/watch?v=xCNz1OHQ30E

More images at: https://www.flickr.com/photos/nihgov/26064937482/in/album-72157659401055954/

NIH director’s blog: https://directorsblog.nih.gov/2016/03/31/snapshots-of-life-fish-awash-in-color/

Duke Today: https://today.duke.edu/2016/03/zebrafish

The Economist: http://www.economist.com/news/science-and-technology/21695380-epidermis-now-comes-technicolor-rainbows-beginning

 

References:

Chen, C.H., Puliafito, A., Cox, B.D., Primo, L., Fang, Y., Di Talia, S., and Poss, K.D. (2016). Multicolor Cell Barcoding Technology for Long-Term Surveillance of Epithelial Regeneration in Zebrafish. Dev Cell 36, 668-680.

Gupta, V., and Poss, K.D. (2012). Clonally dominant cardiomyocytes direct heart morphogenesis. Nature 484, 479-484.

Livet, J., Weissman, T.A., Kang, H., Draft, R.W., Lu, J., Bennis, R.A., Sanes, J.R., and Lichtman, J.W. (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56-62.

Loulier, K., Barry, R., Mahou, P., Le Franc, Y., Supatto, W., Matho, K.S., Ieng, S., Fouquet, S., Dupin, E., Benosman, R., et al. (2014). Multiplex cell and lineage tracking with combinatorial labels. Neuron 81, 505-520.

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Research Assistant (Fixed Term)

Posted by , on 25 April 2016

Closing Date: 15 March 2021

Applications are invited for a 2 year Wellcome Trust funded Research Assistant position to join an international team in the Department of Genetics in central Cambridge. The project is led by Dr Ben Steventon and is aimed towards understanding the role of gene expression heterogeneity in the control of neural/mesodermal cell fate decisions during the elongation of the posterior body axis in zebrafish embryos. For further details on this position please look here.  For further details on our research, please visit steventonlab.wordpress.com

The post-holder will be involved the generation of zebrafish transgenic lines that will enable the imaging of gene-expression dynamics in vivo. In addition, they will utilize cutting-edge imaging techniques to quantify gene expression levels in situ.

The successful candiate will be a highly motivated and well-organised individual with a first degree in biological or biomedical sciences and experience in molecular biology. Experience in zebrafish genetics would be an advantage.

 

Fixed-term: The funds for this post are available for 2 years in the first instance.

The University values diversity and is committed to equality of opportunity.

The University has a responsibility to ensure that all employees are eligible to live and work in the UK.

 

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Categories: Jobs

Bioinformatics postdoctoral position at the Evolutionary and Functional Genomics LAB

Posted by , on 25 April 2016

Closing Date: 15 March 2021

The Evolutionary and Functional Genomics Lab led by Josefa González is seeking a highly motivated postdoctoral researcher to join our research team at the Institute of Evolutionary Biology (CSIC-UPF).
The postdoctoral researcher will work on a project funded by a European Research Council Consolidator Grant that aims at identifying the genetic basis, the molecular mechanisms, and the functional traits relevant for environmental adaptation.
The postdoctoral researcher will be responsible for the in silico characterization of candidate adaptive mutations identified in natural populations of Drosophila melanogaster. Among others, the tasks involved in the postdoctoral research project will be to identify pathways under selection, and to analyze the expression and the epigenetic changes of genes nearby the candidate adaptive insertions.
A PhD in Populations Genetics or a related field, good programming skills, and good writing skills are required. Previous postdoctoral experience will be considered.
We offer a full-time position for 2 years with the possibility of extension. Salary will depend on the experience of the candidate.
Starting date September 2016 but alternative dates can be discussed.

Application
Please send your CV and a brief letter of motivation before the 5th May 2016 to: josefa.gonzalez@ibe.upf-csic.es

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Categories: Jobs