As a neglectful member of this parish over the last few months/years (insert standard academic administration/teaching workload complaints here), I have the great pleasure to come out of my slumber to drum up interest in one of the best things about being (a developmental biologist) in London:
If you have been lots before, you have probably forgotten when it is (I had): this month (27th). If you have never been before, you should come because it is interesting, friendly, and all the lovely things a good conference should be. If you are a PhD student looking for a nice place to give your first talk, YEN is absolutely brilliant. A medium sized, very supportive, and very friendly audience. If you are a desperate postdoc looking to engage in much needed shameless self-promotion, it is good for that too, but you’ll need to get a move on: the abstract deadline is 5TH MAY.
See you there for science and beer (probably in that order).
This post highlights the approach and findings of a new research article published in Disease Models & Mechanisms: ‘Stem cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila’. This feature was written by Elan Strange 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. Elan’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.
A useful approach to investigating the mechanisms underlying complex diseases such as cancer involves exploring common genetic mutations. Understanding the phenotypic impact of such mutations can help to identify risk, estimate prognosis and guide treatment for specific forms of cancer. For example, screening for BRCA1 and BRCA2 mutations has been shown to be effective in determining risk of developing breast and ovarian cancer (Mavaddat et al., 2013). Similarly, Marisa et al. (2013) showed that grouping colon cancer patients into subtypes based on genetic mutations can provide a better indication of prognosis. Researching genetic mutations that correlate with oncogenesis has proven to be an invaluable means of learning more about the causes of cancer and guiding the development of new chemotherapeutics.
UVRAG, the metazoan homolog of yeast Vps38, is well characterized as a regulator of autophagy (Liang et al., 2006), a conserved mechanism by which cells digest and recycle dispensable or dysfunctional organelles and cellular components. Although loss-of-function mutations in UVRAG are known to correlate with tumorigenesis (Ionov et al., 2004) and overexpression of the protein has been shown to reduce cell proliferation (Liang et al., 2006), the precise mechanisms by which UVRAG acts as a tumor suppressor have not yet been elucidated. Given that autophagy has been shown to be involved in several types of cancer (Bento et al., 2016), the most intuitive hypothesis for the role of UVRAG in tumorigenesis implicates its autophagy-regulating function. However, this hypothesis was explored by Knævelsrud et al. (2010), who determined using qualitative and quantitative readouts for autophagy that the tumorigenicity of UVRAG mutations in colorectal cancer cell lines is independent of its role in regulating autophagy. Additionally, UVRAG has functions in DNA repair, maintenance of centrosome stability, and endocytosis (Zhao et al., 2012), all of which are implicated in cancer and could explain the role of UVRAG as a tumor suppressor. In a new study published in DMM, Nagy et al. sought to investigate the role of UVRAG as a tumor suppressor, using the fruit fly Drosophila melanogaster. Drosophila represents a powerful model for exploring the pathology and molecular mechanisms of human intestinal disorders due to the highly similar histological and cellular stress response mechanisms (specifically those involving cell proliferation and renewal) in the guts of mammals and flies.
The authors began by using RNAi silencing to study the effects of adult-onset loss of Uvrag in Drosophila intestinal stem cells (ISCs). They report that the Notch ligand Delta and Wnt ligand Wingless (Wg), two biomolecules known to be trafficked via endosomes, accumulate in intracellular compartments of ISCs lacking UVRAG, thus indicating that these cells are deficient in endosomal trafficking. They validated their RNAi silencing experiments by showing that cells expressing null alleles of Uvrag show similar patterns of Delta and Wg accumulation.
The team then analyzed the effects of Uvrag silencing on ISC proliferation and morphology. The most noteworthy observations made were an increase in ISC number and concomitant thickening of the intestinal wall, both of which are characteristics of intestinal dysplasia (a precancerous lesion, see related DMM Review). To analyze the effects of silencing Uvrag on gut function, the authors fed the flies food containing a blue dye that enabled movement through the gut to be tracked. The feces of flies with UVRAG-deficient ISCs contained less dye, indicating
that these animals retain food more efficiently. Consistent with a previous finding that proper gut function is essential for normal lifespan (Biteau et al., 2010), Nagy et al. found that the fly mutants had significantly reduced lifespan. To look at how flies with UVRAG-deficient guts respond to environmental stress, the authors treated the flies with the toxin dextran sodium sulfate (DSS), and, in a separate experiment, infected them with the pathogen Pseudomonas aeruginosa. Under both treatments, UVRAG-deficient flies were killed faster than control flies. Overall, these experiments show that UVRAG deficiency induces gut dysplasia and sensitizes the gut to external stressors.
ISCs maintain integrity of the gut by proliferating and differentiating via a process dependent on Notch, which induces differentiation by activating the well-studied kinase, target of rapamycin (TOR) (Kapuria et al., 2012). This process involves individual ISCs undergoing asymmetric cell division to produce a new ISC and an enteroblast, the latter of which can then differentiate into an enterocyte (90% of the time) or an enteroendocrine cell (Zeng et al., 2011). The authors wanted to see if Notch signaling can regulate this process in the absence of UVRAG.They report that despite the presence of Notch activity in UVRAG-deficient cells, there is a significant lack of differentiation and active TOR. Interestingly, Uvrag silencing resulted in larger and selective impairment of enteroblast differentiation into enterocytes (but not into enteroendocrine cells).
Based on a previous finding showing that JAK/STAT regulates ISC proliferation in Drosophila (Jiang et al., 2009), the authors sought to determine the activity of key proliferation/differentiation signaling pathways in UVRAG-deficient intestines. They found that while AKT and Ras-MAPK pathways were not involved, JNK activity was misregulated in UVRAG-deficient ISCs. Subsequent knockdown of the JNK homolog Basket or STAT homolog Stat92E suppressed the hyperproliferation seen in UVRAG-deficient ISCs.
The authors then looked at how Notch signaling, which has been implicated in regulating ISC differentiation (Micchelli and Perrimon, 2006), is affected by Uvrag silencing. Silencing of both Uvrag and Notch suppressed ISC differentiation, while Notch overexpression rescued the impaired differentiation phenotype induced by Uvrag silencing. To address the question of whether or not the effects of Uvrag silencing are a consequence of autophagic defects in ISCs, the authors measured levels of the trafficked nucleoporin p62 homolog Ref(2)P. No differences in the endogenous levels of Ref(2)P in UVRAG-deficient and wild-type ISCs were detected, suggesting that autophagy is not impaired in ISCs in the absence of UVRAG.
In perhaps the most revealing experiment presented in the paper, the authors expressed a dominant-negative form of the dynamin homolog (shibire) and silenced Rab7 using RNAi (both in ISCs) to inhibit early and late endocytosis, respectively. Expression of dominant-negative shibire was lethal in young flies; however, silencing of Rab7 induced gut dysplasia in a manner that mimicked Uvrag silencing. This exciting result provides compelling evidence that intestinal dysplasia induced by knocking out Uvrag expression is a result of impaired endocytic trafficking.
The goal of this study was to learn more about the pathogenic role of loss-of-function mutation of UVRAG often observed in human colorectal cancer. The authors determined that deregulation of endocytic trafficking in ISCs, driven by loss of UVRAG, leads to intestinal dysplasia in Drosophila. Given that intestinal dysplasia is a common precancerous lesion in the human gastrointestinal tract, this finding provides important insight into the potential role of UVRAG in colorectal cancer.
References
Bento, C. F., Renna, M., Ghislat, G., Puri, C., Ashkenazi, A., Vicinanza, M., Menzies, F. M. and Rubinsztein, D. C. (2016). Mammalian Autophagy: How Does It Work?Annu. Rev. Biochem.85, annurev–biochem–060815–014556.
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.
Here are the highlights from the current issue of Development:
Making inroads into spermatogonial differentiation
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
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
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
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
In 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
Compared 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
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.
– 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!
– 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.
Also on the Node
– 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!
Here is some developmental biology related content from other journals published by The Company of Biologists.
New neural crest EMT reporter
Stewart 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.
A 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].
CPEB1 and DAZL cooperate in oocytes
Conti 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.
Septate 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.
Surviving hypoxia
Rundle 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.
Planar 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].
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!
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.
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.
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In 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. 
Compared 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. 
– 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 
– Plagiarism, data manipulation, author disputes… what are the 
Stewart 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 
A role for LRP2 in cardiac development
Conti 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 
Septate junction formation in Drosophila midgut
Rundle 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 
Planar Cell Polarity
